Partial Agonist Clonidine Research Articles

Voltage-Dependent Modulation of α2A Adrenergic Receptor Conformations

G protein-coupled receptors (GPCRs) are proteins that span the cell membrane seven times. They are stimulated by extracellular agonists and activate heterotrimeric G proteins to elicit intracellular responses. Recent evidence suggests that GPCRs are voltage-sensitive: They exhibit gating currents similar to ion channels and respond to depolarization of the plasma membrane with changes in agonist affinity. We used a FRET-based biosensor of the α2A adrenergic receptor to analyze voltage dependence of receptor activation in HEK 293 cells by means of voltage-clamp recording. The biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (NE) and receptor activation was measured as decrease in FRET ratio. Receptor stimulation by NE was reduced by membrane depolarization and enhanced by hyperpolarization. This effect was present in wilde-type receptors and transduced to the level of G protein activation, which we determined in a FRET assay that directly detects Gαi protein activation. Depolarization-mediated inhibition of NE activated receptors was strong at low concentrations (500 nM: 60 % inhibition) but almost undetectable at saturating agonist concentrations (100 μM: 9 % inhibition). Both agonist-induced and hyperpolarization-induced receptor activation exhibited a similar monoexponential time course. For both activation modes, speed of activation was primarily dependent on agonist concentration, indicating that depolarization lowers the apparent affinity of the NE receptor interaction and thus causes receptor deactivation by means of NE release. Application of clonidine (1 μM, VM=-90 mV) resulted in a FRET response that was inhibited by 40 % at +60 mV. In contrast to NE, strong receptor-inhibition at +60 mV was present even at super-saturating concentrations of clonidine (100 μM). Therefore we conclude that negative membrane potentials promote active conformations of the α2A adrenergic receptor, increase affinity of full agonists and enhance G protein signaling.

Antagonistic effects of selective alpha1-adrenoceptor antagonists MDL73005EF and tamsulosin and partial agonists clonidine and tizanidine in rat thoracic aorta and rabbit iliac artery.

The antagonistic effects of MDL73005EF and tamsulosin and partial agonists clonidine and tizanidine at rat thoracic aorta and rabbit iliac artery alpha1-adrenoceptors were investigated in this study. Selective alpha1-adrenoceptor antagonists MDL73005EF and tamsulosin dose-dependently shifted the concentration-response curves for noradrenaline to the right. Schild plots of the results obtained from the inhibition by MDL73005EF (pA2 8.30 +/- 0.04) and tamsulosin (pA2 10.51 +/- 0.06) of noradrenaline yielded a straight line with a slope of unity in rat thoracic aorta. The slopes of Schild plots obtained from the inhibition by MDL73005EF and tamsulosin of noradrenaline were significantly different from unity in rabbit iliac artery. Schild plots of the results obtained from the inhibition by clonidine and tizanidine of noradrenaline yielded a straight line with a slope of unity in rat thoracic aorta (pA2 7.08 +/- 0.04 and 7.32 +/- 0.04, respectively). These results suggest that alpha1D-adrenoceptors play a significant role in the alpha1-adrenoceptor-agonist-induced contraction of rat thoracic aorta and rabbit iliac artery, and that clonidine and tizanidine interact with the alpha1D-adrenoceptor subtype as competitive antagonists in rat thoracic aorta.

Fluorescence Resonance Energy Transfer Analysis of α2a-Adrenergic Receptor Activation Reveals Distinct Agonist-Specific Conformational Changes

