The effect of cocaine on kinetics of α<Subscript>1</Subscript>-adrenergic contractile response in different portions of the rat vas deferens

The effect of cocaine (10 μM) on the kinetics of contractile response to noradrenaline (NA) was studied in the rat epididymal and prostatic vas deferens. Cocaine caused an acute increase in vas deferens adrenergic sensitivity primarily due to blockage of NA neuronal capture. The presynaptic action prevailed in the epididymal half: the EC 50 value decreased 32-fold with a slight increase of the maximum adrenergic response more evident in the prostatic half. In the presence of cocaine, the prostatic contraction to NA was mediated not by a single pool of α₁-adrenoceptors like in epididymal vas deferens but by two. Its high affinity pool had the same affinity as α₁-adrenoceptors of the epididymal half, the affinity value of the low one was 36-fold less, and the total maximal response of both pools increased 4.5-fold. The differences in cocaine effect on the rat epididymal and prostatic vas deferens contractions to NA appear to be caused by the different sources of Ca²⁺ involved in these responses.     

Body temperature and sleep: Are they controlled by the same mechanism?

Simultaneous changes in sleep and body temperature, produced either by lesion or by stimulation of the medial preoptic area (mPOA), have given reasons to suggest that thermoregulation and sleep regulation are controlled by the same set of neurons. The reasons for simultaneous changes in these parameters are discussed in the present paper with a view to explaining the relationship between thermoregulation and sleep regulation. Changes in body temperature and sleep on destruction of the preoptic area (POA) neurons and the sequence of these changes, suggest a separate control mechanism in the mPOA for regulation of sleep and body temperature. Evidence is put forward in the present paper to show that the mPOA is not involved in the downregulation or upregulation of changes in body temperature with alteration in the vigilance state. On the other hand, circadian modulation of body temperature is possibly involved in altering sleep propensity. A clear indication regarding separate control of sleep and body temperature came from the studies in which noradrenergic agents were applied into the mPOA of animals with and without lesion of the noradrenergic fibers projecting to the mPOA. Experiments in which sleep was analyzed after experimental manipulations of ambient temperature and body temperature, including peripheral, core and brain temperature, are presented here to show a close relationship between thermoregulation and sleep regulation. Various theories regarding the regulation of body temperature during slow wave sleep and rapid eye movement sleep are also discussed. The functional integrity of the mPOA may be essential not only for the regulation of body temperature and sleep-wakefulness but even for the homeostatic regulation of energy balance of the body in response to alterations in environmental temperature and sleep-wakefulness.

     

Adrenoceptors and signal transduction in neurons

The adrenergic system is an essential regulator of neuronal, endocrine, cardiovascular, vegetative, and metabolic functions. The endogenous catecholamines epinephrine and norepinephrine activate G-protein-coupled receptors to transmit their signal across the plasma membrane. These adrenoceptors can be divided into three different groups: the α₁-receptors (α_1A, α_1B, α_1D), α₂-receptors (α_2A, α_2B, α_2C), and β-receptors (β₁, β₂, β₃). This review summarizes recent findings in the field of adrenoceptor signaling in neurons and includes a discussion of receptor-associated proteins, receptor dimerization, subcellular trafficking, and fluorescence optical methods for studying the kinetics of adrenergic signaling. Spatio-temporal imaging may become an important future tool for identifying the physiological significance of these complex signaling mechanisms in vivo. Gene-targeted mouse models carrying deletions in α₂-adrenoceptor have provided detailed insights into specific neuronal functions of the three α₂-receptor subtypes.     

Protein kinase Cζ regulates phospholipase D activity in rat-1 fibroblasts expressing the α<Subscript>1A</Subscript> adrenergic receptor

Background  

Phenylephrine (PHE), an α₁ adrenergic receptor agonist, increases phospholipase D (PLD) activity, independent of classical and novel protein kinase C (PKC) isoforms, in rat-1 fibroblasts expressing α_1A adrenergic receptors. The aim of this study was to determine the contribution of atypical PKCζ to PLD activation in response to PHE in these cells.     

