Adrenoceptor Activation Research Articles

The relationship between the MMP system, adrenoceptors and phosphoprotein phosphatases

The MMPs and their inhibitors [tissue inhibitor of MMPs (TIMPs)] form the mainstay of extracellular matrix homeostasis. They are expressed in response to numerous stimuli including cytokines and GPCR activation. This review highlights the importance of adrenoceptors and phosphoprotein phosphatases (PPP) in regulating MMPs in the cardiovascular system, which may help explain some of the beneficial effects of targeting the adrenoceptor system in tissue remodelling and will establish emerging crosstalk between these three systems. Although α- and β-adrenoceptor activation increases MMP but decreases TIMP expression, MMPs are implicated in the growth stimulatory effects of adrenoceptor activation through transactivation of epidermal growth factor receptor. Furthermore, they have recently been found to catalyse the proteolysis of β-adrenoceptors and modulate vascular tone. While the mechanisms underpinning these effects are not well defined, reversible protein phosphorylation by kinases and phosphatases may be key. In particular, PPP (Ser/Thr phosphatases) are not only critical in resensitization and internalization of adrenoceptors but also modulate MMP expression. The interrelationship is complex as isoprenaline (ISO) inhibits okadaic acid [phosphoprotein phosphatase type 1/phosphoprotein phosphatase type 2A (PP2A) inhibitor]-mediated MMP expression. While this may be simply due to its ability to transiently increase PP2A activity, there is evidence for MMP-9 that ISO prevents okadaic acid-mediated expression of MMP-9 through a β-arrestin, NF-κB-dependent pathway, which is abolished by knock-down of PP2A. It is essential that crosstalk between MMPs, adrenoceptors and PPP are investigated further as it will provide important insight into how adrenoceptors modulate cardiovascular remodelling, and may identify new targets for pharmacological manipulation of the MMP system.

Protective effect of Chresta martii extract on ethanol-induced gastropathy depends on alpha-2 adrenoceptors pathways but not on nitric oxide, prostaglandins or opioids

Ethnopharmacological relevanceSpecies of Chresta genus- are recognized by the population of northeastern Brazil as traditional herbs used to treat gastric diseases and other disorders. Aim of the studyThis work aimed to find out the action mechanism of Chresta martii hydro alcoholic extract gastro protective effect in the model of ethanol-induced gastropathy. Material and methodsGastropathy was assessed by percentual damaged area determination in photographs of mice opened stomachs. Fasted mice treated with ethanol 99.9% (0.2 ml/animal, p.o.) were pre-treated with Chresta martii hydro alcoholic extract (HAE) (50, 100 or 200 mg/kg, p.o.), ranitidine (80 mg/kg, p.o.) or saline (5 ml/kg; p.o.) in different experimental sets, in which pharmacological tools (naloxone, indomethacin, Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) or yohimbine) were added in order to clarify a possible action mechanism. Animals were sacrificed 30 min after ethanol challenge to stomach analysis. Determination of non-protein sulfhydryl groups and tissue hemoglobin, besides histological assessment (H&E) were taken to fully characterize the HAE gastro protective effect. ResultsHAE (100 and 200 mg/kg) was able to protect mucosa against ethanol gastropathy in presence of three (naloxone, indomethacin and L-NAME) of four antagonist/inhibitor tools. The HAE effect was reversed only by yohimbine, showing the alpha-2 adrenoceptors participation on gastro protective effect of this extract. HAE histological characteristics, NP-SH and Hb were compatible with the protective effects. ConclusionsHAE possesses gastroprotective effects in an ethanol-induced gastropathy model in mice, corroborating the traditional use of this family of plants to treat gastric disorders. This activity is mediated by alpha-2 adrenoceptors activation, but not by nitric oxide release, opioid receptor activation or prostaglandin synthesis. HAE also has antioxidant activity that is thought to either play a role in this biological activity or to be a byproduct of alpha-2 adrenergic complex activation.

