Sympathetic denervation does not alter the density or properties of alpha-1 adrenergic receptors in rat vas deferens

Alpha-1 adrenergic receptors in surgically denervated rat vas deferens were studied using radioligand binding assays of [125I] BE 2254 ([125I]BE) and contraction measurements. Scatchard analysis of saturation isotherms of specific [125I]BE binding showed no change in the affinity or density of binding sites 4, 7 or 14 days after denervation of rat vas deferens. The potency of norepinephrine in inhibiting specific [125I]BE binding was also unchanged 7 days after denervation of vas deferens. The potency of phenylephrine in causing contraction in vitro did not change 4, 7 or 14 days after denervation of vas deferens; however, there was a significant increase in the maximum contractile response to phenylephrine at all time points. After partial inactivation of alpha-1 adrenergic receptors in vitro with phenoxybenzamine, there was an equivalent reduction in the number of [125I]BE binding sites in the control and 14-day denervated vas deferens. The equilibrium dissociation constants calculated from contractile measurements for norepinephrine were the same in the control and denervated tissues. However, there was a 2.2-fold increase in contractile sensitivity to norepinephrine 14 days after denervation and a 3.6-fold increase in contractile sensitivity to methacholine 7 days after denervation. These results show that surgical denervation of the rat vas deferens results in an increase in contractile sensitivity to norepinephrine and methacholine and an increase in maximum contraction. However, there is no change in alpha-1 adrenergic receptor density or properties at any time after denervation. Thus, alterations in alpha-1 adrenergic receptors do not contribute to contractile supersensitivity of denervated rat vas deferens.     

Norepinephrine and potassium induced calcium translocation in rat vas deferens

To understand calcium regulation in smooth muscle, we studied both potassium- and norepinephrine-mediated alterations in the movement of calcium in the smooth muscle of rat vas deferens. We employed 45Ca to measure agonist-mediated calcium influx and efflux, as well as tissue calcium content. In addition we labeled tissues with [3H]myoinositol to measure the effect of norepinephrine on inositol phosphate generation. Stimulation of the vas deferens with 50 mM potassium caused a rapid influx of 45Ca (6-fold). Norepinephrine stimulation, even at a concentration maximal for contraction of the tissue (1 mM), did not result in any alteration in 45Ca influx by itself but inhibited potassium-stimulated 45Ca influx (IC50 = 3 microM). This alpha receptor-mediated effect of norepinephrine was not diminished by either pretreatment with reserpine or adrenergic denervation. Studies of the efflux of 45Ca from vas deferens revealed that efflux was not affected by potassium but was significantly stimulated by norepinephrine. Alpha receptor stimulation of vas deferens smooth muscle caused a marked elevation in the appearance of inositol phosphates, particularly inositol trisphosphate, that was not dependent on extracellular calcium. We conclude that norepinephrine does not stimulate calcium influx in vas deferens smooth muscle but leads to the release of calcium from intracellular stores via formation of inositol trisphosphate and that the resulting increase in intracellular calcium may lead to inactivation of the potential-dependent calcium channel.     

Thyroid status and adrenergic receptor subtypes in the rat: comparison of receptor density and responsiveness

The density and functional responsiveness of adrenergic receptor subtypes were determined in tissues from control, hyperthyroid and hypothyroid rats. There was a decrease in sensitivity to isoproterenol in spontaneously beating right atria, electrically driven left atria and field-stimulated vas deferens associated with hypothyroidism, with no change in maximum response. Hyperthyroidism increased the potency of isoproterenol in right atria, but not in left atria or vas deferens. The maximal response to isoproterenol was greatly reduced in hyperthyroid left atria. The potency of procaterol, a partial agonist at beta adrenergic receptors in right atria, was unaltered in hyper- or hypothyroidism, although the maximum stimulation by procaterol was increased in hyperthyroidism. Scatchard analysis of specific [125I]pindolol binding showed that beta adrenergic receptor density was greater in hyperthyroidism than in hypothyroidism in left atria, right atria, ventricles, vas deferens and cerebral cortex, although the proportions of beta-1 and beta-2 adrenergic receptor subtypes did not change. There was no change in the responsiveness of alpha-1 adrenergic receptors mediating contraction of caudal artery and vas deferens or mediating [3H]inositol phosphate accumulation in cerebral cortex in hyperthyroid or hypothyroid rats, although the maximal contraction of caudal artery was significantly reduced in hyperthyroidism. Scatchard analysis of specific [125I]BE 2254 binding showed that alpha-1 adrenergic receptor density was significantly decreased in the ventricles from hyperthyroid rats and increased in the ventricles of hypothyroid rats, but was unchanged in vas deferens, caudal artery and cerebral cortex. Alpha-2 adrenergic receptor density in cerebral cortex, determined by Scatchard analysis of specific [3H] rauwolscine binding, was not altered in hyperthyroid or hypothyroid rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of alpha-1 adrenergic receptors linked to [3H]inositol metabolism in rat cerebral cortex

