Dexmedetomidine produces its neuroprotective effect via the alpha 2A-adrenoceptor subtype

Which of the three alpha2-adrenoceptor subtypes of alpha2A, alpha2B, or alpha2C mediates the neuroprotective effect of dexmedetomidine was examined in cell culture as well as in an in vivo model of neonatal asphyxia. Dexmedetomidine dose-dependently attenuated neuronal injury (IC50=83+/-1 nM) in neuronal-glial co-cultures derived from wild-type mice; contrastingly, dexmedetomidine did not exert neuroprotection in injured cells from transgenic mice (D79N) expressing dysfunctional alpha2A-adrenoceptors. An alpha2A-adrenoceptor subtype-preferring antagonist 2-[(4,5-Dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole maleate (BRL44408) completely reversed dexmedetomidine-induced neuroprotection, while other subtype-preferring antagonists 2-[2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione dihydrochloride (ARC239) (alpha2B) and rauwolscine (alpha2C) had no significant effect on the neuroprotective effect of dexmedetomidine in neuronal-glial co-cultures. Dexmedetomidine also protected against exogenous glutamate induced cell death in pure cortical neuron cultures assessed by flow cytometry and reduced both apoptotic and necrotic types of cell death. Likewise this neuroprotective effect was antagonised by BRL44408 but not ARC239 or rauwolscine. Dexmedetomidine exhibited dose-dependent protection against brain matter loss in vivo (IC50=40.3+/-6.1 microg/kg) and improved the neurologic functional deficit induced by the hypoxic-ischemic insult. Protection by dexmedetomidine against hypoxic-ischemic-induced brain matter loss was reversed by the alpha2A-adrenoceptor subtype-preferring antagonist BRL44408; neither ARC239 nor rauwolscine reversed the neuroprotective effect of dexmedetomidine in vivo. Our data suggest that the neuroprotective effect of dexmedetomidine is mediated by activation of the alpha2A adrenergic receptor subtype.     

A comparative study of the reversal by different alpha 2-adrenoceptor antagonists of the central sympatho-inhibitory effect of clonidine.

1. The recovery of the clonidine-induced hypotension, bradycardia and sympatho-inhibition produced by several putative alpha 2-adrenoceptor antagonists was investigated in pentobarbitone anaesthetized rats. The activity of four substances containing an imidazoline structure: idazoxan, methoxy-idazoxan, BRL44408 and atipamezole was compared with the effect of fluparoxan, yohimbine and L-657,743; in addition the effect of the alpha 1-adrenoceptor antagonist, prazosin, was also studied. 2. Prazosin (0.03-1 mg kg-1, i.v.) failed to alter the sympatho-inhibitory and hypotensive effects of clonidine (10 micrograms kg-1, i.v.). L-657,743 (0.01-1 mg kg-1, i.v.) induced a recovery of blood pressure, heart rate and renal sympathetic nerve activity. Yohimbine (0.03-3 mg kg-1, i.v.) completely reversed the sympatho-inhibitory effect of clonidine but did not alter its hypotensive effect. 3. The four imidazoline drugs: idazoxan (10-300 micrograms kg-1, i.v.), methoxy-idazoxan (1-100 micrograms kg-1, i.v.), BRL44408 (0.1-3 mg kg-1, i.v.) and atipamezole (0.03-1 mg kg-1, i.v.) and fluparoxan (10-300 micrograms kg-1, i.v.) reversed the clonidine-induced hypotension but produced only a partial recovery of the renal sympathetic nerve activity and of the heart rate. After pretreatment with prazosin (0.1 mg kg-1, i.v.), the recovery of the sympathetic nerve activity elicited by these compounds was significantly higher. In hexamethonium (10 mg kg-1, i.v.) pretreated rats, these five drugs induced dose-related hypertension which was reduced by pretreatment with prazosin (0.1 mg kg-1, i.v.). 4. Our results indicate that the putative alpha 2-adrenoceptor antagonists idazoxan, methoxy-idazoxan, BRL44408, atipamezole and fluparoxan also have a peripheral hypertensive effect which is mediated through activation of vascular alpha 1-adrenoceptors; this property of the compounds may be partly responsible for the reversal of the hypotensive action of clonidine. Considering the structure and the affinities of the drugs tested, our data indirectly suggest that alpha 2A-adrenoceptors may be implicated in the central sympatho-inhibitory effects of clonidine.     

