Adrenoceptor Subtypes Research Articles

Are Alpha-2D Adrenoceptor Subtypes Involved in Rat Mydriasis Evoked by New Imidazoline Derivatives: Marsanidine and 7-Methylmarsanidine?

The imidazoline compounds may produce mydriasis after systemic administration to some species (rats, cats, and mice). In mydriatic activity of imidazolines, α2D-adrenoceptors subtype(s) seems to be involved. In this study, the pupil dilatory effect evoked by 2 newly synthesized imidazoline derivatives—α2-adrenoceptor agonists: marsanidine and 7-methylmarsanidine—was compared. The compounds were tested alone as well as in the presence of α2-adrenoceptor antagonists (nonselective, yohimbine, and selective toward the following α2-adrenoceptor subtypes—α2A-2-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole maleate (BRL44408), α2B-2-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione dihydrochloride (ARC239), α2C-JP1302, α2D-2-(2,3-dihydro-2-methoxy-1,4-benzodioxin-2-yl)-4,5-dihydro-1H-imidazole hydrochloride [RX821002]). The agonists were studied in male Wistar rats and were administered intravenously in cumulative doses. The antagonistic compounds were given in a single dose before the experiment with marsanidine or 7-methylmarsanidine. Pupil diameter was measured with stereoscopic microscope equipped in green light filter. Marsanidine and 7-methylmarsanidine exerted marked mydriatic effects. BRL44408, JP1302, and ARC239 did not cause significant parallel shift to the right of the dose–effect curves obtained for both imidazolines. In case of yohimbine and RX821002, the marked parallel shifts of dose–response curves were observed, with the antagonistic effects of RX821002 more pronounced. In vivo pharmacodynamics experiment suggests that α2D-adrenoceptor subtype is mainly engaged in mydriatic effects evoked in rats by imidazoline derivatives, in particular by clonidine.

Morphological correlates of sex differences in acoustic startle response and prepulse inhibition through projections from locus coeruleus to cochlear root neurons

The noradrenergic locus coeruleus (LC) plays an important role in the promotion and maintenance of arousal and alertness. Our group recently described coerulean projections to cochlear root neurons (CRNs), the first relay of the primary acoustic startle reflex (ASR) circuit. However, the role of the LC in the ASR and its modulation, prepulse inhibition (PPI), is not clear. In this study, we damaged LC neurons and fibers using a highly selective neurotoxin, DSP-4, and then assessed ASR and PPI in male and female rats. Our results showed that ASR amplitude was higher in males at 14days after DSP-4 injection when compared to pre-administration values and those in the male control group. Such modifications in ASR amplitude did not occur in DSP-4-injected females, which exhibited ASR amplitude within the range of control values. PPI differences between males and females seen in controls were not observed in DSP-4-injected rats for any interstimulus interval tested. DSP-4 injection did not affect ASR and PPI latencies in either the male or the female groups, showing values that were consistent with the sex-related variability observed in control rats. Furthermore, we studied the noradrenergic receptor system in the cochlear nerve root using gene expression analysis. When compared to controls, DSP-4-injected males showed higher levels of expression in all adrenoceptor subtypes; however, DSP-4-injected females showed varied effects depending on the receptor type, with either up-, downregulations, or maintenance of expression levels. Lastly, we determined noradrenaline levels in CRNs and other LC-targeted areas using HPLC assays, and these results correlated with behavioral and adrenoceptor expression changes post DSP-4 injection. Our study supports the participation of LC in ASR and PPI, and contributes toward a better understanding of sex-related differences observed in somatosensory gating paradigms.

β2 Adrenoceptors are underexpressed in peripheral blood mononuclear cells and associated with a better metabolic profile in central obesity.

