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.     

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.     

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     

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.     

Antiproliferative and cytotoxic effects of some σ<Subscript>2</Subscript> agonists and σ<Subscript>1</Subscript> antagonists in tumour cell lines

To establish the activity of ligands at ₁ and ₂ receptor, we chose two tumour cell lines, the human SK-N-SH neuroblastoma and the rat C6 glioma lines, which express ₂ receptors at a high density and ₁ receptors in their high-affinity or low-affinity state. We tested the ₂ receptor agonist PB28 and the ₂ antagonist AC927, and (+)-pentazocine and NE100 as agonist and antagonist, respectively, at ₁ receptors, with regard to antiproliferative and cytotoxic effects. In addition, 1,3-di(2-tolyl)guanidine (DTG) and haloperidol were tested as reference compounds displaying nearly equipotent affinity (₂>₁ and ₁>₂, respectively). In both SK-N-SH and C6 cells, PB28 and NE100 displayed the most potent results both in antiproliferative and cytotoxic assay while AC927 and (+)-pentazocine were inactive in both assays. The cytotoxic and antiproliferative effects of DTG and haloperidol reflected their ₁ antagonist activity and ₂ agonist activity. Moreover, our results in the tumour cell lines correlated well with those for ₂ activity found previously in a functional assay in the guinea-pig bladder. These findings establish a new model for evaluating both ₂ and ₁ receptor activity of ligands, which could be useful for developing new ligands having mixed ₂ agonist/₁ antagonist activity as potential antineoplastic agents.     

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.     

Molecular characterization of specific H<Subscript>1</Subscript>-receptor agonists histaprodifen and its N<Superscript>α</Superscript>-substituted analogues on bovine aortic H<Subscript>1</Subscript>-receptors

We determined the molecular properties of the selective and potent H(1)-receptor agonist histaprodifen and its N(alpha) substituted analogues: methyl-, dimethyl-, and imidazolylethyl-histaprodifen (suprahistaprodifen). All derivatives show high affinity for (3)H-mepyramine labeled bovine aortic H(1)-receptor binding sites with the following order of potency: suprahistaprodifen > dimethylhistaprodifen > methylhistaprodifen > histaprodifen > histamine. Suprahistaprodifen and dimethylhistaprodifen were the most potent displacers of (3)H-mepyramine binding (K(i)=4.3 and 4.9 nM, respectively). Histaprodifen, methylhistaprodifen and suprahistaprodifen binding was differentially influenced by GTP, whereas dimethylhistaprodifen was not affected. All drugs, except dimethylhistaprodifen, were activators of G-proteins. Their order of potency was suprahistaprodifen > histamine > histaprodifen > methylhistaprodifen. Their effect on G-protein activation was abolished by the addition of the H(1)-receptor antagonist triprolidine (10 microM), which given alone did not activate G-proteins. Our data suggest that histaprodifens are potent but heterogeneous H(1)-receptor ligands with diverse effects on the molecular level in our model system. While the histaprodifen, methylhistaprodifen and suprahistaprodifen data are in agreement with their agonistic nature, as shown in the functional studies performed on different species (rat and guinea pig H(1)-receptor), dimethylhistaprodifen behaved as an antagonist in our study.     

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.     

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     

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.     

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     

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     

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.     

Binding of (−)-[<Superscript>3</Superscript>H]-CGP12177 at two sites in recombinant human β<Subscript>1</Subscript>-adrenoceptors and interaction with β-blockers

