A Gαs DREADD Mouse for Selective Modulation of cAMP Production in Striatopallidal Neurons

Here, we describe a newly generated transgenic mouse in which the Gs DREADD (rM3Ds), an engineered G protein-coupled receptor, is selectively expressed in striatopallidal medium spiny neurons (MSNs). We first show that in vitro, rM3Ds can couple to Gα_olf and induce cAMP accumulation in cultured neurons and HEK-T cells. The rM3Ds was then selectively and stably expressed in striatopallidal neurons by creating a transgenic mouse in which an adenosine2A (adora2a) receptor-containing bacterial artificial chromosome was employed to drive rM3Ds expression. In the adora2A-rM3Ds mouse, activation of rM3Ds by clozapine-N-oxide (CNO) induces DARPP-32 phosphorylation, consistent with the known consequence of activation of endogenous striatal Gα_s-coupled GPCRs. We then tested whether CNO administration would produce behavioral responses associated with striatopallidal G_s signaling and in this regard CNO dose-dependently decreases spontaneous locomotor activity and inhibits novelty induced locomotor activity. Last, we show that CNO prevented behavioral sensitization to amphetamine and increased AMPAR/NMDAR ratios in transgene-expressing neurons of the nucleus accumbens shell. These studies demonstrate the utility of adora2a-rM3Ds transgenic mice for the selective and noninvasive modulation of Gα_s signaling in specific neuronal populations in vivo.This unique tool provides a new resource for elucidating the roles of striatopallidal MSN Gα_s signaling in other neurobehavioral contexts.     

Gαi2 signaling: friend or foe in cardiac injury and heart failure?

Receptors coupled to G proteins have many effects on the heart. Enhanced signaling by Gα_s and Gα_q leads to cardiac injury and heart failure, while Gα_i2 signaling in cardiac myocytes can protect against ischemic injury and β-adrenergic-induced heart failure. We asked whether enhanced Gα_i2 signaling in mice could protect against heart failure using a point mutation in Gα_i2 (G184S), which prevents negative regulation by regulators of G protein signaling. Contrary to our expectation, it worsened effects of a genetic dilated cardiomyopathy (DCM) and catecholamine-induced cardiac injury. Gα_i2^G184S/+/DCM double heterozygote mice (TG9⁺Gα_i2^G184S/+) had substantially decreased survival compared to DCM animals. Furthermore, heart weight/body weight ratios (HW/BW) were significantly greater in TG9⁺Gα_i2^G184S/+ mice as was expression of natriuretic peptide genes. Catecholamine injury in Gα_i2^G184S/G184S mutant mice produced markedly increased isoproterenol-induced fibrosis and collagen III gene expression vs WT mice. Cardiac fibroblasts from Gα_i2^G184S/G184S mice also showed a serum-dependent increase in proliferation and ERK phosphorylation, which were blocked by pertussis toxin and a mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor. Gα_i2 signaling in cardiac myocytes protects against ischemic injury but enhancing Gα_i2 signaling overall may have detrimental effects in heart failure, perhaps through actions on cardiac fibroblasts.     

α-Terpineol reduces nociceptive behavior in mice

Context: α-Terpineol (TPN) is a monoterpenoid alcohol present in the essential oils of several species of the Eucalyptus genus (Myrtaceae).

Objective: TPN was assessed for its antinociceptive activity in rodents.

Materials and methods: The antinociceptive effect of TPN was examined using the acetic acid writhing reflex, formalin, glutamate, and capsaicin-induced nociception tests.

Results: TPN produced a significant (P?<?0.01 or P?<?0.001) analgesic effect by reduction at the early and late phases of paw licking and reduced the writhing reflex in mice (formalin and writhing tests, respectively). In the glutamate test, all doses of TPN produced significant (P?<?0.01) nociceptive protection. When the capsaicin-induced nociception test was conducted, TPN produced dose-related inhibition of the nociceptive behavior. In addition, the results of a hot plate test showed central analgesic properties for TPN (P?<?0.01 or P?<?0.001). Such results were unlikely to be provoked by motor abnormality.

Conclusion: Our results suggest that TPN might represent an important tool for management and/or treatment of painful conditions.     