Several lines of evidence suggest that G-protein-coupled receptors can adopt different active conformations, but their direct demonstration in intact cells is still missing. Using a fluorescence resonance energy transfer (FRET)-based approach we studied conformational changes in alpha(2A)-adrenergic receptors in intact cells. The receptors were C-terminally labeled with cyan fluorescent protein and with fluorescein arsenical hairpin binder at different sites in the third intracellular loop: N-terminally close to transmembrane domain V (I3-N), in the middle of the loop (I3-M), or C-terminally close to transmembrane domain VI (I3-C). All constructs retained normal ligand binding and signaling properties. Changes in FRET between the labels were determined in intact cells in response to different agonists. The full agonist norepinephrine evoked similar FRET changes for all three constructs. The strong partial agonists clonidine and dopamine induced partial FRET changes for all constructs. However, the weak partial agonists octopamine and norphenephrine only induced detectable changes in the construct I3-C but no change in I3-M and I3-N. Dopamine-induced FRET-signals were approximately 1.5-fold slower than those for norepinephrine in I3-C and I3-M but >3-fold slower in I3-N. Our data indicate that the different ligands induced conformational changes in the receptor that were sensed differently in different positions of the third intracellular loop. This agrees with X-ray receptor structures indicating larger agonist-induced movements at the cytoplasmic ends of transmembrane domain VI than V and suggests that partial agonism is linked to distinct conformational changes within a G-protein-coupled receptor.

Partial agonism at the human α2A-autoreceptor: role of binding duration

Temperature-induced changes of affinity and efficacy of the alpha2-adrenoceptor full agonist UK14,304 and the partial agonists clonidine and guanfacine were investigated to elucidate the mechanism of partial agonism at the terminal alpha2-autoreceptor. The effect of temperature on the efficacy of the substances was tested in 3H-noradrenaline release experiments at 37 degrees C and at room temperature. Human neocortical slices were prelabeled with 3H-noradrenaline, superfused, and stimulated electrically under autoinhibition-free conditions. Furthermore, saturation binding experiments with human neocortical synaptosomes were performed at 37 degrees C and 17 degrees C to evaluate the influence of temperature on the affinity of 3H-clonidine and 3H-UK14,304. Temperature-induced changes of the association and dissociation rate constants of 3H-UK14,304 and 3H-clonidine were assessed in corresponding kinetic binding experiments. Our experiments reveal that clonidine and guanfacine lose efficacy when the temperature is lowered, whereas no change was noted for the full agonist UK14,304. Moreover, the affinity of clonidine and guanfacine was shown to decrease at lower temperature. Kinetic experiments indicated that the loss of affinity observed for 3H-clonidine at 17 degrees C is due to a marked reduction of the association rate. The loss of efficacy of the partial agonists is most likely related to the short binding duration; partial agonists do not bind long enough to the receptor to mediate a maximum response. The discrepancy between the time required to elicit a maximum response and the actual binding time may be greater for partial agonists at lower temperatures, thus, causing the intrinsic activity to decline.

Direct optical recording of intrinsic efficacy at a G protein-coupled receptor

Full and partial agonists activate receptors to varying degrees, presumably by inducing full or partial conformational changes in the receptor protein. Varying degrees of partial agonism corresponding to varying intrinsic efficacies have been demonstrated for many compounds acting at G-protein-coupled receptors, but a method to determine intrinsic efficacies directly at the receptor level has so far been lacking. Here we describe a method that allows the direct monitoring of agonist-induced conformational changes in G-protein-coupled receptors. The cyan (CFP) and yellow (YFP) variants of the green fluorescent protein were fused to the receptors. This resulted in fluorescence resonance energy transfer (FRET) between the CFP- and YFP-moieties. The extent of FRET was reduced in the presence of an agonist. The FRET signal strictly followed agonist occupancy of the receptor. Using the α 2-adrenergic receptor as a model system, the full agonist noradrenaline produced a full signal, the partial agonist clonidine produced only a partial signal, and the antagonist phentolamine had no effect. Thus, optical recording of the agonist-induced conformational change in a G-protein-coupled receptor allows the direct analysis of the intrinsic efficacies of agonists.