Functional selectivity of central Gα‐subunit proteins in mediating the cardiovascular and renal excretory responses evoked by central α2‐adrenoceptor activation in vivo

Activation of brain α(2) -adrenoceptors in conscious rodents decreases heart rate (HR) and mean arterial blood pressure (MAP) and increases urine output and urinary sodium excretion. In vitro, α(2) -adrenoceptor stimulation activates Gα(i(1-3)) , Gα(o) and Gα(s) -subunit protein-gated signal transduction pathways. Here we have investigated whether these same Gα-subunit protein-gated pathways mediate the cardiovascular and renal excretory responses to central α(2) -adrenoceptor activation in conscious Sprague-Dawley rats. Rats were pre-treated by intracerebroventricular injection (i.c.v.) with an oligodeoxynucleotide (ODN) targeted to a Gα(i1) , Gα(i2) , Gα(i3) , Gα(o) , Gα(s) or a scrambled (SCR) ODN sequence (25 µg, 24 h). On the day of study, the α(2) -adrenoceptor agonist guanabenz (50 µg) or saline vehicle, was injected i.c.v. into ODN-pre-treated conscious rats. MAP and HR were recorded, and urine was collected for 150 min. In vehicle- and SCR ODN-pre-treated rats, i.c.v. guanabenz decreased MAP and HR, and produced marked diuretic and natriuretic responses. Selective ODN-mediated down-regulation of brain Gα(i2) -subunit proteins abolished the central guanabenz-induced hypotension and natriuresis. In contrast, following selective Gα(s) down-regulation, the characteristic hypotensive response to i.c.v. guanabenz was converted to an immediate increase in MAP. The bradycardic and diuretic responses to i.c.v. guanabenz were not blocked by pre-treatment with any ODN. There was functional selectivity of Gα(i2) and Gα(s) subunit protein-gated signal transduction pathways in mediating the hypotensive and natriuretic, but not bradycardic or diuretic, responses evoked by central α(2) -adrenoceptor activation in vivo.

Brain pathways involved in the modulatory effects of noradrenaline in lateral septal area on cardiovascular responses.

We have previously reported that stimulation of alpha-1 adrenoceptors by noradrenaline (NA) injected into the lateral septal area (LSA) of anaesthetized rats causes pressor and bradycardic responses that are mediated by acute vasopressin release into the circulation through activation of the paraventricular nucleus (PVN). Although the PVN is the final structure of this pathway, the LSA has no direct connections with the PVN, suggesting that other structures may connect these areas. To address this issue, the present study employed c-Fos immunohistochemistry to investigate changes caused by NA microinjection into the LSA in neuronal activation in brain structures related to systemic vasopressin release. NA microinjected in the LSA caused pressor and bradycardic responses, which were blocked by intraseptal administration of α-1 adrenoceptor antagonist (WB4101, 10 nmol/200 nL) or systemic V-1 receptor antagonist (dTyr(CH2)5(Me)AVP, 50 μg/kg). NA also increased c-Fos immunoreactivity in the prelimbic cortex (PL), infralimbic cortex (IL), dorsomedial periaqueductal gray (dmPAG), bed nucleus of the stria terminalis (BNST), PVN, and medial amygdala (MeA). No differences in the diagonal band of Broca, cingulate cortex, and dorsolateral periaqueductal gray (dlPAG) were found. Systemic administration of the vasopressin receptor antagonist dTyr AVP (CH2)5(Me) did not change the increase in c-Fos expression induced by intra-septal NA. This latter effect, however, was prevented by local injection of the alpha-1 adrenoceptor antagonist WB4101. These results suggest that areas such as the PL, IL, dmPAG, BNST, MeA, and PVN could be part of a circuit responsible for vasopressin release after activation of alpha-1 adrenoceptors in the LSA.

Involvement of descending serotonergic and noradrenergic pathways in CB1 receptor-mediated antinociception

Cannabinoids produce antinociceptive and antihyperalgesic effects mainly through activation of the inhibitory CB1 receptors. The demonstration that antinociceptive effects of systemic cannabinoids are significantly diminished following surgical dorsolateral funiculus lesion provides evidence that supraspinal sites and descending pain modulatory pathways play crucial roles in systemic cannabinoid analgesia. In this review, we will firstly provide a background, brief overview of descending modulatory pathways followed by descending pathways implicated in cannabinoid analgesia. We will then describe the recent evidence of the involvement of descending serotonergic and noradrenergic pathways in CB1 receptor-mediated antinociception. This review will provide evidences that systemically administered cannabinoids reinforce the descending serotonergic and noradrenergic pathways to produce acute antinociceptive effects via spinal 5-HT7, 5-HT2A and alpha-2 adrenoceptors activation.