The properties of alpha-1 adrenergic receptors in rat cerebral cortex were examined by measuring increases in [3H]inositol metabolism in brain slices. Slices of rat cerebral cortex were incubated in the presence of 0.23 microM [3H]inositol in Krebs-Ringer-bicarbonate buffer containing 10 mM lithium chloride, and the production of water-soluble [3H]inositol phosphates was monitored after extraction and anion-exchange chromatography. Norepinephrine caused a 4- to 6-fold increase in [3H]inositol metabolism in cerebral cortical slices, and this response was blocked much more potently by the alpha-1-selective antagonist prazosin than by the alpha-2-selective antagonist yohimbine. Epinephrine and norepinephrine were both full agonists and stimulated [3H]inositol metabolism to the same extent in this system. The synthetic drugs phenylephrine and methoxamine were both partial agonists at these receptors, with intrinsic activities only 56 to 58% of epinephrine and norepinephrine. A variety of imidazoline and other partial agonists caused no measurable stimulation of [3H]inositol metabolism in this preparation. The response to norepinephrine was completely blocked by alpha adrenergic receptor antagonists with the potency order prazosin greater than BE 2254 greater than indoramin = phentolamine greater than azapetine greater than piperoxan greater than yohimbine. In the absence of calcium, basal [3H]inositol metabolism was increased, but norepinephrine caused the same 5-fold stimulation as in the presence of 2.5 mM CaCl2. The potencies of both antagonists and agonists in inhibiting or activating [3H]inositol metabolism in slices of rat cerebral cortex were highly correlated with their ability to displace the alpha-1 adrenergic receptor selective radioligand [125I]BE 2254 from specific binding sites in membrane preparations of rat cerebral cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of alpha-1 adrenergic receptor density and functional responsiveness in rat brain

The effects of changes in norepinephrine availability on alpha-1 adrenergic receptor density and responsiveness were determined in several regions of rat brain. Receptor density was determined from specific 125I-BE 2254 binding, and responsiveness was determined by norepinephrine-stimulated increases in inositol phosphate accumulation and in cyclic AMP accumulation in the absence or presence of adenosine in slices where beta adrenergic receptors had been inactivated. Adrenergic input was reduced by destroying noradrenergic neurons with DSP-4, depleting amines with chronic reserpine treatment or blocking alpha-1 adrenergic receptors with chronic prazosin treatment. All three treatments caused similar increases in alpha-1 adrenergic receptor density in cerebral cortex but not in striatum or hippocampus. DSP-4 treatment increased the maximal cyclic AMP response to both norepinephrine and phenylephrine but did not alter the maximal inositol phosphate response or the maximal potentiation of the cyclic AMP response to adenosine. DSP-4 treatment also increased the potency of norepinephrine in activating all three responses in cerebral cortex. Adrenergic input was increased by chronic treatment with desmethylimipramine to block norepinephrine reuptake, chronic i.c.v. infusion of norepinephrine or chronic yohimbine treatment to block presynaptic autoreceptors. None of these treatments caused significant changes in alpha-1 adrenergic receptor density or functional responsiveness in any region studied. The results suggest that alpha-1 adrenergic receptor density and responsiveness in rat brain can be increased but not easily decreased. They also suggest that different brain regions are affected differently by alterations of adrenergic input and that the different responses are not coregulated.     