Partial agonist clonidine mediates alpha(2)-AR subtypes specific regulation of cAMP accumulation in adenylyl cyclase II transfected DDT1-MF2 cells.

alpha2-Adrenergic receptor (alpha(2)-AR) activation in the pregnant rat myometrium at midterm potentiates beta(2)-AR stimulation of adenylyl cyclase (AC) via Gbetagamma regulation of the type II isoform of adenylyl cyclase. However, at term, alpha(2)-AR activation inhibits beta(2)-AR stimulation of AC. This phenomenon is associated with changes in alpha(2)-AR subtype expression (midterm alpha(2A/D)-AR >> alpha(2B)-AR; term alpha(2B) >or =alpha(2A/D)-AR), without any change in ACII mRNA, suggesting that alpha(2A/D)- and alpha(2B)-AR differentially regulate beta(2)-cAMP production. To address this issue, we have stably expressed the same density of alpha(2A/D)- or alpha(2B)-AR with AC II in DDT1-MF2 cells. Clonidine (partial agonist) increased beta(2)-AR-stimulated cAMP production in alpha(2A/D)-AR-ACII transfectants but inhibited it in alpha(2B)-AR-ACII transfectants. In contrast, epinephrine (full agonist) enhanced beta(2)-stimulated ACII in both alpha(2A)- and alpha(2B)-ACII clonal cell lines. 4-Azidoanilido-[alpha-(32)P]GTP-labeling of activated G proteins indicated that, in alpha(2B)-AR transfectants, clonidine activated only Gi(2), whereas epinephrine, the full agonist, effectively coupled to Gi(2) and Gi(3). Thus, partial and full agonists selectively activate G proteins that lead to drug specific effects on effectors. Moreover, these data indicate that Gi(3) activation is required for potentiation of beta(2)-AR stimulation of AC by alpha(2A/D) and alpha(2B)-AR in DDT1-MF2 cells. This may reflect an issue of the amount of Gbetagamma released upon receptor activation and/or betagamma composition of Gi(3) versus Gi(2).

Moxonidine, a selective imidazoline-1 receptor agonist, suppresses the effects of ethanol withdrawal on the acoustic startle response in rats

Background: There is a need for improved treatments for ethanol withdrawal in humans. Previously, ethanol withdrawal has been shown to enhance the acoustic startle response in rats. Because many ethanol withdrawal symptoms are caused by autonomic hyperactivity, we examined the effects of two antihypertensives, the imidazoline(I) 1 agonist moxonidine and the α 2-adrenergic partial agonist clonidine, on the ethanol-withdrawal-enhanced acoustic startle response in rats. d-amphetamine-enhanced startle served as a positive control. Methods: Male, Long-Evans rats were made ethanol-dependent through unlimited access to liquid diet containing 6.7% v/v ethanol for 10 days. The concentration of ethanol was reduced to 3.3% v/v on the 11th day. On the 12th day, the rats received control diet. The acoustic startle response was tested 24 hours following the withdrawal of ethanol. Control rats were maintained on control liquid diet throughout the experiment. Results: As has been shown previously, withdrawal from the chronic ingestion of ethanol significantly enhanced the acoustic startle response. Pretreatment with moxonidine (0.01, 0.1, and 1.0 mg/kg, subcutaneously), but not clonidine (0.3, 1.0, and 3.0 mg/kg, subcutaneously), significantly attenuated the ethanol withdrawal-induced elevation of the acoustic startle response. Moxonidine did not suppress the elevation in the startle response caused by d-amphetamine. Conclusions: These results indicate that I 1 receptors can play an important role in ethanol withdrawal and that moxonidine may be useful for the treatment of ethanol withdrawal in humans.

Effects of the selective cyclooxygenase-2 inhibitor nimesulide on vascular contractions in endothelium-denuded rat aorta