Regulation of catecholamine release in human adrenal chromaffin cells by β-adrenoceptors

The adrenal gland plays a fundamental role in the response to a variety of stress situations. After a stress condition, adrenal medullary chromaffin cells release, by exocytosis, high quantities of catecholamine (epinephrine, EP; norepinephrine, NE), especially EP. Once in the blood stream, catecholamines reach different target organs, and induce their biological actions through the activation of different adrenoceptors. Adrenal gland cells may also be activated by catecholamines, through hormonal, paracrine and/or autocrine system. The presence of functional adrenoceptors on human adrenal medulla and their involvement on catecholamines secretion was not previously evaluated. In the present study we investigated the role of β1-, β2- and β3-adrenoceptors on catecholamine release from human adrenal chromaffin cells in culture.We observed that the β-adrenoceptor agonist (isoproterenol) and β2-adrenoceptor agonist (salbutamol) stimulated catecholamine (NE and EP) release from human adrenal chromaffin cells. Furthermore, the β2-adrenoceptor antagonist (ICI 118,551; 100nM) and β3-adrenoceptor antagonist (SR 59230A; 100nM) inhibited the catecholamine release stimulated by isoproterenol and nicotine in chromaffin cells. The β1-adrenoceptor antagonist (atenolol; 100nM) did not change the isoproterenol- neither the nicotine-evoked catecholamine release from human adrenal chromaffin cells. Moreover, our results show that the protein kinase A (PKA), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and phospholipase C (PLC) are intracellular mechanisms involved in the catecholamine release evoked by salbutamol. In conclusion, our data suggest that the activation of β2- and β3-adrenoceptors modulate the basal and evoked catecholamine release, NE and EP, via an autocrine positive feedback loop in human adrenal chromaffin cells.

Emotional modulation of the synapse

Acute stress and emotional arousal can enhance the consolidation of long-term memories in a manner that is dependent on β -adrenoceptor activation in the basolateral complex of the amygdala (BLA). The BLA interacts with multiple memory systems in the brain to modulate a variety of classes of memory. However, the synaptic mechanisms of this interaction remain unresolved. This review describes the evidence of modulation of memory and synaptic plasticity produced by emotional arousal,stress hormones, and pharmacological or electrophysiological stimulation of the amygdala. The amygdala modulation of local translation and/or degradation of the synaptic plasticity-related proteins, activity-regulated cytoskeletal-associated protein and calcium/calmodulin dependent protein kinase II α , is offered as a potential mechanism for the rapid memory consolidation that is associated with emotionally arousing events. This model shares features with synaptic tagging and the emotional tagging hypotheses.