Background: Central obesity (CO) is an inflammatory disease. Because immune cells and adipocytes are catecholamines(CA)-producing cells, we studied the expression of adrenoceptors (AR) in peripheral blood mononuclear cells (PBMCs) hypothesizing a distinct adrenergic pattern in inflammatory obesity.Methods: AR expression was assessed in blood donors categorized by waist circumference (WC) (CO: WC≥0.80 m in women and ≥0.94 m in men). Following a pilot study for all AR subtypes, we measured β2AR expression in fifty-seven individuals and correlated this result with anthropometric, metabolic and inflammatory parameters. A ratio (R) between AR mRNA of CO and non-CO<0.5 was considered under and >2.0 over expression.Results: The pilot study revealed no differences between groups, except for β2AR mRNA. CO individuals showed underexpression of β2AR relatively to those without CO (R=0.08; p=0.009). β2AR expression inversely correlated with triacylglycerol (r=-0.271; p=0.041), very low-density lipoprotein-cholesterol (r=-0.313; p=0.018) and leptin (r=-0.392; p=0.012) and positively with high-density lipoprotein-cholesterol (r=0.310: p=0.045) plasma levels. Multiple logistic regression analysis showed a protective effect of β2AR expression (≥2x10-6) [odds ratio (OR) 0.177 with respective confidence interval of 95% (95% CI) (0.040- 0.796)] for the occurrence of CO. A higher association was found for women as compared to men (Ξ9:1) [OR 8.972 (95% CI) (1.679-47.949)].Conclusion: PBMCs β2AR, underexpressed in centrally obese, are associated with a better metabolic profile and showed a protective role for the development of CO. The discovery of β2AR as a new molecular marker of obesity subphenotypes in PBMCs might contribute to clarify the adrenergic immunomodulation of inflammatory obesity.

Noradrenergic modulation of evoked dopamine release and pH shift in the mouse dorsal hippocampus and ventral striatum

Rapid monoamine release in the dorsal hippocampus is not well characterized, despite its postulated role in modulating fast hippocampal circuit dynamics. We measured monoamine release in the dorsal hippocampus upon stimulation of the ventral tegmental area (VTA) with fast-scan cyclic voltammetry in anesthetized norepinephrine-depleted and non-depleted mice. Within the hippocampus, norepinephrine depletion altered the ability of α2 adrenergic compounds and transporter blockers to modulate the small, evoked monoamine signal. These manipulations also affected the pH shifts observed after stimulation in a drug-dependent manner. The evoked signal was potentiated by α2C adrenoceptor subtype antagonism, but was not affected by or α2A adrenoceptor antagonism. The same subtype-specific pattern was observed on evoked dopamine release in the ventral striatum. The pharmacological and anatomical evidence supports a contribution by dopamine to the VTA-evoked hippocampal monoamine signal, and confirms the interaction between the mesohippocampal and coeruleohippocampal systems. These results also reinforce the notion that α2C, but not α2A adrenoceptors regulate endogenous dopaminergic activity. We believe our findings hold implications for understanding the efficacy of α2 adrenergic agonists and antagonists that are used widely for therapeutic purposes.

α2 Adrenoceptor: a Target for Neuropathic Pain Treatment.

Neuropathic pain is originated from different alterations of the nervous system. The difficulty of treatment strongly impairs quality of life of affected people. It is associated with severe, chronic sensory disturbances characterized by spontaneous pain, increased responsiveness to painful stimuli and pain perceived in response to normally non-noxious stimuli. The underlying mechanisms are complex and involve both peripheral and central nervous components. The noradrenergic system plays a pivotal role in the control of pain since its widespread distribution in the "pain matrix" representing a valuable therapeutic target. This review focused on the α2 adrenoceptor subtype modulation as strategy for neuropathic pain relief. Drugs acting as direct α2 adrenoceptor agonists (clonidine and dexmedetomidine) were analyzed as well as the indirect α2 adrenoceptor modulators. The overview included norepinephrine reuptake inhibitors (reboxetine, maprotiline), serotonin/norepinephrine reuptake inhibitors (venlafaxine, milnacipran, amitriptyline, duloxetine, bicifadine) and the compounds characterized by a double pharmacodynamic mechanism combining the norepinephrine reuptake inhibition and the μ opioid agonist profile (tramadol and tapentadol). A summary of recent compounds was illustrated.

Pharmaceutical evaluation of naftopidil enantiomers: Rat functional assays in vitro and estrogen/androgen induced rat benign prostatic hyperplasia model in vivo

Naftopidil (NAF) is a α1D/1A adrenoceptor selective drug used for the treatment of both benign prostatic hyperplasia and lower urinary tract symptoms (BPH/LUTS). However, NAF is used as a racemate in clinic. To compare the differences and similarities among two enantiomers and racemate, pharmacological activities were evaluated through rat functional assays in vitro and estrogen/androgen (E/T) induced rat BPH model in vivo. NAF and the two enantiomers showed similar blocking activity on α1 receptor. S-NAF exhibited more α1D/1A adrenoceptor subtype selectivity than R-NAF and the racemate. The selectivity ratios pA2 (α1D)/pA2 (α1B) and pA2 (α1A)/pA2 (α1B) were 40.7- and 16.2-fold, respectively. NAF and its enantiomers effectively prevented the development of rat prostatic hyperplasia via suppressing the increase of the prostatic wet weight, visually. The quantitative analysis of the relative acinus volume, relative stroma volume, relative epithelial volume, epithelial height and expression of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA) were carried out. S-NAF showed an advantage on the effect of inhibiting prostate wet weight and stroma volume over R-NAF and racemate NAF (P<0.05). Nevertheless, no other significant difference was observed between these two enantiomers. In conclusion, both R-NAF and S-NAF not only relax prostate muscle but also inhibit the prostate growth, thus relieve BPH.