Site-dependent inhibition of neuronal c-jun in the brainstem elicited by imidazoline I1 receptor activation: role in rilmenidine-evoked hypotension

Clonidine (a mixed alpha2-adrenoceptor and imidazoline I1 receptor agonist)-evoked hypotension was associated with dissimilar reductions in c-jun gene expression in the rostral ventrolateral medulla (RVLM) and the nucleus tractus solitarius (NTS) in normotensive rats. In the present study, we investigated the relative contribution of the alpha2-adrenoceptor vs. the imidazoline I1 receptor to the reduction in c-jun gene expression in these two brainstem areas. In conscious spontaneously hypertensive rats (SHRs), equihypotensive doses of three centrally acting hypotensive drugs with different selectivity for the two receptors were administered intracisternally (4 microl) to limit their actions to the brain. As a control, a similar hypotensive response was elicited by i.v. hydralazine. Clonidine (0.5 microg), or alpha-methylnorepinephrine (alpha-MNE, 4 microg), a highly selective alpha2-adrenoceptor agonist, similarly reduced c-jun mRNA expression in the NTS and rostral ventrolateral medulla. In contrast, a similar hypotensive response (-37+/-3.5 mm Hg) caused by the selective imidazoline I1 receptor agonist rilmenidine (25 microg) was associated with reduction in c-jun mRNA expression in the rostral ventrolateral medulla, but not in the NTS. Further, intra-rostral ventrolateral medulla rilmenidine (40 nmol) reduced c-Jun protein expression in rostral ventrolateral medulla and blood pressure and both responses were antagonized by selective imidazoline I1 receptor (efaroxan, 4 nmol), but not alpha2-adrenoceptor (SK&F 86466, 10 nmol) blockade. These results suggest: (1) the c-jun containing neurons in the brainstem are involved in the centrally mediated hypotension elicited by centrally acting antihypertensive agents, and (2) the alpha2-adrenoceptor modulates c-jun gene expression in the NTS and rostral ventrolateral medulla implicated in centrally mediated hypotension, and (3) the imidazoline I1 receptor mediated inhibition of c-jun gene expression in the rostral ventrolateral medulla, but not in the NTS, contributes to the centrally mediated hypotension by the second generation drugs.     

Pressor response to pulsatile compression of the rostral ventrolateral medulla mediated by nitric oxide and c-fos expression

It has been reported that neurovascular compression of the rostral ventrolateral medulla might be causally related to essential hypertension. Recently, we found that pulsatile compression of the rostral ventrolateral medulla increases sympathetic nerve activity and elevates arterial pressure via activation of glutamate receptors in rats. We also found that increases in sympathetic and cardiovascular activities by microinjection of L-glutamate into the rostral ventrolateral medulla are mediated by c-fos expression-related substance(s) following activation of the nitric oxide-cyclic GMP pathway. Herein, we investigated whether responses to pulsatile compression are mediated by local activation of the nitric oxide-cyclic GMP pathway and/or c-fos expression-related substance(s) in rats. Increases in arterial pressure (15+/-1 mmHg), heart rate (9+/-1 b.p.m.), and sympathetic nerve activity (% change: 8.5+/-1.1%) induced by pulsatile compression were partially but significantly inhibited after local microinjection of a nitric oxide synthase inhibitor, L-N(G)-nitroarginine methyl ester (8+/-2 mmHg, 1+/-1 b.p.m., 4.0+/-1.3%; P<0.05 vs compression without pretreatment) or 7-nitroindazole (7+/-2 mmHg, 2+/-1 b.p.m., 4.0+/-1. 5%; P<0.05), or a soluble guanylate cyclase inhibitor, methylene blue (9+/-1 mmHg, 4+/-1 b.p.m., 4.1+/-1.4%; P<0.05). In addition, increases in arterial pressure, heart rate, and sympathetic nerve activity by pulsatile compression were significantly reduced 6 h after microinjection of antisense oligodeoxynucleotide to c-fos mRNA (2+/-2 mmHg, 2+/-1 b.p.m., 1.0+/-1.0%; P<0.05 vs sense oligodeoxynucleotide). These results suggest that increases in sympathetic and cardiovascular activities induced by pulsatile compression of the rostral ventrolateral medulla are mediated, at least in part, by local activation of the nitric oxide-cyclic GMP pathway and c-fos expression-related substance(s) in rats.     

Interactions of chloroethylclonidine with rauwolscine- and prazosin-sensitive adrenoceptors in dog saphenous vein.

