Effects of imidazoline derivatives on cholinergic motility in guinea-pig ileum: involvement of presynaptic alpha2-adrenoceptors or imidazoline receptors?

The present study investigates the possibility that imidazoline receptors mediate modulation of cholinergic motor functions of the guinea-pig ileum. For this purpose, the effects of a series of compounds with known affinity for alpha2-adrenoceptors and/or imidazoline recognition sites were examined on the cholinergic twitch contractions evoked by electrical field stimulation (0.1 Hz) of longitudinal muscle-myenteric plexus preparations. Additional experiments were carried out on ileal strips preincubated with [3H]choline, superfused with physiological salt solution containing hemicholinium-3, and subjected to electrical field stimulation (1 Hz). The stimulation-induced outflow of radioactivity was taken as an index of endogenous acetylcholine release. Alpha-methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline and xylazine caused a concentration-dependent inhibition of twitch responses (IC50 from 0.13 to 1.05 microM; Emax from 85.9 to 92.5%). Rilmenidine and agmatine were less potent in reducing the twitch activity, and the latter compound acted also with low intrinsic activity (IC50=44.9 microM; Emax=35.5%). In interaction experiments, the inhibitory action of clonidine on twitch responses was competitively antagonized by RX 821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline), idazoxan, rauwolscine, yohimbine and BRL 44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)-methyl] -4,5-dihydroimidazoline), whereas prazosin (10 microM), ARC 239 (2-(2,4-(O-methoxy-phenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl- 1,3-(2H,4H)-isoquinolindione; 10 microM) and BRL 41992 (1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo[c,f]imidazol[1,5-a]a zepine; 10 microM) were without effect. Rauwolscine antagonized the inhibitory effects of various agonists on ileal twitch activity in a competitive manner and with similar potency. Agmatine and idazoxan did not significantly modify the twitch contractions when tested in the presence of alpha2-adrenoceptor blockade by rauwolscine (3 microM) or RX 821002 (1 microM). Linear regression analysis showed that the affinity values of antagonists correlated with their affinity at the alpha2A and alpha2D binding sites as well as at previously classified alpha2A/D adrenoceptor subtypes, whereas no significant correlation was obtained when comparing the potency estimates of agonists and antagonists with the affinity at I1 or I2 binding sites. When tested on the electrically induced outflow of tritium, alpha-methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline, xylazine and rilmenidine yielded inhibitory concentration-response curves which were shifted rightward to a similar extent in the presence of rauwolscine (3 microM). In the absence of further drugs, agmatine significantly reduced the evoked tritium outflow at the highest concentrations tested (10 and 100 microM), whereas idazoxan (up to 100 microM) was without effect. When RX 821002 (1 microM) was added to the superfusion medium, neither agmatine nor idazoxan modified the evoked outflow of radioactivity. The results argue against modulation by imidazoline receptors of acetylcholine release from myenteric plexus nerve terminals. They provide evidence that compounds endowed with imidazoline-like structures affect the cholinergic motor activity of the guinea-pig ileum by interacting with presynaptic alpha2-adrenoceptors belonging to the alpha2D subtype.     

Characterization of alpha 2-adrenoceptors mediating contraction of dog saphenous vein: identity with the human alpha 2A subtype

1. In the dog saphenous vein alpha 1- and alpha 2-adrenoceptors mediate noradrenaline-induced contractions in vitro. In order to study the alpha 2-adrenoceptor in isolation, alpha 1-adrenoceptors were inactivated by treatment of tissues with the alkylating agent phenoxybenzamine (3.0 microM for 30 min) in the presence of rauwolscine (1 microM) to protect alpha 2-adrenoceptors. 2. Noradrenaline-induced contractions of tissues treated with phenoxybenzamine were antagonized competitively by the selective alpha 2-adrenoceptor antagonist rauwolscine, pKB = 8.63 +/- 0.07 (means +/- s.e. mean; n = 3), consistent with an interaction at alpha 2-adrenoceptors. 3. Noradrenaline was a full agonist at alpha 2-adrenoceptors in dog saphenous vein. By use of the method of partial receptor alkylation and analysis of concentration-effect curve data by direct, operational model fitting methods, the affinity (pKA) and efficacy (tau) were 5.74 +/- 0.07 and 7.50 +/- 1.05, respectively (n = 6). Nine other agonists which were examined each had affinities higher than noradrenaline, but with the exception of the imidazoline, A-54741 (5,6-dihydroxy-1,2,3,4-tetrahydro-1-naphthyl-imidazoline) had relatively lower efficacies. 4. To compare the alpha 2-adrenoceptor in dog saphenous vein to the human recombinant subtypes, the affinities of twenty-one compounds were estimated in functional studies in the dog saphenous vein and in radioligand binding studies for the human alpha 2A, alpha 2B and alpha 2C receptor subtypes expressed in Chinese hamster lung (CHL) cells. 5. Of twenty-one compounds examined in ligand binding studies, only nine had greater than ten fold selectivity for one human receptor subtype over either of the other two. These compounds were A-54741, oxymetazoline, guanfacine, guanabenz, prazosin, spiroxatrine, tolazoline, WB 4101 and idazoxan. In dog saphenous vein, their affinities (pKA and pKB for agonists and antagonists respectively) were: A-54741 (pKA = 8.03 +/- 0.05), oxymetazoline (pKA = 7.67 +/- 0.09), guanfacine (pKA = 6.79 +/- 0.03); guanabenz (pKA = 7.02 +/- 0.13); prazosin (pKB = 5.19 +/- 0.08), spiroxatrine (pKB = 6.59 +/- 0.04), tolazoline (pKB = 6.21 +/- 0.07), WB 4101 (pKB = 7.42 +/- 0.09) and idazoxan (pKB = 7.11 +/- 0.08). 6. Comparisons of affinity estimates for these nine compounds at the receptor in dog saphenous vein and at the human recombinant subtypes suggest that the vascular receptor is most similar to the h alpha 2A subtype; correlation coefficients (r) were 0.82 (h alpha 2A), 0.24 (h alpha 2B) and 0.04 (h alpha 2C).     