To verify the hypothesis that the non-conventional partial agonist (–)-CGP12177 binds at two ₁-adrenoceptor sites, human ₁-adrenoceptors, expressed in CHO cells, were labelled with (–)-[³H]-CGP12177. We compared the binding affinity and antagonist potency of 12 clinically used -blockers against the cyclic AMP-enhancing effects of (–)-isoprenaline and (–)-CGP12177.(–)-[³H]-CGP12177 bound to a high affinity site (H; K_H=0.47 nM) and low affinity site (L); K_L=235 nM). (–)-[³H]-CGP12177 dissociated from the ₁-adrenoceptors with a fast component (k_off=0.45 min^–1), consistent with the L-site, and a slow component (k_off=0.017–0.033 min^–1), consistent with the H-site. (–)-Isoprenaline and (–)-CGP12177 caused 96-fold and 12-fold maximal increases in cyclic AMP levels with –logEC₅₀M of 8.2 and 7.6. (–)-CGP12177 antagonised the effects of (–)-isoprenaline with a pK_B of 9.9. The -blockers antagonised the effects of (–)-isoprenaline more than the effects of (–)-CGP12177 with potency ratios: (–)-atenolol 1,000, (±)-metropolol 676, (–)-pindolol 631, (–)-timolol 589, (±)-carvedilol 204, (±)-oxprenolol 138, (±)-sotalol 132, (–)-propranolol 120, (±)-bisoprolol 95, (±)-alprenolol 81, (±)-nadolol 68 and (–)-bupranolol 56. In intact cells the binding constants of -blockers, estimated from competition with 3–5 nM (–)-[³H]-CGP12177 (binding to the H-site), correlated with the corresponding affinities estimated from antagonism of the (–)-isoprenaline effects.We conclude that (–)-[³H]-CGP12177 binds at two sites in the recombinant ₁-adrenoceptor. (–)-CGP12177 is an antagonist of catecholamine effects through the H-site and a non-conventional partial agonist through the L-site. -blockers are more potent antagonists through the H-site than the L-site.     

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.     

Molecular structure of the rabbit α<Subscript>2A</Subscript>-adrenoceptor: a contribution to the α<Subscript>2A</Subscript>-adrenoceptor versus I<Subscript>1</Subscript> imidazoline receptor controversy

In view of the high structural and pharmacological similarities between the alpha(2A)-adrenoceptors of humans and other mammalian species, it has been concluded, in particular, from experiments in rabbits that the (2A)-adrenoceptor is the exclusive site of action of central antihypertensive drugs, although the amino acid sequence of the alpha(2A)-adrenoceptor of just this species was unknown. Therefore, the aim of the present investigation was to determine the complete nucleotide sequence of the coding region of the rabbit alpha(2A)-adrenoceptor gene. Degenerate oligonucleotides corresponding to regions of the alpha(2A)-adrenoceptor conserved between rat and man were used in a polymerase chain reaction with genomic DNA prepared from rabbit. A 1,356-base pair product with an open reading frame of 1,353 base pairs was obtained that encodes a protein of 451 amino acids which is similar to the alpha(2A)-adrenoceptors of other mammals (man, pig, rat, mouse, guinea-pig and cattle) but not to their alpha(2B)- and alpha(2C)-adrenoceptor subtypes suggesting its classification as an alpha(2A)-adrenoceptor. However, the degree of amino acid sequence identity is, at best, only 80% and, thus, about 10% less than between the other mammalian species. Compared with the human sequence there are 81 substantial changes of amino acids. In conclusion, rabbit and human alpha(2A)-adrenoceptors substantially differ in their amino acid sequence which may explain the opposite pharmacodynamic properties of the central antihypertensive drug rilmenidine (alpha(2)-adrenoceptor agonism and antagonism, respectively) reported in the literature. Hence, the present study supports the view that experiments with central antihypertensive drugs in rabbits are not reliably predictive for the site of action of such drugs in man.     

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     

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.     

Efficacy and safety of the novel α<Subscript>4</Subscript>β<Subscript>2</Subscript> neuronal nicotinic receptor partial agonist ABT-089 in adults with attention-deficit/hyperactivity disorder: a randomized,double-blind,placebo-controlled crossover study

Rationale  

α₄β₂ Neuronal nicotinic receptors (NNRs) are implicated in the pathophysiology of attention-deficit/hyperactivity disorder (ADHD).