Potentiation of the relaxing action of isoproterenol by forskolin in rabbit aortic rings: The involvement of β2-adrenoceptors

• 1.1. Forskolin, an activator of adenylate cyclase, potentiated the relaxing response to isoproterenol in rabbit aortic rings precontracted by phenylephrine (PE).
• 2.2. The potentiating effect of forskolin was inhibited by propranolol, a β-adrenoceptor inhibitor, but not by methylene blue, a guanylate cyclase inhibitor.
• 3.3. The relaxing response to terbutaline, a β₂-adrenoceptor agonist, but not lower concentrations of dobutamine, a β₁-adrenoceptor agonist, also was potentiated by forskolin. Forskolin, however, potentiated the relaxing response to high concentrations of dobutamine, which activates both β₁- and β₂-adrenoceptors.
• 4.4. Yohimbine, an α₂-adrenoceptor inhibitor, glyburide, an ATP-sensitive K⁺ channel inhibitor, iberiotoxin, a Ca²⁺-activated K⁺ channel inhibitor, or endothelium-removal failed to affect the potentiating effect of forskolin.
• 5.5. Dibutyryl cyclic AMP (cAmp) also potentiated the relaxing response to terbutaline.
• 6.6. These results suggest that in rabbit aortic rings forskolin causes the apparent potentiation of isoproterenol-induced relaxation by mainly affecting the relaxing response due to the activation of β₂-adrenoceptors by the forskolin-induced increase in the level of cAMP.

     

Effect of a prostaglandin antagonist, N-0164, on cAMP generation and hydrolysis in the rat uterus

N-0164 (sodium-p-benzyl-4-[1-oxo-2-(4-chlorobenzyl)-3-phenyl propyl] phenylphosphonate) (20–100 μM), an antagonist of the contractile effect of prostaglandins, reversed the prostaglandin E₂ (PGE₂) inhibition of isoproterenol-induced cAMP accumulation in rat uterus. N-0164, at the same concentrations, was a potent cAMP-phosphodiesterase inhibitor in broken cell preparations and potentiated the cAMP response to isoproterenol in intact tissue. The potency of N-0164 to inhibit cAMP-phosphodiesterase and to reverse the effect of PGE₂ on the cAMP response to isoproterenol were comparable (ec₅₀:50 and 60 μM respectively). In the presence of 10 mM theophylline, N-0164 did not affect the inhibitory effect of PGE₂. Furthermore, N-0164 produced similar proportional increases in the cAMP response to isoproterenol in the presence and absence of PGE₂. These results suggest that the apparent reversal by N-0164 of the PGE₂ effect on the cAMP response to isoproterenol is not due to its prostaglandin antagonistic action but to inhibition of cAMP-phosphodiesterase. N-0164, at concentrations lower than those inhibiting cAMP-phosphodiesterase, selectively inhibited the PGE₂-induced contractions of the rat uterus (ec₅₀, 4 μM), while at higher concentrations it also diminished carbachol-induced contractions. These results indicate that in the rat uterus N-0164 has at least two effects, prostaglandin antagonism and cAMP-phosphodiesterase inhibition, and suggest that the contractile effect of PGE₂ is independent of the effect of PGE₂ on the isoproterenol-induced rise in cAMP.     

Interaction of morphine but not fentanyl with cerebral α2‐adrenoceptors in α2‐adrenoceptor knockout mice

Objectives α₂‐Adrenergic and μ‐opioid receptors belong to the rhodopsin family of G‐protein coupled receptors and mediate antinociceptive effects via similar signal transduction pathways. Previous studies have revealed direct functional interactions between both receptor systems including synergistic and additive effects. To evaluate underlying mechanisms, we have studied whether morphine and fentanyl interacted with α₂‐adrenoceptor‐subtypes in mice lacking one individual α₂‐adrenoceptor‐subtype (α₂‐adrenoceptor knockout). Methods Opioid interaction with α₂‐adrenoceptors was investigated by quantitative receptor autoradiography in brain slices of α_2A‐, α_2B‐ or α_2C‐adrenoceptor deficient mice. Displacement of the radiolabelled α₂‐adrenoceptor agonist [¹²⁵I]paraiodoclonidine from α₂‐adrenoceptors in different brain regions by increasing concentrations of morphine, fentanyl and naloxone was analysed. The binding affinity of both opioids to α₂‐adrenoceptor subtypes in different brain regions was quantified. Key findings Morphine but not fentanyl or naloxone provoked dose‐dependent displacement of [¹²⁵I]paraiodoclonidine from all α₂‐adrenoceptor subtypes in the brain regions analysed. Binding affinity was highest in cortex, medulla oblongata and pons of α_2A‐adrenoceptor knockout mice. Conclusions Our results indicated that morphine interacted with α₂‐adrenoceptors showing higher affinity for the α_2B and α_2C than for the α_2A subtype. In contrast, fentanyl and naloxone did not show any relevant affinity to α₂‐adrenoceptors. This effect may have an impact on the pharmacological actions of morphine.     