We have examined the effects of the selective cyclooxygenase-2 inhibitor nimesulide and the non-selective cyclooxygenase inhibitor indomethacin on vascular responsiveness of endothelium-denuded rat aorta. Isometric contractions were obtained to the α-adrenoceptor agonists phenylephrine (full agonist) and clonidine (partial agonist relative to phenylephrine) and to endothelin-1 and KCl. Maximum contractile responses to the partial agonist clonidine were significantly reduced by nimesulide (10 μM) and by indomethacin (10 μM) to 60.8±8.5% ( n=8) and 69.0±9.6% ( n=12) of control, respectively, as compared with the effects of vehicle (99.0±5.8%; n=17). The inhibitors had lesser effects against contractions to phenylephrine: nimesulide had no significant effect, whereas indomethacin caused a small but significant reduction in the maximum contraction to phenylephrine to 90.3±5.0% ( n=12) of control (vehicle: 108.0±5.2%, n=15 nimesulide: 111.8±5.9%, n=5). Neither nimesulide nor indomethacin had any effect on contractions to endothelin-1 or KCl. These actions differed from the effects of the Ca 2+ entry blocker nifedipine, which significantly reduced contractions to clonidine and KCl to a similar extent. The maximum contraction to clonidine was also significantly reduced by the thromboxane receptor antagonist SQ 29548 (1 μM) to 83.4±6.4% of control ( n=7) (vehicle 115.5±7.5%, n=7). It is concluded that the cyclooxygenase inhibitors nimesulide or indomethacin reduce vascular responsiveness to α-adrenoceptor agonists in endothelium-denuded rat aorta, presumably by preventing the formation of vasoconstrictor prostaglandins in aortic smooth muscle by cyclooxygenase-2. This reduced vascular responsiveness was most clearly seen with the partial agonist clonidine.

Real-time monitoring of endogenous noradrenaline release in rat brain slices using fast cyclic voltammetry. 2. Operational characteristics of the α 2 autoreceptor in the bed nucleus of stria terminalis, pars ventralis

Fast cyclic voltammetry (FCV) at carbon fibre microelectrodes was used to monitor stimulated noradrenaline (NA) efflux in slices of the ventral part of the rat bed nucleus of stria terminalis (BSTV) superfused with artificial cerebrospinal fluid at 32°C. NA efflux was evoked by local electrical stimulation (trains of 10–50 pulses, 0.2 ms duration, 10 mA constant current at 10–500 Hz). The effects of four α 2 antagonists (yohimbine, rauwolscine, parazosin and WB 4101) and three α 2 agonits (clonidine, oxymetazoline and UK 14304) were examined. All drugs (1 μM) were added via the superfusate. Yohimbine and rauwolscine increased NA efflux on the lower but not the higher frequency trains: maximum increases (on 20 Hz, 50 pulse stimulation) were to 392±63% (yohimbine) and 243±7% (rauwolscine). There was a threshold train duration for demonstration of autoreceptor antagonism of 500–1000 ms. Prazosin and WB 4101 did not increase NA efflux but caused a modest decrease at the higher (100–500 Hz) frequencies. The effects of the α 2 agonists were also affected by stimulus train duration. Longer trains reduced agonist (clonidine) effects. When tested on pseudo-one pulse (POP) stimulations (less than 100 ms duration), the α 2 agonists decreased NA efflux. UK 14304 reduced NA efflux on 20 pulse/200 Hz stimulation to a g reater degree ( 86±7%) than the partial agonists clonidine ( 39±3%) or oxymetazoline ( 40±8%). The present results demonstrate α 2 autoreceptors are a major mechanism in the control of NA efflux in the BSTV. The lack of potentiation of NA efflux by prazosin suggests that the autoreceptor is not of the α 2B or α 2C subtype while the clear inhibitory action of oxymetazoline suggests an α 2A or α 2D receptor. With appropriate selection of stimulation conditions, the actions, of both antagonists and agonists (partial and full) can be examined.

Prejunctional alpha 2-adrenoceptors in mouse atria function through G-proteins which are sensitive to N-ethylmaleimide, but not pertussis toxin.