Effects of iodoproxyfan, a potent and selective histamine H3 receptor antagonist, on alpha 2 and 5-HT3 receptors

We determined the affinity and/or potency of the novel H3 receptor antagonist iodoproxyfan at alpha 2 and 5-HT3 receptors. Iodoproxyfan and rauwolscine (a reference alpha 2 ligand) (i) monophasically displaced 3H-rauwolscine binding to rat brain cortex membranes (pKi 6.79 and 8.59); (ii) facilitated the electrically evoked tritium overflow from superfused mouse brain cortex slices preincubated with 3H-noradrenaline (pEC50 6.46 and 7.91) and (iii) produced rightward shifts of the concentration-response curve (CRC) of (unlabelled) noradrenaline for its inhibitory effect on the evoked overflow (pA2 6.65 and 7.88). In the guinea-pig ileum, iodoproxyfan 6.3 mumol/l failed to evoke a contraction by itself but depressed the maximum of the CRC of 5-hydroxytryptamine (pD'2 5.24). Tropisetron (a reference 5-HT3 antagonist) produced rightward shifts of the CRC of 5-hydroxytryptamine (pA2 7.84). In conclusion, the affinity/potency of iodoproxyfan at H3 receptors (range 8.3-9.7 [1]) exceeds that at alpha 2 receptors by at least 1.5 log units and that at 5-HT3 receptors by at least 3 log units.     

In vitro evidence for increased facilitation of striatal acetylcholine release via pre- and postsynaptic NMDA receptors in hemiparkinsonian rats

The NMDA-evoked acetylcholine release from striatal slices and synaptosomes was investigated in rats subjected to unilateral injection of 6-hydroxydopamine into the substantia nigra. In slices prepared from the striatum contralateral to the lesion, the NMDA-evoked endogenous acetylcholine release was not significant at 10 microM NMDA and maximal at 100 microM NMDA (124 +/- 19%). Conversely, in slices taken from the dopamine-depleted striatum, NMDA was effective even at 10 microM (41 +/- 4%), and at 100 microM (196 +/- 24%) efficacy was nearly doubled. In synaptosomes prepared from the contralateral striatum, NMDA maximally stimulated 20 mM KCl-induced endogenous acetylcholine release at 1 microM (66 +/- 5.1%), with lower concentrations (0.01-0.1 microM) being ineffective. Conversely, in synaptosomes prepared from the dopamine-depleted striatum, NMDA maximally enhanced the K+/--evoked acetylcholine release at 0.1 microM (118 +/- 12.4%). Concentration-response curves of NMDA-evoked acetylcholine release in sham-operated rats could be superimposed on those observed in the contralateral striatum of the 6-hydroxydopamine-lesioned animals. The present data support the view of an increased glutamatergic regulation of striatal acetylcholine release via pre- and postsynaptic NMDA receptors during Parkinson's disease.     