1. alpha 1-Adrenoceptors have been classified pharmacologically into four subtypes (alpha 1A, alpha 1B, alpha 1C and alpha 1D) on the basis of their differential affinity for novel antagonists such as chloroethylclonidine (CEC). While CEC is considered an alpha 1B-adrenoceptor antagonist, our earlier studies revealed that it also acted like an agonist in the dog saphenous vein (DSV). The present study characterized the contraction induced by CEC in endothelium-denuded rings from DSV. 2. Concentration-response curves for CEC were constructed in the absence (EC50 value of 11.13 +/- 3.6 microM, n = 8) and presence of propranolol (beta-adrenoceptor antagonist, 30 nM), rauwolscine (alpha 2-adrenoceptor antagonist, 30 nM), prazosin (alpha 1-adrenoceptor antagonist, 30 nM) or methysergide (5HT2 antagonist, 30 nM) or both prazosin and rauwolscine. Pretreatment with methysergide (9.83 +/- 5.14 microM, n = 4) or propranolol (23.78 +/- 12.32 microM, n = 4) had no consistent effect. In the presence of rauwolscine, the concentration-response curve for CEC was significantly shifted to the right with an EC50 value of 48.82 +/- 13.2 microM (n = 8). In the presence of prazosin, the CEC concentration-response curve had an EC50 value of 29.12 +/- 6.42 microM (n = 8). Pretreatment with both prazosin and rauwolscine shifted the concentration-response curve for CEC to the right with an EC50 value of 72.67 +/- 10.69 microM (n = 8, P < 0.05). Maximum responses were significantly reduced only in tissues that were treated with both prazosin and rauwolscine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Examination of the role of inhibition of cyclic AMP in alpha 2-adrenoceptor mediated contractions of the porcine isolated palmar lateral vein.

1. We have examined the effect of elevation of cellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) on alpha 1- and alpha 2-adrenoceptor-mediated contraction of the isolated palmar lateral vein of the pig. Cellular cyclic AMP was increased by either inhibition of phosphodiesterase by rolipram, or direct activation of adenylyl cyclase by forskolin. 2. Noradrenaline (1 nM-10 microM) caused concentration-dependent contractions of the porcine isolated palmar lateral vein (pD2 7.32 +/- 0.07, n = 10). The selective alpha 1-adrenoceptor antagonist, prazosin (0.1 microM) and the selective alpha 2-adrenoceptor antagonist, rauwolscine (1 microM) caused a 10 fold rightward displacement of the concentration-response curve and a combination of the two antagonists caused a 200 fold rightward displacement of the concentration-response curve. The selective alpha 2-adrenoceptor agonist, UK-14304, also produced concentration-dependent contractions of the palmar lateral vein (pD2 7.70 +/- 0.15, n = 5), but the maximum response was 55.5 +/- 7.6% (n = 5) of that produced by noradrenaline. Prazosin (0.1 microM) failed to affect responses to UK-14304 but rauwolscine, 1 microM, caused a 200 fold rightward displacement. The estimated pKB value for rauwolscine (8.28 +/- 0.19, n = 10) is consistent with inhibition of alpha 2-adrenoceptors. Thus, the porcine isolated palmar lateral vein has a population of alpha 1- and alpha 2-adrenoceptors capable of producing a contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological profile of the mechanisms involved in the external carotid vascular effects of the antimigraine agent isometheptene in anaesthetised dogs

The present study set out to investigate the external carotid vascular effects of isometheptene in vagosympathectomised dogs, anaesthetised with pentobarbital. One-minute intracarotid (intra-arterial; i.a.) infusions of isometheptene (10, 30, 100 and 300 microg/min) produced dose-dependent decreases in external carotid blood flow, without affecting blood pressure or heart rate. The vasoconstrictor responses to 100 microg/min and 300 microg/min of isometheptene were clearly attenuated in animals pretreated with reserpine (5,000 microg/kg). Moreover, after prazosin (an alpha1-adrenoceptor antagonist; 100 microg/kg), the responses to isometheptene remained unaltered in untreated as well as reserpine-pretreated dogs. In contrast, the responses to isometheptene were attenuated by rauwolscine (an alpha2-adrenoceptor antagonist; 300 microg/kg) in untreated animals, and were practically abolished in reserpine-pretreated dogs. Further investigation into the specific alpha2-adrenoceptor subtypes, using selective antagonists, showed that BRL44408 (alpha2A) and MK912 (alpha2C) markedly attenuated this response, while imiloxan (alpha2B) was ineffective. The involvement of 5-HT1B and 5-HT1D receptors seems highly unlikely since antagonists at 5-HT1B (SB224289) and 5-HT1D (BRL15572) receptors (both at 300 microg/kg) were ineffective. On this basis, it is concluded that isometheptene-induced canine external carotid vasoconstriction is mediated by both indirect (a tyramine-like action) and direct (acting at receptors) mechanisms, which mainly involve alphaA- and alpha2C-adrenoceptors, while the involvement of alpha1-adrenoceptors seems rather limited.     