1. The identity of the G-proteins involved in prejunctional alpha 2-adrenoceptor signal transduction in mouse atria was examined by use of the G-protein inactivators N-ethylmaleimide and pertussis toxin. 2. The alpha 2-adrenoceptor partial agonist clonidine (0.03 microM) inhibited the electrical stimulation-induced (S-I) outflow of radioactivity from mouse atria which were incubated with [3H]-noradrenaline and stimulated at 5 Hz. The partial alpha 2-adrenoceptor agonist St 363 (10 microM) inhibited the S-I outflow of radioactivity at the lower stimulation frequency of 2.5 Hz. The inhibitory effects of these compounds were not altered in mice pretreated with pertussis toxin (1.5 micrograms, i.v.). 3. The alpha 2-adrenoceptor antagonist, idazoxan (0.1 microM), increased the S-I outflow of radioactivity from mouse atria stimulated at 5 Hz, and this effect was not altered in atria from mice pretreated with pertussis toxin. 4. The inhibitory effects of clonidine and St 363 and the facilitatory effect of idazoxan on the S-I outflow of radioactivity from mouse atria were significantly less in atria incubated with N-ethylmaleimide (NEM, 3 microM) for 60 min before the [3H]-noradrenaline incubation. 5. The results suggest that prejunctional alpha 2-adrenoceptors in mouse atria function through G-proteins which are NEM-sensitive, but pertussis toxin insensitive.

Influence of some α2‐‐receptor agonists and antagonists on the excitatory non‐adrenergic, non‐cholinergic neurotransmission in the airways of guinea‐pigs in vivo

Neural control of smooth muscle tension in the airways of guinea-pigs can be subdivided into at least four components: an excitatory cholinergic, an inhibitory noradrenergic, an excitatory non-adrenergic, non-cholinergic and an inhibitory non-adrenergic, non-cholinergic component. The existence of alpha 2-adrenoceptors that modulate the excitatory non-adrenergic, non-cholinergic nerve activity has also been demonstrated. The aim of the present study was to investigate the dose-dependence of the selective alpha 2-receptor agonists UK 14,304, dexmedetomidine and clonidine on these alpha 2-adrenoceptors in the airway of guinea-pigs in vivo. Electrical stimulation of the cervical vagus nerves in anaesthetized guinea-pigs resulted in a biphasic response in insufflation pressure: an immediate increase due to excitation of cholinergic nerve fibres and a slower, longer-lasting increase due to activation of the excitatory non-adrenergic, non-cholinergic fibres to be studied. The cholinergic compound was abolished by atropine, leaving the excitatory non-adrenergic, non-cholinergic component as the sole response recorded during vagal stimulation. UK 14,304 and dexmedetomidine attenuated the response in a dose-dependent manner, whereas the effect of the partial agonist clonidine was less dose-related. UK 14,304 and dexmedetomidine reduced the insufflation pressure about 60 and 53% respectively at the highest dose. In conclusion, the neurotransmission in the excitatory non-adrenergic, non-cholinergic nerves is attenuated by selective alpha 2-receptors in a dose-dependent manner. The present study adds support to earlier observations that the excitatory non-adrenergic, non-cholinergic neural system in the airways of guinea-pigs is modulated by alpha 2-adrenoceptors.

Involvement of presynaptic imidazoline receptors in the ?2-adrenoceptor-independent inhibition of noradrenaline release by imidazoline derivatives

An involvement of imidazoline recognition sites in the modulation of transmitter release was investigated in the rabbit pulmonary artery and aorta preincubated with [3H]noradrenaline and superfused with physiological salt solution containing cocaine, corticosterone and propranolol. Electrical impulses were applied transmurally at 0.66 or 2 Hz. In the absence of further drugs, rauwolscine as well as the imidazoline derivatives BDF 6143 [4-chloro-2-(2-imidazoline-2-ylamino)-isoindoline], idazoxan and phentolamine increased the 3H overflow from the pulmonary artery, evoked by electrical stimulation at 2 Hz; the effect was due to the alpha 2-autoreceptor blocking property of these drugs. The maximum increase in overflow obtainable with the imidazolines was considerably lower than with rauwolscine. The concentration-response curves of the imidazolines were bell-shaped. At 0.66 Hz, BDF 6143 did not facilitate, but concentration-dependently inhibited, whereas idazoxan failed to change the evoked 3H overflow. When, at the stimulation frequency of 2 Hz, presynaptic alpha 2-adrenoceptors were blocked by rauwolscine and/or pre-exposure to phenoxybenzamine, the electrically evoked 3H overflow from the pulmonary artery and/or aorta was inhibited by the following imidazoline derivatives: the alpha 2-adrenoceptor antagonists BDF 6143, idazoxan and phentolamine, the alpha 1-adrenoceptor agonist with alpha 2-blocking property cirazoline as well as the alpha 2-adrenoceptor agonists clonidine and moxonidine. The maximum inhibition caused by BDF 6143 was greater than that due to clonidine and moxonidine; the latter two, hence, behaved as partial agonists. At the stimulation frequency of 0.66 Hz, the imidazolines exhibited a higher potency than, but a similar intrinsic activity to that at 2 Hz. Noradrenaline did not affect the evoked 3H overflow. The BDF 6143-induced inhibition of evoked 3H overflow was not modified by metitepine, atropine, theophylline, dipyridamole and indometacin, but was counteracted by the partial agonists clonidine and moxonidine. The results exclude the possibility that alpha 1- and alpha 2-adrenoceptors, 5-HT1 receptors, muscarine receptors, P1 purinoceptors and prostaglandin receptors are involved in the imidazoline-induced inhibition of noradrenaline release. They provide evidence indicating that the inhibitory effect is mediated by imidazoline receptors on the postganglionic sympathetic nerve terminals of the rabbit pulmonary artery and aorta.

