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     

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.     

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     

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.     

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.     

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.     

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.     

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     

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.     

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     

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.     

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     

Modulation of tnf-α-induced icam-1 expression,no and h<Subscript>2</Subscript>0<Subscript>2</Subscript> production by alginate,allicin and ascorbic acid in human endothelial cells

Plant nutrients are believed to provide protection against various diseases including inflammation. Since interactions of the cell adhesion molecules are known to play important roles in mediating inflammation, inhibiting adhesion protein upregulation is a possible therapeutic target. In this study, the interacellular adhesion molecule-1 (ICAM-1) was induced in human umbilical endothelial cells (HUVECs) after stimulation with TNF-alpha. In addition, alginate, ascorbic acid and allicin were demonstrated to inhibit the TNF-alpha induced expression of ICAM-1 on the HUVECs in a dose-dependent manner. These compounds also inhibited the production of NO and H2O2 induced by TNF-alpha, which suggests that the inhibition of ICAM-1 expression by the three compounds may be due to the modulated production of the reactive oxygen/nitrogen components. Overall, these results indicate that these dietary components have a therapeutic potential in the treatment of various inflammatory disorders associated with an increase in endothelial leukocyte adhesion molecules.     

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.     

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.     

Functional characterisation of α<Subscript>1</Subscript>-adrenoceptors in denervated rat vas deferens

We investigated whether or not surgical denervation of the rat vas deferens changes the ₁-adrenoceptor subtypes involved in the contractions to noradrenaline. Denervated vas deferens was 22 times more sensitive to noradrenaline (pD₂=7.35±0.04) than control vas (pD₂=6.01±0.03). This difference in noradrenaline potency was eliminated when cocaine (6 M) was added to control vas (pD₂=7.22±0.04). The noradrenaline-induced contractions of control and denervated vas deferens were insensitive to the _1B/_1D-adrenoceptor alkylating agent chloroethylclonidine (100 M, 45 min). The concentration-response curves to noradrenaline in control and denervated vas deferens were competitively antagonised by prazosin (pA₂9.6), WB-4101 (pA₂9.5), 5-methyl urapidil (pA₂8.4), phentolamine (pA₂8.7), yohimbine (pA₂6.9), BMY 7378 (pA₂6.9) and indoramin (pA₂8.7). After the treatment of control and denervated vas deferens with phenoxybenzamine, the partial agonist oxymetazoline antagonised competitively the concentration-response curves to noradrenaline showing pA₂ values 7.4 in both groups. We conclude that noradrenaline-induced contractions in control and denervated rat vas deferens are mediated by _1A-adrenoceptors and that surgical denervation of the rat vas deferens is not able to change the ₁-adrenoceptor subtypes involved in the contractions to noradrenaline.     

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.