Comparison of propranolol and metoprolol on isolated frog heart

Sympathomimetic drugs and drugs that block adrenoceptors have important effects, some of which are of great clinical value. These effects vary dramatically according to the drug's selectivity for  and  receptors. 1 and 2 adrenoceptors coexist in the heart of various animal species, including man. Competitive radio-ligand binding studies performed in membranes from homogenized hearts have shown that only 20-30% of the total β-adrenoceptors are of the β2-subtype in adult mammalian ventricular tissue. This number is even further reduced when purified cardiac myocytes rather than homogenized tissues are used. Unlike the mammalian heart, β-adrenoceptor population in the frog heart is composed of a majority (~ 80%) of β2-receptors. Frogs, weighing 150 - 250g, double pith a frog and fasten it to a frog board, ventral side up, midline incision was given on the abdomen. Pectoral girdle was removed and the heart was exposed, Pericardium was removed carefully 'v' shaped cut was given in inferior vena cava and the tip of syme's cannula was passed into it. It was tied firmly with inferior vena cava to assure the cannula in place. Immediately the aorta were cutted and carefully heart along with cannula were isolated from the animal, The effect of cardio-selective  adrenergic blocker Metoprolol was added to the biophase in addition to 1 g of adrenaline and the contraction of heart till the 70-80% of inhibition is produced and the difference from normal contraction (inhibition in height of contraction) was recorded. The procedure was repeated by adding -blocker Metoprolol in the dose of 2.5 g, 5 g, 10 g, 20 g, 40 g, 80 g, 160 g and 320 g respectively. Individual findings are recorded on smoked drum cylinder, fixed with resin (colophony) and the recordings are measured. The ID50 of propranolol and metoprolol was found to be 1.3 and 44 g respectively and interestingly propranolol was more efficient in inhibiting the contraction of adrenaline than metoprolol. These interesting facts can be supported by the finding that 2 receptors predominate in the myocytes of Rana tigrina and this overwhelms the more efficient post-receptor coupling of 1 blockers. Competitive radio-ligand binding studies performed in membranes from homogenized hearts have shown that only 20-30% of the total β-adrenoceptors are of the β2-subtype in adult mammalian ventricular tissue. This number is even further reduced when purified cardiac myocytes rather than homogenized tissues are used. Yet, selective activation of β2-adrenoceptors produces a large increase in the amplitude of contraction in intact mammalian cardiac muscle as well as in isolated ventricular myocytes. Moreover, selective β2- adrenoceptor activation was found to produce a stimulation of the L-type Ca

Abstract P144: ER Stress-Mediated Reduction of SERCA2a Protein Is Not Involved in Increase in Lethal Heart Failure After Myocardial Infarction in Obese Type 2 Diabetes

Rationale: Our previous studies showed that increased endoplasmic reticulum (ER) stress in the myocardium impairs cytoprotective signaling in obese type 2 diabetes mellitus (T2DM). Objective: We examined roles of ER stress in alterations of ventricular function and mortality after myocardial infarction by T2DM. Methods and Results: SERCA2a protein level was significantly lower by 45% and Ser16- and Thr17-phosphorylated phospholamban levels tended to be lower in a rat model of T2DM (OLETF) than those in non-diabetic control rats (LETO). Treatment with an ER stress modulator, 4-phenylbutyric acid (4-PBA) or tauroursodeoxycholic acid (TUDCA), restored SERCA2a protein in OLETF to the level of that in LETO. Although left ventricular (LV) ejection fractions were comparable in OLETF and LETO, LV end-systolic elastance (1685±86 vs. 1953±68 mmHg/ml) and LVdP/dtmax (5886±337 vs. 7389±457 mmHg/s) were lower and tau was larger (12.1±0.9 vs. 8.9±0.8 msec) in OLETF. These impairments of LV function in OLETF were restored by treatment with 4-PBA (LV end-systolic elastance: 1813±58 mmHg/ml, LVdP/dtmax: 7053±546 mmHg/s and tau: 8.5±0.5 msec). Elevation of LV systolic pressure and dP/dtmax levels by dobutamine infusion was reduced, and inhibitory effects of verapamil on the dobutamine-induced positive inotropism were blunted in OLETF. In patch-clamp experiments using isolated cardiomyocytes, increase in L-type Ca 2+ channel current after isoproterenol treatment was markedly attenuated in OLETF as compared with the change in LETO. Mortality rate within 48 hrs after myocardial infarction was significantly higher in OLETF than in LETO (64.0% vs. 7.7%). Telemetric recording indicated that rapid progression of heart failure but not arrhythmia was responsible for the high mortality rate in OLETF. In contrast to the improvement of baseline ventricular dysfunction, neither 4-PBA nor TUDCA significantly reduced mortality rate after infarction in OLETF. Conclusions: ER stress plays a major role in reduction of SERCA2a by T2DM. Blunted response of cardiac L-type Ca 2+ channels to adrenoceptor activation, rather than reduced SERCA2a, may contribute to increase in lethal heart failure after myocardial infarction in T2DM.

