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.     

Role of β<Subscript>3</Subscript>-adrenoceptors in regulation of retinal vascular tone in rats

The aim of this study was to determine the role of β₃-adrenoceptors in the action of endogenous catecholamines (adrenaline and noradrenaline) on rat retinal arterioles in vivo. Using an original high-resolution digital fundus camera, the rat ocular fundus images were captured. The diameter of retinal arterioles contained in the images was measured. Both systemic blood pressure and heart rate were recorded continuously. Adrenaline (0.3–5.0 μg/kg/min, i.v.) increased the diameter of retinal arterioles, mean blood pressure and heart rate in a dose-dependent manner. Under blockade of β₁/β₂-adrenoceptors with propranolol (2 mg/kg, i.v. bolus followed by 100 μg/kg/min infusion), adrenaline decreased the diameter of retinal arterioles. Similar observation was made under treatment with the β₃-adrenoceptor antagonist L-748337 (50 μg/kg, i.v.). The pressor response to adrenaline was enhanced by propranolol, but not by L-748337. The positive chronotropic action of adrenaline was markedly prevented by propranolol, whereas it was unaffected by L-748337. Noradrenaline (0.03–1.0 μg/kg/min, i.v.) decreased the diameter of retinal arterioles but increased the mean blood pressure and heart rate. The effects of noradrenaline on retinal arteriolar diameter and blood pressure were unaffected by propranolol or L-748337. The positive chronotropic action of noradrenaline was almost completely abolished by propranolol. These results suggest that β₃-adrenoceptors play crucial roles in vasodilator responses to adrenaline of retinal arterioles but have minor or no effect on noradrenaline-induced responses. The results also indicate that the functional role of β₃-adrenoceptors may be more important than that in peripheral resistance vessels.     

Cardiostimulant and cardiodepressant effects through overexpressed human β<Subscript>2</Subscript>-adrenoceptors in murine heart: regional differences and functional role of β<Subscript>1</Subscript>-adrenoceptors

(-)-Isoprenaline enhances cardiac contractility through beta-adrenoceptors. However, in cardiac tissue from transgenic mice with a 200-400-fold cardiac overexpression of the human beta(2)-adrenoceptor (TG4) we observed a pronounced cardiodepression at high (-)-isoprenaline concentrations. Here, we investigated the functional role of the coexisting beta(1)-, beta(2)-, and beta(3)-adrenoceptor subtypes in several regions of the TG4 heart, and in particular their contribution to the negative inotropic effect. In paced TG4 left atria, (-)-isoprenaline produced bell-shaped concentration-effect curves increasing (-logEC(50)M=9.0) and decreasing (-logIC(50)M=6.4) contractile force. These effects were unaffected by the beta(1)-selective CGP 20712A (300 nM). The beta(2)-selective inverse agonist ICI 118,551 (30-1,000 nM) antagonised in surmountable manner both the positive and negative inotropic effects of (-)-isoprenaline with similar concentration-dependence, consistent with an exclusive mediation through beta(2)-adrenoceptors. The beta(3)-adrenoceptor-selective agonist BRL37344 (1 nM-10 microM) failed to produce significant inotropic effects in TG4 left atria. Subsequently, we measured left atrial action potentials accompanying the inotropic changes induced by (-)-isoprenaline. Action potentials tended to have shorter duration in left atria from TG4 mice than from non-transgenic littermate mice. However, (-)-isoprenaline prolonged the duration of 30% repolarisation in atria from non-transgenic littermate but not from TG4 mice, while 90% repolarisation was abbreviated in both groups of atria. Negative inotropic effects of (-)-isoprenaline were also observed in right ventricular preparations. Pertussis toxin-treatment of the mice abolished the negative inotropic effects in left atria and reduced cardiodepression in right ventricle, indicating an involvement of beta(2)-adrenoceptor coupling to PTX-sensitive G-proteins. In additional experiments, designed to study the native murine beta(1)-adrenoceptor function, we used the physiological beta(1)-adrenoceptor agonist (-)-noradrenaline. In the presence of 600 nM ICI 118,551 we failed to find a functional role of the beta(1)-adrenoceptors in left atria, and detected only a marginal contribution to the positive chronotropic effect in right atria. We also investigated the effects of the non-conventional partial agonist (-)-CGP 12177 (0.2 nM-6 microM), which in wild-type mice causes tachycardia through beta(1)-adrenoceptors. In TG4 right atria, however, (-)-CGP 12177-evoked tachycardia was resistant to blockade by CGP 20712A but antagonised by ICI 118,551, consistent with mediation through human beta(2)-adrenoceptors.The results from TG4 mice suggest that the positive and negative inotropic effects of (-)-isoprenaline are mediated through human overexpressed beta(2)-adrenoceptors coupled to G(s) protein and G(i) protein, respectively. The (-)-isoprenaline-evoked shortening of the atrial action potential combined with reduced responses of L-type Ca(2+) current may contribute to the negative inotropic effects. The function of murine cardiac beta(1)-adrenoceptors is suppressed by overexpressed human beta(2)-adrenoceptors.     