Reserpine-induced postjunctional supersensitivity in rat vas deferens and caudal artery without changes in alpha adrenergic receptors

Caudal artery and vas deferens from rats treated chronically with reserpine (1 mg/kg/day i.p.) were used to study drug-induced postjunctional supersensitivity in smooth muscle. There was no change in contractile sensitivity of the rat vas deferens after 1 day of reserpine treatment; however, there were similar increases in sensitivity 4 and 7 days after reserpine treatment. The potencies of phenylephrine, methacholine and potassium chloride in causing contraction of the vas deferens were significantly increased by 4.9-, 19.5- and 1.23-fold, respectively, after 7 days of chronic treatment. This treatment also increased the potencies of phenylephrine, serotonin and potassium chloride in causing contraction of rat caudal artery by 1.8-, 1.7- and 1.23-fold, respectively; however, the potency of clonidine was unchanged after 7 days of reserpine treatment. There was no change in sensitivity to phenylephrine 1 day after reserpine treatment but sensitivity was significantly increased after 4 days. There was no significant change in maximum contractile response in either tissue to any of these agents after chronic reserpine treatment. Scatchard analysis of saturation isotherms of specific [125I]BE 2254 binding to membrane fractions from rat vas deferens and caudal artery showed no change in the density or affinity of alpha-1 adrenergic receptors after 1, 4 or 7 days of chronic reserpine treatment. In addition, no change was observed in specific [3H]rauwolscine binding to either tissue after 7 days of chronic reserpine treatment, suggesting no change in alpha-2 adrenergic receptors. These data indicate that changes in alpha adrenergic receptors are not involved in postjunctional supersensitivity of smooth muscle caused by chronic reserpine treatment.     

Napamezole, an alpha-2 adrenergic receptor antagonist and monoamine uptake inhibitor in vitro

Napamezole (2-[3,4-dihydro-2-naphthalenyl)methyl]-4,5-dihydro-1H- imidazole-monohydrochloride) is a selective alpha-2 adrenergic receptor antagonist and a monoamine re-uptake inhibitor in vitro. The alpha adrenergic antagonist activity of napamezole was determined in vitro in rat brain receptor binding assay using [3H]clonidine and [3H]prazosin for alpha-2 and alpha-1 receptors, respectively. The Ki values for napamezole were 28 nM (alpha-2) and 93 nM (alpha-1). The relative potencies for inhibiting [3H]clonidine binding were: phentolamine greater than idazoxan greater than napamezole greater than mianserin greater than yohimbine greater than piperoxan greater than rauwolscine greater than tolazoline much greater than prazosin; and for inhibition [3H]prazosin binding they were: prazosin greater than phentolamine greater than mianserin greater than napamezole greater than yohimbine greater than idazoxan greater than tolazoline. Alpha adrenoceptor antagonism was also assessed in the isolated rat vas deferens. Napamezole reversed clonidine-induced decreased in twitch height in the electrically stimulated rat vas deferens (alpha-2 antagonism with a Kb of 17 nM). The rank order of potency as an alpha-2 antagonist relative to other compounds was phentolamine greater than idazoxan greater than yohimbine greater than piperoxan = napamezole greater than mianserin much greater than prazosin. Napamezole also antagonized methoxamine-induced contractions (alpha-1) of the rat vas deferens with a Kb of 135 nM. The rank order of potency of these compounds as alpha-1 antagonists was prazosin greater than phentolamine greater than mianserin greater than yohimbine greater than napamezole greater than idazoxan.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacologic characterization of SK&F 104078, a novel alpha-2 adrenoceptor antagonist which discriminates between pre- and postjunctional alpha-2 adrenoceptors