Pre‐ and postsynaptic effects of norepinephrine on γ‐aminobutyric acid‐mediated synaptic transmission in layer 2/3 of the rat auditory cortex

Noradrenergic terminals from the locus coeruleus release norepinephrine (NE) throughout most brain areas, including the auditory cortex, where they affect neural processing by modulating numerous cellular properties including the inhibitory γ-aminobutyric acid (GABA)ergic transmission. We recently demonstrated that NE affects GABAergic signaling onto cortical pyramidal cells in a complex manner. In this study, we used a combination of patch-clamp recording and immunohistochemical techniques to identify the synaptic site and the location of the adrenergic receptors involved in the modulation of GABAergic signaling in cortical layer 2/3 of the rat. Our results showed that NE increases the frequency of spike-independent miniature inhibitory postsynaptic currents (mIPSCs), as well as the probability of release of unitary inhibitory postsynaptic currents (IPSCs) obtained with patch-clamp pair-recordings. The pharmacology of mIPSCs and the identification of adrenergic receptors in neurons containing the GABAergic marker parvalbumin (PV) suggest that NE increases the presynaptic probability of GABA release by activating α(2) - and β-receptors on PV-positive neurons. On the contrary, bath-applied NE or phenylephrine, decreased the current mediated by pressure application of the GABA(A) -receptor agonist muscimol, as well as the amplitude-but not the frequency-of mIPSCs, indicating that activation of postsynaptic α(1) adrenoceptors reversibly depressed GABAergic currents. We speculate that while a generalized postsynaptic decrease of GABAergic inhibition might decrease the synaptic activation threshold for pyramidal neurons corresponding to an alert state, NE might promote perception and sensory binding by facilitating lateral inhibition as well as the production of γ-oscillations by a selective enhancement of perisomatic inhibition.

Regulation of ocular adrenoceptor genes expression by 5-MCA-NAT

We have demonstrated that 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT), reduces intraocular pressure (IOP) in rabbits. In addition, we have reported a link between hypotensive effect of 5-MCA-NAT and sympathetic nervous system. Moreover, it is known that aqueous humour production is controlled by the activation of adrenoceptors (ADRs) present in the ocular ciliary epithelium. Thus, the aim of this study is to investigate if the hypotensive effect of 5-MCA-NAT is due to a regulation of ciliary ADR genes expression. To confirm this we followed the effect of 5-MCA-NAT on rabbit IOP for 144 consecutive hours. A sustained IOP reduction for up to 72 h (P<0.01) was seen. In addition, changes in ADRB2 and ADRA2A mRNA were measured in cultured rabbit nonpigmented ciliary epithelial cells. After 5-MCA-NAT treatment, a significant downregulation of ADRB2 and upregulation of ADRA2A was observed. These results provide the regulation of ADRs mRNA by 5-MCA-NAT.

Adrenoceptor activity of muscarinic toxins identified from mamba venoms

Muscarinic toxins (MTs) are snake venom peptides named for their ability to interfere with ligand binding to muscarinic acetylcholine receptors (mAChRs). Recent data infer that these toxins may have other G-protein-coupled receptor targets than the mAChRs. The purpose of this study was to systematically investigate the interactions of MTs with the adrenoceptor family members. We studied the interaction of four common MTs, MT1, MT3, MT7 and MTα, with cloned receptors expressed in insect cells by radioligand binding. Toxins showing modest to high-affinity interactions with adrenoceptors were additionally tested for effects on functional receptor responses by way of inhibition of agonist-induced Ca²⁺ increases. All MTs behaved non-competitively in radioligand displacement binding. MT1 displayed higher binding affinity for the human α(2B)-adrenoceptor (IC₅₀ = 2.3 nM) as compared with muscarinic receptors (IC₅₀ ≥ 100 nM). MT3 appeared to have a broad spectrum of targets showing high-affinity binding (IC₅₀ = 1-10 nM) to M₄ mAChR, α(1A)-, α(1D)- and α(2A)-adrenoceptors and lower affinity binding (IC₅₀ ≥ 25 nM) to α(1B)- and α(2C)-adrenoceptors and M₁ mAChR. MT7 did not detectably bind to other receptors than M₁, and MTα was specific for the α(2B)-adrenoceptor. None of the toxins showed effects on β₁- or β₂-adrenoceptors. Some of the MTs previously found to interact predominantly with mAChRs were shown to bind with high affinity to selected adrenoceptor subtypes. This renders these peptide toxins useful for engineering selective ligands to target various adrenoceptors.