A STUDY ON THE POSTJUNCTIONAL EXCITATORY α-ADRENORECEPTOR SUBTYPES IN THE MESENTERIC ARTERIAL BED OF THE RAT

• 1 The nature of the postsynaptic adrenoreceptor subtypes which mediate vasoconstriction in the mesenteric arterial bed of the rat was investigated using mixed and selective α₁, α₂ and β-agonists and antagonists.
• 2 Phenylephrine (PE) an α₁selective agonist and noradrenaline (NA) a mixed α₁and α₂-agonist, produced a rise in perfusion pressure (vasoconstriction). The responses to NA remained stable with time whereas responses to PE considerably increased.
• 3 UK14304 an α₂-selective agonist at low doses (10^?8 ?10^?7 moles), caused small, slow contractions in most preparations. Repeated administration of these doses or slightly higher ones, desensitized the tissue to this compound but not to NA or PE. Finally, UK14304 given simultaneously with NA or PE, at doses higher than 5 × 10^?7 moles, reduced contractions to the latter compounds and this effect was not altered by 10^?7 M rauwolscine, an α₂-selective antagonist.
• 4 Prazosin, an α₁-selective antagonist, as expected, reduced contractions to NA considerably at 10^?10 ?10^?8 M and abolished contractions to UK14304 at 2 × 10^?9 M.
• 5 Rauwolscine, at 10^?8 M, potentiated contractions to NA and at 10^?6 M reduced contractions to both NA and PE (when compared to time controls).
• 6 When propranolol (10^?6 M), a β-antagonist was included in the perfusion fluid, rauwolscine no longer potentiated responses to NA but reduced them at all concentrations. Under the same conditions rauwolscine affected the responses to PE in a similar direction to that observed in the absence of propranolol.
• 7 These results suggest that in the rat mesenteric arterial bed:

• a. rauwolscine exerts an effect additional to α₂-adrenoreceptor antagonism. Modification of this effect by propranolol indicates an interaction between this effect of rauwolscine and the β-adrenoreceptor.
• b. vasoconstriction in the mesenteric arterial bed of the rat is mainly mediated postsynaptically by α₁-adrenoreceptors although the contribution of an α₂-mediated component cannot be excluded.
• c. UK14304 is an α₁-partial agonist as well as an α₂-agonist.

     

Nefopam but not physostigmine affects the thermoregulatory response in mice via α2-adrenoceptors

Nefopam, a non-opioid, centrally acting benzoxazocine analgesic, proved to be as efficient in treatment of postanaesthetic thermoregulatory shivering as clonidine or meperidine. However, its exact mechanism of action is still unclear. Potent anti-shivering activity was also demonstrated for physostigmine primarily based on cholinergic but probably also different additional mechanisms of action. Hypothesizing an involvement of α₂-adrenoceptors we studied their role in nefopam- and physostigmine-mediated thermoregulation in a mouse model of nonshivering thermogenesis. To differentiate possible α₂-adrenoceptor subtype-specific interactions, we analysed wildtype mice and mice with deletion of the α_2A-, α_2B- or α_2C-adrenoceptor (knock out).Ten mice of each genotype (n = 40) were administered saline, saline plus atipamezole, 1 mg/kg nefopam, 25 mg/kg nefopam, 25 mg/kg nefopam plus atipamezole, physostigmine and physostigmine plus atipamezole intraperitoneally. Each mouse was randomly subjected to each of the seven different treatments. Afterwards, the mice were positioned into a plexiglas chamber where rectal temperature and mixed expired carbon dioxide were measured during following whole body cooling. Thermoregulatory threshold temperature of nonshivering thermogenesis and maximum response intensity were analysed.Nefopam decreased the thermoregulatory threshold temperature in wildtype, α_2B- and α_2C-adrenoceptor mice. This effect was partially abolished by additional administration of the α₂-adrenoceptor antagonist atipamezole. In α_2A-adrenoceptor knock out mice, nefopam did not affect the thermoregulatory threshold. In contrast, physostigmine decreased the thermoregulatory threshold in wildtype and all α₂-adrenoceptor knock out mice independently from additional atipamezole administration.Our results indicate an important role of the α_2A-adrenoceptor in the thermoregulatory response induced by nefopam but not by physostigmine in mice.     