Structure determination, synthesis, and biological evaluation of a metabolite of the selective α1D adrenoceptor antagonist TAK-259

5-Chloro-1-(5-chloro-2-(methylsulfonyl)benzyl)-2-imino-1,2-dihydropyridine-3-carboxamide hydrochloride (TAK-259) is a novel, selective, and orally active α1D adrenoceptor antagonist with anti-urinary frequency effect. A metabolite (2) of TAK-259 was identified from monkey urine samples. To elucidate the structure of 2, extraction and purification of the metabolite from TAK-259-treated monkey urine was conducted. Structural analysis of the purified compounds using NMR indicated the compound to be 2-amino-5-chloro-1-[5-chloro-2-(methylsulfonyl)benzyl]-4-oxo-1,4-dihydropyridine-3-carboxamide (2a). An authentic sample of compound 2a was synthesized via regioselective alkylation of 4-methoxypyridin-2-amine. The biological activity of metabolite 2a was also evaluated, and the compound found not to possess affinity toward any known α1 adrenoceptor subtype.

Age-related effects of dexmedetomidine on myocardial contraction and coronary circulation in isolated guinea pig hearts

Dexmedetomidine is a selective α2 adrenergic agonist. Although dexmedetomidine is widely used for sedation and analgesia, it frequently produces hypotension and bradycardia. The present study aimed to evaluate the effects of dexmedetomidine on cardiac function and coronary circulation using Langendorff-perfused guinea pig hearts. Coronary perfusion pressure (CPP) and left ventricular pressure (LVP) were continuously monitored, and electric field stimulation (EFS) was applied to stimulate sympathetic nerve terminals. Dexmedetomidine almost completely inhibited the EFS-induced increase in LVP at all ages. The effect of dexmedetomidine on coronary artery resistance varied according to postnatal age, i.e., dexmedetomidine had little effect on CPP in young hearts (<4 weeks) but increased CPP by 10 mmHg at 4–8 weeks and by 15 mmHg at >8 weeks. The increase in CPP in adult hearts was inhibited by imiloxan, an α2B antagonist, and prazosin, an α1 antagonist. The results suggest that dexmedetomidine acts on α2 adrenergic receptors at sympathetic nerve terminals to suppress the release of norepinephrine. In addition, the findings suggest that dexmedetomidine directly affects α1 adrenoceptors and/or α2B adrenoceptors on coronary smooth muscles to increase CPP. The age-related changes in α adrenoceptor subtypes may be linked to the cardiodepressant effects of dexmedetomidine.

Both Alpha and Beta Adrenergic Receptor Expression Is Increased in the Renal Medulla of Spontaneously Hypertensive African Green Monkeys