Chemical stimulation of the ST36 acupoint reduces both formalin-induced nociceptive behaviors and spinal astrocyte activation via spinal alpha-2 adrenoceptors

Spinal astrocytes have emerged as important mechanistic contributors to pathological and chronic pain. Recently, we have demonstrated that injection of diluted bee venom (DBV) into the Zusanli (ST36) acupoint produces a potent anti-nociceptive effect via the activation of spinal alpha-2 adrenoceptors. However, it is unclear if this anti-nociceptive effect is associated with alterations in spinal astrocytes. Thus, the present study was designed to determine: (1) whether DBV's anti-nociceptive effect in the formalin test involves suppression of spinal astrocyte activation; (2) whether DBV-induced astrocyte inhibition is mediated by spinal alpha-2 adrenoceptors; and (3) whether this glial modulation is potentiated by intrathecal administration of the glial metabolic inhibitor, fluorocitrate (FC) in combination with DBV injection. DBV was injected directly into the ST36 acupoint, and spinal expression of the astrocytic marker, glial fibrillary acidic protein (GFAP), was assessed together with effects on formalin-induced nociception. DBV treatment reduced pain responses in the late phase of the formalin test and significantly blocked the formalin-evoked increase in spinal GFAP expression. These effects of DBV were prevented by intrathecal pretreatment with selective alpha-2A and alpha-2C adrenoceptor antagonists. Moreover, low dose intrathecal injection of FC in conjunction with low dose DBV injection into the ST36 acupoint synergistically suppressed pain responses and GFAP expression. These results demonstrate that DBV stimulation of the ST36 acupoint inhibits the formalin-induced activation of spinal astrocytes and nociceptive behaviors in this inflammatory pain model and this inhibition is associated with the activation of spinal alpha-2 adrenoceptors.

α2-Adrenoceptor-Mediated Inhibition of Catecholamine Release from the Adrenal Medulla of Spontaneously Hypertensive Rats is Preserved in the Early Stages of Hypertension

In this study, we evaluated the effect of α(2) -adrenoceptor activation on catecholamine release from the adrenal medulla of pre-hypertensive (6-week-old) and hypertensive (16-week-old) spontaneously hypertensive rats (SHR) and of age-matched normotensive control Wistar Kyoto (WKY) rats. Catecholamine overflow from isolated adrenal medullae was evoked by the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) in the absence and presence of the α(2) -adrenoceptor agonist medetomidine (MED). The spontaneous outflow of adrenaline was similar between age-matched SHR and WKY rats. However, the spontaneous outflow of noradrenaline was significantly lower in SHR compared with age-matched WKY rats. DMPP (0.1-3 mM) increased the outflow of noradrenaline and adrenaline in a concentration-dependent manner. The E(max) values for adrenaline overflow were similar between strains, but the E(max) values for noradrenaline overflow were significantly lower in SHR. The EC(50) values for noradrenaline and adrenaline overflow were significantly higher in SHR compared with age-matched WKY rats. MED (0.1-300 nM) reduced the DMPP-evoked overflow (DMPP 500 μM) of noradrenaline and adrenaline in a concentration-dependent manner and was capable of totally inhibiting this effect. The inhibitory action of MED was similar between age-matched SHR and WKY rats. In the adrenals, the α(2A)- and α(2B)-adrenoceptor subtypes had the highest mRNA expression levels; the α(2C)-adrenoceptor subtype had the lowest mRNA expression levels. The mRNA levels for the three subtypes were similar between strains. In conclusion, in SHR during the development of hypertension, adrenal α(2) -adrenoceptor inhibitory function is conserved, accompanied by reduced noradrenaline release and unchanged adrenaline release.