5-HT1B receptors and alpha 2A/2C-adrenoceptors mediate external carotid vasoconstriction to dihydroergotamine

Dihydroergotamine produces external carotid vasoconstriction in vagosympathectomized dogs by 5-HT(1B/1D) receptors and alpha(2)-adrenoceptors. This study identified the specific subtypes involved in this response. One-minute intracarotid infusions of dihydroergotamine (5.6-10 microg/min) dose-dependently decreased external carotid blood flow without affecting blood pressure or heart rate. This response was: (1) partly blocked in dogs pretreated intravenously with the antagonists SB224289 (5-HT(1B); 2,3,6,7-tetrahydro-1'-methyl-5-[2'-methyl-4' (5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-carbonyl]furo[2,3-f]indole-3-spiro-4'-piperidine hydrochloride), rauwolscine (alpha(2)), BRL44408 (alpha(2A); 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole) or MK912 (alpha(2C); (2S,12bS)-1'3'-dimethylspiro(1,3,4,5',6,6',7,12b-octahydro-2Hbenzo[b]furo[2,3-a]quinazoline)-2,4'-pyrimidin-2'-one); (2) markedly blocked after SB224289 plus rauwolscine; and (3) unaffected after BRL15572 (5-HT(1D); 1-(3-chlorophenyl)-4-[3,3-diphenyl (2-(S,R) hydroxypropanyl) piperazine] hydrochloride) or imiloxan (alpha(2B)). Therefore, the above response involves 5-HT(1B) receptors and alpha(2A/2C)-adrenoceptors.     

3H]rauwolscine behaves as an agonist for the 5-HT1A receptors in human frontal cortex membranes.

The alpha 2 adrenergic antagonist [3H]rauwolscine binds with comparable nanomolar affinity to alpha 2 adrenoceptors and the nonadrenergic 5-HT1A receptors sites in human frontal cortex membranes. Addition of 0.5 mM GTP into the incubation medium produces a significant decrease in the amount of [3H]rauwolscine binding sites (Bmax = 230 +/- 16 and 115 +/- 11 fmol/mg protein in the absence and presence of GTP, respectively). The affinity for [3H]rauwolscine remains unchanged (i.e. KD = 40 +/- 0.9 nM and 4.1 +/- 1 nM). This effect of GTP can be attributed to decreased binding of the radioligand to the 5-HT1A receptors. GTP decreases binding of [3H]rauwolscine to nearly the same level as the one corresponding to the alpha 2 adrenoceptors in membranes from both the human frontal cortex and hippocampus. The venom of the marine cone snail, Conus tessulatus, preferentially inhibits [3H]rauwolscine binding to 5-HT1A receptors as compared with the alpha 2 adrenoceptors. Following complete masking of the 5-HT1A receptors by this venom. GTP no longer affects the saturation binding characteristics of [3H]rauwolscine for the remaining alpha 2 adrenoceptors. Nucleotides decrease the binding of [3H]rauwolscine to the 5-HT1A receptors with an order of potencies (i.e. GTP gamma S greater than GPP(NH)P much greater than GDP greater than GTP much greater than ATP) that is typical for nucleotide-mediated receptor-G protein dissociation. This suggests that [3H]rauwolscine is a 5-HT1A receptor agonist and this conclusion is compatible with earlier functional studies, indicating that rauwolscine (as well as yohimbine) has agonistic properties at the level of 5-HT autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological profile of the clonidine-induced inhibition of vasodepressor sensory outflow in pithed rats: correlation with alpha(2A/2C)-adrenoceptors