Direct fusion of subunits of heterodimeric nitric oxide sensitive guanylyl cyclase leads to functional enzymes with preserved biochemical properties: Evidence for isoform specific activation by ciguates

Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme consisting of an α and a β subunit. Two heterodimeric enzymes are known to be important for NO-signalling in humans: α₁/β₁ and α₂/β₁. No difference had so far been detected with respect to their pharmacological properties, but as we show in the present paper the new drugs cinaciguat and ataciguat activate the α₁/β₁ form more effectively. Recent evidence suggests that homodimeric complexes of α and β subunits exist in vivo and that these non-heterodimerizing subunits have a separate function from cGMP signaling. To isolate the effect of the α₁/β₁ or α₂/β₁ heterodimeric enzyme in overexpression experiments from potential effects of non-heterodimerizing α₁, β₁ or α₂ subunits, we cloned constructs that guarantee a 1:1 stochiometry between α and β subunits and rule out the presence of homodimers. The carboxy-terminus of the β₁ subunit was directly fused to the amino-terminus of either the α₁ or α₂ subunit. The two different “conjoined” NOsGCs faithfully reproduced the biochemical and pharmacological properties of the α₁/β₁ and α₂/β₁ heterodimeric enzymes including the differential activation by ciguat-activators. Conjoined NOsGCs can be used for isoform specific overexpression in transgenic animals and therapeutic overexpression may be an application in the future. In both cases possible side effects of homodimeric α or β subunits are avoided. Crystallization with the goal of structure determination may also be easier for conjoined NOsGCs because enzyme preparations are more homogenous and are free of “contaminating” homodimers.     

Noradrenergic regulation of itch transmission in the spinal cord mediated by α-adrenoceptors

It has recently been shown that clonidine suppresses itch-related responses via its action on α₂-adrenoceptors in the spinal cord, raising the possibility that the descending noradrenergic system regulates itch signaling in the spinal cord. In this study, we investigated whether the transmission of itch signals in the spinal cord is under tonic inhibition by the descending noradrenergic system. An intraplantar injection of serotonin in mice induced biting of the treated paw (an itch-related response). An intrathecal injection of 6-hydroxydopamine (catecholaminergic neurotoxin) enhanced the itch-related response. There was a significant inverse correlation between the response and noradrenaline content. An intrathecal injection of phentolamine (α-adrenoceptor antagonist) enhanced serotonin-induced biting, although prazosin (α₁-, α_2B-, and α_2C-adrenoceptor antagonist) and yohimbine (α₂-adrenoceptor antagonist) had no effects. Intrathecal injections of phenylephrine (α₁-adrenoceptor agonist) and clonidine (α₂-adrenoceptor agonist) inhibited serotonin-induced biting. The action of phenylephrine was antagonized by intrathecal prazosin but not 5-methylurapidil (α_1A-adrenoceptor antagonist), cyclazosin (α_1B-adrenoceptor antagonist), and BMY 7378 (α_1D-adrenoceptor antagonist). mRNAs encoding α_1A-, α_1B-, α_2A-, α_2B-, and α_2C-adrenoceptor subtypes were expressed in the dorsal root ganglion and spinal dorsal horn. These results suggest that the descending noradrenergic system exerts tonic inhibition on itch signaling in the spinal cord. Both α₁- and α₂-adrenoceptors may be involved in the tonic inhibition of itch signaling and the stimulation of either α-adrenoceptor subtype may result in the inhibition of itch.     