Noradrenergic afferents and receptors in the medial preoptic area: neuroanatomical and neurochemical links between the regulation of sleep and body temperature

Several studies have shown the importance of the medial preoptic area in the regulation of sleep-wakefulness and of body temperature. The medial preoptic area has a rich noradrenergic innervation, coming mostly from the lateral tegmental noradrenergic system. The accumulating evidences show that the noradrenergic afferents to the medial preoptic area are involved in the induction of sleep. This hypnogenic mechanism operates through the postsynaptic alpha1 and alpha2-adrenergic receptors. Noradrenergic afferents are also involved in the thermoregulatory mechanisms, and the activation of these fibers brings about a fall in body temperature. Though the body temperature changes are brought about by the same receptor subtypes as those involved in hypnogenesis, observations suggest the possibility of separate sets of noradrenergic afferents in the medial preoptic area for sleep regulation and thermoregulation. In this review, we present the compelling evidences, which showed that the noradrenergic afferents of the medial preoptic area bring about a fall in body temperature and other thermoregulatory behavioral alterations associated with sleep.     

Role of alpha adrenoceptors and nitric oxide on cardiovascular responses in acute and chronic hypertension

The contribution of α-adrenoceptors and nitric oxide (NO) on the alterations of sympathetically mediated cardiovascular responses after acute (AcH) and chronic (ChH) hypertension was evaluated in pithed aortic coarcted hypertensive rats. Pressor and tachycardia response produced by electrical stimulation of preganglionic sympathetic fibers or exogenous noradrenaline (NA) were recorded in the absence and presence of prazosin (α₁-antagonist), rauwolscine (α₂-antagonist), or N ^G-nitro-l-arginine methyl ester (l-NAME; an inhibitor of NO synthase). Compared with age-matched sham-operated rats (Nt), the pressor response produced by electrical stimulation or NA was smaller in AcH rats and larger in ChH rats. Prazosin caused a decrease of pressor response elicited by electrical stimulation or NA in all groups. However, this effect was higher in ChH. Rauwolscine produced a similar increase of sympathetically mediated pressor response in Nt and AcH rats. Nevertheless, this antagonist did not affect the sympathetically mediated pressor response in ChH rats. In addition, rauwolscine did not affect the NA-induced pressor response in all groups. The pressor response elicited by l-NAME was larger in all groups compared without l-NAME and in presence of l-arginine. Moreover, l-NAME in the presence of NA increased sympathetically mediated pressor response is in all groups, compared without it or in the presence of l-arginine. Compared with Nt, basally produced NO in aortic rings was increased in AcH but decreased in ChH. Collectively, our data suggest that decreased cardiovascular reactivity in AcH is due to an increase in basally produced NO. In ChH, enhanced cardiovascular response appears to be associated with a decrease in produced NO and an increase in released NA from sympathetic nerves.     

Regeneration of cardiomyocytes from bone marrow: Use of mesenchymal stem cell for cardiovascular tissue engineering

We have isolated a cardiomyogenic cell line (CMG cell) from murine bone marrow mesenchymal stem cells. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine exposure. They began spontaneous beating after 2 weeks, and expressed ANP and BNP. Electron microscopy revealed a cardiomyocyte-like ultrastructure. These cells had several types of action potentials; sinus node-like and ventricular cell-like action potentials. The isoform of contractile protein genes indicated that their muscle phenotype was similar to fetal ventricular cardiomyocytes. They expressed α_1A, α_1B, α_1D, β₁, and β₂ adrenergic and M₁ and M₂ muscarinic receptors. Stimulation with phenylephrine, isoproterenol and carbachol increased ERK phosphorylation and second messengers. Isoproterenol increased the beating rate, which was blocked with CGP20712A (β₁-selective blocker). These findings indicated that cell transplantation therapy for the patients with heart failure might possibly be achieved using the regenerated cardiomyocytes from autologous bone marrow cells in the near future. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular cloning, stable expression and cellular localization of human α1-adrenergic receptor subtypes: effect of charcoal/dextran treated serum on expression and localization of α1D -adrenergic receptor

