Neurotensin agonists block the prepulse inhibition deficits produced by a 5-HT<Subscript>2A</Subscript> and an α<Subscript>1</Subscript> agonist

Rationale Neurotensin (NT) agonists have been proposed as potential antipsychotics based exclusively upon their ability to inhibit dopamine-2 (D₂) receptor transmission. Several other pharmacological mechanisms have been implicated in enhancing the antipsychotic profile produced by D₂ inhibition alone. These include inhibition of 5-HT_2A and ₁-adrenoceptors. Recently, we reported that systemic administration of the neurotensin agonist PD149163 blocks deficits in prepulse inhibition (PPI) of the startle reflex produced by the 5-HT_2A receptor agonist DOI. This suggested that NT agonists could inhibit 5-HT_2A modulation of neurotransmission.Objective To determine if other peripherally administered NT agonists shared this effect, we examined the effects of NT69L, another NT agonist, on DOI-induced PPI deficits. In addition, to determine if NT agonists also inhibit ₁-adrenoceptor neurotransmission, we examined the effects of PD149163 and NT69L on PPI deficits induced by the ₁-adrenoceptor agonist, cirazoline.Methods In the NT69L/DOI study, rats received subcutaneous (SC) injections of NT69L (0, 0.1, 1, or 2 mg/kg) followed 30 min later by SC saline or DOI (0.5 mg/kg). In the NT agonist/cirazoline studies, animals received SC injections of either PD149163 (0, 0.01, 0.1, or 1 mg/kg) or NT69L (0, 0.01, 0.1, or 1 mg/kg) followed 30 min later by SC saline or cirazoline (0.7 mg/kg). Animals were tested in startle chambers 20 min later.Results In all three experiments the PPI disruption produced by DOI and cirazoline was blocked by the NT agonists.Conclusions These findings provide strong evidence that NT agonists inhibit 5-HT_2A and ₁-adrenoceptor modulation of neurotransmission, pharmacological effects that, in conjunction with their known inhibition of dopamine transmission, strengthen the antipsychotic potential of NT agonists.     

All three α<Subscript>2</Subscript>-adrenoceptor types serve as autoreceptors in postganglionic sympathetic neurons

Postganglionic sympathetic neurons and brain noradrenergic neurons use _2A- and _2C-adrenoceptors as presynaptic autoreceptors. The present experiments were carried out in order to see whether they possess presynaptic _2B-autoreceptors as well. Pieces of atria, vasa deferentia, the occipito-parietal cortex and the hippocampus were prepared from either wildtype (WT) mice or mice in which both the _2A- and the _2C-adrenoceptor gene had been disrupted (_2ACKO). The pieces were incubated with ³H-noradrenaline and then superfused and stimulated electrically. In a first series of experiments, single pulses or brief, autoinhibition-poor pulse trains were used for stimulation. The ₂-adrenoceptor agonist UK 14,304 (brimonidine) reduced the evoked overflow of tritium in all four tissues from WT mice but did not change it in any tissue from _2ACKO mice. A different pattern was obtained with medetomidine as ₂ agonist. Like UK 14,304, medetomidine reduced the evoked overflow of tritium in all four tissues from WT mice and did not affect overflow in brain slices from _2ACKO mice; however, in contrast to UK 14,304, medetomidine reduced evoked overflow also in atrial and vas deferens pieces from _2ACKO mice, although with a lower maximum and potency than in WT preparations. The -adrenoceptor antagonists rauwolscine, phentolamine, prazosin, spiroxatrine and WB 4101 shifted the concentration-response curve of medetomidine in _2ACKO atria and vasa deferentia to the right. The pK_d values of the five antagonists against medetomidine in _2ACKO atria and vasa deferentia correlated with pK_d values at prototypical _2B radioligand binding sites but not at _2A or _2C binding sites. In a second series of experiments, autoinhibition-rich pulse trains were used for stimulation. Under these conditions, rauwolscine and phentolamine increased the evoked overflow of tritium from _2ACKO atrial and vas deferens pieces but not from _2ACKO brain slices. The increase was smaller (by 40% in atria and by 70% in the vas deferens) than previously observed in WT preparations (by 200–400%). In a last series of experiments, mRNA for the _2B-adrenoceptor was demonstrated by RT-PCR in thoracolumbar sympathetic ganglia from WT, _2AKO, _2CKO and _2ACKO mice but not from _2BKO mice. The results show that brain noradrenergic neurons express only _2A- and _2C-adrenoceptors as autoreceptors. Postganglionic sympathetic neurons, however, can express _2B-adrenoceptors as presynaptic autoreceptors as well. The _2B-autoreceptors are activated by medetomidine but not by UK 14,304. They are also activated by previously released noradrenaline. The two-₂-autoreceptor hypothesis has to be replaced by a three-autoreceptor hypothesis for postganglionic sympathetic neurons.     