FUNCTIONAL ASSESSMENT OF α1-ADRENOCEPTOR SUBTYPES IN PORCINE CORONARY ARTERY

1.α₁-Adrenoceptors are known to play an important role in vasoconstriction in response to adrenergic stimulation. However, the functional importance of α₁-adrenoceptor subtypes at the epicardial coronary artery remains unclear. We examined α-adrenoceptor subtypes by comparing functional affinities for α-adrenoceptor antagonists on noradrenaline (NA)-induced vasoconstriction in porcine denuded right coronary arteries. 2. Noradrenaline induced a dose-dependent vasoconstriction in incubated vessel rings. Prazosin and phentolamine were potent and competitive antagonists for NA-induced contraction (pA₂ 10.27 and 9.03, respectively). In contrast, the selective α₂-adrenoceptor antagonist yohimbine had a low affinity (pA₂ 6.13). Two selective α_1A-adrenoceptor antagonists, WB 4101 and 5-methyl urapidil, were potent and competitive antagonists of α₁-adrenoceptor-induced contraction (pA₂ 10.67 and 8.90, respectively) and the selective α_1D-adrenoceptor antagonist BMY 7378 had a low affinity (pA₂ 6.06). Noradrenaline-induced contraction was insensitive to the alkylating effects of chlorethyl-clonidine. These observations indicate that the vasoconstriction is predominantly mediated by the α_1A-adrenoceptor subtype. This was also supported by a good correlation between pA₂ values from the present study and reported binding affinities (pK_i) of various α-adrenoceptor antagonists with cloned human α_1A-adrenoceptors (r= 0.98), but not for α_1B- or α_1D-adreno-ceptor subtypes (r= 0.77 and 0.41, respectively). 3. Our results indicate that the α_1A-adrenoceptor is the main functional receptor subtype in porcine denuded coronary arteries.     

Pharmacological Characterisation of a Structurally Novel α2C ‐Adrenoceptor Antagonist ORM‐10921 and its Effects in Neuropsychiatric Models

The α₂‐adrenoceptors (ARs) are important modulators of a wide array of physiological responses. As only a few selective compounds for the three α₂‐AR subtypes (α_2A, α_2B and α_2C) have been available, the pharmacological profile of a new α_2C‐selective AR antagonist ORM‐10921 is reported. Standard in vitro receptor assays and antagonism of α₂, and α₁‐AR agonist ‐evoked responses in vivo were used to demonstrate the α_2C‐AR selectivity for ORM‐10921 which was tested in established behavioural models related to schizophrenia and cognitive dysfunction with an emphasis on pharmacologically induced hypoglutamatergic state by phencyclidine or MK‐801. The Kb values of in vitro α_2C‐ AR antagonism for ORM‐10921 varied between 0.078–1.2 nM depending on the applied method. The selectivity ratios compared to α_2A‐AR subtype and other relevant receptors were 10‐100 times in vitro. The in vivo experiments supported its potent α_2C‐antagonism combined with only a weak α_2A‐antagonism. In the pharmacodynamic microdialysis study, ORM‐10921 was found to increase extracellular dopamine levels in prefrontal cortex in the baseline conditions. In the behavioural tests, ORM‐10921 displayed potent antidepressant and antipsychotic‐like effects in the forced swimming test and prepulse‐inhibition models analogously with the previously reported results with structurally different α_2C‐selective AR antagonist JP‐1302. Our new results also indicate that ORM‐10921 alleviated the NMDA‐antagonist‐induced impairments in social behaviour and watermaze navigation. This study extends and further validates the concept that α_2C‐AR is a potential therapeutic target in CNS disorders such as schizophrenia or Alzheimer's disease and suggests the potential of α_2C‐antagonism to treat such disorders .     

Antinociceptive Synergism of MD‐354 and Clonidine. Part II. The α2‐Adrenoceptor Component