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.     

A comparative autoradiographic study of the density of [<Superscript>3</Superscript>H]SR95531, [<Superscript>3</Superscript>H]MK-801 and [<Superscript>3</Superscript>H]cGMP binding in the locus coeruleus and central pontine grey of spontaneously hypertensive and Wistar–Kyoto rats

The Spontaneously Hypertensive rat (SHR) has been previously shown to have a host of neurochemical differences compared with their normotensive counterpart, the Wistar–Kyoto (WKY) rat. Using quantitative receptor autoradiography, the density of GABA_A and NMDA receptors and [³H]cGMP binding within the locus coeruleus (LC) and central pontine grey (CGPn) were compared in the SHR and WKY rat using the radioligands [³H]SR95531, [³H]MK-801 and [³H]cGMP respectively. It was found that [³H]SR95531 binding was significantly greater in both the LC and CGPn of the SHR compared with the WKY rat (unpaired t test; P<0.05). Greater binding densities of [³H]MK-801 and [³H]cGMP were also observed in the LC of the SHR compared with the WKY rat; however, no differences in the binding density of these two ligands were observed in the CGPn. It is suggested that these neurochemical differences within the LC of the SHR may relate to phenotypic differences between SHR and WKY rats that have previously been reported.     

β<Superscript>2</Superscript>/β<Superscript>3</Superscript>-di- and α/β<Superscript>3</Superscript>-tetrapeptide derivatives as potent agonists at somatostatin sst<Subscript>4</Subscript> receptors

Four linear beta(2)/beta(3)-di- and alpha/beta(3)-tetrapeptides (1-4) were investigated as somatostatin sst(4) receptor agonists on recombinant human and mouse somatostatin receptors. Human somatostatin receptor subtypes 1-5 (sst(1-5)), and mouse somatostatin receptor subtypes 1,3,4 and 5, were characterised using the agonist radioligands [(125)I]LTT-SRIF-28, [(125)I][Tyr(10)]CST(14) and [(125)I]CGP 23996 in stably transfected Chinese hamster lung fibroblast (CCL39) cells. The peptides bound selectively to sst(4) receptors with nanomolar affinity (pK(d)=5.4-7.8). The peptides were investigated on second messenger systems both as agonists, and as antagonists to SRIF-14-mediated effects in CCL39 cells expressing mouse sst(4 )receptors, via measurement of inhibition of forskolin-stimulated adenylate cyclase activity, and stimulation of luciferase expression. The peptides showed full agonism or pronounced partial agonism (40 to 100% relative intrinsic activity) in both inhibition of forskolin-stimulated adenylate cyclase activity (pEC(50)=5.5-6.8), and luciferase expression (pEC(50)=5.5-6.5). The agonist potential was confirmed since antagonism was very difficult to establish. The data show that beta(2)/beta(3)-di- and alpha/beta(3)-tetrapeptide derivatives have agonist potential at recombinant somatostatin sst(4) receptors. Therefore, they may be used to elucidate physiological and biochemical effects mediated by sst(4), and may also have potential as therapeutic agents.     