Comparison of the α-adrenoceptor-mediated effects of β<Subscript>3</Subscript>-adrenoceptor ligands in rat pulmonary artery

We have recently shown that the -adrenoceptor ligands CGP 12177, bupranolol, and SR 59230A (aryloxypropanolamines), but not BRL 37344 and CL 316243 (phenylethanolamines), exhibit significant affinity for ₁-adrenoceptors and that CGP 12177 displays partial agonist properties at -adrenoceptors in rat pulmonary artery. In this study, bupranolol and SR 59230A were further evaluated for their potential -adrenoceptor mediated effects (i.e., agonist and/or antagonist properties) in rat intralobar pulmonary artery and compared with BRL 37344 and CL 316243. Bupranolol induced a relaxation in phenylephrine-precontracted arteries, but had no effect in prostaglandin -precontracted ones. SR 59230A also elicited a relaxation in phenylephrine-precontracted arteries. In -precontracted arteries, SR 59230A induced a contractile response that was insensitive to the irreversible -adrenoceptor antagonist phenoxybenzamine. BRL 37344 at high concentrations, but not CL 316243, produced slight relaxation in both phenylephrine- and -precontracted arteries. The contractile response to phenylephrine was antagonized by bupranolol and SR 59230A in a competitive manner (pA₂: 6.38 and 7.08 respectively). The concentration–response curve to phenylephrine was also shifted to the right by BRL 37344 (mean pK_b: 4.45), but not by CL 316243 (100 M). This study indicates that the aryloxypropanolamine derivatives bupranolol and SR 59230A exhibit competitive antagonist, but no agonist properties on ₁-adrenoceptors, SR 59230A also inducing -adrenoceptor-independent contraction. Among the phenylethanolamines, BRL 37344 but not CL 316243, also exerts an antagonist effect on ₁-adrenoceptors, with a much lower potency than the aryloxypropanolamines studied.     

Effect of the blockade of μ<Subscript>1</Subscript>-opioid and 5HT<Subscript>2A</Subscript>-serotonergic/α<Subscript>1</Subscript>-noradrenergic receptors on sweet-substance-induced analgesia

Rationale Sweet-substance-induced analgesia has been widely studied, and the investigation of the neurotransmitters involved in this antinociceptive process is an important way for understanding the involvement of the neural system controlling this kind of antinociception.Objective The aim of this study was to investigate the involvement of opioid and monoaminergic systems in sweet-substance-induced analgesia.Methods The present work was carried out in an animal model with the aim of investigating whether acute (24 h) or chronic (14 days) intake of a sweet substance, such as sucrose (250 g/l), is followed by antinociception. Tail withdrawal latencies in the tail-flick test were measured before and immediately after this treatment. Immediately after the recording of baseline values, independent groups of rats were submitted to sucrose or tap-water intake and, after chronic treatment, they were pretreated with intraperitoneal administration of (1) naltrexone at 0.5, 1, 2 or 3 mg/kg; (2) naloxonazine at 5, 10, 20 or 30 mg/kg; (3) methysergide at 0.5, 1, 2 or 3 mg/kg; (4) ketanserin at 0.5, 1, 2 or 3 mg/kg; or (5) physiological saline.Results Naltrexone and methysergide at two major doses decreased sweet-substance-induced analgesia after chronic intake of a sweet substance. These effects were corroborated by peripheral administration of naloxonazine and ketanserin.Conclusions These data give further evidence for: (a) the involvement of endogenous opioids and a ₁-opioid receptor in the sweet-substance-induced antinociception; (b) the involvement of monoamines and 5HT_2A serotonergic/₁-noradrenergic receptors in the central regulation of the sweet-substance-produced analgesia.     