Abstract: Previously, we reported that antinociceptive synergism of a 5‐HT₃/α₂‐adrenoceptor ligand MD‐354 (m‐chlorophenylguanidine) and clonidine combination occurs, in part, through a 5‐HT₃ receptor antagonist mechanism. In the present investigation, a possible role for α₂‐adrenoceptors was examined. Mechanistic studies using yohimbine (a subtype non‐selective α₂‐adrenoceptor antagonist), BRL 44408 (a preferential α_2A‐adrenoceptor antagonist) and imiloxan (a preferential α_2B/C‐adrenoceptor antagonist) on the antinociceptive actions of a MD‐354/clonidine combination were conducted. Subcutaneous pre‐treatment with all three antagonists inhibited the antinociceptive synergism of MD‐354 and clonidine in the mouse tail‐flick assay in a dose‐dependent manner (AD₅₀ = 0.33, 2.1, and 0.17 mg/kg, respectively). Enhancement of clonidine antinociception by MD‐354 did not potentiate clonidine’s locomotor suppressant activity in a mouse locomotor assay. When [ethyl‐³H]RS‐79948‐197 was used as radioligand, MD‐354 displayed almost equal affinity to α_2A‐ and α_2B‐adrenoceptors (K_i = 110 and 220 nM) and showed lower affinity at α_2C‐adrenoceptors (K_i = 4,700 nM). MD‐354 had no subtype‐selectivity for the α₂‐adrenoceptor subtypes as an antagonist in functional [³⁵S]GTPγS binding assays. MD‐354 was a weak partial agonist at α_2A‐adrenoceptors. Overall, in addition to the 5‐HT₃ receptor component, the present investigation found MD‐354 to be a weak partial α_2A‐adrenoceptor agonist that enhances clonidine’s thermal antinociceptive actions through an α₂‐adrenoceptor‐mediated mechanism without augmenting sedation.     

The C-terminal half of the α2C-adrenoceptor determines the receptor's membrane expression level and drug selectivity

In tissues as well as in transfected cells, α_2C-adrenoceptors show poorer expression levels compared to α_2A-adrenoceptors. In order to characterize which regions of the α_2C-adrenoceptor are involved in regulating the expression of binding-competent receptors at the plasma membrane, six chimeric α_2A-/α_2C-adrenoceptors were constructed. The wild-type α_2A- and α_2C-adrenoceptors and the six chimeric α_2A-/α_2C-adrenoceptors were transiently transfected into human embryonic kidney 293 (HEK293) cells, and the expression levels were investigated by radioligand binding. The results show that the C-terminal half of the α_2C-adrenoceptor, ranging from the second extracellular loop to the C-terminus, is the main determinant of the low expression level of binding-competent α_2C-adrenoceptors in HEK293 cell membranes. The so-called retention signal in the N-terminus of the α_2C-adrenoceptor had a less profound effect on the expression levels of the chimeric receptors. For seven drugs competing for [³H]-RX821002 binding, the K _i values were determined at the wild-type α_2A- and α_2C-adrenoceptors and at four of the chimeric α_2A-/α_2C-adrenoceptors. The results show that the α_2C- over α_2A-selectivity of spiroxatrine, spiperone, clozapine, MK912, and chlorpromazine, as well as the α_2A- over α_2C-selectivity of BRL44408, resides mainly in the C-terminal half of the receptors. To some extent, the region comprising the N-terminal half of the receptors contributed to the α_2C-selectivity of spiperone, clozapine, and chlorpromazine.     

In rats, oral oleoyl-DHEA is rapidly hydrolysed and converted to DHEA-sulphate

Background

In addition to the regulation of blood pressure, α₂- and β-adrenoceptor (AR) subtypes play an important role in the modulation of noradrenergic neurotransmission in the human CNS and PNS. Several studies suggest that the α₂-AR responsiveness in cells and tissues after chronic epinephrine (EPI) or norepinephrine (NE) exposure may vary, depending on the β-AR activity present there. Recently, we reported that in BE(2)-C human neuroblastoma cells (endogenously expressing α_2A- and β₂-AR), chronic EPI treatment (300 nM) produced a dramatic β-adrenoceptor-dependent desensitization of the α_2A-AR response. The aim of this study is to determine if stable addition of a β₂-AR to a second neuroblastoma cell line (SH-SY5Y), that normally expresses only α_2A-ARs that are not sensitive to 300 nM EPI exposure, would suddenly render α_2A-ARs in that cell line sensitive to treatment with the same EPI concentration.

Methods

These studies employed RT-PCR, receptor binding and inhibition of cAMP accumulation to confirm α₂-AR subtype expression. Stable clones of SH-SY5Y cells transfected to stably express functional β₂-ARs (SHβ₂AR4) were selected to compare sensitivity of α₂-AR to EPI in the presence or absence of β₂-ARs.