BACKGROUND AND PURPOSE: Resistance blood vessels are innervated by sympathetic and primary sensory nerves, which modulate vascular tone through the release of noradrenaline and calcitonin gene-related peptide (CGRP), respectively. Moreover, electrical stimulation of the perivascular sensory outflow in pithed rats results in vasodepressor responses which are mainly mediated by CGRP release. The present study has investigated the role of alpha(2)-adrenoceptors in the inhibition of these vasodepressor responses. EXPERIMENTAL APPROACH: 144 pithed male Wistar rats were pretreated with hexamethonium (2 mg kg(-1) min(-1)) followed by i.v. continuous infusions of either methoxamine (15 and 30 microg kg(-1) min(-1)) or clonidine (3, 10 and 30 microg kg(-1) min(-1)). Under these conditions, electrical stimulation (0.56-5.6 Hz; 50 V and 2 ms) of the spinal cord (T(9)-T(12)) resulted in frequency-dependent decreases in diastolic blood pressure. KEY RESULTS: The infusion of clonidine (10 microg kg(-1) min(-1)), as compared to those of methoxamine (15 or 30 microg kg(-1) min(-1)), inhibited the vasodepressor responses to electrical stimulation without affecting those to i.v. bolus injections of alpha-CGRP (0.1-1 microg kg(-1)). This inhibition by clonidine was: (i) antagonized by 300 microg kg(-1) rauwolscine (alpha(2A/2B/2C)), 300 and 1000 microg kg(-1) BRL44408 (alpha(2A)), or 10 and 30 microg kg(-1) MK912 (alpha(2C)); and (ii) unaffected by 1 ml kg(-1) saline, 100 microg kg(-1) BRL44408, 3000 and 10,000 microg kg(-1) imiloxan (alpha(2B)) or 3 microg kg(-1) MK912. CONCLUSIONS AND IMPLICATIONS: The inhibition produced by 10 microg kg(-1) min(-1) clonidine on the vasodepressor (perivascular) sensory outflow in rats may be mainly mediated by prejunctional alpha(2A)/alpha(2C)-adrenoceptors.     

Adenosine, dipyridamole and isosorbide dinitrate are ineffective to prevent the sympathetic initiation of poststenotic myocardial ischemia

An activation of cardiac sympathetic nerves increases coronary vascular resistance distal to severe stenoses and induces ischemia of the dependent myocardium. The selective alpha 2-adrenoceptor antagonist rauwolscine and the calcium antagonist nifedipine prevent both poststenotic vasoconstriction and ischemia. To exclude the possibility that the beneficial action of nifedipine is based on unspecific coronary dilation rather than a functional antagonism against alpha 2-adrenoceptor mediated poststenotic vasoconstriction we now tested coronary dilatory drugs with a different underlying mechanism. The left ventrolateral cervical cardiac sympathetic nerve was stimulated in 12 anesthetized, vagotomized dogs. A severe stenosis of left circumflex coronary artery was defined by the absence of a postocclusive reactive hyperemia. Sympathetic stimulation increased end-diastolic poststenotic resistance from 0.45 +/- 0.10 to 0.83 +/- 0.18 mmHg X min X 100 g/ml and induced a net lactate production of the poststenotic myocardium. Adenosine (50 micrograms/kg X min i.c., n = 5), dipyridamole (0.2 mg/kg i.v., n = 3) and isosorbide-dinitrate (1 mg i.c., n = 4) did not prevent the increase in resistance and the net lactate production. Thus the effectiveness to prevent alpha 2-adrenergic poststenotic coronary constriction appears to be specific for alpha 2-antagonists and calcium antagonists.     

Differential neural activation of vascular alpha-adrenoceptors in oral tissues of cats

The aim of this study was to determine the relative contribution of alpha(1)- and alpha(2)-adrenoceptors involved in sympathetic-evoked vasoconstrictor responses in tissues perfused by the lingual arterial circulation in pentobarbital anesthetized cats. Blood flow in the lingual artery was measured by ultrasonic flowmetry. Laser-Doppler flowmetry was utilized to measure oral tissue vasoconstrictor responses in the maxillary gingiva and from the surface of the tongue. Electrical stimulation of the preganglionic superior cervical sympathetic nerve resulted in frequency-dependent blood flow decreases at all three sites. These responses were stable over time and were uniformly antagonized by administration of phentolamine (0.3 - 3.0 mg kg(-1)). The selective alpha(1)-adrenoceptor antagonist, prazosin (10 - 300 microg kg(-1)), attenuated vasoconstriction in the lingual artery and gingiva, but was ineffective in blocking vasoconstriction in the tongue. Subsequent administration of rauwolscine (300 microg kg(-1)) antagonized remaining vasoconstrictor responses. In contrast, rauwolscine (10 - 300 microg kg(-1)), given alone, blocked evoked vasoconstriction in the tongue, and was without effect on gingival or lingual artery vasoconstrictor responses. Subsequent administration of prazosin (300 microg kg(-1)) largely antagonized remaining neurally elicited responses. These results suggest that neural vasoconstrictor responses in some regional vascular beds in the cat oral cavity are mediated by both alpha(1)- and alpha(2)-adrenoceptors. In contrast, tongue surface vasoconstrictor responses to sympathetic nerve activation appear to be mediated primarily by alpha(2)-adrenoceptors.     