SK&F 104078 has been reported to be a moderately potent antagonist at postjunctional alpha-2 adrenoceptors in canine saphenous vein, rabbit saphenous vein and canine saphenous artery (KB = 76-150 nM). In contrast, SK&F 104078 has been found to have essentially no affinity for prejunctional alpha-2 adrenoceptors in the guinea pig atrium. To characterize further the pharmacology of SK&F 104078 we have examined its effects in several additional alpha-2 adrenoceptor models and on several non alpha adrenoceptor-mediated vascular responses. SK&F 104078 does not block the neuroinhibitory effect of alpha methylnorepinephrine in the guinea pig ileum. In contrast, in the rat vas deferens, high concentrations of SK&F 104078 (3-30 microM) antagonized the neuroinhibitory effect of UK 14,304; however, the antagonism was not competitive. At concentrations up to 1 microM, SK&F 104078 did not potentiate [3H]overflow from guinea pig vas deferens or guinea pig atrium prelabeled with [3H]norepinephrine, indicating no prejunctional alpha-2 adrenoceptor blocking activity in these tissues. SK&F 104078 is a competitive antagonist at alpha-1 adrenoceptors, and at 5-hydroxytryptamine2 receptors, as demonstrated by blockade of norepinephrine- and serotonin-induced contraction in the rabbit aorta, with KB values of 155 and 20 nM, respectively. At concentrations up to 10 microM, SK&F 104078 does not depress angiotensin II-induced contraction of rabbit aorta. At 1 microM, no depression of the response to Ca++ in depolarized rabbit aorta is observed, although a significant inhibition of this response is seen at 10 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-1 adrenergic receptor: binding and phosphoinositide breakdown in human myometrium.

Alpha-1 adrenergic receptors were examined in both inner and outer layers of human pregnant myometrium using radioligand binding of [3H]prazosin. [3H]prazosin bound rapidly and reversibly to a single class of high affinity binding sites in myometrial membrane preparations. Scatchard analysis gave similar values of equilibrium dissociation constants in both myometrial layers. In contrast, more alpha-1 adrenergic receptors were detected in the outer layer than in the inner layer. Antagonist inhibited [3H]prazosin binding with an order of potency of prazosin greater than phentolamine greater than idazoxan. Competition experiments have also revealed that a stable guanine nucleotide decreases the apparent affinity of norepinephrine for myometrial [3H]prazosin binding sites. The functional status of these alpha-1 adrenergic receptors was also assessed by measuring the norepinephrine-induced accumulation of inositol phosphates in myometrial tissue. Norepinephrine produced a concentration-dependent accumulation of inositol phosphates in both myometrial layers. However, norepinephrine-induced increases in inositol 1,4,5-triphosphate were only observed in the outer layer. These results indicate that alpha-1 adrenergic receptors in human myometrium at the end of pregnancy are linked to phosphoinositide hydrolysis and that this response occurs mainly in the outer layer.     

CH-38083, a selective, potent antagonist of alpha-2 adrenoceptors

The selectivity and specificity of CH-38083 [7,8-(methylenedioxi)-14-alpha-hydroxyalloberbane HCl], a berbane derivative for alpha adrenoceptors has been studied and compared with yohimbine and idazoxan in peripheral tissues and in the central nervous system. In isolated tissue experiments CH-38083 was a competitive antagonist at presynaptic alpha-2 adrenoceptors on the axon terminals of the rat vas deferens (pA2 against xylazine = 8.17 +/- 0.06) and of the longitudinal muscle strip of guinea pig ileum (pA2 against xylazine = 8.07 +/- 0.20). As far as its postsynaptic alpha-2 adrenoceptor antagonistic activity is concerned its affinity in rat vas deferens (pA2 = 4.95 +/- 0.11) against l-phenylephrine and in rabbit pulmonary artery (pA2 = 5.38 +/- 0.33 against l-norepinephrine) was markedly less than that displayed for presynaptic sites. From pA2 values obtained in rat vas deferens the calculated alpha-1/alpha-2 adrenoceptor selectivity ratios for yohimbine, idazoxan and CH-38083 were 4.7, 117.5 and 1659, respectively. CH-38083 failed to show any affinity for histamine and muscarinic receptors and it even potentiated the effect of serotonin on atropinized longitudinal muscle strip of guinea pig ileum. It enhanced the release of [3H]norepinephrine from electrically stimulated mouse vas deferens loaded previously with labeled [3H]norepinephrine. In binding studies carried out in rat brain membrane preparations using [3H]prazosin and [3H]idazoxan, the selectivity ratios (Ki alpha-1/Ki alpha-2) proved to be 32.5, 289.5 and 1368 for yohimbine, idazoxan and CH-38083, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific changes in the cholinergic system in guinea-pig vas deferens after denervation