The cDNAs encoding for three subtypes of adrenergic receptors, α_1A-, α_1B- and α_1D-ARs, were cloned and expressed in HEK 293 cells. Expression of α_1A- and α_1B-AR subtypes in HEK 293 cells was stable even with increased passages but that of α_1D-AR was not. Cellular localization studies using immunofluorescence and flow cytometry revealed that expression of α_1A- and α_1B-ARs was primarily localized on the cell membrane whereas expression of α_1D-AR was␣predominantly intracellular. Our studies clearly demonstrated that the culturing of the recombinant cell lines expressing α_1D-AR in charcoal/dextran treated fetal bovine serum (FBS) resulted in targeting of α_1D-AR to the cell membrane and thus, significantly improving its stability and availability for ligand binding studies.Sunil M. Khattar, Roop Singh Bora and Priyanka Priyadarsiny contributed equally to this work.     

Cardiac α1-Adrenoceptors that regulate contractile function: Subtypes and subcellular signal transduction mechanismsthat regulate contractile function: Subtypes and subcellular signal transduction mechanisms

Activation of α₁-adrenoceptors as well as endothelin (ET) and angiotensin II (Ang II) receptors in cardiac muscle is coupled to acceleration of the hydrolysis of phosphoinositide (PI), with resultant production of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol. There is an excellent correlation between the extent of acceleration of the PI hydrolysis and the positive inotropic effect (PIE) under most experimental conditions after the administration of α-adrenoceptor agonists, ET and Ang II in the rabbit ventricular muscle. The PIE of the α-adrenoceptor agonists, ET and Ang II is associated with a negative lusitropic effect and an increase in the sensitivity of myofilaments to Ca²⁺ ions. The PIE can be selectively inhibited by inhibitors of protein kinase C (PKC) such as staurosporine, NA 0345 and H-7, with little effect on the PI hydrolysis and the PIE of isoproterenol and Bay k 8644. Surprisingly, an activator of PKC, phorbol 12,13-dibutyrate (PDBu), selectively and more completely inhibited the PIE and acceleration of PI hydrolysis induced by the α-adrenoceptor agonists as well as by ET and Ang II in the rabbit. These receptor agonists consistently cause intracellular alkalinization by activation of Na⁺−H⁺ exchange, while the effects on membrane ion channel activities are divergent. For example, α-adrenoceptor agonists cause monophasic prolongation of the action potential, the time course of which coincides well with that of the PIE, while ET and Ang II produce a biphasic change in action potential duration, i.e., the long-lasting prolongation preceded by a transient abbreviation. α-Adrenoceptor agonists scarcely affect I_Ca, whereas ET elicits a biphasic alteration of the current. In addition, the potassium current, I_Kl, is markedly suppressed by α-adrenoceptor agonists, but this effect is not revealed with Ang II under the same experimental condition. These results indicate that the effects of α₁-adrenoceptors stimulation are partially shared by those of ET and Ang II receptor activation in the heart. Approximately 60% of the total population of α₁-adrenoceptors in the rabbit ventricle are composed of α_1B subtype, which is susceptible to chlorethylclonidine (CEC) and is predominantly responsible for the α₁-mediated PIE and PI hydrolysis. The remaining fraction that belongs to α_1A-adrenoceptors subtype is further subclassified into the WB 4101-sensitive (partly coupled to PI hydrolysis) and the niguldipinesensitive (PI hydrolysis-unrelated) subtypes. Special issue dedicated to Dr. Kinya Kuriyama.     

Possible Effect of Activation of α7-Nicotinic Acetylcholine Receptors in the Mitochondrial Membrane on the Development of Apoptosis

Using flow cytometry and sandwich-immunoenzyme assay, we showed that nicotinic acetylcholine receptors with a subunit α7 (nAChRs α7) expressed in the outer mitochondrial membrane are involved in the control of mitochondria-dependent apoptosis. Pre-incubation of the mitochondria with an nAChRs α7 agonist, choline, decreased dissipation of the membrane potential of these organelles induced by the action of 0.5 mM hydrogen peroxide (H₂O₂) but did not influence the analogous effect of a high Ca²⁺ concentration (90 μM). Agonists of nAChRs α7 (choline, acetylcholine, and PNU 282987), or an inhibitor of voltage-dependent anion channels, DIDS, prevented the release of cytochrome c from the intermembrane mitochondrial space under the action of H₂O₂. In contrast, an antagonist of nAChRs α7, methyllycaconitine, promoted the release of cytochrome c and prevented the effects of agonists. The obtained data confirm the active involvement of nAChRs α7 and voltage-dependent anion channels in the process of formation of mitochondrial pores. In this case, agonists of mitochondrial nAChRs α7 subunits exert an antiapoptotic effect, while antagonists of mitochondrial nAChRs α7 subunits manifest a proapoptotic action.     