The African Green Monkey (Chlorocebus aethiops sabaeus), the Caribbean vervet, is a highly translational model of spontaneous hypertension (HT). The Caribbean vervet has a very close phylogenetic relationship with humans with an estimated divergence time of ~12 million years ago. Within a cohort of adult animals, 36% (124 of 345) of African Green Monkeys exhibit systolic blood pressures (SBP)&gt; 140 mmHg, as measured by forearm cuff plethysmography. Heart rate is elevated in HT vs. normotensive (NT) animals (121±2 bpm vs. 136±2 bpm, p&lt;0.05) suggesting that sympathetic nerve activity is likely elevated in the HT African Green Monkey that behaviorally maintains upright posture throughout the day and night. This study tested the hypothesis that adrenergic receptor subtypes are upregulated in the renal cortex, outer medulla, and inner medulla of adult male HT African Green Monkeys. Primers were custom designed and evaluated for appropriate specificities and efficiencies (95–105%) for α1a, α1d, α2a, α2c, β1 and β2 adrenoceptors. Gene expression was measured with qRT‐PCR and normalized to RPS32 or RPS13A expression and RQs compared with the Mann‐Whitney U Test. The HT cohort of African Green Monkeys (n=18) averaged SBP of 168.24 ± 7.25mmHg compared with NT animals (n=18) that averaged 96.61 ± 3.20mmHg. Expression of each adrenoceptor subtype was similar in the renal cortex between the two groups. Expression of α1a, α1d, α2a, and α2c genes were increased in the renal outer medulla of HT animals (α1a: NT RQ 1 ± 0.43 vs. HT RQ 2.26 ± 0.50, p&lt;0.05; α1d: NT RQ 1 ± 0.34 vs. HT RQ 2.25 ± 0.32, p&lt;0.05; α2a: NT RQ 1 ± 0.24 vs. HT RQ 1.92 ± 0.19, p&lt;0.05; and α2c: NT RQ 1 ± 0.15 vs HT RQ 1.65 ± 0.15, p&lt;0.05). β1 and β2 adrenergic receptor expression in the outer medulla was also upregulated in HT animals (β1: NT RQ 1 ± 0.24 vs. HT RQ 1.88 ± 0.18, p&lt;0.05; β2: NT RQ 1 ± 0.21 vs HT RQ 1.87 ± 0.24; p&lt;0.05). Expression of adrenergic receptors in the inner medulla of HT animals was similar (p&gt;0.05), with the exception of the β2 adrenergic receptor, which was upregulated in the HT group (NT RQ 1 ± 0.25 vs HT RQ 1.62 ± 0.10, p&lt;0.05). These data suggest that the renal outer medulla of HT African Green Monkey may be sensitized to sympathetic nerve activity via elevated adrenergic receptor gene expression. Thus, subtle changes in renal sympathetic outflow may elicit renal medullary vasoconstriction and sodium retention and contribute to the development of the spontaneous hypertensive phenotype in the African Green Monkey. This study further characterizes a unique non‐human primate model of spontaneous hypertension with genetic, behavioral, and social similarities to humans, which may lead to the identification of novel therapies for the treatment of idiopathic hypertension.

Sympathectomy and Sympathetic Blockade Reduce Pain Behavior Via Alpha-2 Adrenoceptor of the Dorsal Root Ganglion Neurons in a Lumbar Radiculopathy Model.

Animal experimental study with intervention. We investigated whether sympathectomy and pharmacological sympathetic blockade reduced pain behavior and reversed adrenoceptor mRNA expression of the dorsal root ganglion (DRG) in a lumbar radiculopathy model. The abnormal sympathetic-somatosensory interaction may underlie some forms of neuropathic pain. There are several reports that sympathectomy and pharmacological sympathetic blockades are often effective to treat neuropathic pain. However, its pathophysiological mechanisms remain obscure. We used 91 male Sprague-Dawley rats. Just after root constriction (RC), the rats underwent sympathectomy or received 3 local injections of subtype-specific α-adrenergic receptor antagonists around the DRG. We evaluated the analgesic effects of sympathectomy and sympathetic blockade using behaviors indicative mechanical allodynia and thermal hyperalgesia. We estimated the mRNA expression levels of the DRG adrenoceptor subtypes using real time reverse transcription polymerase chain reaction. Sympathectomy and α2-antagonist significantly reduced the mechanical allodynia and thermal hyperalgesia after RC. Real time reverse transcription polymerase chain reaction analysis indicated that sympathectomy possibly reversed α2A- and α2B-adrenoceptors mRNA overexpression in the DRG after RC. We considered that pain behaviors of neuropathic pain are due, at least in part, to enhanced sympathetic noradrenergic transmission within the DRG. Suppression of sympathetic activity by reducing adrenergic release, α2-adrenoceptor stimulation, and/or α2-adrenoceptor upregulation in the DRG may relieve neuropathic pain. 3.

Norepinephrine Modulates Pyramidal Cell Synaptic Properties in the Anterior Piriform Cortex of Mice: Age-Dependent Effects of β-adrenoceptors.

Early odor preference learning in rodents occurs within a sensitive period [≤postnatal day (P)10–12], during which pups show a heightened ability to form an odor preference when a novel odor is paired with a tactile stimulation (e.g., stroking). Norepinephrine (NE) release from the locus coeruleus during stroking mediates this learning. However, in older pups, stroking loses its ability to induce learning. The cellular and circuitry mechanisms underpinning the sensitive period for odor preference learning is not well understood. We first established the sensitive period learning model in mice – odor paired with stroking induced odor preference in P8 but not P14 mice. This learning was dependent on NE-β-adrenoceptors as it was prevented by propranolol injection prior to training. We then tested whether there are developmental changes in pyramidal cell excitability and NE responsiveness in the anterior piriform cortex (aPC) in mouse pups. Although significant differences of pyramidal cell intrinsic properties were found in two age groups (P8–11 and P14+), NE at two concentrations (0.1 and 10 μM) did not alter intrinsic properties in either group. In contrast, in P8–11 pups, NE at 0.1 μM presynaptically decreased miniature IPSC and increased miniature EPSC frequencies. These effects were reversed with a higher dose of NE (10 μM), suggesting involvement of different adrenoceptor subtypes. In P14+ pups, NE at higher doses (1 and 10 μM) acted both pre- and postsynaptically to promote inhibition. These results suggest that enhanced synaptic excitation and reduced inhibition by NE in the aPC network may underlie the sensitive period.