A small rapid phase (1st phase) was distinguished from a concomitant phase (2nd phase) in contraction of guinea-pig vas deferens. The vas deferens 4 days after denervtion exhibited supersensitivity to stimulants. The supersensitivity in the 2nd phase of contraction was nonspecific, but that in the 1st phase was specific to muscarinic cholinergic agonists. The increase in sensitivity resulted in a shift of the dose-response curve to lower concentrations without significant change in the maximal response. Muscarinic cholinergic receptors in the vas deferens, determined by measuring binding of [3H]quinuclidinyl benzilate, changed after denervation. The maximal binding sites increased from 115 to 165 fmol/mg of protein with no significant change in the dissociation constant. The affinity of the receptor for agonist also did not change significantly. Studies on [3H]WB4101 binding indicated no significant change in alpha adrenergic receptors after denervation. Thus, specific supersensitivity of the cholinergic mechanism mediated by muscarinic acetylcholine receptors occurred after denervation of guinea-pig vas deferens. This increased sensitivity is discussed in relation to the amount of receptor.     

Alpha-1 adrenergic stimulation of 1,4,5-inositol trisphosphate formation in ventricular myocytes

We demonstrated previously that alpha-1 adrenergic catecholamines modulate cardiac automaticity in a manner that is dependent upon the function of a pertussis toxin sensitive guanine nucleotide binding protein (G protein). Furthermore, we demonstrated that alpha-1 adrenergic receptor stimulation promotes the accumulation of inositol monophosphate (IP1). In the present study we used high-pressure liquid chromatography to resolve individual inositol phosphate isomers formed in norepinephrine-stimulated cultured rat ventricular myocytes. Norepinephrine stimulated a rapid, transient increase in 1,4,5-inositol trisphosphate (1,4,5-IP3) which was followed by slower, sustained increases in 1,3,4-IP3, inositol bisphosphate (IP2) and IP1. IP1 was composed of two major isomers with retention times characteristic of 1-IP1 and 4-IP1. 4-IP1 was the predominant IP1 isomer formed during stimulation with norepinephrine suggesting that the polyphosphoinositides rather than phosphatidylinositol are the principal targets of norepinephrine-stimulated phospholipase C activity in the heart. This was confirmed in studies performed on myocyte membranes which demonstrated proportionately greater IP2 and IP3 (relative to IP1) accumulation in response to norepinephrine. G protein regulation of alpha-1 adrenergic-dependent inositol phospholipid hydrolysis also was examined. In myocyte membranes, guanosine-5'-0-(3-thiotriphosphate) induced the accumulation of IP2 and IP3 and was required for the stimulatory effect of norepinephrine. This response was not impaired after pretreatment with pertussis toxin. These results indicate that the myocyte alpha-1 adrenergic receptor is coupled to a polyphosphoinositide-specific phospholipase C by a pertussis toxin insensitive G protein and suggest that under certain conditions IP3 may serve an important role in alpha-1 adrenergic modulation of cardiac function.     

Pre- and postjunctional actions of hydralazine in vascular and nonvascular smooth muscle in vitro