Calcium channel function and regulation in β<Subscript>1</Subscript>- and β<Subscript>2</Subscript>-adrenoceptor transgenic mice

Cardiac effects of catecholamines on the L-type calcium channel depend on -adrenoceptor subtype (₁- vs. ₂-adrenoceptor). Chronic overexpression of these receptors leads to hypertrophy and early death at moderate (₁) or excessive (₂) levels of overexpression respectively. In order to examine the role of L-type calcium channels in altered cardiomyocyte calcium homeostasis found with ₁-adrenoceptor overexpression, and to understand the quantitative differences between -adrenoceptor subtypes regarding calcium channel regulation, we examined single channels in myocytes obtained from ₁- and ₂-adrenoceptor transgenic mice. The effects of the agonist isoproterenol were investigated and compared with acute receptor stimulation in the respective non-transgenic littermates.Channels from ₁-adrenoceptor transgenic mice have normal baseline activity, and channel number is not reduced. This contrasts to previous findings with ₂-adrenoceptor transgenic mice, where channel activity is depressed. Isoproterenol is unable to stimulate channel activity in both transgenic models.In conclusion, the L-type calcium channel is not likely to be involved in alterations of calcium handling of ₁-adrenoceptor transgenic myocytes. Furthermore, chronic ₁-adrenoceptor overexpression does not depress channel activity, giving another example of the difference between ₁- and ₂-adrenoceptor signal transduction.K.F. and T.K. equally contributed to this work     

Yohimbine disrupts prepulse inhibition in rats via action at 5-HT<Subscript>1A</Subscript> receptors,not α<Subscript>2</Subscript>-adrenoceptors

RATIONALE: Prepulse inhibition (PPI) of the acoustic startle response is an operational measure of sensorimotor gating that can be assessed in both humans and animals. The noradrenergic system appears to play a role in PPI as the alpha1 agonist cirazoline disrupts PPI and the alpha1 antagonist prazosin blocks the disruptions in PPI produced by phencyclidine. OBJECTIVES: To better understand the role of adrenergic receptors in the modulation of PPI, we assessed the effects of the alpha2 adrenergic antagonist yohimbine (2.5, 5.0, and 7.5 mg/kg) on PPI. RESULTS: Yohimbine reduced PPI at the 5.0 and 7.5 mg/kg doses, without significantly affecting startle magnitude. In separate experiments, we examined whether adrenergic or serotonergic compounds blocked this disruption in PPI produced by yohimbine. There was a trend for the alpha2 agonist clonidine (0.01, 0.02 mg/kg) to attenuate the PPI disruption produced by yohimbine. However, other alpha2 agonists (guanfacine, medetomidine) and an alpha1 antagonist (prazosin) failed to prevent the disruption. The alpha2 antagonist atipamezole weakly decreased PPI in a narrow dose range (0.3-1.0 mg/kg). The 5-HT1A antagonist WAY100,635 (0.1, 0.3 mg/kg) significantly prevented the yohimbine-induced disruption of PPI. CONCLUSIONS: These findings indicate that (1) yohimbine disrupts PPI in rats and (2) the yohimbine-induced disruption of PPI is largely due to the 5-HT1A partial agonist properties of yohimbine.     

Binding of [<Superscript>3</Superscript>H]prazosin to α<Subscript>1A</Subscript>- and α<Subscript>1B</Subscript>-adrenoceptors,and to a cimetidine-sensitive non-α<Subscript>1</Subscript> binding site in rat kidney membranes

[(3)H]Prazosin bound to alpha(1A)- and alpha(1B)-adrenoceptors, as well as to a cimetidine-sensitive non-alpha(1)-adrenoceptor binding site in rat kidney membranes. An experimental design is presented where the alpha(1)-adrenoceptors are selectively exposed by blocking the non-alpha(1) binding site with 60 microM cimetidine. Conversely, the non-alpha(1) binding site can be selectively exposed by blocking the alpha(1)-adrenoceptors with 600 nM metitepine. The identity of the non-alpha(1) binding site for [(3)H]prazosin in the rat kidney, herein pharmacologically characterized by 33 competing substances, is still unknown.     