Stimulatory effect of Coca-Cola<Superscript>TM</Superscript> on gastroduodenal HCO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> secretion in rats

We examined the effect of various carbonated beverages, especially Coca-Cola^TM, on the HCO₃⁻ secretion in the rat stomach and duodenum. Under urethane anaesthesia, a chambered stomach or a proximal duodenal loop was perfused with saline, and HCO₃⁻ secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. The amount of CO₂ contained in these beverages was about 4–7 g/mL. Coca-Cola^TM topically applied to the mucosa for 10 min significantly increased the HCO₃⁻ secretion in both the stomach and the duodenum. The HCO₃⁻ response in the duodenum was totally abolished by indomethacin and also partially inhibited by acetazolamide, an inhibitor of carbonic anhydrase. Likewise, the response in the stomach was also markedly inhibited by either acetazolamide or indomethacin. The mucosal application of Coca-Cola^TM increased the PGE₂ contents in both the stomach and the duodenum. Other carbonated beverages, such as sparkling water, Fanta Grape^TM or cider, also increased the HCO₃⁻ secretion in these tissues. These results suggest that Coca-Cola^TM induces HCO₃⁻ secretion in both the stomach and the duodenum, and these responses may be attributable to both the intracellular supply of HCO₃⁻ generated via carbonic anhydrase, and endogenous PGs, probably related to the acidic pH of the solution. Received 4 August 2006; accepted 10 November 2006     

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     

Modulation of 5-HT<Subscript>1A</Subscript> receptor-mediated Ca<Superscript>2+</Superscript> responses by co-expression with various recombinant G<Subscript>α</Subscript> proteins in CHO-K1 cells

The ability of the human 5-HT_1A receptor to activate different recombinant G proteins was investigated in CHO-K1 cells by monitoring 5-HT ligand-mediated Ca²⁺ responses upon co-expression with either G_q, G₁₅ or chimeric G_q/i3 proteins. Each G protein yielded a typical 5-HT-dependent Ca²⁺ response with different kinetic parameters both for the onset-time of maximal Ca²⁺ response (21 to 30 s) and time-dependent attenuation (43 to 73% of residual activity at 1 min upon peak Ca²⁺ response). Pertussis toxin-treatment fully abolished the Ca²⁺ responses mediated by both the endogenous G_i/o and the chimeric-PTX-sensitive G_q/i3 proteins. In contrast, Ca²⁺ responses driven by recombinant G_q and G₁₅ proteins were decreased by PTX, respectively by 52% and 35%, corresponding to the level of endogenous G protein activation. The pharmacology of the 5-HT ligand-mediated Ca²⁺ responses was highly affected by both the presence and nature of the co-expressed G protein. This influence was more pronounced for the partial agonists L 694247, 8-OH-DPAT, flesinoxan and buspirone in contrast to ipsapirone. The G protein rank order for apparent increase of ligands' intrinsic activity was: G_q <G_q/i3 <G₁₅ protein. Each of the 5-HT-mediated Ca²⁺ responses could be antagonised by WAY 100635, buspirone and methiothepin regardless of the absence or presence of a G_q, G_q/i3 or G₁₅ protein. In conclusion, these data reinforce that depending on the presence and nature of the G protein environment, 5-HT_1A ligands may display a large spectrum of activities.Abbreviations AFU Arbitrary fluorescence unit - 5-CT 5-Carboxamidotryptamine - 5-HT 5-Hydroxytryptamine (serotonin) - 8-OH-DPAT 8-(Hydroxy-2-(di-n-propylamino)tetralin - CHO Chinese hamster ovary - PLC Phospholipase C - WAY 100635 N-[2-[4-(2-methoxyphenyl)1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide - PTX Bordetella pertussis toxin - wt Wild-type     

The effects of hydrocortisone on rat heart muscarinic and adrenergic α<Subscript>1</Subscript>, β<Subscript>1</Subscript> and β<Subscript>2</Subscript> receptors,propranolol-resistant binding sites and on some subsequent steps in intracellular signalling