This study investigated the in vitro prejunctional and postjunctional actions of hydralazine in vascular and nonvascular smooth muscle. Low concentrations (micromolar) of hydralazine blocked phenylephrine-induced increases in perfusion pressure in the innervated rat kidney, whereas high concentrations (greater than 10 microM) were required in the perfused, innervated rabbit ear artery. High concentrations of hydralazine were required to block phenylephrine-induced contractions of innervated rat vas deferens and anococcygeus muscle. After in vitro denervation, rabbit ear arteries became sensitive to low concentrations of hydralazine, but this was not observed in the rat vas deferens or anococcygeus muscle. Hydralazine (1-3 microM) was without effect on 3H-release from rat vas deferens, anococcygeus muscle and kidney previously incubated with [3H]norepinephrine. Hydralazine (1 microM) decreased field stimulation-induced 3H-release from [3H]norepinephrine-loaded rabbit ear arteries. The results from the rabbit ear artery confirm that in some vessels the presence of sympathetic nerve terminals can modify the postjunctional actions of low concentrations of hydralazine. However, the other vascular tissue studied (rat renal vascular bed) was sensitive to low concentrations of hydralazine while innervated. In conclusion, the existence of a postjunctional relaxant effect of hydralazine, observed in vitro at concentrations compatible with therapeutic blood levels found in humans, has been confirmed using two different vascular preparations. The relevance of the prejunctional effect of hydralazine remains to be ascertained.     

Adrenergic nerves mediate angiotensin-induced prostaglandin release in the rabbit vas deferens

The effect of sympathectomy and norepinephrine depletion on prostaglandin (PG) synthesis in response to angiotensins II and III was examined in isolated vasa deferentia of the rabbit. Either 6-hydroxydopamine or surgical denervation significantly depressed norepinephrine concentrations in the vas deferens relative to contralateral controls, thus confirming an effective sympathectomy. Guanethidine also resulted in a significant reduction in norepinephrine concentrations in the vas deferens. Base-line PGE production by the vasa deferentia was not altered by guanethidine but was attenuated by 6-hydroxydopamine and increased by surgical denervation. All the treatments reduced angiotensin-induced PGE synthesis. The effect of denervation on PGE synthesis was greater than that of norepinephrine depletion. We interpret these results to indicate that angiotensins stimulate PGE production by adrenergic nerves in the vas deferens and that released norepinephrine mediates a part of the PGE production in response to the angiotensins. The 6-hydroxydopamine experiments are consistent with the adrenergic nerves being the predominant source of PGE in this preparation.     

Receptor reserve at the alpha-2 adrenergic receptor in the rat cerebral cortex

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), an irreversible alpha-2 antagonist, was used to establish and quantitate the receptor reserve at the alpha-2 adrenergic autoreceptor mediating inhibition of [3H]norepinephrine ([3H]NE) release in rat cerebral cortical slices. EEDQ treatment had no effect on [3H]NE uptake or base-line release. Four hours after EEDQ treatment (0.8 mg/kg i.p.), the EC50 was shifted 7-fold to the right and there was a 21.5% decrease in the maximal response to the full alpha-2 agonist UK-14304. Using the double-reciprocal plot analysis, the equilibrium activation constant (KA) was calculated to be 1.41 +/- 0.8 microM. Similar analysis of alpha-2 autoreceptor response at various times after 1.6 mg/kg of EEDQ gave similar values for the KA. Therefore, evaluation of either the response of the remaining native receptors after partial irreversible inactivation or the response of newly synthesized receptors after nearly complete irreversible inactivation can be used to determine the KA of the receptor. Comparison of repopulation kinetics analyses for alpha-2 receptor response and estimated receptor number revealed that recovery of maximal response was much faster than actual receptor recovery. By examining the relationship between alpha-2 autoreceptor occupancy and response it was possible to determine that there is approximately a 60 to 70% receptor reserve; only 1.5% of the receptors need to be occupied by UK-14304 in order to obtain 50% of the maximal inhibition of [3H]NE release. The presence of a large receptor reserve must be taken into account when evaluating alpha-2 adrenergic autoreceptor regulation in the rat cerebral cortex.     