Anticataleptic properties of α<Subscript>2</Subscript> adrenergic antagonists in the crossed leg position and bar tests: differential mediation by 5-HT<Subscript>1A</Subscript> receptor activation

Rationale Recent studies suggest that ₂ adrenoceptor blockade may improve the antipsychotic-like effects of neuroleptics and attenuate dopamine D₂ receptor antagonist-induced catalepsy. However, several ₂ adrenergic antagonists also display serotonin 5-HT_1A receptor agonist activity, which may contribute to anticataleptic actions.Objectives In this study, we examined a series of ₂ adrenergic antagonists to determine the role of activity at serotonin 5-HT_1A receptors in their anticataleptic effects.Methods Catalepsy in rats induced by the antipsychotic haloperidol (2.5 mg/kg, SC) was measured using the cross-legged position (CLP) and bar tests. The compounds examined in this study, in decreasing rank order of ₂ adrenergic versus 5-HT_1A receptor selectivity, were atipamezole, methoxy-idazoxan (RX821002), efaroxan, idazoxan, and yohimbine. Antagonism studies were conducted using the selective 5-HT_1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide dihydrochloride (WAY100635).Results Idazoxan, efaroxan, and yohimbine significantly attenuated the cataleptic effects of haloperidol (2.5 mg/kg, SC) in the CLP test and the actions of their highest doses were significantly blocked by pre-treatment with WAY100635 (0.63 mg/kg, SC). In contrast to the other compounds, methoxy-idazoxan was ineffective in the CLP test. Atipamezole exhibited anticataleptic effects in the bar and CLP tests which were not blocked by WAY100635. Similarly, the anticataleptic effects of methoxy-idazoxan and idazoxan in the bar test were not blocked by WAY100635.Conclusions Serotonin 5-HT_1A receptors play a prominent role in anticataleptic effects of certain ₂ adrenergic antagonists in the CLP test, whereas ₂-adrenergic mechanisms are likely to be primarily responsible for the anticataleptic effects of these ligands in the bar test.     

The α<Subscript>2</Subscript>-adrenoceptor antagonist atipamezole reduces the development and expression of <Emphasis Type="SmallCaps">d</Emphasis>-amphetamine-induced behavioural sensitization

The possible effect of atipamezole, a potent and specific alpha(2)-adrenoceptor antagonist, on the development and expression of d-amphetamine-induced behavioural sensitization was evaluated in mice. Male (C57Bl/6J) mice were given daily doses of d-amphetamine (2 mg/kg). In addition, groups of mice received injections of atipamezole (0.3 or 1 mg/kg) 20 min before d-amphetamine or vehicle administration. Idazoxan (1 mg/kg) was used in some experiments to extend the results to other alpha(2)-adrenoceptor antagonists. Challenge doses of d-amphetamine were administered to the mice on days 7-9 to evaluate the effects of alpha(2)-adrenoceptor antagonists on the d-amphetamine sensitization, evidenced by increased locomotor activation.Mice treated repeatedly with d-amphetamine developed strong locomotor sensitization that was reduced by pretreatment with alpha(2)-adrenoceptor antagonists. Acute atipamezole at both doses attenuated the expression of d-amphetamine-induced sensitization. Atipamezole at 1 mg/kg alone had no effect on locomotor activity, but the lower dose (0.3 mg/kg) increased locomotor activity after repeated administration.These results indicate that alpha(2)-adrenoceptor antagonists modulate the actions of d-amphetamine in a manner not explicable by their enhancing actions on noradrenaline and dopamine release, and may thus provide a novel approach to the treatment of motor complications caused by dopaminergic agents, such as dyskinesias, and perhaps also drug dependence.     

Discriminative stimulus effects of zolpidem in squirrel monkeys: role of GABA<Subscript>A</Subscript>/α<Subscript>1</Subscript> receptors

Abstract Rationale. The discriminative stimulus effects of zolpidem in squirrel monkeys trained at doses greater than or equal to 3.0 mg/kg differ from those of conventional benzodiazepines (BZs), but the extent to which these effects reflect the selectivity of zolpidem for GABA_A/α₁ receptors is not known. Objectives. The present study investigated the ability of GABA_A/α₁-preferring agonists to substitute for training doses of zolpidem greater than or equal to 3.0 mg/kg and the ability of GABA_A/α₁-preferring antagonists to block zolpidem's discriminative stimulus effects. Methods. Squirrel monkeys were trained to discriminate intravenous injections of zolpidem (3.0 or 5.6 mg/kg) from saline and tested with BZ agonists differing in selectivity and efficacy at GABA_A/α₁ receptors. Antagonism of the effects of zolpidem was studied using the GABA_A/α₁-preferring antagonists β-carboline-3-carboxylate-t-butyl ester (β-CCT) and 3-propyloxy-β-carboline (3-PBC). Results. Zolpidem and quazepam (GABA_A/α₁-preferring agonist) engendered full substitution for zolpidem, whereas CL 218,872 (GABA_A/α₁-preferring partial agonist) and the non-selective BZ agonists alprazolam and flunitrazepam engendered low and variable levels of zolpidem-lever responding (35–58%). Both β-CCT and 3-PBC antagonized the discriminative stimulus effects of zolpidem in a surmountable fashion. Conclusions. Our findings provide evidence for a key role of GABA_A/α₁ receptors in the discriminative stimulus effects of zolpidem at relatively high training doses, and suggest that selectivity and relatively high efficacy at GABA_A/α₁ receptors is required for BZ agonists to reproduce these discriminative stimulus effects. Electronic Publication     