The Inhibition of the Dorsal Paragigantocellular Reticular Nucleus Induces Waking and the Activation of All Adrenergic and Noradrenergic Neurons: A Combined Pharmacological and Functional Neuroanatomical Study

GABAergic neurons specifically active during paradoxical sleep (PS) localized in the dorsal paragigantocellular reticular nucleus (DPGi) are known to be responsible for the cessation of activity of the noradrenergic neurons of the locus coeruleus during PS. In the present study, we therefore sought to determine the role of the DPGi in PS onset and maintenance and in the inhibition of the LC noradrenergic neurons during this state. The effect of the inactivation of DPGi neurons on the sleep-waking cycle was examined in rats by microinjection of muscimol, a GABA_A agonist, or clonidine, an alpha-2 adrenergic receptor agonist. Combining immunostaining of the different populations of wake-inducing neurons with that of c-FOS, we then determined whether muscimol inhibition of the DPGi specifically induces the activation of the noradrenergic neurons of the LC. Slow wave sleep and PS were abolished during 3 and 5 h after muscimol injection in the DPGi, respectively. The application of clonidine in the DPGi specifically induced a significant decrease in PS quantities and delayed PS appearance compared to NaCl. We further surprisingly found out that more than 75% of the noradrenergic and adrenergic neurons of all adrenergic and noradrenergic cell groups are activated after muscimol treatment in contrast to the other wake active systems significantly less activated. These results suggest that, in addition to its already know inhibition of LC noradrenergic neurons during PS, the DPGi might inhibit the activity of noradrenergic and adrenergic neurons from all groups during PS, but also to a minor extent during SWS and waking.     

Distinct interactions between the human adrenergic β2 receptor and Gαs—an in silico study

The aim of this study was to perform an in silico analysis of the interaction of the human β₂ adrenergic receptor with Gα_s. In a first step, a systematic surface-interaction-scan between the inactive or active human β₂ adrenergic receptor and Gα_s was performed in order to gain knowledge about energetically preferred areas on the potential energy surface. Subsequently, two energetically favored regions for the active human β₂ adrenergic receptor–Gα_s complex were identified. Two representative complex structures were put into a POPC (1-palmitoyl-2-oleoyl-phosphatidylcholine) bilayer and solvated in order to perform molecular dynamic simulations. The simulations revealed that both conformations, which have comparable potential energy, are stable. A mean number of about 14 hydrogen bonds was observed between the active receptor and Gα_s for both conformations. Based on these results, two energetically favored β₂–Gα_scomplexes can be proposed.     

Effect of corticosterone on noradrenergic nuclei in the pons-medulla and [3H]NA release from terminals in hippocampal slices

The aim of the present study was to investigate possible membrane and genomic effects of corticosterone on the noradrenergic system of the rat brain. Corticosterone effects were studied in vivo by treating rats s.c. with 10 mg/kg corticosterone for 7 or 14 days. In the first two experiments corticosterone significantly decreased th noradrenaline (NA) and dopamine (DA) levels in the pons-medulla, an area which contains the A1-A7 noradrenergic cell groups, while the NA and DA levels in the dorsal hippocampus remained unchanged. In a third experiment where the locus coeruleus (LC) and the A1 and A2 nuclei (A1,A2) were analysed separately, NA levels were unchanged but total MHPG levels and the total MHPG/NA ratio were decreased in the A1,A2 area. Chronic corticosterone treatment (14 days) did not alter the ₂-adrenoceptor-mediated modulation of [³H]NA release from dorsal hippocampal slices. Neither the spontaneous outflow nor the electrically stimulated release of [³H]NA from dorsal hippocampal slices of untreated rats was affected by exposure of the slices to corticosterone (10^–7 M–10^–4 M) in the superfusion buffer. Thus, chronic corticosterone treatment of rats altered the noradrenergic system of the pons-medulla, but did not change the ₂-adrenoceptor-mediated modulation of NA release in the dorsal hippocampus, a major terminal area of the LC neurons. Corticosterone also did not appear to have a direct membrane effect on the NA terminals in the dorsal hippocampus of the rat.     