Attenuation of pain behaviour by local administration of alpha-2 adrenoceptor antagonists to dorsal root ganglia in a rat radiculopathy model.

There were several reports suggesting α-adrenoceptor antagonists are effective to treat neuropathic pain. The aims of this study were as follows: (1) to introduce drug delivery system for dorsal root ganglion (DRG) neurons; (2) to elucidate the effects of α-adrenoceptor antagonists in acute, subacute or chronic phase and (3) to determine which subtype of adrenoceptor was mainly involved. We used 130 male Sprague-Dawley rats. After root constriction, rats received three local injections of α-adrenoceptor antagonists around DRG. We administered the non-selective α-adrenoceptor antagonist phentolamine for 3 consecutive days from day 0, 4 or 11 after the surgery, and the α1-adrenoceptor antagonist prazosin, the α1-adrenoceptor antagonist silodosin, the more preferred α1-adrenoceptor than prazosin and the α2-adrenoceptor antagonist yohimbine for 3 consecutive days from day 0 after the surgery. Phentolamine and yohimbine continually attenuated pain behaviour. Prazosin at high dose attenuated pain behaviour, however, prazosin at low dose did not attenuate pain behaviour every experimental day. Silodosin had no analgesic effect. Phentolamine injections from day 4 after surgery attenuated pain behaviour that had been established on the 3rd experimental day until the 28th post-operative day, although effect of phentolamine wore off. Phentolamine injections from day 11 after surgery temporarily attenuated pain behaviour that had been established on the 3rd, 7th and 10th experimental days. This study showed α-adrenoceptor antagonists could suppress pain behaviour via α2-adrenoceptor in acute phase and temporary attenuate pain behaviour in chronic phase. These findings presented potentials sympathetic nerve blockade contributed to treat neuropathic pain.

Abstract P010: Specific Renal Outer Medulla Upregulation of α-and β-Adrenergic Receptor Subtypes in Spontaneously Hypertensive African Green Monkeys

The African Green Monkey ( Chlorocebus aethiops sabaeus ) is a highly translational model of spontaneous hypertension (HT). Within the cohort of measured colony animals, 33% (98 of 300) of adult African Green Monkeys exhibit systolic blood pressures (SBP)&gt; 140 mmHg, assessed by forearm cuff plethysmography. Heart rate is elevated in HT vs. normotensive (NT) animals (121±2 bpm vs. 134±2 bpm, p&lt;0.05) suggesting that sympathetic nerve activity is likely elevated in the HT African Green Monkey, similar to patients with essential hypertension. This study assessed mRNA expression of adrenergic receptor subtypes in the renal cortex, outer medulla, and inner medulla of male HT and NT African Green Monkeys. Primers were custom designed and evaluated for appropriate specificities and efficiencies (95-105%) for α 1a , α 1d , α 2c , β 1 and β 2 adrenoceptors. Gene expression was measured with qRT-PCR and normalized to RPS32 expression in the liver of each animal. The HT cohort of African Green Monkeys (n=18) assessed an averaged SBP of 168.24 ± 7.25mmHg compared with NT animals (n=18) that averaged 96.61 ± 3.20mmHg. Expression of α 1a , α 1d and α 2c genes were all increased in the renal outer medulla of HT animals (α 1a : NT RQ 0.24 ± 0.15 vs. HT RQ 0.89 ± 0.22, p&lt;0.05; α 1d : NT RQ 0.43 ± 0.24 vs. HT RQ 1.99 ± 0.74, p&lt;0.05; and α 2c : NT RQ 1.11 ± 0.53 vs HT RQ 1.41 ± 0.35, p&lt;0.05). β 1 adrenergic receptor expression in the outer medulla was similar in NT and HT animals (β 1 : NT RQ 0.75 ± 0.30 vs. HT RQ 1.82 ± 0.64, p&gt;0.05), but β 2 adrenoceptor expression was upregulated in HT animals (β 2 : NT RQ 0.36 ± 0.13 vs HT RQ 2.05 ± 0.82; p&lt;0.05). Expression of each adrenoceptor subtype was similar in the renal cortex and inner medulla of HT animals (p&gt;0.05, all groups compared with Mann-Whitney U Test). These data suggest that the renal outer medulla of HT African Green Monkey may be sensitized to sympathetic nerve activity via elevated adrenergic receptor gene expression. Thus, subtle changes in renal sympathetic outflow may elicit renal medullary vasoconstriction and contribute to the development of the spontaneous hypertensive phenotype in the African Green Monkey.