Effect of maturation on alpha-adrenoceptor activity in newborn piglet mesentery

To characterize the mesenteric alpha1- and alpha2-adrenoceptor populations in newborn piglets, an extracorporeal circuit was established to control intestinal blood flow in 0- to 2-day old and 10- to 14-day old animals. In both groups, alpha-adrenoceptor activation was first documented by observing dose-dependent increases in mesenteric perfusion pressure after intramesenteric arterial injection of alpha-adrenoceptor agonists. In the 10- to 14-day old piglets, mesenteric vasoconstrictor responses to alpha1-adrenoceptor agonists (methoxamine and norepinephrine) and an alpha2-adrenoceptor agonist (BHT-933) were each blunted (P < 0.05, analysis of variance) by peripheral intravenous injections of prazosin (an alpha1-adrenoceptor antagonist) and yohimbine (an alpha2-adrenoceptor antagonist), respectively. The mesenteric vasoconstrictor responses to those agonists were not significantly attenuated by prazosin or yohimbine in 0- to 2-day old animals, nor were they blunted by YM-12617 (alpha1-adrenoceptor antagonist) or idazoxan (alpha2-adrenoceptor antagonist)--compounds that are structurally unrelated to prazosin and yohimbine, respectively. In addition, mesenteric vasoconstrictor responses to other known vasoconstrictor agents--angiotensin II, neuropeptide Y, and a thromboxane A2 mimic (U-46619)--were not effected in either age group by prazosin or yohimbine, implying these agents act independently of alpha-adrenoceptor mechanisms. These data suggest that (1) there exists functional mesenteric alpha1- and alpha2-adrenoceptor-like activity in 10- to 14-day old piglets that, in 0- to 2-day old animals, is not specifically expressed; and (2) mesenteric alpha-adrenoceptor function becomes more selective as newborn piglets mature.     

The interaction between enalaprilat and selected alpha-adrenoceptor antagonists in isolated rat tail arteries

1. Isolated perfused rat tail artery preparations were used to investigate the effects of the angiotensin converting enzyme inhibitor enalaprilat on the actions of a series of alpha-adrenoceptor antagonists. The agonist used was phenylephrine. 2. Enalaprilat (1 mumol/L) potentiated the competitive alpha 1-adrenoceptor antagonist actions of phentolamine (10-100 nmol/L) and yohimbine (0.3-3.0 mumol/L) as well as the non-competitive antagonist action of phenoxybenzamine (50-100 pmol/L). 3. The competitive alpha 1-adrenoceptor antagonist action of prazosin (1-10 nmol/L) was not affected by enalaprilat. 4. For the competitive alpha 1-adrenoceptor antagonists, including prazosin, there appeared to be an inverse relationship between antagonist potency and the extent of potentiation by enalaprilat. 5. The results support the hypothesis and angiotensin II modulates vascular smooth muscle alpha 1-adrenoceptor function.     

Pentobarbital differentially inhibits N-methyl-D-aspartate and kainate-stimulated [3H]noradrenaline overflow in rat cortical slices

1. This study examined the ability of pentobarbital to inhibit NMDA and kainate-stimulated [3H]noradrenaline ([3H]NA) overflow in rat brain cortical slices. 2. Pentobarbital inhibited NMDA-evoked [3H]NA overflow at 100 microM and greater and inhibited kainate-evoked [3H]NA overflow at 10 microM and greater. 3. The ability of pentobarbital to inhibit concentration-response curves for NMDA and kainate-evoked overflow of [3H]NA were also examined. Pentobarbital (300 microM) caused a 20% reduction in NMDA and a 50% reduction in kainate-induced maximal responses.     

The expression of functional postsynaptic alpha2-adrenoceptors in the corpus cavernosum smooth muscle

1. The purpose of this study was to determine if corpus cavernosum smooth muscle expresses functional postsynaptic alpha2-adrenoceptors (AR). 2. The alpha2-adrenoceptor agonist UK 14,304 elicited concentration-dependent contractions in rabbit corpus cavernosum smooth muscle (CCSM). The half-maximal response occurred at 0.32+/-0.03 microM and the maximum contraction at 10 microM UK 14,304. 3. Pretreatment of CCSM strips with selective alpha2-adrenoceptor antagonists, rauwolscine and RS-15385, produced rightward shifts in the dose-response curves to UK 14,304 (pA2 values 7.1 and 8.5, respectively). In contrast, these antagonists did not alter contraction induced by the alpha1-adrenoceptor agonist phenylephrine (PE) or oxymetazoline. UK 14,304-induced contractions were also inhibited by prazosin (pA2 = 9.08). 4. UK 14,304-induced contractions, unlike those to PE, were highly dependent on the presence of extracellular Ca2+. 5. [3H]-rauwolscine bound to CCSM membranes with high affinity (Kd = 1.5 nM). [3H]-rauwolscine binding was displaced by unlabelled rauwolscine, RS-15385, UK 14,304 and prazosin, but not by PE. 6. UK 14,304 inhibited forskolin and prostaglandin E1 (PGE1)-induced increases in intracellular cyclic AMP concentration in primary cultures of rabbit CCSM cells. 7. These results demonstrate that CCSM expresses Gi-coupled postsynaptic alpha2-adrenoceptors, and activation of these receptors causes contraction of trabecular smooth muscle.     