Noradrenergic regulation of period1 expression in spinal astrocytes is involved in protein kinase A, c-Jun N-terminal kinase and extracellular signal-regulated kinase activation mediated by α1- and β2-adrenoceptors

Our recent data suggest that noradrenaline (NA) regulates expression of Per1 mRNA in rat C6 cells, as a model of brain astrocytes, by two distinct NA-mediating pathways. Although C6 cells possess potential astrocyte-type characteristics, we hypothesize that astrocytes located in a distinct tissue or organ play specific roles consistent with their own unique functions in response to the surrounding environment. We have herein found in primary rat spinal astrocytes using real-time RT-PCR that NA induced robust transient increases in Per1, Cry1, Cry2 and Bmal1 mRNA expression. Cry1, Cry2 and Bmal1 expressions induced by NA were attenuated by transfection of Per1 small interference RNA (siRNA). The effect of NA on Per1 expression was partially blocked by either prazosin (a selective antagonist of α1-adrenoceptor) or ICI118551 (a selective antagonist of β2-adrenoceptor), and completely blocked by the combination of both antagonists. Treatment with H89 (a protein kinase A [PKA] inhibitor), SP600125 (a c-Jun N-terminal kinase [JNK] inhibitor), or PD98059 (an extracellular signal-regulated kinase [ERK] inhibitor), partially inhibited NA-induced Per1 mRNA expression, and the combination of these three inhibitors inhibited expression to nearly a non-stimulated level. Furthermore, NA phosphorylated not only ERK but also JNK1, an effect that was detected by western blotting. These actions were inhibited only by prazosin, and not by ICI118551. In addition, we found that NA induced phosphorylation of transcription-related proteins such as cAMP response element binding protein (CREB) and c-Jun. These phosphorylation processes were regulated through distinct pathways: CREB phosphorylation was dependent on the PKA and JNK pathways but c-Jun phosphorylation was mediated by the ERK and JNK pathways. These results suggest that Per1 plays a key role in noradrenergic regulation on clock gene expression in spinal astrocytes and activation of α1 and β2 adrenoceptors are of importance in regulation of Per1 mRNA expression via PKA/JNK-CREB and ERK/JNK-c-Jun cascades.

Ethanol- and isoprenaline-induced responses in pig parenchymal lung tissue.

Ethanol (8.7-34.8 mM; 0.5-0.20% v/v) had no significant effect on spontaneously developed tension in pig lung parenchyma strips, although higher cumulative concentrations caused relaxation (at 34.8-174 mM) and contraction (at 348-1392 mM). Ethanol-induced effects were not caused by activation of adrenoceptors or of cholinoceptors, since blockade of these receptors did not alter those effects. Single low concentrations of ethanol (8.7-34.8 mM) which can occur in-vivo in man, caused increases in the relaxant potency of isoprenaline in pig lung strips, although the sizes of these increases varied widely. In addition, ethanol-induced potentiation of isoprenaline was not concentration-related. This effect was not caused by inhibition of isoprenaline uptake since [3H]isoprenaline transport was found not to be significantly altered in the presence of ethanol (8.7-34.8 mM).

Prolonged effects of reserpine administration on adrenoceptor activity in dogs.

Prolonged effects of reserpine administration on adrenoceptor activity in dogs.

Evidence for alpha 2-adrenoceptor agonist activity of minoxidil.

The present investigation was undertaken to study the mechanism of action of minoxidil using various smooth muscle preparations. Minoxidil (4.7 x 10(-6) M to 4.7 x 10(-4) M) produced a concentration-dependent inhibition of field stimulation-evoked responses in rat anococcygeus muscle and vas deferens. The inhibition produced by minoxidil was antagonized by yohimbine (2.5 x 10(-7) M). Minoxidil (1.4 x 10(-5) M to 4.7 x 10(-4) M) also produced a concentration-dependent relaxation in oestrogen-primed potassium chloride-depolarized rat uterus. These responses were blocked not only by yohimbine but also by glibenclamide (2.02 x 10(-8) M). Our results suggest that minoxidil possesses alpha 2-adrenoceptor agonist activity in addition to potassium-channel-opening activity.