Norepinephrine responses in rat renal and femoral veins are reinforced by vasoconstrictor prostanoids

Norepinephrine (NE) responses are larger in renal and femoral veins compared to phenylephrine (PE). These differences may be due to the subtypes of adrenoceptor involved in these responses or to the involvement of local modulatory mechanisms. Therefore, the present study investigated in organ bath the adrenoceptor subtypes involved in the NE and PE responses in both renal and femoral veins as well as the influence of local mechanisms related to NO and to prostanoids upon these responses. The obtained data showed that the NE responses in these veins were not significantly modified by the selective inhibition of β1 or β2-adrenoceptors as well as AT1 or AT2 receptors. However, yohimbine reduced the NE Rmax in renal veins and, in parallel, right shifted the NE concentration–response curves in femoral veins. In both veins, prazosin reduced the NE Rmax and the clonidine induced a measurable contraction. The endothelium removal attenuated the NE responses in femoral veins, thereby abolishing the differences of NE and PE responses. Furthermore, the NE responses in renal and femoral veins were attenuated by indomethacin, which suppressed the statistical difference in relation to the PE response. In conclusion, a synergism between α1- and α2-adrenoceptors is essential to assure full NE contractile responses in both renal and femoral veins. Thus, by acting simultaneously in these adrenoceptors, NE induces more pronounced contractile responses, in comparison to PE, not only in renal but also in femoral veins. Moreover, this pronounced NE response in both renal and femoral veins appears to involve endothelium-derived vasoconstrictor prostanoids.

Cyclic Adenosine Monophosphate-Mediated Enhancement of Vascular Endothelial Growth Factor Released by Differentiated Human Monocytic Cells: The Role of Protein Kinase A

Objective: Our investigation was designed to examine the signaling pathway involved in the enhancement of vascular endothelial growth factor (VEGF) release by β-adrenoceptor agonists. Materials and Methods: Human U937 cells differentiated into macrophages were primed with lipopolysaccharide (LPS) in the absence or presence of β-adrenoceptor agonists and antagonists. The VEGF released and the intracellular cyclic adenosine monophosphate (cAMP) generated were assayed by ELISA. Where necessary, differences between mean values were tested for significance using Student's t test. Results: Isoprenaline, procaterol and salbutamol concentration-dependently enhanced the release of VEGF induced by LPS in U937 cells. R*,R*-(±)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid (BRL 37344), a selective β₃-adrenoceptor agonist, did not enhance VEGF release. Using isoprenaline as an agonist, propranolol, ICI 118551 and atenolol produced a parallel rightward shift of the concentration-response curve with no reduction in the maximum response. The -logK_B values were 8.12 ± 0.17, 8.03 ± 0.05 and 7.23 ± 0.05 for propranolol, ICI 118551 and atenolol, respectively, indicating the possible involvement of both β₁- and β₂-adrenoceptor subtypes. Isoprenaline and prostaglandin E₂ concentration-dependently increased cAMP generation in U937 cells. Isoprenaline, db-cAMP and 6-Bnz-cAMP, a protein kinase A (PKA) activator, all enhanced VEGF release induced by LPS, and this effect was abolished by KT 5720 and Rp-cAMPS, which are both selective PKA inhibitors, suggesting that PKA is the downstream effector of cAMP activity. 8-CPT-cAMP, a selective activator of the Epac system, had no effect on VEGF release induced by LPS, indicating that the Epac pathway played no role in the release process. Conclusion: In this study, we established that β₁- and β₂- but not β₃-adrenoceptors mediated cAMP-dependent enhancement of VEGF release induced by LPS in differentiated U937 cells, and that PKA was the downstream effector of cAMP activity.