Effects of morphine and methadone treatment on mRNA expression of Gα(i) subunits in rat brains

Methadone is clinically effective as substitution therapy in patients with opioid dependence. The diversity of methadone and morphine in their intracellular activity is postulated. We compared the effects of repeated daily treatment of Sprague-Dawley rats with morphine (10 mg/kg) and methadone (1 mg/kg) on the expression of the Gα(i1-i3) mRNAs in several rat brain areas using RT-qPCR. We found that both opioid receptor agonists decreased Gα(i3) mRNA in only the nucleus accumbens. Although there was no difference in the influence of morphine and methadone on Gα(i), our results indicate that among Gα(i) subunits, the Gα(i3) is specifically involved in the mechanism of action of both drugs.     

Effect of Gαq／11 protein and ATP-sensitive potassium channels on prostaglandin E1 preconditioning in rat hearts

To investigate the effect of G~11 signaling pathway and ATP-sensitive potassium channels (KAxP channels) on prostaglandin E1 (PGE0 induced early and delay-preconditioning protection in rat hearts. METHODS: Two series of experiments were performed in Wistar rat hearts. In the first series of experiment, all rats were pretreated with PGE 40 min or 23 h 20 min before the experiment. Ischemia-reperfusion injury was induced by 30 min coronaryartery occlusion followed by 90 min reperfusion. Hemodynamics, infarct size, and scores of ventricular arrhythmias were measured. The expression of G~q/H protein in the heart was measured by Western blot analysis in the second series. RESULTS: Preconditioning with PGE~ (25 lag/kg ) markedly reduced infarct size, left ventricular enddiastolic pressure, and scores of ventricular arrhythmia. The effect of PGE1 was significantly attenuated by glibenclamide (1 mg/kg, ip), a nonselective KA~ channel inhibitor. PGE~ caused a significant increase in the expression of G~/~ protein. CONCLUSION: Activations of G~/H signal pathway and Kgrp channel played significantroles in the cardioprotection of PGE~ preconditioning in rat heart and might be an important mechanism of signal transduction pathway during the PGEj preconditioning.     

α-Monofluoromethyldopa (MFMD) in combination withl-DOPA

Summary We have investigated the interaction of -monofluoromethyldopa (MFMD) with the effects of i.p. injectedl-DOPA (200 mg·kg^–1) on blood pressure and tissue catecholamines in normal and spontaneously hypertensive rats (SHR). MFMD 10 mg·kg^–1 (i.p.) effectively antagonizes thel-DOPA induced increase in heart dopamine (DA). This action is also seen after 15 or 50 mg·kg^–1. The accumulation of DA in the brain is very much reduced by MFMD 50 mg ·kg^–1 while after 15 or, especially, 10 mg·kg^–1 more DA is formed in the rrain than afterl-DOPA alone, probably due to the peripheral decarboxylase inhibition which presents morel-DOPA to the brain. We conclude that MFMD 10 mg ·kg^–1 gives a relatively selective peripheral inhibition of the decarboxylation ofl-DOPA and this dose combination was accordingly found to result in a reduction of blood pressure in conscious animals. This hypotensive response tol-DOPA was attenuated after MFMD 15 mg·kg^–1 and was absent after MFMD 50 mg·kg^–1. Interestingly, the hypotensive effect ofl-DOPA after MFMD 10 or 15 mg·kg^–1 was more pronounced in SHR.     

α7 nAChR in airway respiratory epithelial cells

This review examines the role of α7 nAChR in different types of airway epithelial cells of the normal human bronchial tree. In each of these cells α7 nAChR activation elicits a specific effect. The effect is essentially mitogenic, whereas in the airway basal cells are antiproliferative. It is postulated that α7 nAChR may have mitogenic or antiproliferative signals differentially activated in different types of airway cells and under exposure to exogenous stimuli such as nicotine.     

Functional selectivity of central Gα-subunit proteins in mediating the cardiovascular and renal excretory responses evoked by central α(2) -adrenoceptor activation in vivo

BACKGROUND AND PURPOSE

Activation of brain α₂-adrenoceptors in conscious rodents decreases heart rate (HR) and mean arterial blood pressure (MAP) and increases urine output and urinary sodium excretion. In vitro, α₂-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 α₂-adrenoceptor activation in conscious Sprague-Dawley rats.