Results

A series of molecular, biochemical and pharmacological studies indicated that the difference between the cell lines could not be attributed to α₂-AR heterogeneity. We now report that after transfection of functional β₂-AR into SH-SY5Y cells (SHβ₂AR4), chronic treatment with modest levels of EPI desensitizes the α_2A-AR. This effect results from a β₂-AR dependent down-regulation of native α_2A-ARs by EPI accompanied by enhanced translocation of GRK2 and GRK3 to the membrane (required for GRK-mediated phosphorylation of agonist-occupied receptors).

Conclusion

This study further supports the hypothesis that the presence of the β-AR renders the α_2A-AR more susceptible to desensitization with physiological levels of EPI.     

Comparison of the Binding Activities of Some Drugs on α2A, α2B and α2C-Adrenoceptors and Non-Adrenergic Imidazoline Sites in the Guinea Pig

Abstract: Simultaneous computer modelling of control and guanfacine-masked [³H]-MK 912 saturation curves as well as guanfacine competition curves revealed that both α_2A- and α_2C-adrenoceptor subtypes were present in the guinea pig cerebral cortex. The K_d value of [₃H]-MK 912 determined for the α_2A-subtype was 403 pM and for the α_2C-subtype 79.8 pM; the receptor sites showing capacities 172 and 19.5 fmol/mg protein, respectively. The K_ds of guanfacine were 20 and 880 nM for the α_2A- and α_2C-adrenoceptor, respectively. In the guinea pig kidney [₃H]-MK 912 bound to a single saturable site with K_d 8.34 nM and capacity 285 fmol/mg protein, the site showing pharmacological properties like an α_2B-adrenoceptor. Binding constants of 22 compounds for the three guinea pig α₂-adrenoceptor subtypes were determined by computer modelling competition curves using for the cerebral cortex a “3-curve assay”, for the kidney an “1-curve assay”, and using [³H]-MK 912 as labelled ligand. Of the tested drugs guanfacine and BRL 44408 were found to be clearly α_2A-selective. Spiroxatrine, yohimbine, rauwolscine and WB 4101, as well as [₃H]-MK 912 itself, were found to be α_2C-selective. The most selective compounds for α_2B-adrenoceptors, when compared to α_2A-adrenoceptors, were ARC 239 and prazosin. In the guinea pig kidney [₃H]-p-aminoclonidine bound to α₂-adrenoceptors as well as to non-adrenergic imidazoline sites. The α₂-adrenoceptors could be completely blocked using 10 μM (-)-adrenaline without the non-adrenergic sites being affected. During these conditions the analysis of combined saturation and competition studies using labelled and unlabeled p-aminoclonidine with computer modelling revealed that the ligand labelled two different sites with K_ds of 310 and 47,000 nM, respectively. Competition curves of 16 compounds for the non-adrenergic [₃H]-p-aminoclonidine sites were shallow and resolved into two-site fits. For the high affinity [₃H]-p-aminoclonidine site the highest affinities were shown by 1-medetomidine, UK-14,304, guanabenz and detomidine; the K_ds of these drugs ranging 26–72 nM. All drugs tested showed low but varying affinities for the low affinity [₃H]-p-aminoclonidine site. These data indicated that the [³H]-p-aminoclonidine binding sites of the guinea pig kidney are grossly different from the [₃H]-idazoxan binding I₂-receptors previously demonstrated also to be present in the guinea pig kidney.     

Clonidine inhibits itch-related response through stimulation of α(2)-adrenoceptors in the spinal cord in mice

The present study investigated whether clonidine – an α₂-adrenoceptor agonist known to relieve pain – is able to suppress itch-related behavior in mice. An intraplantar injection of serotonin induced biting (an itch-related response), which was inhibited by intraperitoneal and intrathecal, but not intraplantar or intracisternal, clonidine injections. The effect of intrathecal clonidine was inhibited by intrathecal injections of phentolamine (a non-selective α-adrenoceptor antagonist) and yohimbine (a selective α₂-adrenoceptor antagonist), but not by prazosin (a selective α₁-adrenoceptor antagonist). The effect of intraperitoneal clonidine was also inhibited by intrathecal yohimbine. These results suggest that clonidine is an effective antipruritic agent and that the effect is mainly mediated by the stimulation of α₂-adrenoceptors in the dorsal horn.     

Evaluation of a new beta-adrenoceptor agonist, procaterol, based on metabolic responses in rats.