Desmoplastic cerebral astrocytoma of infancy: a case report

Strips of human right atrial appendages were preincubated with [3H]noradrenaline and then superfused with physiological salt solution containing inhibitors of uptake1 and uptake2. Tritium overflow was evoked by transmural electrical stimulation (standard frequency: 2 Hz). Prostaglandin E2 (PGE2) inhibited the electrically evoked tritium overflow; at the highest concentration investigated, tritium overflow was reduced by about 80% and the pIC50% value was 7.14. The effect of PGE2 was not affected by rauwolscine, which, by itself, increased the evoked overflow. Naproxen failed to affect the evoked tritium overflow and its inhibition by PGE2. The inhibitory effect of PGE2 on the electrically evoked tritium overflow was mimicked by prostaglandin E1, the EP1/EP3-receptor agonist sulprostone and the EP2/EP3-receptor agonist misoprostol with the rank order of potency (pEC50%): sulprostone (7.68) > misoprostol (7.10) > PGE1 (6.39). In contrast, PGF2alpha, the IP/EP1-receptor agonist iloprost and the stable thromboxane A2 analogue U46619 (9,11-dideoxy-11alpha,9alpha-epoxy-methanoprostaglandin++ + F2alpha) did not change evoked tritium overflow. PGD2 caused facilitation. These results suggest that the sympathetic nerve fibres innervating human atrial appendages are endowed with presynaptic inhibitory EP3 and facilitatory DP-receptors. The EP3-receptors appear not to be tonically activated and do not interact with the alpha2-autoreceptors.     

Monoaminergic control of the release of calcitonin gene-related peptide- and substance P-like materials from rat spinal cord slices

The possible control by monoamines of the spinal release of substance P- and calcitonin gene-related peptide-like materials (SPLM and CGRPLM, respectively) was investigated in vitro, using slices of the dorsal half of the rat lumbar enlargement superfused with an artificial cerebrospinal fluid. Whereas the spontaneous outflow of SPLM and CGRPLM was changed by none of the agonists/antagonists of monoamine receptors tested, the overflow of both peptide-like materials due to 30 mM K+ was differentially affected by alpha 2-adrenoreceptor and dopamine D-1 receptor ligands. Noradrenaline (10 microM to 0.1 mM) and clonidine (0.1 mM) significantly reduced the K(+)-evoked overflow of SPLM, and both effects could be prevented by idazoxan (10 microM) and prazosin (10 microM) as expected from their mediation through the stimulation of alpha 2B-adrenoreceptors. In contrast, CGRPLM overflow remained unaffected by alpha 2-adrenoreceptor ligands. Dopamine D-1 receptor stimulation by SKF 82958 (10-100 nM) significantly increased the K(+)-evoked overflow of both SPLM and CGRPLM, and this effect could be prevented by the selective D-1 antagonist SCH 39166 (1 microM). Further studies with selective ligands of other monoamine receptors indicated that neither alpha 1- and beta-adrenergic receptors, dopamine D-2, nor serotonin 5-HT1A and 5-HT3 receptors are apparently involved in some control of the spinal release of CGRPLM and SPLM. These data are discussed in line with the postulated presynaptic control by monoamines of primary afferent fibres conveying nociceptive messages within the dorsal horn of the spinal cord.     

Characterization of the endothelin receptor subtype mediating epithelium-derived relaxant nitric oxide release from guinea-pig trachea

The coupling of the endogenously expressed alpha2A-adrenoceptors in human erythroleukemia cells (HEL 92.1.7) to Ca2+ mobilization and inhibition of forskolin-stimulated cAMP production was investigated. The two enantiomers of medetomidine [(+/-)-[4-(1-[2, 3-dimethylphenyl]ethyl)-1H-imidazole]HCl] produced opposite responses. Dexmedetomidine behaved as an agonist in both assays (i.e. , it caused Ca2+ mobilization and depressed forskolin-stimulated cAMP production). Levomedetomidine, which is a weak agonist in some test systems, reduced intracellular Ca2+ levels and further increased forskolin-stimulated cAMP production and therefore can be classified as an inverse agonist. A neutral ligand, MPV-2088, antagonized responses to both ligands. Several other, chemically diverse alpha2-adrenergic ligands also were tested. Ligands that could promote increases in Ca2+ levels and inhibition of cAMP production could be classified as full or partial agonists. Their effects could be blocked by the alpha2-adrenoceptor antagonist rauwolscine and by pertussis toxin treatment. Some typical antagonists such as rauwolscine, idazoxan, and atipamezole had inverse agonist activity like levomedetomidine. The results suggest that the alpha2A-adrenoceptors in HEL 92.1.7 cells exist in a precoupled state with pertussis toxin-sensitive G proteins, resulting in a constitutive mobilization of intracellular Ca2+ and inhibition of cAMP production in the absence of agonist. This constitutive activity can be antagonized by inverse agonists such as levomedetomidine and rauwolscine. Levomedetomidine can be termed a "protean agonist" because it is capable of activating uncoupled alpha2-adrenoceptors in other systems and inhibiting the constitutive activity of precoupled alpha2-adrenoceptors in HEL 92.1. 7 cells. With this class of compounds, the inherent receptor "tone" could be adjusted, which should provide a new therapeutic principle in receptor dysfunction.     