Hypotensive effect of urapidil: CNS site and relative contribution

The purposes of our study were to determine the contribution of the CNS to the hypotensive effect of urapidil in the cat and the specific brain site of action of this agent. For the first purpose, urapidil was studied on preganglionic sympathetic nerve activity, arterial pressure, and heart rate. Three systemic bolus doses of urapidil were administered (0.22, 0.44, and 1.3 mg/kg). All three doses lowered arterial pressure, and the highest dose produced a significant decrease in sympathetic nerve discharge in five of six animals studied. The lower two doses had no significant effect on sympathetic activity, and none of the doses altered heart rate. These results suggest that a high i.v. dose of urapidil is required to evoke hypotension by an action in the central nervous system (CNS). For the second purpose, urapidil was applied bilaterally to the intermediate area of the ventral surface of the medulla in doses of 25 and 50 micrograms. These doses caused decreases in arterial pressure of -6.1 +/- 2.2 (p less than 0.05) and -21.0 +/- 5.9 (p less than 0.05) mm Hg, respectively, but no change in heart rate. In addition, respiratory stimulation also occurred with the higher dose as respiratory minute volume increased by 81 +/- 14 ml/min (p less than 0.05). The highest dose of urapidil had no effect on arterial pressure when applied to other chemosensitive areas of the ventral surface of the brain. Comparative studies with prazosin (10 micrograms applied bilaterally to the intermediate area) indicated no hypotensive effect of this alpha 1-adrenoceptor blocking agent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Respective contributions of alpha-adrenergic and non-adrenergic mechanisms in the hypotensive effect of imidazoline-like drugs

The hypotensive effect of imidazoline-like drugs, such as clonidine, was first attributed to the exclusive stimulation of central alpha2-adrenoceptors (alpha2ARs). However, a body of evidence suggests that non-adrenergic mechanisms may also account for this hypotension. This work aims (i) to check whether imidazoline-like drugs with no alpha2-adrenergic agonist activity may alter blood pressure (BP) and (ii) to seek a possible interaction between such a drug and an alpha2ARs agonist alpha-methylnoradrenaline (alpha-MNA). We selected S23515 and S23757, two imidazoline-like drugs with negligible affinities and activities at alpha2ARs but with high affinities for non-adrenergic imidazoline binding sites (IBS). S23515 decreased BP dose-dependently (-27+/-5% maximal effect) when administered intracisternally (i.c.) to anaesthetized rabbits. The hypotension induced by S23515 (100 microg kg(-1) i.c.) was prevented by S23757 (1 mg kg(-1) i.c.) and efaroxan (10 microg kg(-1) i.c.), while these compounds, devoid of haemodynamic action by themselves, did not alter the hypotensive effect of alpha-MNA (3 and 30 microg kg(-1) i.c.). Moreover, the alpha2ARs antagonist rauwolscine (3 microg kg(-1) i.c.) did not prevent the effect of S23515. Finally, whilst 3 microg kg(-1) of S23515 or 0.5 microg kg(-1) of alpha-MNA had weak hypotensive effects, the sequential i.c. administration of these two drugs induced a marked hypotension (-23+/-2%). These results indicate that an imidazoline-like drug with no alpha2-adrenergic properties lowers BP and interacts synergistically with an alpha(ARs agonist.     

Relevance of the use of [3H]-clonidine to identify imidazoline receptors in the rabbit brainstem.

1. [3H]-clonidine binding was investigated in membranes isolated from the ventral medulla oblongata of the rabbit, where clonidine produced a hypotensive effect which was not mediated by adrenoceptors. [3H]-clonidine specific binding, as defined by the difference between the binding of [3H]-clonidine in the presence and in the absence of 10 microM cirazoline, occurred at two sites: a high affinity site with a KD = 2.9 +/- 0.7 nM and a Bmax of 40 +/- 8 fmol mg-1 protein and a low affinity site with a KD = 18.2 +/- 0.4 nM and a Bmax of 66 +/- 14 fmol mg-1 protein. 2. The high affinity sites being catecholamine-sensitive were identified as alpha 2-adrenoceptors. The low affinity binding of [3H]-clonidine was insensitive to catecholamines, as well as to other alpha 2-adrenoceptor specific probes, and could be inhibited with high affinity only by compounds which lowered blood pressure when directly injected in the nucleus reticularis lateralis of the ventral brainstem, or by antagonists. 3. It was concluded that in the ventral medulla of the rabbit, [3H]-clonidine labelled alpha 2-adrenoceptors and imidazoline receptors (IRs). Only the latter were related to the hypotensive effects of clonidine and rilmenidine directly injected into the rostroventrolateral medulla oblongata (RVLM) of the rabbit. The methodological problems regarding the study of IRs with [3H]-clonidine are discussed.     