Effects of β3-adrenoceptor activation on expression of pancreatic adrenoceptors and angiotensin II receptors in ApoE−/− mice

Hyperlipidemia can be harmful to the pancreas and β3-adrenoceptor agonist can improve lipid metabolism disorder. We aimed to study the effects of β3-adrenoceptor activation on glucose, insulin and the expression of pancreatic adrenoceptors and angiotensin II receptors. Ten C57BL/6J mice at the age of 10 weeks served as normal control, and forty age-matched apolipoprotein E knockout (ApoE−/−) mice were randomly divided into hyperlipidaemia model group, low-dose and high-dose β3-adrenoceptor agonist group and β3-adrenoceptor antagonist group. After 26 weeks of high-fat diet, treatments were given for 12 weeks. Serum glucose and insulin levels in 48 weeks old mice were measured using an automatic biochemical detector. Quantitative rt-PCR and Western blot were used to analyze the mRNA and protein expression of α1A-, α2A-, β2-, β3-adrenoceptors and angiotensin II type 1 and type 2 receptors in pancreas. We found that β3-adrenoceptor agonist could decrease serum glucose and insulin levels in aged ApoE−/− mice (P<0.01) and down-regulate the expression of α1A-adrenoceptor and angiotensin II type 1 receptor which were significantly increased in model mice (P<0.05, P<0.01). Compared with the model mice, α2A-, β2-, β3-adrenoceptor and angiotensin II type 2 receptor expression were up-regulated in β3-adrenoceptor agonist treat mice (P<0.05, P<0.01). These results suggest that chronic β3-adrenoceptor activation regulated the expression of adrenoceptors and angiontensin II receptors towards contrary direction, which indicates that there are interactions between β3-adrenoceptor and subtypes of adrenoceptor and angiotensin II receptor, and these interactions may play a protective role in pancreas and improve glucose metabolism disorders.

Functional and radioligand binding characterization of the α1L-adrenoceptor subtype of the human vas deferens.

Alpha1 -adrenoceptor antagonists can cause ejaculatory dysfunction as an adverse effect. Contractions of the human vas deferens are mediated via α1A -adrenoceptors, and this study investigated whether the low affinity state of this receptor (α1L -adrenoceptor) is involved in mediating contractions of this tissue. The potency of agonists and the affinity of receptor subtype selective antagonists were determined in functional experiments and in [(3) H]tamsulosin binding experiments to identify the α1 -adrenoceptor subtype population present in the human vas deferens. The α1A -adrenoceptor selective agonist A61603 was a full agonist and was 250-fold more potent than noradrenaline. Prazosin antagonized contractile responses to phenylephrine with a low affinity (pKd = 8.6). Only high concentrations of RS17053 antagonized responses to phenylephrine and yielded a relatively low affinity estimate of 7.0. BMY7378 (α1D -adrenoceptor selective) gave a low affinity estimate (pKd = 6.7), whilst tamsulosin (α1A - and α1D -adrenoceptor selective) had a high affinity (pKd = 9.9). [(3) H]Tamsulosin bound to human vas deferens membranes with a high affinity (pKd = 10.0). Prazosin, RS17053 and BMY7378 competed with [(3) H]tamsulosin with low affinities for a single population of binding sites (pKd values of 8.5, 7.2 and 6.3, respectively). These functional and radioligand binding data indicate that the human vas deferens possesses a homogeneous population of α1 -adrenoceptors which have the pharmacological properties of the putative α1L -adrenoceptor, the same functional receptor previously identified in the human prostate.

Antihyperalgesic and antiallodynic effects of mianserin on diabetic neuropathic pain: A study on mechanism of action

This study used various experimental pain methods to investigate the effects of subacute mianserin administration on diabetes-induced neuropathic pain in rats. The effect of mianserin on hyperalgesia occurring in connection with peripheral diabetic neuropathy was examined using the Randall–Selitto (mechanical nociceptive stimulus), Hargreaves (thermal nociceptive stimulus), and cold-plate (4°C, thermal nociceptive stimulus) tests. The dynamic plantar aesthesiometer, which measures the threshold values for mechanical stimuli, was used for allodynia studies. Thermal allodynia was evaluated with the warm-plate (38°C) test. At 30 and 45mg/kg, mianserin effectively improved mechanical and thermal hyperalgesia occurring in connection with diabetic neuropathy. Subacute administration of mianserin also reduced diabetes-associated mechanical and thermal allodynia. The ability of mianserin to reduce diabetic neuropathic pain was comparable to that of pregabalin (10mg/kg). The antihyperalgesic and antiallodynic effects of mianserin were reversed with α-methyl-para-tyrosine methyl ester (AMPT, an inhibitor of catecholamine synthesis), phentolamine (a non-selective α-adrenoceptor antagonist), propranolol (a non-selective β-adrenoceptor antagonist), and naloxone (a non-selective opioid receptor antagonist) administrations. The same effects were not reversed, however, by para-chlorophenylalanine methyl ester (PCPA; an inhibitor of serotonin synthesis). These results suggest that the beneficial effect of mianserin on diabetic neuropathic pain is mediated through an increase in catecholamine levels in the synaptic cleft as well as through interactions with both subtypes of adrenoceptors and opioid receptors. Considering that mianserin exhibits simultaneous antidepressant and antinociceptive effects, this drug could provide a good alternative for treating the pain associated with diabetic neuropathy and the mood disorders caused directly by diabetes.