Glucocorticoids affect the expression and density of neurotransmitter receptors in many tissues but data concerning the heart are contradictory and incomplete. We injected rats with hydrocortisone for 1–12 days and measured the densities of cardiac muscarinic receptors, ₁-, ₁- and ₂-adrenoceptors and propranolol-resistant binding sites (formerly assumed to be the putative ₄-adrenoceptor). Some aspects of intracellular signalling were also evaluated: we measured adenylyl cyclase activity (basal, isoprenaline- and forskolin-stimulated and carbachol-inhibited), the coupling between muscarinic receptors and G proteins and basal and isoprenaline-stimulated heart rate. The density of cardiac muscarinic receptors increased (in both the atria and the ventricles). The density of ₁-adrenoceptors increased in the atria and was little changed in the ventricles. The density of ₂-adrenoceptors increased in both the atria and the ventricles. The number of ₁-adrenoceptors decreased initially, followed by a transient increase in the atria and did not change in the ventricles. The density of propranolol-resistant binding sites first increased and then diminished in the atria and did not change in the ventricles. Although there were noticeable changes in receptor densities, the stimulatory and inhibitory effects on adenylyl cyclase, basal and isoprenaline-stimulated heart rate and the coupling between muscarinic receptors and G proteins were not significantly altered. This may indicate that changes in receptor densities might be one of the mechanisms maintaining stable functional output. Deceased     

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     

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.     

Loss of [<Superscript>3</Superscript>H]4-DAMP binding to muscarinic receptors in the orbitofrontal cortex of Alzheimer’s disease patients with psychosis

Rationale Neuropsychiatric behaviours in Alzheimer’s disease (AD) patients have been associated with neocortical alterations of presynaptic cholinergic and muscarinic M2 receptor markers. In contrast, it is unclear whether non-M2 muscarinic receptors have a role to play in AD behavioural symptoms. Objectives To correlate the alterations of neocortical postsynaptic muscarinic receptors with clinical features of AD. Materials and methods [³H]4-DAMP were used in binding assays with lysates of Chinese hamster ovary (CHO) cells stably transfected with M1–M5 receptors. [³H]4-DAMP was further used to measure muscarinic receptors in the postmortem orbitofrontal cortex of aged controls and AD patients longitudinally assessed for cognitive decline and behavioural symptoms. Results [³H]4-DAMP binds to human postmortem brain homogenates and M1-, M3-, M4- and M5-transfected CHO lysates with subnanomolar affinity. Compared to the controls, the [³H]4-DAMP binding density is reduced only in AD patients with significant psychotic symptoms. The association between reduced [³H]4-DAMP binding and psychosis is independent of the effects of dementia severity or neurofibrillary tangle burden. Conclusions This study suggests that the loss of non-M2 muscarinic receptors in the orbitofrontal cortex may be a neurochemical substrate of psychosis in AD and provides a rationale for further development of muscarinic receptor ligands in AD pharmacotherapy.     

Pharmacological profile of the cyclic nociceptin/orphanin FQ analogues c[Cys<Superscript>10,14</Superscript>]N/OFQ(1–14)NH<Subscript>2</Subscript> and c[Nphe<Superscript>1</Superscript>,Cys<Superscript>10,14</Superscript>]N/OFQ(1–14)NH<Subscript>2</Subscript>