Functional selectivity of brain Gα‐subunit proteins in the cardiovascular and renal excretory responses evoked by central α2‐adrenoceptor activation in vivo

HypothesisBrain Gα‐subunit proteins signal in a functionally selective manner to mediate the integrated cardiovascular depressor and renal excretory responses evoked by pharmacological activation of central α2‐adrenoceptors in vivo.MethodsThe α2‐agonist guanabenz (50 μg) was administered by intracerebroventricular (i.c.v.) injection to conscious instrumented Sprague‐Dawley rats pre‐treated (24‐h) i.c.v. with saline or oligodeoxynucleotides (ODN) targeted to Gαi1,Gi2, Gi3, Gαo, Gαq, Gαz or a scrambled (S) sequence (25 μg, 24‐h, N=5/group). Mean arterial pressure (MAP) and heart rate (HR) were recorded and was urine collected for 150‐min (10‐min periods).Results i.c.v. ODN pre‐ treatment (24h; 5 μg) Peak Δ HR (bpm) Peak Δ MAP (mmHg) Peak Δ urine output (μl/min) Peak Δ UNaV (μeq/min) Saline Vehicle −92 ± 9 −19 ± 2 +136 ± 9 +6.2 ± 0.8 S −94 ± 12 −18 ± 1.2 +132 ± 11 +5.9 ± 0.6 Gαi1 −88 ± 11 −19 ± 2 +153 ± 16 +6.1 ± 0.8 Gαi2 −90 ± 13 −1 ± 1** +125 ± 11 +0.6 ± 0.3** Gαi3 −66 ± 9 −17 ± 1.3 +143 ± 16 +6.2 ±0.9 Gαo −47 ± 5* −16 ± 1.6 +127 ± 10 +6.2 ± 0.5 Gαq −106 ± 7 −15 ± 2 +214 ± 7** +7.2 ± 1.2 Gαz −92 ± 8 −16 ± 2 +110 ± 6* +5.8 ± 1.2 p&lt;0.05, p&lt;0.01 vs. S ODN group Brain Gαi2 subunit protein‐gated signal‐transduction pathways selectively mediate the hypotensive and natriuretic, but not bradycardic or diuretic, responses evoked by central α2‐adrenoceptor activation in vivo. In contrast, the profound α2‐agoinst‐stimulated diuretic response is mediated, in part, via opposing Gαz and Gαq subunit protein gated pathways (stimulatory versus inhibitory, respectively).ConclusionsThese studies demonstrate that in vivo, brain GPCR Gα‐subunit protein pathways play an essential and physiologically important gating role in mediating functionally selective changes in systemic cardiovascular function and the renal excretion of water and sodium post‐ligand binding at central α2‐adrenoceptors. RDW‐AHA 2250585; DRK‐DK43337, HL071212, AHA 0855293E, and RDW/DRK‐ P20 RR018766

Functional antagonism of β‐adrenoceptor subtypes in the catecholamine‐induced automatism in rat myocardium

Myocardial automatism and arrhythmias may ensue during strong sympathetic stimulation. We sought to investigate the relevant types of adrenoceptor, as well as the role of phosphodiesterase (PDE) activity, in the production of catecholaminergic automatism in atrial and ventricular rat myocardium. The effects of adrenoceptor agonists on the rate of spontaneous contractions (automatic response) and the amplitude of electrically evoked contractions (inotropic response) were determined in left atria and ventricular myocytes isolated from Wistar rats. Catecholaminergic automatism was Ca(2+) -dependent, as it required a functional sarcoplasmic reticulum to be exhibited. Although both α- and β-adrenoceptor activation caused inotropic stimulation, only β(1) -adrenoceptors seemed to mediate the induction of spontaneous activity. Catecholaminergic automatism was enhanced and suppressed by β(2) -adrenoceptor blockade and stimulation respectively. Inhibition of either PDE3 or PDE4 (by milrinone and rolipram, respectively) potentiated the automatic response of myocytes to catecholamines. However, only rolipram abolished the attenuation of automatism produced by β(2) -adrenoceptor stimulation. α- and β(2) -adrenoceptors do not seem to be involved in the mediation of catecholaminergic stimulation of spontaneous activity in atrial and ventricular myocardium. However, a functional antagonism of β(1) - and β(2) -adrenoceptor activation was identified, the former mediating catecholaminergic myocardial automatism and the latter attenuating this effect. Results suggest that hydrolysis of cAMP by PDE4 is involved in the protective effect mediated by β(2) -adrenoceptor stimulation.