Down-regulation of P2U-purinergic nucleotide receptor messenger RNA expression during in vitro differentiation of human myeloid leukocytes by phorbol esters or inflammatory activators

To determine why renal vasoconstriction elicited by periarterial nerve stimulation (RNS) at lower frequencies (< 4 Hz) is resistant to alpha-adrenergic receptor blockade in the rat kidney, we reevaluated the effect of alpha-receptor antagonists on the vasoconstrictor response to norepinephrine (NE) and to RNS and on the release of adrenergic transmitter. The alpha-receptor antagonist prazosin (PZ) at 0.2 and 7 nM reduced the vasoconstrictor response to NE, and 2.4 microM PZ abolished it. PZ (0.2 or 7 nM) reduced RNS-induced vasoconstriction without altering the fractional tritium overflow. PZ (2.4 microM) enhanced fractional tritium overflow and reduced the vasoconstrictor response to RNS at 2-10 Hz, but not at 0.5 or 1 Hz. The effect of 0.2 nM PZ to reduce RNS-induced vasoconstriction was reversed by increasing the concentration to 2.4 microM. Corynanthine (COR; 2.6 microM), a preferential alpha-receptor blocker, or phenoxybenzamine (PBZ; 30 nM) abolished the vasoconstrictor response to NE but only partially reduced response to RNS and enhanced the fractional tritium overflow. Rauwolscine (RW; 2.5 nM), a preferential alpha 2-receptor antagonist, did not alter the vasoconstrictor response to NE but potentiated RNS-induced vasoconstriction and fractional tritium overflow. RW (7.7 microM) inhibited NE-induced vasoconstriction but potentiated the vasoconstrictor response to RNS and fractional tritium overflow. PZ (7 nM) abolished the potentiation by RW and reduced the vasoconstrictor response to RNS. These data suggest that a component of RNS-induced vasoconstriction in the rat kidney is attributable to co-release of a nonadrenergic transmitter with NE. The diminished effect of alpha-receptor antagonists at higher concentrations (e.g., PZ 2.4 microM) to reduce RNS-induced vasoconstriction is caused by their prejunctional action to enhance co-release of the nonadrenergic transmitter.     

Adrenergic responses in silent and putative inhibitory pacemaker-like neurons of the rat rostral ventrolateral medulla in vitro

Noradrenaline and adrenergic agonists were tested on pacemaker-like and silent neurons of the rat rostral ventrolateral medulla using intracellular recording in coronal brainstem slices as well as in punches containing only the rostral ventrolateral medullary region. Noradrenaline (1-100 microM) depolarized or increased the frequency of discharge of all cells tested in a dose-dependent manner. The noradrenaline-induced depolarization was associated with an apparent increase in cell input resistance at low concentrations and a decrease or no significant change at higher concentrations. Moreover, it was voltage dependent and its amplitude decreased with membrane potential hyperpolarization. Noradrenaline caused a dose-related increase in the frequency and amplitude of spontaneous inhibitory postsynaptic potentials. The alpha 1-adrenoceptor antagonist prazosin (0.5 microM) abolished the noradrenaline depolarizing response as well as-the noradrenaline-evoked increase in synaptic activity and unmasked an underlying noradrenaline dose-dependent hyperpolarizing response associated with a decrease in cell input resistance and sensitive to the alpha 2-adrenoceptor/antagonist yohimbine (0.5 microM). The alpha 1-adrenoceptor agonist phenylephrine (10 microM) mimicked the noradrenaline depolarizing response associated with an increase in membrane resistance as well as the noradrenaline-induced increase in synaptic activity. The alpha 2-adrenoceptor agonists UK-14,304 (1-3 microM) and clonidine (10-30 microM) produced only a small hyperpolarizing response, whereas the beta-adrenoceptor agonist isoproterenol (10-30 microM) had no effect. Baseline spontaneous postsynaptic potentials were abolished by strychnine (1 microM), bicuculline (30 microM) or both. However, only the strychnine-sensitive postsynaptic potentials had their frequency increased by noradrenaline or phenylephrine and they usually occurred with a regular pattern. Tetrodotoxin (1 microM) eliminated 80-95% of baseline spontaneous postsynaptic potentials and prevented the increase in synaptic activity evoked by noradrenaline and phenylephrine. Similar results were obtained in rostral ventrolateral medulla neurons impaled in both coronal slices and punches of the rostral ventrolateral medulla. It is concluded that noradrenaline could play an important inhibitory role in the rostral ventrolateral medulla via at least two mechanisms: an alpha 2-adrenoceptor-mediated hyperpolarization and an enhancement of inhibitory synaptic transmission through activation of alpha 1-adrenoceptors located on the somatic membrane of glycinergic interneurons. Some of these interneurons exhibit a regular discharge similar to the pacemaker-like neurons and might, at least in part, constitute a central inhibitory link in the baroreceptor-vasomotor reflex pathway.     