In this study we describe the activity of two cyclic nociceptin/orphanin FQ (N/OFQ) peptides; c[Cys^10,14]N/OFQ(1–14)NH₂ (c[Cys^10,14]) and its [Nphe¹] derivative c[Nphe¹,Cys^10,14]N/OFQ(1–14)NH₂ (c[Nphe¹,Cys^10,14]) in native rat and mouse and recombinant human N/OFQ receptors (NOP). Cyclisation may protect the peptide from metabolic degradation.In competition binding studies of rat, mouse and human NOP the following rank order pK_i was obtained: N/OFQ(1–13)NH₂(reference agonist)>N/OFQ=c[Cys^10,14]>>c[Nphe¹Cys^10,14]. In GTP³⁵S studies of Chinese hamster ovary cells expressing human NOP (CHO_hNOP) c[Cys^10,14] (pEC₅₀ 8.29) and N/OFQ(1–13)NH₂ (pEC₅₀ 8.57) were full agonists whilst c[Nphe¹Cys^10,14] alone was inactive. Following 30 min pre-incubation c[Nphe¹Cys^10,14] competitively antagonised the effects of N/OFQ(1–13)NH₂ with a pA₂ and slope factor of 6.92 and 1.01 respectively. In cAMP assays c[Cys^10,14] (pEC₅₀ 9.29, E_max 102% inhibition of the forskolin stimulated response), N/OFQ(1–13)NH₂ (pEC₅₀ 10.16, E_max 103% inhibition) and c[Nphe¹Cys^10,14] (~80% inhibition at 10 M) displayed agonist activity. In the mouse vas deferens c[Cys^10,14] (pEC₅₀ 6.82, E_max 89% inhibition of electrically evoked contractions) and N/OFQ(1–13)NH₂ (pEC₅₀ 7.47, E_max 93% inhibition) were full agonists whilst c[Nphe¹Cys^10,14] alone was inactive. c[Nphe¹Cys^10,14] (10 M) competitively antagonised the effects of N/OFQ(1–13)NH₂ with a pK_B of 5.66. In a crude attempt to assess metabolic stability, c[Cys^10,14] was incubated with rat brain membranes and then the supernatant assayed for remaining peptide. Following 60 min incubation 64% of the 1 nM added peptide was metabolised (compared with 54% for N/OFQ-NH₂).In summary, we report that c[Cys^10,14] is a full agonist with a small reduction in potency but no improvement in stability whilst c[Nphe¹Cys^10,14] displays tissue (antagonist in the vas deferens) and assay (antagonist in the GTP³⁵S assay and agonist in cAMP assay) dependent activity.Presented in part to The British Pharmacological Society at the Brighton, UK Meeting January 2003     

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     

Discrimination between α2-adrenoceptors and [3H]idazoxan-labelled non-adrenergic sites in rabbit white fat cells

Previous results indicated that the rabbit could represent a suitable model for investigations on the functional role of α₂-adrenoceptors in fat cells, but the characterization of these receptors was not resolved yet. In the present report, imidazoline compounds were used to attempt a better definition of rabbit adipocyte α₂-receptivity by means of lipolysis and binding studies. Lipolysis data showed that UK14304 is a full α₂-adrenoceptor agonist promoting a strong antilipolysis in rabbit fat cells. Moreover, the methoxy derivative of idazoxan, RX821002, is a more potent antagonist of UK14304-induced antilipolysis than idazoxan or yohimbine. Whereas [³H]yohimbine failed to bind at rabbit adipocyte α₂-adrenoceptors, [³H]UK14304 and [³]RX821002 are valuable tools to study this receptor. Analysis of binding data demonstrated that [³H]UK14304 labels the high-affinity-state receptor while [³H]RX821002 binds to the whole α₂-adrenergic population. Inhibition studies [³H]RX821002 and [³H]UK14304 binding by various compounds confirmed the α₂-adrenergic nature of the sitesl labelled by both radioligands. The other α₂-adrenoceptor radioligand, [³H]idazoxan, labelled binding sites which are insentitive to catecholamine. Competition studies of [³H]idazoxan binding with imidazoline derivatives revealed structure-activity relationships different from those of α₂-adrenoceptors. The most striking observation is that substitutions in the 2-position of idazoxan markedly reduce the affinity for the non-adrenergic sites, whereas the α₂-potency is increased or unchanged.     