The α<Subscript>2</Subscript>-adrenoceptor antagonist atipamezole potentiates anti-Parkinsonian effects and can reduce the adverse cardiovascular effects of dopaminergic drugs in rats

The present experiments investigated the effects of the specific ₂-adrenoceptor antagonist atipamezole, alone and in combination with a dopamine agonist, on motor function in rats with a unilateral 6-hydroxydopamine lesion of the nigro-striatal pathway and on exploratory behaviour and cardiovascular function in rats equipped with telemetry transmitters. Dexmedetomidine, an ₂-adrenoceptor agonist and the ₂-adrenoceptor antagonists idazoxan and yohimbine were used as reference compounds. In the unilaterally lesioned animals, direct dopamine agonists, such as apomorphine, induce contralateral turning behaviour. Indirect agonists, such as amphetamine, induce ipsilateral circling in the animals. Atipamezole (0.3 mg/kg s.c) potentiated and dexmedetomidine (10 µg/kg s.c.) decreased contralateral circling evoked by apomorphine (50 µg/kg s.c.) and by l-3,4-dihydroxyphenylalanine (L-DOPA, 5 mg/kg i.p.). Atipamezole also prolonged the duration of action of L-DOPA. Atipamezole dose-dependently induced ipsilateral turning behaviour and potentiated turning induced by amphetamine (1 mg/kg i.p.). The ₁-adrenoceptor antagonist prazosin (0.1 mg/kg i.p.) partially antagonised the effect of amphetamine and had a strong inhibitory effect on the atipamezole-induced potentiation of the amphetamine response. Prazosin did not have any major effect on either the apomorphine response itself or on the potentiation of the apomorphine response by atipamezole. This suggests that atipamezole can modulate motor function both indirectly, by stimulating the release of noradrenaline and directly, by blocking postsynaptic ₂-adrenoceptors in neurones other than noradrenergic nerves. The ₂-adrenoceptor antagonists, when tested at comparably effective central ₂-adrenoceptor antagonising doses in a rat mydriasis model: atipamezole 0.3 mg/kg s.c., idazoxan 1 mg/kg s.c. and yohimbine 3 mg/kg s.c., all induced ipsilateral turning behaviour and potentiated apomorphine-induced contralateral circling. The effects of the ₂-adrenoceptor antagonists were in general similar in these experiments. In habituated non-lesioned rats equipped with telemetry transmitters, apomorphine (50 µg/kg s.c.) decreased blood pressure in the home cage and in an open-field test. It also decreased spontaneous motor activity in the open field. Neither atipamezole (0.3 mg/kg s.c.) nor idazoxan (1 mg/kg s.c.) had any effect on blood pressure when given alone, but reversed the apomorphine-induced decrease in blood pressure. Atipamezole also diminished apomorphine-induced sedation in the open-field test. In conclusion, atipamezole improved the efficacy of L-DOPA and apomorphine in an animal model of Parkinsons disease and also reduced adverse dopaminergic effects on vigilance and on cardiovascular function. These results suggest that an investigation of the effects of specific ₂-adrenoceptor antagonists in Parkinsons disease patients is warranted.     

Positive allosteric modulatory effects of ajulemic acid at strychnine-sensitive glycine α<Subscript>1</Subscript>- and α<Subscript>1</Subscript>β-receptors