ORL1 receptor-mediated inhibition by nociceptin of noradrenaline release from perivascular sympathetic nerve endings of the rat tail artery

In the present study the effect of the opioid heptadecapeptide nociceptin, also termed orphanin FQ, an endogenous ligand for the orphan receptor named ORL1 (opioid receptor-like 1) receptor, was investigated on [3H]noradrenaline release induced by electrical field stimulation (24 pulses at 0.4 Hz, 200 mA, 0.3 ms duration) in the rat tail artery in the absence and presence of an alpha2-adrenoceptor antagonist, rauwolscine 3 microM. Nociceptin inhibited the electrically-evoked tritiated noradrenaline release in a concentration-dependent manner from rat tail arteries. This inhibitory effect of nociceptin was enhanced in the presence of the alpha2-adrenoceptor antagonist rauwolscine (maximum inhibition by 25% and 50% in the absence and presence of rauwolscine, respectively). At a supramaximal concentration (10 microM), the inhibitory action of DAGO, a selective micro-opioid receptor agonist, was less pronounced than that of nociceptin. The inhibitory effect of nociceptin was counteracted by naloxone benzoylhydrazone (3 microM) which by itself did not change the stimulation-evoked noradrenaline overflow. Naloxone (10 microM), a non-selective opioid receptor antagonist, did not affect the inhibitory effect of nociceptin whereas it abolished that of DAGO. In conclusion, these results suggest that nociceptin modulates noradrenergic neurotransmission by acting on prejunctional ORL1 receptors located on nerve terminals innervating the rat tail artery. They also demonstrate that prejunctional ORL1 receptors interact with prejunctional alpha2-adrenoceptors. The physiological significance of this phenomenon remains to be determined.     

American Pediatric Society John Howland Award 1998: presentation and acceptance

In the dog saphenous vein (DSV), phenylephrine (PE) responses through alpha-1 adrenoceptors receptors are antagonized by both alpha-1 and alpha-2 receptor antagonists. Furthermore, pretreatment with chloroethylclonidine (CEC) eliminates prazosin binding but reduces rauwolscine binding by half (). In new functional experiments, the effects of preincubation with phenoxybenzamine (PBZ), an irreversible alpha adrenoceptor antagonist, on responses to PE and two selective alpha-2 adrenoceptor agonists were evaluated. Also, the ability of prazosin or rauwolscine to prevent irreversible losses of responses to these agonists when coincubated with PBZ was determined. Preincubation in PBZ (10-300 nM) concentration dependently reduced PE Emax and the calculated fraction of residual receptors (q). Preincubation in PBZ (10-300 nM) increased KB values for prazosin (30 and 100 nM) but did not alter the KB value for rauwolscine (50 nM) acting at the residual receptors from control values. Coincubation of PBZ with prazosin partially prevented these PBZ actions (Emax partly restored) on responses to PE, but coincubation of rauwolscine (/=300 nM caused >50% reduction in Emax values of responses but did not alter the EC50 values for either agonist. Coincubation of rauwolscine with PBZ protected responses to alpha-2 agonists against PBZ (1 microM) effects. This study shows that PE initiates contractions at atypical alpha-1 adrenoceptors represented by all sites of PE action. Rauwolscine antagonizes PE actions but does not protect against PBZ inactivation. Typical alpha-2 adrenoceptors are distinguished from the unusual alpha-1 adrenoceptors by their lesser sensitivity to PBZ and their protection by rauwolscine from PBZ.     