Evidence against serotonin as a vasoconstrictor neurotransmitter in the rabbit basilar artery

It has been suggested recently that serotonergic nerves distinct from the known adrenergic innervation are present in cerebral blood vessels. We have confirmed that serotonin is present in the wall of rabbit cerebral arteries using a high-performance liquid chromatography technique, with levels in the basilar artery of 0.27 +/- 0.04 microgram/g wet wt. Furthermore, adrenergic denervation in vitro with 6-hydroxydopamine, while substantially reducing norepinephrine content, did not alter serotonin levels in the basilar artery. However, it was not possible to demonstrate specific accumulation of [3H]serotonin into distinct serotonergic nerves. Both the basilar artery and ear artery (which has been shown not to be innervated with serotonergic nerves) accumulated [3H]serotonin when incubated with a low concentration (10(-8) M). However, [3H]serotonin accumulation was reduced markedly in 6-hydroxydopamine-treated vessels as well as in the presence of the norepinephrine uptake blocker, desmethylimipramine. Furthermore, pretreatment with the serotonin uptake blocker, fluoxetine, did not inhibit selectively [3H]serotonin accumulation in the basilar artery. Thus, the majority of [3H]serotonin accumulation can be attributed to adrenergic nerves. The possibility that serotonergic nerves contribute to the neurogenic constrictor response of the rabbit basilar artery was also tested. Adrenergic denervation with 6-hydroxydopamine in vitro abolished the constrictor response to transmural nerve stimulation completely, but levels of endogenous serotonin were not affected. Thus, although the presence of endogenous serotonin in cerebral arteries has been confirmed, this substance does not appear to contribute to the neurogenic vasoconstriction seen in this vessel.     

Selectivity of (+)-4-propyl-9-hydroxynaphthoxazine [(+)-PHNO] for dopamine receptors in vitro and in vivo

The intrinsic activity of the potent dopamine (DA) agonist 4-propyl-9-hydroxynaphthoxazine [(+)-PHNO] was examined in receptor binding assays for the following receptors: DA, alpha-1 and alpha-2 adrenergic, serotonin-1 and -2, neuroleptic, beta adrenergic, anxiolytic, adenosine A-1, gamma-aminobutyric acid, muscarinic and opiate. (+)-PHNO exhibited strong to moderate potencies [IC50 (nanomolars) in parentheses] in binding to DA (24), "neuroleptic" (67), alpha-2 adrenergic (77) and serotonin-1 (277) sites. The pharmacological activity of the naphthoxazine both in vivo and in vitro was contrasted with the known DA agonists apomorphine, pergolide, lisuride and 6-ethyl-9-oxaergoline in tests of DA, alpha-2 adrenergic and serotonergic function. Each compound was examined in vitro in receptor binding assays for interactions with DA, alpha-2 adrenergic, serotonin-1 and serotonin-2 receptors and for alpha-2 adrenergic activity in inhibiting field-stimulated contractions of the vas deferens of the rat. In vivo, alpha-2 adrenergic activity was assayed via measurement of mydriasis after i.v. injections in the rat, whereas serotonin activity was assayed by measuring drug-induced inhibition of 5-hydroxytryptophan accumulation, and DA activity was assessed by quantifying stereotyped behavior after both i.p. and i.v. injections. Selectivity ratios for the DA receptor were derived from effective dose values determined in these tests and demonstrated that only apomorphine was more selective as a DA agonist than (+)-PHNO in vivo. (+)-PHNO was the least active agent at the alpha-2 receptors in the vas deferens and with serotonergic mechanisms in vivo to reduce 5-hydroxytryptophan accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of sigma and phencyclidine receptor ligands with the norepinephrine uptake carrier in both rat brain and rat tail artery.