The synthetic cannabinoid ajulemic acid (CT-3) is a potent cannabinoid receptor agonist which was found to reduce pain scores in neuropathic pain patients in the absence of cannabis-like psychotropic adverse effects. The reduced psychotropic activity of ajulemic acid has been attributed to a greater contribution of peripheral CB receptors to its mechanism of action as well as to non-CB receptor mechanisms. Loss of inhibitory synaptic transmission within the dorsal horn of the spinal cord plays a key role in the development of chronic pain following inflammation or nerve injury. Inhibitory postsynaptic transmission in the adult spinal cord involves mainly glycine. As we hypothesised that additional non-CB receptor mechanisms of ajulemic acid might contribute to its effect in neuropathic pain, we investigated the interaction of ajulemic acid with strychnine-sensitive α₁- and α₁β-glycine receptors by using the whole-cell patch clamp technique. Ajulemic acid showed a positive allosteric modulating effect in a concentration range which can be considered close to clinically relevant concentrations (EC₅₀ values: α₁ = 9.7 ± 2.6 μM and α₁β = 12.4 ± 3.4 μM). Direct activation of glycine receptors was observed at higher concentrations above 100 μM (EC₅₀ values: α₁ = 140.9 ± 21.5 μM and α₁β = 154.3 ± 32.1 μM). These in vitro results demonstrate that ajulemic acid modulates strychnine-sensitive glycine receptors in clinically relevant concentrations.     

Cross-talk between β<Subscript>1</Subscript>-adrenoceptors and ET<Subscript>A</Subscript> receptors in modulation of the slow component of delayed rectifier K<Superscript>+</Superscript> currents

Delayed rectifier K⁺ currents (I_K) play a critical role in determining cardiac action potential duration (APD). Modulation of I_K affects cardiac excitability critically. There are three components of cardiac delayed rectifier, and the slowly activating component (I_Ks) is influenced strongly by a variety of stimuli. Plasma levels of noradrenaline and endothelin are elevated in heart failure, and arrhythmias are promoted by such humoral abnormalities through modulation of ion channels. It has been reported that protein kinase A (PKA) and protein kinase C (PKC) modulate I_Ks from human minK in a complex manner. In the present study, we coexpressed human minK with the human ₁-adrenoceptor (h₁AR) and the endothelin receptor subtype A (hET_AR) in Xenopus oocytes and investigated the effects of receptor activation on the currents (I_Ks) flowing through the oocytes. ET-1 modulated I_Ks biphasically: a transient increase followed by a decrease. The PKC inhibitor chelerythrine completely inhibited the effects of ET-1. Intracellular EGTA abolished the transient increase by ET-1 and partially inhibited the subsequent decrease in the currents. When I_Ks was increased by 10^–6 M isoproterenol (ISO), ET-1 did not increase but rather decreased the current to an even greater extent than under control conditions. In addition, the effects of ISO on I_Ks were suppressed by ET_AR stimulation. These data indicate that I_Ks can be regulated by cross-talk between the ET_AR and ₁AR systems in addition to direct regulation by each receptor system.     

Selective antagonism of the ataxic effects of zolpidem and triazolam by the GABA<Subscript>A</Subscript>/α<Subscript>1</Subscript>-preferring antagonist β-CCt in squirrel monkeys

Abstract Rationale. Delineation of the receptor mechanisms underlying the behavioral effects of benzodiazepines should allow for the development of drugs with improved clinical utility and reduced side effects. Objectives. The purpose of the present study was to investigate the role of GABA_A/α₁ receptors in the sedative and motor-impairing effects of benzodiazepines. Methods. Squirrel monkeys were tested with the GABA_A/α₁-preferring agonist zolpidem and the nonselective benzodiazepine agonist triazolam alone and in combination with the GABA_A/α₁-preferring antagonist β-CCt and the nonselective benzodiazepine antagonist flumazenil. During 30-min experimental sessions, all occurrences of normal behaviors like locomotion, environment- and self-directed behaviors, as well as side effects such as ataxia, rest and procumbent postures were scored. Results. Zolpidem and triazolam produced dose-dependent reductions in locomotion and environment-directed behavior and increased ataxia and procumbent posture. Triazolam, but not zolpidem, also engendered species-typical rest posture at some doses. Flumazenil antagonized all of the behavioral effects of zolpidem and triazolam, whereas β-CCt antagonized only zolpidem- and triazolam-induced ataxia. Conclusions. GABA_A/α₁ receptor mechanisms appear to play a key role in the ataxic effects of benzodiazepine agonists in squirrel monkeys, similar to recent results with transgenic mice. In contrast to the findings of these recent studies, GABA_A mechanisms other than or in addition to those mediated at the α₁ subunit may play a more important role in the sedative/hypnotic effects of benzodiazepines in squirrel monkeys. Electronic Publication     

α<Subscript>1</Subscript>-Adrenergic and α<Subscript>2</Subscript>-adrenergic balance in the dorsal pons and gross behavioral activity of mice in a novel environment