Broad-spectrum beta-lactamases: profile and perspectives after 3 years of systematic detection at the Main Hospital of Dakar (1 February 1992 to 1 February 1995)

In the present study, the effects of adrenoceptor agonists and antagonists on baclofen-induced antinociception was investigated. Intraperitoneal administration of different doses of baclofen (2.5, 5 and 10 mg/kg) induced antinociception in the tail-flick test. The response was dose-dependent. The alpha 2-adrenoceptor agonist, clonidine, increased, while the alpha 1-adrenoceptor agonist, phenylephrine, decreased the baclofen response. In reserpine-treated animals, alpha 2-adrenoceptor, clonidine, induced antinociception and increased that of baclofen. Yohambine and propranolol but not prazosin decreased the baclofen effect. Although phenoxybenzamine itself induced antinociception it did not alter the baclofen response significantly. Reserpine treatment decreased the response induced by single administration of baclofen or phenoxybenzamine and that induced by a combination of baclofen with either phenoxybenzamine or prazosin. It may be concluded that alpha 2-adrenoceptor stimulation increases, while alpha 1-adrenoceptor activation decreases the baclofen-induced antinociception, and aminergic mechanism(s) may have a positive influence on baclofen response.     

Characterization of postsynaptic alpha-1 and alpha-2 adrenoceptors in the feline saphenous and femoral veins [corrected

Experiments were designed to characterize the effects mediated by alpha-1 and alpha-2 adrenoceptors in saphenous and femoral veins of the cat. Ring segments of saphenous and femoral veins were mounted for isometric tension recording in modified Krebs-bicarbonate solution, gassed with 95% O2-5% CO2 and maintained at 37 degrees C. Norepinephrine (a mixed alpha 1 and alpha 2 agonist), phenylephrine (a preferential alpha 1 agonist) and clonidine (a preferential alpha 2 agonist) caused dose (concentration)-dependent contractions in saphenous and femoral veins. The maximal contractions produced by clonidine were significantly less than those produced by norepinephrine or phenylephrine in both veins. However, threshold dose and EC50 values indicated that clonidine was more potent than norepinephrine and phenylephrine. Contractile responses to these agonists were attenuated when the veins were pretreated with alpha 1-or alpha 2-adrenoceptor antagonists, prazosin and yohimbine, respectively. The contractile responses to norepinephrine and tyramine were inhibited to a greater extent by yohimbine than by prazosin in both saphenous and femoral veins, suggesting that norepinephrine released from perivascular nerve terminals activates preferentially postsynaptic alpha 2-adrenoceptors. Further examination of alpha-adrenoceptor subtypes was achieved by comparing pA2 values of prazosin and yohimbine from Arunlakshana and Schild plots. Chronic sympathetic denervation by removing lumbar sympathetic chain significantly reduced the contractile responses evoked by tyramine. Denervation did not significantly affect the concentration-response curve to phenylephrine but significantly augmented the contractile responses evoked by clonidine in both veins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha 1-adrenoceptors and calcium sources in adrenergic neurogenic contractions of rat vas deferens

1. The involvement of alpha 1-adrenoceptor subtypes in adrenergic neurogenic contractions of different type was studied in epididymal and prostatic portions of the rat vas deferens. 2. The adrenergic component of neurogenic contractions was isolated by suramin (300 microM). Twitch-like and tonic contractions were elicited by appropriate pulse patterns of electrical field stimulation, and contractions relying on intracellular calcium mobilization and calcium entry were isolated by means of nifedipine (10 microM) and ryanodine (20 microM), respectively. Increasing concentrations of 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)- amino)-propyl)benzeneacetonitrile (HV 723), prazosin and 5-methylurapidil progressively, monophasically and with potency decreasing in that order reduced and finally abolished all types of contraction, with one exception: concentration-effect curves of 5-methylurapidil in epididymal segments in the presence of ryanodine levelled off at about 75% inhibition. In the presence of both nifedipine (10 microM) and ryanodine (20 microM), contractions were abolished. 3. Contractions elicited by exogenous noradrenaline were also studied in the presence of either nifedipine 10 microM (prostatic segments) or ryanodine 20 microM (epididymal segments). Increasing concentrations of tamsulosin, WB 4101, benoxathian, HV 723, prazosin, 5-methylurapidil and urapidil progressively, monophasically and with potency decreasing in that order reduced and eventually abolished both kinds of contraction, with two exceptions: in epididymal segments in the presence of ryanodine, the concentration-effect curve of 5-methylurapidil was biphasic and the curve of urapidil levelled off at only partial inhibition. 4. In slices prepared from the prostatic end and preincubated with [3H]-noradrenaline, WB 4101, HV 723, prazosin and 5-methylurapidil, at the highest concentrations tested against neurogenic contractions, increased only slightly the overflow of tritium elicited by trains of 50 pulses at 5 Hz. 5. It is concluded that two alpha l-adrenoceptor subtypes mediate adrenergic neurogenic contractions of rat vas deferens. The main one, pharmacologically alpha 1A, activates both calcium mobilization and entry. In addition there is a second receptor, not previously detected in the vas deferens and not corresponding to any named alpha l subtype, characterized by high and similar affinity for tamsulosin, WB 4101, benoxathian,HV 723 and prazosin and very low affinity for 5-methylurapidil and urapidil, and linked exclusively to calcium entry. Both subtypes and their respective transduction pathways also contribute to contractions elicited by exogenous noradrenaline. An alpha 1B-adrenoceptor-mediated contraction was not found under any experimental conditions.     