Noradrenergic modulation of serotonin release in rat dorsal and median raphé nuclei via alpha(1) and alpha(2A) adrenoceptors.

The rat rostral raphé nuclei receive catecholaminergic innervation from the locus coeruleus and other areas. In the present study, we investigated noradrenergic modulation of 5-HT release in rat dorsal and median raphé nuclei (DRN and MRN) slices (350 microm thick) superfused with artificial cerebrospinal fluid (aCSF). The raphé was locally stimulated (0.1 ms pulses, 10 mA) and 5-HT release was monitored at carbon fibre microelectrodes using fast cyclic voltammetry. The selective noradrenaline reuptake inhibitor desipramine (50 nM) did not increase stimulated (20 pulses, 100 Hz) 5-HT release but significantly slowed 5-HT reuptake in both DRN and MRN. On short stimulus trains (10 pulses, 200 Hz), the alpha(2)-selective agonist dexmedetomidine (10nM) decreased evoked 5-HT release in DRN and MRN (to 44+/-3 and 43+/-7% of pre-drug values, respectively, at minimum). In both nuclei, this response was antagonised by the selective alpha(2A)-antagonist BRL 44408 (1 microM: P<0.001 vs. dexmedetomidine) but not by the selective alpha(2B/C)-adrenoceptor antagonist ARC 239 (500 nM), the selective 5-HT(1A) antagonist WAY 100635 (100 nM) or the alpha(1)-selective antagonist prazosin (1 microM), suggesting that the effect of dexmedetomidine is wholly attributable to alpha(2A)-receptor activation. The alpha(1)-adrenoceptor agonist phenylephrine (5 microM) significantly decreased 5-HT release (to 49+/-7 and 41+/-4% of pre-drug values in DRN and MRN, respectively). The response was blocked by prazosin (P<0.001) and BRL 44408 (P<0.01) in DRN and by prazosin, BRL 44408 and WAY 100635 (all P<0.05) in MRN, suggesting that the effect of phenylephrine is, under these conditions, only partly mediated via alpha(1)-adrenoceptors. On long stimuli (30 pulses, 10 Hz), BRL 44408 (1 microM) increased evoked 5-HT efflux to 187+/-17 and 178+/-2% of pre-drug values in DRN and MRN, respectively (both P<0.001 vs. vehicle). Collectively, these data show that activation of both alpha(1) and alpha(2A)-adrenoceptors can decrease stimulated 5-HT release in the rostral raphé nuclei. Since the effect of dexmedetomidine was not antagonised by prazosin, we suggest that its effect was mediated directly, possibly through alpha(2A) receptors located on 5-HT cell elements, and not transduced indirectly through alpha(1)-adrenoceptor activation, as previously suggested by others.     

Pharmacological evidence that alpha2A- and alpha2C-adrenoceptors mediate the inhibition of cardioaccelerator sympathetic outflow in pithed rats

It has been suggested that the alpha(2)-adrenoceptors mediating cardiac sympatho-inhibition in pithed rats closely resemble the pharmacological profile of the alpha(2A)-adrenoceptor subtype. However, several lines of evidence suggest that more than one subtype may be involved. Thus, the present study has pharmacologically re-evaluated the receptor subtype(s) involved in the inhibitory effect of the alpha(2)-adrenoceptor agonist, B-HT 933, on the tachycardic responses elicited by selective cardiac sympathetic stimulation (0.03, 0.1, 0.3, 1 and 3 Hz) in desipramine-pretreated pithed rats. I.v. continuous infusions of B-HT 933 (30 microg/kg min), which failed to modify the tachycardic responses to exogenous noradrenaline, inhibited those induced by preganglionic (C(7)-T(1)) stimulation of the cardiac sympathetic outflow at all frequencies of stimulation (0.03-3 Hz). This cardiac sympatho-inhibitory response to B-HT 933 was: (1) unaltered by saline (1 ml/kg) or the antagonists BRL44408 (100 microg/kg; alpha(2A)) or imiloxan (3000 and 10,000 microg/kg; alpha(2B)); (2) partially antagonized by BRL44408 (300 microg/kg) or MK912 (10 microg/kg; alpha(2C)) given separately; and (3) completely antagonized by rauwolscine (300 microg/kg; alpha(2)), MK912 (30 microg/kg) or the combination of BRL44408 (300 microg/kg) plus MK912 (10 microg/kg). Moreover, the above doses of antagonists, which are high enough to block their respective receptors, failed to block per se the tachycardic responses to sympathetic stimulation. These results suggest that the cardiac sympatho-inhibition induced by B-HT 933 in pithed rats is mainly mediated by stimulation of alpha(2A)- and alpha(2C)-adrenoceptors.     