Intravenous injection of 5-(1-hydroxy-2-isopropylaminobutyl)-8-hydroxycarbostyril hydrochioride hemihydrate (procaterol) or isoproterenol into fasted rats caused increases in blood levels of glucose, lactate, free fatty acids (FFA), glycerol, immunoreactive insulin and cyclic AMP. Procaterolinduced alterations of these metabolic parameters, other than FFA, were durable; the increases were observable over a period longer than 2 hr, in contrast to a much shorter duration of isoproterenol-induced metabolic changes. These actions of procaterol were antagonized by propranolol, were observed in adrenodemedullated rats, and were enhanced by theophylline. It is thought therefore, that metabolic changes induced by procaterol are actually mediated via β-adrenoceptors, as are its pharmacological actions. Procaterol caused hyperlactacidemia at molar doses ten to one hundred times lower than those required for isoproterenol, trimetoquinol or salbutamol. A much higher dose of procaterol was required to increase blood levels of FFA, glycerol and insulin. In view of our findings that butoxamine, a selective β₂-adrenoceptor antagonist, antagonized the isoproterenol-induced blood lactate, while practolol, a selective ₁-antagonist, effectively inhibited the stimulatory actions of isoproterenol on FFA, glycerol and insulin, it is concluded that procaterol is a more selective ₂-adrenoceptor agonist than isoproterenol or trimetoquinol, and is more potent than salbutamol.     

Characterization of α-Adrenoceptor Activity in Term-newborn Piglet Mesentery

For further characterization of neonatal mesenteric α₁-adrenoceptor populations, an extracorporeal perfusion circuit was established to control intestinal blood flow in 0-2 day old piglets. Activation of α₁-adrenoceptors was first documented by observing dose-dependent increases in mesenteric perfusion pressure after intra-mesenteric arterial injection of methoxamine and noradrenaline. Peripheral intravenous injections of WB 4101 (a competitive α_1A-adrenoceptor antagonist), but not clorethylclonidine (CEC, an α_1B-adrenoceptor antagonist), significantly (P < 0.05, analysis of variance) blunted mesenteric vasoconstrictor responses to those agonists. That the mesenteric vasoconstrictor response to mesenteric plexus stimulation was unaltered by CEC, but was muted by both WB 4101 and SK&F 104856 (a post-junctional α₁- and α₂-adrenoceptor antagonist) suggests that pre- and post-junctional α_1A-adrenoceptors are present and functional at birth.     

Pretreatment with aspirin does not influence the pressor response to α2-adrenoceptor agonists in conscious rabbits

It has proved relatively easy to demonstrate pressor responses mediated via α₂-adrenoceptors in-vivo and in whole blood perfused vascular beds, but not in in-vitro work. The possibility that platelet α₂-adrenoceptor activation, with subsequent release of vasoactive prostanoids, contributes to the pressor response to α₂-adrenoceptor agonists was investigated. The pressor response to the α₂-adrenoceptor agonist BHT 920 (Alefexole) was compared in rabbits pretreated with distilled water or aspirin (3 times 200 mg kg⁻¹ by gavage). Aspirin pretreatment had no significant effect on responses to BHT 920; thus, prostanoid formation does not appear to be an important mechanism contributing to postsynaptic α₂-adrenoceptor activation.     

α-Elapitoxin-Aa2a, a long-chain snake α-neurotoxin with potent actions on muscle (α1)(2)βγδ nicotinic receptors, lacks the classical high affinity for neuronal α7 nicotinic receptors