EXPERIMENTAL APPROACH

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 α₂-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.

KEY RESULTS

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.

CONCLUSIONS AND IMPLICATIONS

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 α₂-adrenoceptor activation in vivo.     

α(1)-Adrenoceptors in the urinary tract

α(1)-Adrenoceptors have been identified and characterized extensively by functional, radioligand-binding, and molecular biological techniques. Molecular clones have been isolated for three α(1)-subtypes (α(1a), α(1b), and α(1d)), and these subtypes are also functionally characterized. α(1)-Adrenoceptors are present in the prostate, urethra, bladder (urothelium, smooth muscle, and afferent nerves), ureter, vas deferens, peripheral ganglia, nerve terminals, vascular tissues, and central nervous system (CNS), and they could all potentially influence overall urinary function and contribute to both the therapeutic and adverse effects of α(1)-adrenoceptor antagonists in patients with benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS). This review aimed to discuss the relevant physiological and pharmacological roles and molecular biology of α(1)-adrenoceptor subtypes in the prostate, urethra, bladder, ureter, and CNS.     

Adenosinergic modulation of 3α-hydroxy-5α-pregnan-20-one induced catalepsy in mice

  Rationale: Neurosteroid 3α, 5α THP, a positive allosteric modulator of the GABA_A receptor Cl^– ionophore complex, induces catalepsy-like dopamine antagonists, adenosine agonists or GABA agonists. Adenosine and dopamine receptors are co-localized on GABAergic neurons in the striatum and regulate GABA-mediated neurotransmission. Moreover, the antagonistic interactions between specific subtypes of adenosine and dopamine receptors are involved in motor depressant or motor stimulant effects of adenosine receptor agonists or antagonists, respectively. Such interaction may modulate neurosteroid-induced catalepsy. Objective: This study examined the modulation of 3α, 5α THP-induced catalepsy by adenosinergic agents. Methods: Catalepsy induced by 3α, 5α THP (2–8 μg, ICV) was assessed by bar test periodically up to 3 h in mice. Adenosine A₁, A_2A or A₃ receptor agonists or antagonists were given IP or ICV prior to 3α, 5α THP. Some animals received IP dopamine D₂ receptor agonist or antagonist 30 min prior to above combination treatment. Results: Adenosine A₁, A_2A, and A₃ receptor agonists potentiated, whereas adenosine A_2A receptor antagonists, but not A₁ antagonists, reversed 3α, 5α THP-induced catalepsy. These effects of adenosine agonists and antagonists were abolished by prior administration of bromocriptine, the dopamine D₂ receptor agonist and spiperone, the dopamine D₂ receptor antagonist, respectively. Conclusions: These findings suggest specific adenosine-dopamine receptor interaction in the striatum to modulate 3α, 5α THP-induced catalepsy. Received: 1 November 1998 / Final version: 5 January 1999     

The neurosteroid 3α-hydroxy-5α-pregnan-20-one affects dopamine-mediated behavior in rodents