&lt;b&gt;α1-Adrenoceptors relate Ca&lt;/b&gt;&lt;sup&gt;&lt;b&gt;2+&lt;/b&gt;&lt;/sup&gt;&lt;b&gt; modulation and protein secretions in rat lacrimal &lt;/b&gt;&lt;b&gt;gland &lt;/b&gt;

Noradrenaline (NA) is a catecholamine with multiple roles including as a hormone and a neurotransmitter. Cellular secretory activities are enhanced by adrenergic stimuli as well as by cholinergic stimuli. The present study aimed to determine which adrenoceptors play a role in controlling intracellular calcium ion ([Ca(2+)]i) level in acinar cells of rat lacrimal glands. Expression of mRNA for adrenoceptor subtypes in the acinar cells was assessed using RT-PCR. All types except α2c, β1, and β3 were detected. NA induced a [Ca(2+)]i increase with a biphasic pattern in the acinar cells. Removal of extracellular Ca(2+) and use of Ca(2+)-channel blockers did not inhibit the NA-induced [Ca(2+)]i increases. In contrast, U73122 and suramin almost blocked these increases. The α1-adrenoceptor agonist phenylephrine induced a strong increase in [Ca(2+)]i. However, clonidine and isoproterenol failed to induce a [Ca(2+)]i increase. The peroxidase activity was quantified as a measure of mucin secretion. Ca(2+)-dependent exocytotic secretion of peroxidase was detected in rat lacrimal glands. The RT-PCR results showed that MUC1, MUC4, MUC5AC, MUC5B, and MUC16 were expressed in acinar cells. These findings indicated that NA activates α1-adrenoceptors, which were found to be the main receptors in Ca(2+)-related cell homeostasis and protein (including mucin) secretion in lacrimal glands.

Specific role of α2A - and α2B -, but not α2C -, adrenoceptor subtypes in the inhibition of the vasopressor sympathetic out-flow in diabetic pithed rats.

Several lines of evidence have shown an association of diabetes with a catecholamines' aberrant homeostasis involving a drastic change in the expression of adrenoceptors. This homeostatic alteration includes, among other things, atypical actions of α2 -adrenoceptor agonists within central and peripheral α2 -adrenoceptors (e.g. profound antinociceptive effects in diabetic subjects). Hence, this study investigated the pharmacological profile of the α2 -adrenoceptor subtypes that inhibit the vasopressor sympathetic out-flow in streptozotocin-pre-treated (diabetic) pithed rats. For this purpose, B-HT 933 (up to 30 μg/kg min) was used as a selective α2 -adrenoceptor agonist and rauwolscine as a non-selective α2A/2B/2C -adrenoceptor antagonist; in addition, BRL 44408, imiloxan and JP-1302 were used as subtype-selective α2A -, α2B - and α2C -adrenoceptor antagonists, respectively (all given i.v.). I.v. continuous infusions of B-HT 933 inhibited the vasopressor responses induced by electrical sympathetic stimulation without affecting those by i.v. bolus injections of noradrenaline in both normoglycaemic and diabetic rats. Interestingly, the ED50 for B-HT 933 in diabetic rats (25 μg/kg min) was almost 1-log unit greater than that in normoglycaemic rats (3 μg/kg.min). Moreover, the sympatho-inhibition induced by 10 μg/kg min B-HT 933 in diabetic rats was (i) abolished by 300 μg/kg rauwolscine or 100 and 300 μg/kg BRL 44408; (ii) partially blocked by 1000 μg/kg imiloxan; and (iii) unchanged by 1000 μg/kg JP-1302. Our findings, taken together, suggest that B-HT 933 has a less potent inhibitory effect on the sympathetic vasopressor responses in diabetic (compared to normoglycaemic) rats and that can probably be ascribed to a down-regulation of α2C -adrenoceptors.