In contrast to all classical long-chain α-neurotoxins possessing the critical fifth disulfide bond, α-elapitoxin-Aa2a (α-EPTX-Aa2a), a novel long-chain α-neurotoxin from the common death adder Acanthophis antarcticus, lacks affinity for neuronal α7-type nicotinic acetylcholine receptors (nAChRs). α-EPTX-Aa2a (8850 Da; 0.1–1 μM) caused a concentration-dependent inhibition of indirect twitches, and blocked contractures to cholinergic agonists in the isolated chick biventer cervicis nerve-muscle preparation, consistent with a postsynaptic curaremimetic mode of action. α-EPTX-Aa2a (1–10 nM) produced a potent pseudo-irreversible antagonism of chick muscle nAChRs, with an estimated pA₂ value of 8.311 ± 0.031, which was not reversed by monovalent death adder antivenom. This is only 2.5-fold less potent than the prototypical long-chain α-neurotoxin, α-bungarotoxin. In contrast, α-EPTX-Aa2a produced complete, but weak, inhibition of ¹²⁵I-α-bungarotoxin binding to rat hippocampal α7 nAChRs (pK_I = 3.670), despite high sequence homology and similar mass to a wide range of long-chain α-neurotoxins. The mostly likely cause for the loss of α7 binding affinity is a leucine substitution, in loop II of α-EPTX-Aa2a, for the highly conserved Arg³³ in long-chain α-neurotoxins. Arg³³ has been shown to be critical for both neuronal and muscle activity. Despite this substitution, α-EPTX-Aa2a retains high affinity for muscle (α1)₂βγδ nAChRs. This is probably as a result of an Arg²⁹ residue, previously shown to be critical for muscle (α1)₂βγδ nAChR affinity, and highly conserved across all short-chain, but not long-chain, α-neurotoxins. α-EPTX-Aa2a therefore represents a novel atypical long-chain α-neurotoxin that includes a fifth disulfide but exhibits differential affinity for nAChR subtypes.     

3'-Fluoro substitution in the pyridine ring of epibatidine improves selectivity and efficacy for alpha4beta2 versus alpha3beta4 nAChRs

The analog of epibatidine having a fluoro substituent at the 3′ position of the pyridine ring has been recently developed and shown to possess binding affinity in the pM range to α4β2 nAChRs and in the nM range to α7 nAChRs and to exhibit potent agonist activity in nicotine-induced analgesia tests. Here we used patch-clamp technique in a whole-cell configuration to compare functional activity of 3′-fluoroepibatidine to that of epibatidine by itself on recombinant α4β2, α7 and α3β4 neuronal nAChRs. The agonist effect of (±)-epibatidine was partial and yielded comparable EC₅₀s of 0.012 μM (72% efficacy) and 0.027 μM (81% efficacy) at α4β2 and α3β4 nAChRs, respectively, but was full at α7 nAChRs with an EC₅₀ of 4.8 μM. Testing of the analog at different concentrations revealed that it acts as a full agonist with an EC₅₀ of 0.36 μM at α4β2 nAChRs and induces partial agonist effect (66% efficacy) at α7 nAChRs with an EC₅₀ of 9.8 μM and an IC₅₀ corresponding to 225 μM. In contrast, the analog caused only 24% maximal activation at the range of concentrations from 0.1 to 100 μM and, in addition, induced an inhibition of α3β4 nAChR function with an IC₅₀ of 8.3 μM. Our functional data, which are in agreement with previous binding and behavioral findings, demonstrate that 3′-fluoro substitution in the pyridine ring of epibatidine results in an improved pharmacological profile as observed by an increased efficacy and selectivity for α4β2 versus α3β4 nAChRs.     

The three-finger toxin MTα is a selective α2B-adrenoceptor antagonist

Muscarinic toxins (MTs) are three-finger folded peptides isolated from mamba snake venoms. In this report we describe a selective antagonistic interaction of MTα with the human α_2B-adrenoceptor. In a functional assay, measuring the α_2B-adrenoceptor-induced increase in intracellular [Ca²⁺], we found that both venomous MTα and synthetic MTα inhibited the response in a concentration-dependent way. MTα did not affect the responses of α_2A-, α_2C-, α_1A- or α_1B-adrenoceptors. To further explore the binding of MTα to the α_2B-adrenoceptor, we performed ligand binding experiments on Sf9 cell homogenates with [³H]RX821002 as reporter ligand. MTα bound to the receptor rather slowly requiring about 60 min to reach equilibrium. In equilibrium binding experiments, MTα displaced the radioligand with an IC₅₀ of 3.2 nM, but was not able to displace all bound radioligand. Using a saturation binding protocol, we found that MTα suppressed the maximum binding without any greater impact on the affinity of the radioligand, indicating a non-competitive mode of inhibition. The toxin bound reversibly to α_2B-adrenoceptor, but extensive washing was needed for full recovery of binding sites at high toxin concentrations. Surprisingly, MTα did not affect [³H]-N-methylscopolamine binding to the muscarinic receptor subtypes at concentrations found to fully block α_2B-adrenoceptors, showing that the toxin is a more potent antagonist for the α_2B-adrenoceptor than for muscarinic receptors. These findings should open up new views in terms of selective adrenoceptor drug design as well as in elucidation of α₂-adrenoceptor physiology.