Acute cardiovascular effects of the alpha2-adrenoceptor antagonist, idazoxan, in rats: influence of the basal sympathetic tone

Intravenous administration of the alpha2-adrenoceptor antagonist, idazoxan, elicits variable cardiovascular effects, depending on experimental conditions. In this study, the effects of idazoxan were investigated in rats with high, low, or no basal sympathetic tone. In a group of conscious Sprague-Dawley rats (n = 9), mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nervous activity (RSNA) were recorded. Idazoxan (250 microg/kg, i.v.) induced a transient decrease in MAP (-12+/-3 mm Hg) that was accompanied by increases in HR (49+/-14 beats/min) and RSNA (53+/-14%). In six of nine rats, a light pentobarbitone anesthesia was given. Basal RSNA was decreased (6.0+/-1.3 microV from 12.8+/-4.1 microV; p<0.05), and the depressor effect of idazoxan was reversed to a pressor effect (21+/-6 mm Hg) associated with bradycardia (-16+/-8 beats/min) and sympathoinhibition (-56+/-15%). In eight conscious intact rats, idazoxan (250 microg/kg, i.v.) attenuated by approximately 40% the pressor response to the selective alpha1-adrenoceptor agonist, cirazoline (0.5 microg/kg, i.v.). In three groups of six to seven ganglion-blocked (chlorisondamine, 2.5 mg/kg, i.v.) conscious rats, idazoxan dose-dependently increased mean arterial pressure (MAP: 39+/-2, 55+/-3, and 69+/-4 mm Hg at 125, 250, and 500 microg/kg, i.v., respectively) with minimal changes in HR. In contrast, the noradrenaline-releasing agent, tyramine (62.5, 125, and 250 microg/kg, i.v.), dose-dependently increased both MAP and HR. The alpha1-adrenoceptor antagonist, prazosin (1 mg/kg, i.v.; n = 8) blunted by approximately 70% (p<0.01) the pressor effect of 250 microg/kg idazoxan. It is concluded that in rats with high sympathetic tone, idazoxan has depressor effects, most likely related to its peripheral alpha-adrenoceptor antagonist properties. In rats with low or no sympathetic tone, idazoxan induced pressor responses mainly secondary to its partial agonist activity at vascular postjunctional alpha1-adrenoceptors.     

Partial inhibition by epithelium of tracheal smooth muscle relaxation induced by the potassium channel activator, BRL 38227.

1. A method is described whereby either the serosal (Out) or epithelial (In) sides of rat isolated tracheae were selectively perfused. Perfusion with BRL 38227 (10(-8)-5 x 10(-6) M; In/Out) of preparations with intact epithelium (+ EP) precontracted with carbachol (10(-6) M; Out/In) produced complete relaxation. Perfusion with aminophylline (10(-5)-10(-3) M; In) of + EP preparations precontracted with carbachol (10(-6) M; Out) also produced complete relaxation. 2. In preparations precontracted with carbachol (10(-6) M) epithelium removal (- EP) increased the sensitivity to the relaxant effect of BRL 38227 (In), but not BRL 38227 (Out) [- log EC50, + EP/- EP; carbachol (In), BRL 38227 (Out): 6.76 +/- 0.11 vs 6.67 +/- 0.15; carbachol (Out), BRL 38227 (In): 5.93 +/- 0.06 vs 6.25 +/- 0.07]. Removal of the epithelium increased also the sensitivity to BRL 38227 (In) of preparations precontracted with a lower concentration (5 x 10(-7) M) of carbachol (Out). [- log EC50, + EP/- EP, carbachol (Out), BRL 38227 (In): 6.19 +/- 0.14 vs 6.58 +/- 0.17]. 3. Removal of the epithelium did not affect the sensitivity to BRL 38227 (In) of preparations precontracted with a higher concentration (5 x 10(-6) M) of carbachol (Out). 4. In both + EP and - EP preparations precontracted with carbachol (10(-6) M; Out), BRL 38227 (In) had a more potent relaxant effect than aminophylline (In) (EC50, BRL 38227 vs aminophylline, + EP/- EP: 5.93 +/- 0.06 vs 3.66 +/- 0.11/6.25 +/- 0.07 vs 3.77 +/- 0.11).(ABSTRACT TRUNCATED AT 250 WORDS)