RATIONALE: The neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) has been previously shown to induce catalepsy in mice that is modified by GABAergic, dopaminergic, adenosinergic and serotonergic agents. In light of the interaction of this endogenous neurosteroid with GABAergic and dopaminergic transmission, there is potential interest in the possible role of 3alpha,5alpha-THP in psychotic disorders. OBJECTIVE: This study assessed the effect of 3alpha,5alpha-THP in certain dopamine-mediated behavioral paradigms that are widely used to predict antipsychotic-like activity. METHODS: 3alpha,5alpha-THP (1-8 microg per animal, i.c.v.), the classic neuroleptic (dopamine receptor antagonist) haloperidol (0.25 mg/kg, i.p.), and the benzodiazepine diazepam (7 mg/kg, i.p.) were injected into different groups of animals, and their behavior was screened using the following animal tests: conditioned avoidance response, apomorphine-induced climbing, and amphetamine-induced motor hyperactivity. Separate groups of mice that received 3alpha,5alpha-THP (1-8 microg per animal, i.c.v.) were screened for catalepsy. Furthermore, the effect of a sub-cataleptic dose (0.1 microg per mouse, i.c.v.) of 3alpha,5alpha-THP, either alone or in combination with the GABA(A) receptor antagonist picrotoxin (0.8 mg/kg, i.p.) was measured on haloperidol-induced catalepsy. RESULTS: 3alpha,5alpha-THP like haloperidol reduced conditioned avoidance, apomorphine-induced cage climbing and amphetamine-induced motor hyperactivity. Diazepam only affected conditioned avoidance. 3alpha,5alpha-THP also induced dose-dependent catalepsy. Furthermore, sub-cataleptic doses of 3alpha,5alpha-THP potentiated haloperidol-induced catalepsy. This potentiation was blocked by prior treatment with the GABA(A) receptor antagonist picrotoxin. CONCLUSION: These findings suggest that 3alpha,5alpha-THP, by its action at the GABA(A) receptors, increases GABAergic tone leading to a behavioral profile similar to that of dopamine receptor antagonists.     

α2-Adrenoceptor-mediated inhibition of platelet adenylate cyclase activity in heroin addicts in abstinence

The effects of corticotropin-releasing factor (CRF) and previously published effect of stress on the locomotor activity of mice in different regions of an open field were compared. Intracerebroventricular (ICV) administration of 0.2 g CRF, like stress, significantly increased center region activity; this effect was reversed by the benzodiazepine diazepam (DZP) at a dose of DZP having no significant effect alone. A dose of DZP that antagonized CRF-potentiated center region activity did not block amphetamine-stimulated center area activity. These results suggest that CRF may normally be responsible for many behavioral changes during conditions of stress.     

Evidence for a dopaminergic dominance in the 4,α-dimethyl-m-tyramine-induced hypermotility in mice

The effects of various drugs on the hypermotility induced in mice by 4,-dimethyl-m-tyramine (H 77/77) was investigated.The H 77/77-induced hypermotility was strongly inhibited by centrally acting dopamine-(DA-) receptor blockers (e.g., benperidol, fluspirilene, haloperidol) and potentiated by drugs increasing central DA activity by release of DA, by uptake inhibition (d- and l-amphetamine, nomifensine), or by inhibition of monoamine oxidase (e.g., parglyline, chlorgyline). While DA-receptor agonists (e.g., bromocriptine, ergocornine) potentiated hypermotility, the effects of those having a short duration of action (apomorphine, piribedil) were partly obscured. The catecholamine precursor L-dopa increased hypermotility only slightly, but its effect was markedly potentiated after inhibition of dopa decarboxylase.Inhibition of tyrosine hydroxylase with -methyltyrosine methyl ester HCl (H 44/68) totally blocked H 77/77 hypermotility. Inhibition of noradrenaline (NA) synthesis with bis-(4-methyl-l-homopiperazinyl thiocarbonyl)-disulphide (FLA 63) had no significant effect, whereas after sodium diethyldithiocarbamate (DDC) there was a moderate decrease in motility of about 25%.Blockers of - and -adrenoceptors either did not affect H 77/77 hypermotility (e.g., WB 4101, pindolol) or inhibited it only at very high doses (e.g., phentolamine, propranolol). -Adrenoceptor agonists (clonidine, guanfacine) had no clear effects.No correlation was found between the H 77/77-inhibiting effect of antidepressant drugs and their ability to inhibit NA uptake.Several antiserotoninergic compounds (pizotifen, 27-096, 29-245) blocked the effects of H 77/77, and a 65% drop in hypermotility was obtained after inhibition of 5-HT-synthesis with 6-flourotryptophan. Substances that increase central serotoninergic activity (l-5-HTP, fenfluramine), however, were inactive.Anticholinergic drugs (e.g., atropine, dexetimide) increased H 77/77 hypermotility, whereas cholinergic drugs (e.g., arecoline, physostigmine) produced no dose-related effect.It is cocluded that DA plays a dominant role in the H 77/77-induced hypermotility in mice, although functional NA and 5-HT systems appear to be a prerequisite for the full H 77/77 effect.