Rationale Central α₁- and α₂-adrenoceptors in a number of different brain regions are known to have opposing actions on gross behavioral activity, with the former stimulating and the latter inhibiting activity. Therefore, blockade of α₁-receptors may induce inactivity by leading to unopposed α₂ activity.Objective The aim of this study was to test if central blockade of α₂-receptor function restores behavioral activity in α₁-receptor-blocked mice.Methods Dose-response studies were undertaken on the effects of α₁- and α₂-agonists and antagonists microinjected into the dorsal pons on gross behavioral activity in a novel cage test.Results The behavioral inactivity resulting from blockade of α₁-receptors in the pons with the antagonist, terazosin, was reversed by either a low dose of an α₂-antagonist, atipamezole, or a low dose of an α₂-agonist, dexmedetomidine, but was exacerbated by a high dose of the α₂-agonist.Conclusion The results support the hypothesis that blockade of α₁-receptors in the dorsal pons of mice produces inactivity by causing unopposed activity of α₂-receptors. This condition may be relevant to inactive states seen after stress or during depressive illness.     

5-HT<Subscript>1B</Subscript> receptors, α<Subscript>2A/2C</Subscript>- and,to a lesser extent, α<Subscript>1</Subscript>-adrenoceptors mediate the external carotid vasoconstriction to ergotamine in vagosympathectomised dogs

It has previously been suggested that ergotamine produces external carotid vasoconstriction in vagosympathectomised dogs via 5-HT_1B/1D receptors and ₂-adrenoceptors. The present study has reanalysed this suggestion by using more selective antagonists alone and in combination. Fifty-two anaesthetised dogs were prepared for ultrasonic measurements of external carotid blood flow. The animals were divided into thirteen groups (n=4 each) receiving an i.v. bolus injection of, either physiological saline (0.3 ml/kg; control), or the antagonists SB224289 (300 g/kg; 5-HT_1B), BRL15572 (300 µg/kg; 5-HT_1D), rauwolscine (300 µg/kg; ₂), SB224289 + BRL15572 (300 µg/kg each), SB224289 + rauwolscine (300 µg/kg each), BRL15572 + rauwolscine (300 µg/kg each), rauwolscine (300 µg/kg) + prazosin (100 µg/kg; ₁), SB224289 (300 µg/kg) + prazosin (100 µg/kg), SB224289 (300 µg/kg) + rauwolscine (300 µg/kg) + prazosin (100 µg/kg), SB224289 (300 µg/kg) + prazosin (100 µg/kg) + BRL44408 (1,000 µg/kg; _2A), SB224289 (300 µg/kg) + prazosin (100 µg/kg)+ imiloxan (1,000 µg/kg; _2B), or SB224289 (300 µg/kg) + prazosin (100 µg/kg) + MK912 (300 µg/kg; _2C). Each group received consecutive 1-min intracarotid infusions of ergotamine (0.56, 1, 1.8, 3.1, 5.6, 10 and 18 µg/min), following a cumulative schedule. In saline-pretreated animals, ergotamine induced dose-dependent decreases in external carotid blood flow without affecting arterial blood pressure or heart rate. These control responses were: unaffected by SB224289, BRL15572, rauwolscine or the combinations of SB224289 + BRL15572, BRL15572 + rauwolscine, rauwolscine + prazosin, SB224289 + prazosin, or SB224289 + prazosin + imiloxan; slightly blocked by SB224289 + rauwolscine; and markedly blocked by SB224289 + rauwolscine + prazosin, SB224289 + prazosin + BRL44408 or SB224289 + prazosin + MK912. Thus, the cranio-selective vasoconstriction elicited by ergotamine in dogs is predominantly mediated by 5-HT_1B receptors as well as _2A/2C-adrenoceptor subtypes and, to a lesser extent, by ₁-adrenoceptors.In memoriam: Luis F. Valdivia died on 26 May 2004     

Novel human α<Subscript>1a</Subscript>-adrenoceptor single nucleotide polymorphisms alter receptor pharmacology and biological function