Co-release of endogenous ATP and [3H]noradrenaline from rat hypothalamic slices: origin and modulation by alpha2-adrenoceptors

The release of endogenous ATP, measured by the luciferin-luciferase assay, and of [3H]noradrenaline from the in vitro superfused rat hypothalamic slices were studied. ATP and [3H]noradrenaline were released simultaneously during resting conditions and in response to low and high frequency field electrical stimulation; the release of both substances were frequency dependent between 2 Hz and 16 Hz. The stimulation-induced release of ATP and [3H]noradrenaline was diminished by more than 80% under Ca2+-free conditions. Tetrodotoxin inhibited the majority of the evoked release of both ATP and [3H]noradrenaline, however, it was less effective in reducing the release of [3H]noradrenaline, than that of ATP. Bilateral stereotaxic injection of 6-hydroxydopamine (4 microg/side) to the ventral part of the ventral noradrenergic bundle, originating from the A1 cell group in the brainstem, resulted in a 55% reduction of endogenous noradrenaline content of the hypothalamic slices, and the tritium uptake and the stimulation-evoked release of [3H]noradrenaline was also markedly reduced. While the basal release of ATP was not affected, the evoked release was diminished by 72% by this treatment. Perfusion of the slices with noradrenaline (100 microM) initiated rapid and continuous tritium release; on the other hand, it did not release any ATP. In contrast, 6 min perfusion of (-)nicotine and 1,1-dimethyl-4-phenyl-piperazinium iodide evoked parallel release of ATP and [3H]noradrenaline which was inhibited by the nicotinic receptor antagonist mecamylamine; 6-hydroxydopamine lesion of the ventral part of the ventral noradrenergic bundle did not affect the nicotine-evoked ATP and [3H]noradrenaline release. While CH 38083, a non subtype-selective alpha2-antagonist and BRL44408, the subtype-selective alpha2AD antagonist augmented the evoked release of [3H]noradrenaline, ARC239, a selective alpha2BC antagonist was without effect. In contrast, neither of the alpha2-antagonists significantly affected the evoked-release of ATP. In summary, we report here that endogenous ATP and [3H]noradrenaline are co-released stimulation-dependently from superfused rat hypothalamic slices. A significant part of the release of both compounds is derived from the nerve terminals, originating from the A1 catecholaminergic cell group of brainstem nuclei. Unlike that from the peripheral sympathetic transmission, noradrenaline and alpha1-adrenoceptor agonists were unable to promote the release of ATP. Conversely, parallel ATP and noradrenaline release could be induced by nicotine receptor activation, but this release does not originate from the same nerve endings. The evoked-release of [3H]noradrenaline is inhibited by endogenous noradrenaline via alpha2AD subtype of adrenoreceptors, while the release of ATP is not subject to this autoinhibitory modulation. In conclusion, our results support the view that ATP is involved in the neurotransmission in the hypothalamus, but the sources of the released ATP and noradrenaline seem to be not identical under different stimulatory and modulatory conditions.     

Alpha-adrenergic reactivity of the microcirculation in conscious spontaneously hypertensive rats

The goal of this study was to determine the functional distribution of alpha 1- and alpha 2-adrenoceptors in the striated muscle microcirculation. Experiments were performed in intact conscious spontaneously hypertensive rats (SHR) that were provided with a dorsal microcirculatory chamber to allow microvascular diameter measurements. Administration of selective alpha 1- and alpha 2-agonists, phenylephrine and azepexole, respectively, induced different patterns of microvascular constriction. alpha 1-Adrenoceptor stimulation showed a preferential constriction of large arteries and venules. The entire arteriolar microvasculature was sensitive to alpha 2-adrenoceptor stimulation, whereas the venular vessels did not respond to azepexole. The selective alpha 1- and alpha 2-antagonists prazosin and yohimbine showed patterns of vasodilator activity comparable to those of the corresponding agonists. The specificity of the drug-induced effects was verified by comparing their effects with those of graded hemorrhage, a non-pharmacological method for blood pressure lowering. In the range of blood pressure decreases comparable to that obtained by alpha-adrenoceptor antagonists, graded hemorrhage did not influence microvascular diameters. These results show a differential functional distribution of alpha 1- and alpha 2-adrenoceptors along the microvascular tree in striated muscle of conscious SHR.