The interaction of several sigma and PCP receptor ligands with the norepinephrine uptake carrier was investigated in the rat tail artery and brain. These ligands include haloperidol; (+)- and (-) 3-(3-hydroxy- phenyl)-N-(1-propyl)piperidine (3-PPP), (+/-)-BMY 14802, [(+/-) alpha-(4-fluoro- phenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazine butanol]; (+)-SKF 10047, [(+/-)-N-allyl-N-normetazocine]; 1,3-di-ortho-tolyl-guanidine; rimcazole (BW 234U), [cis-9-[3-(3,5-dimethyl-1-piperazinyl)propyl] carbazole dihydrochloride]; and MK 801, [(+)-5-methyl-10,1 1-dihydro-5H-dibenzo[a, d]cyclohepten-5, 10-imine maleate]. Our results show that all ligands used, except 1,3-di-ortho-tolyl-guanidine, inhibit both neuronal [3H]norepinephrine accumulation in the rat tail artery and specific [3H] desmethylimipramine binding in the rat brain. Except for (+)-SKF 10047, the order of potency of the ligands used for inhibiting neuronal [3H]norepinephrine accumulation in the rat tail artery was similar to that measured for inhibition of specific [3H]desmethylimipramine binding in the rat brain. Despite these similarities, our results also suggest that haloperidol, (+)- and (-)3-PPP, MK 801, rimcazole and cocaine interact with the [3H]norepinephrine site in the rat tail artery and with the [3H]desmethylimipramine binding site in the rat brain in a complex fashion. These studies demonstrate an important action on the norepinephrine carrier by these sigma and PCP ligands in the rat tail artery and brain.     

Alpha-2 adrenergic inhibition of Ca(++)-evoked [3H]norepinephrine release from synaptosomes is blocked by depolarization

The Ca(++)-evoked release of [3H]norepinephrine was used in these studies to investigate presynaptic regulation of norepinephrine release. In hippocampal synaptosomes, previously unexposed to Ca++ during isolation and superfusion, 1.25 mM Ca++ evoked a modest (4 to 7% of total stores) release of [3H]norepinephrine with 4.5 mM [K+] present. The alpha-2 adrenergic agonist clonidine inhibited 60% of the Ca(++)-evoked [3H]norepinephrine release. The alpha-2 adrenergic antagonists idazoxan and yohimbine reversed clonidine inhibition of release whereas the alpha-1 antagonist prazosin did not. Increasing the [K+] before Ca++ exposure increased [3H]norepinephrine release, and at 20 [K+] the release increased to over 20% of total stores. However, at [K+] above 9 mM, inhibition of Ca(++)-evoked release by clonidine decreased, and by 20 mM [K+] clonidine no longer inhibited release. Release was unaffected by 5 microM idazoxan or the opiate antagonist naloxone at 15 or 20 mM [K+]. The K+ channel blockers tetraethylammonium (5 mM) and 4-aminopyridine (0.1 mM) increased Ca(++)-evoked release almost 4-fold above control (4.5 mM [K+] present). Neither clonidine nor idazoxan affected Ca(++)-evoked release with the K+ channel blockers present. Therefore, even though K+ channel blockers and 20 mM [K+] increase neurotransmitter release, it is not autoreceptor activation by released endogenous norepinephrine that is responsible for blocking alpha-2 inhibition, but the depolarization produced by these treatments. The 20 mM [K+] blockade of alpha-2 inhibition was decreased by lowering the [Ca++] in the superfusion buffer. Therefore, synaptosomal accumulation of Ca++ may partially explain the loss of alpha-2 inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological analysis of alpha-2 adrenergic mechanisms in nociception and ataxia

In order to assess the involvement of alpha-2 adrenergic receptors in nociception, the in vitro potencies of seven alpha-2 adrenergic agonists (clonidine, guanabenz, guanfacine, BHT-920, ICI 106270, xylazine and lofexidine) were compared with their ability to prevent the writhing response elicited by i.p. administration of phenyl-p-guinone. Administration of each compound elicited antinociception, and this effect was attenuated by pretreatment with the alpha-2 adrenergic antagonists, yohimbine. The potency of these compounds to cause antinociception was correlated with their potency to displace [3H]clonidine from its binding site on brain membranes and with their ability to inhibit the twitch of the electrically stimulated vas deferens, suggesting an alpha-2 involvement in the antinociceptive action. In addition to causing antinociception, administration of these agonists also impaired rotorod performance in mice. These agonists were 2.5 to 72 times more potent in inhibiting writhing than in impairing rotorod performance, and, except for ICI 106270, there was a correlation between antinociceptive and ataxic potency. ICI 106270 was a notable exception to this correlation, however, producing only minimal ataxia, which unlike the other agonists was not reversed by yohimbine. These results indicate that alpha-2 adrenergic agonists can produce antinociception and further suggest that this may be dissociable from the ataxia.