Blunted Growth Hormone Response to Clonidine in Patients With Generalized Anxiety Disorder

Patients with panic disorder or depression have abnormal responses to the alpha 2-adrenergic receptor partial agonist clonidine. Evidence linking anxiety to noradrenergic dysfunction and the presence of anxiety symptoms in both depression and panic suggest that abnormal responses to clonidine in these disorders could be due to the anxiety symptoms. To explore a possible link between "nonspecific" anxiety symptoms and abnormal responses to clonidine, patients with DSM-III-defined generalized anxiety disorder were given intravenous infusions of clonidine hydrochloride. Responses of plasma growth hormone, 3-methoxy-4-hydroxyphenylglycol, heart rate, blood pressure, and psychological states were determined in 11 patients with generalized anxiety disorder and 14 healthy subjects. Clonidine produced significantly smaller growth hormone responses in patients than in healthy controls. The two groups did not differ in 3-methoxy-4-hydroxyphenylglycol, heart rate, blood pressure, or psychological responses to clonidine. These results are compared with data from similar studies on patients with panic disorder and depression. The blunting of the growth hormone response to clonidine in all three disorders could be due to the presence of generalized anxiety symptoms. Subsensitivity of postsynaptic alpha 2-adrenoreceptors may be present in all three disorders; however, there are alternative interpretations of growth hormone blunting in response to clonidine. Blunting was observed in DSM-III-defined generalized anxiety disorder, whether or not the DSM-III-R criterion of excessive worry was also present.

Relationship between alpha 1-adrenoceptor density and functional response of rat vas deferens. Studies with phenoxybenzamine.

The relationship between the density of alpha 1-adrenoceptors and the longitudinal contractile response of rat vas deferens was examined by using phenoxybenzamine to irreversibly decrease alpha 1-adrenoceptor density. Receptor density was measured by Scatchard plots of saturation analysis of specific 125I-BE 2254 (125IBE) binding and compared to the potency of alpha-adrenoceptor agonists in causing contraction and the maximum contraction elicited by these agonists. Treatment of isolated vasa deferentia in organ baths with phenoxybenzamine caused a dose-dependent decrease in the density of 125IBE binding sites, the potency of the full agonist phenylephrine in activating contraction, and the maximum contractile response. The percentage of functional receptors remaining after phenoxybenzamine treatment (q value) was calculated from the contraction data and compared to the percentage of 125IBE binding sites remaining. The reduction in the number of functional receptors was much greater than the reduction in the number of 125IBE binding sites at all doses of phenoxybenzamine. Since the magnitude of the contractile response caused by a weak partial agonist should be approximately proportional to the density of functional receptors in the tissue, partial agonists were used as an independent measure of the degree of functional receptor inactivation. Bath application of two different doses of phenoxybenzamine caused a decrease in the maximal contraction caused by the partial agonists clonidine and ephedrine which was similar to the calculated decrease in the q value, but not to the observed decrease in the density of 125IBE binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)