Comparison of the subjective effects of Δ<Superscript>9</Superscript>-tetrahydrocannabinol and marijuana in humans

RATIONALE: There has been controversy about whether the subjective, behavioral or therapeutic effects of whole plant marijuana differ from the effects of its primary active ingredient, Delta(9)-tetrahydrocannabinol (THC). However, few studies have directly compared the effects of marijuana and THC using matched doses administered either by the smoked or the oral form.OBJECTIVE: Two studies were conducted to compare the subjective effects of pure THC to whole-plant marijuana containing an equivalent amount of THC in normal healthy volunteers. In one study the drugs were administered orally and in the other they were administered by smoking.METHODS: In each study, marijuana users (oral study: n=12, smoking study: n=13) participated in a double-blind, crossover design with five experimental conditions: a low and a high dose of THC-only, a low and a high dose of whole-plant marijuana, and placebo. In the oral study, the drugs were administered in brownies, in the smoking study the drugs were smoked. Dependent measures included the Addiction Research Center Inventory, the Profile of Mood States, visual analog items, vital signs, and plasma levels of THC and 11-nor-9-carboxy-THC.RESULTS: In both studies, the active drug conditions resulted in dose-dependent increases in plasma THC levels, and the levels of THC were similar in THC-only and marijuana conditions (except that at the higher oral dose THC-only produced slightly higher levels than marijuana). In both the oral study and the smoking study, THC-only and whole plant marijuana produced similar subjective effects, with only minor differences.CONCLUSION: These results support the idea that the psychoactive effects of marijuana in healthy volunteers are due primarily to THC.     

Gender comparison of muscarinic receptor expression and function in rat and human urinary bladder: differential regulation of M<Subscript>2</Subscript> and M<Subscript>3</Subscript> receptors?

Since symptoms of bladder dysfunction occur more frequently in women than in men and since muscarinic receptors are the physiologically most important system to mediate bladder contraction, we have compared the number, subtype distribution and function of muscarinic receptors in bladders from male and female rats. Muscarinic receptor function was also assessed in bladder strips from male and female human bladder. Male and female rats expressed a similar number of muscarinic receptors (144+/-5 vs. 140+/-6 fmol/mg protein in saturation radioligand binding). While competition binding curves for the moderately M(2)-selective methoctramine were not consistently better fitted by a two-site model, most competition curves for the M(3)-selective darifenacin were biphasic and yielded 29+/-10% and 31+/-7% high affinity sites (corresponding to M(3) receptors) in male and females, respectively. Immunoreactivity of alpha-subunits of the G-proteins G(q/11), G(i1/2), G(i3) and G(s) did not significantly differ between both genders. The muscarinic receptor agonist carbachol similarly stimulated inositol phosphate accumulation in bladder slices from male and female rats with calculated maximum responses of 69+/-17 and 77+/-18% over basal and pEC(50) values of 4.90+/-0.45 and 4.40+/-0.46, respectively. While darifenacin inhibited carbachol-stimulated inositol phosphate formation approximately 100-fold more potently than methoctramine, each antagonist was similarly potent in both genders. Carbachol concentration-dependently contracted bladder strips with a pEC(50) of 5.66+/-0.05 and 5.72+/-0.06 and maximum effects of 4.3+/-0.1 and 4.2+/-0.2 mN/mg wet weight in male and female rats, respectively. The contractile effect of carbachol was concentration-dependently antagonised by the non-selective atropine (1-30 nM), the M(1)-selective pirenzepine (1-30 M), the M(2)-selective methoctramine (1-10 microM) and the M(3)-selective darifenacin (10-100 nM), with the latter exhibiting a partly unsurmountable antagonism. The overall potency of all four antagonists suggested that contraction was mediated predominantly if not exclusively by M(3) receptors with no appreciable differences between both male and female rats. Similarly, the maximum effects (4.4+/-0.6 vs. 4.4+/-2.4 mN/mg) and pEC(50) (6.07+/-0.05 vs. 6.32+/-0.14) of carbachol did not differ between genders in bladder samples from 25 consecutive patients. We conclude that number und function of muscarinic receptors and the relative roles of their M(2) and M(3) subtypes do not differ between urinary bladders of male and female rats; at least with regard to overall muscarinic responsiveness this situation appears to be similar in humans.