We identified nine naturally-occurring human single nucleotide polymorphisms (SNPs) in the _1a-adrenoceptor (_1aAR) coding region, seven of which result in amino acid change. Utilizing rat-1 fibroblasts stably expressing wild type _1aAR or each SNP at both high and low levels, we investigated the effect of these SNPs on receptor function. Compared with wild type, two SNPs (R166K, V311I) cause a decrease in binding affinity for agonists norepinephrine, epinephrine, and phenylephrine, and also shift the dose–response curve for norepinephrine stimulation of inositol phosphate (IP) production to the right (reduced potency) without altering maximal IP activity. In addition, SNP V311I and I200S display altered antagonist binding. Interestingly, a receptor with SNP G247R (located in the third intracellular loop) displays increased maximal receptor IP activity and stimulates cell growth. The increased receptor signaling for _1aAR G247R is not mediated by altered ligand binding or a deficiency in agonist-mediated desensitization, but appears to be related to enhanced receptor–G protein coupling. In conclusion, four naturally-occurring human _1aAR SNPs induce altered receptor pharmacology and/or biological activity. This finding has potentially important implications in many areas of medicine and can be used to guide _1aAR SNP choice for future clinical studies.     

Involvement of α<Subscript>1</Subscript> and β-adrenoceptors in adrenaline stimulation of the glucagon-secreting mouse α-cell

Stimulation of glucagon release and inhibition of insulin secretion from the islets of Langerhans are important for the blood-glucose-elevating effect of adrenaline. The mechanisms by which adrenaline accomplishes these actions may involve direct effects and indirect ones mediated by altered release of other islet hormones. In the present study we investigated how adrenaline affects the cytoplasmic Ca²⁺ concentration, which controls glucagon secretion from the pancreatic -cell. The studies were performed on isolated mouse -cells, which were identified by immunocytochemistry.The adrenaline effects consisted of initial mobilisation of intracellular Ca²⁺, accompanied by voltage-dependent influx of the ion. Part of the effect could be attributed to -adrenoceptor activation, as it was mimicked by the rise in cAMP and inhibited by the antagonist propranolol as well as the protein kinase A inhibitor adenosine 3,5-cyclic monophosphorothioate Rp-isomer. ₁-Adrenoceptors were also involved, since the antagonists phentolamine and prazosin completely abolished the effects of adrenaline. Experiments with clonidine and yohimbine gave little evidence of a role of ₂-adrenoceptors. The results indicate that ₁- and -adrenoceptors on the -cells mediate adrenaline-stimulated glucagon secretion. The complete inhibition of the adrenaline response after blocking ₁-adrenoceptors indicates an interaction with the -adrenergic pathway.Drs. Vieria and Liu contributed equally to the article     

GABA<Subscript>A</Subscript>/α<Subscript>1</Subscript> receptor agonists and antagonists: effects on species-typical and heightened aggressive behavior after alcohol self-administration in mice

Rationale The positive modulation of gamma-aminobutyric acid type-A (GABA_A) receptors is a putative mechanism via which alcohol escalates aggressive behavior. Broad-spectrum benzodiazepine antagonists block alcohol-heightened aggression in rats and monkeys. However, the degree to which GABA_A subunit composition plays a role in heightened aggressive behavior induced by self-administration of a moderate alcohol dose remains unresolved.Objective -Carboline-3-carboxylate-t-butyl ester (-CCt) and zolpidem act preferentially at GABA_A receptors containing the ₁ subunit as antagonist and agonist, respectively, and serve as useful tools to evaluate the role of GABA_A receptor subtypes in self-administered alcohol on aggression.Methods Male resident mice, housed in breeding pairs, were conditioned to nose-poke in a removable panel in their home cage, with each fifth poke being reinforced by the delivery of 0.05 ml of 6% ethanol (EtOH). After consuming EtOH, the resident mice were given the antagonists -CCt and flumazenil or agonists zolpidem and triazolam, and then confronted an intruder male in their home cage for a 5-min period.Results Following self-administration of EtOH (1.0 g/kg, 1.7 g/kg), 14 of 37 resident mice displayed unusually large increases in the frequency of attack bites and sideways threats. Flumazenil or -CCt decreased alcohol-heightened and non-heightened aggression in a dose-dependent manner. Administration of 3 mg/kg -CCt lowered the aggression-heightening effects of 1 g/kg and 1.7 g/kg EtOH, but did not antagonize the sedative effects of 3.0 g/kg EtOH. Triazolam and zolpidem decreased alcohol-heightened and non-heightened aggressive behavior, and these antiaggressive effects were accompanied by reduced motor activity, indicating sedation.Conclusions Benzodiazepine antagonists, particularly those acting preferentially at GABA_A/₁ subunit-containing receptors, decrease alcohol-heightened and species-typical aggressive behavior, but are ineffective in attenuating the sedative effects of alcohol.