Effects on sleep of microdialysis of adenosine A1 and A2a receptor analogs into the lateral preoptic area of rats

Evidence suggests that adenosine (AD) is an endogenous sleep factor. The hypnogenic action of AD is mediated through its inhibitory A1 and excitatory A2A receptors. Although AD is thought to be predominantly active in the wake-active region of the basal forebrain (BF), a hypnogenic action of AD has been demonstrated in several other brain areas, including the preoptic area. We hypothesized that in lateral preoptic area (LPOA), a region with an abundance of sleep-active neurons, AD acting via A1 receptors would induce waking by inhibition of sleep-active neurons and that AD acting via A2A receptors would promote sleep by stimulating the sleep-active neurons. To this end, we studied the effects on sleep of an AD transport inhibitor, nitrobenzyl-thio-inosine (NBTI) and A1 and A2A receptor agonists/antagonists by microdialyzing them into the LPOA. The results showed that, in the sleep-promoting area of LPOA: 1) A1 receptor stimulation or inhibition of AD transport by NBTI induced waking and 2) A2A receptor stimulation induced sleep. We also confirmed that NBTI administration in the wake promoting area of the BF increased sleep. The effects of AD could be mediated either directly or indirectly via interaction with other neurotransmitter systems. These observations support a hypothesis that AD mediated effects on sleep-wake cycles are site and receptor dependent.     

α2A-Adrenoceptor-specific Stimulation of [35S]GTPγS Binding to Membrane Preparations of Rat Frontal Cortex

Functional activation of α_2A adrenergic receptors in the crude membranes from rat frontal cortex was studied by a [³⁵S]-guanosine 5′-O-(γ-thiotriphosphate) ([³⁵S]GTPγS) binding assay. α_2A agonists UK14304 and guanfacine decreased the ability of GDP to compete with [³⁵S]GTPγS binding to the membranes and 0.1 mM GDP was found to be optimal for the following functional experiments. However, even after careful optimization of experimental conditions the specificity of ligands for rat α₂ adrenoceptors were not sufficient, as agonists as well as antagonists became activators of other signal transduction systems before achieving their maximal effect in the α_2A-adrenergic system. Only using compromising concentration of agonist (up to 1 μM UK14304) and antagonist (up to 1 μM RS79948) to inhibit agonist’s effect, allowed us to filtrate out α_2A specific effect for characterization of signal transduction in rat frontal cortex membranes for the comparison efficacies of this system for different animals from behavioral experiments.     

GABAA receptor diversity and pharmacology

Because of its control of spike-timing and oscillatory network activity, γ-aminobutyric acid (GABA)-ergic inhibition is a key element in the central regulation of somatic and mental functions. The recognition of GABA_A receptor diversity has provided molecular tags for the analysis of distinct neuronal networks in the control of specific pharmacological and physiological brain functions. Neurons expressing α₁GABA_A receptors have been found to mediate sedation, whereas those expressing α₂GABA_A receptors mediate anxiolysis. Furthermore, associative temporal and spatial memory can be regulated by modulating the activity of hippocampal pyramidal cells via extrasynaptic α₅GABA_A receptors. In addition, neurons expressing α₃GABA_A receptors are instrumental in the processing of sensory motor information related to a schizophrenia endophenotype. Finally, during the postnatal development of the brain, the maturation of GABAergic interneurons seems to provide the trigger for the experience-dependent plasticity of neurons in the visual cortex, with α₁GABA_A receptors setting the time of onset of a critical period of plasticity. Thus, particular neuronal networks defined by respective GABA_A receptor subtypes can now be linked to the regulation of various clearly defined behavioural patterns. These achievements are of obvious relevance for the pharmacotherapy of certain brain disorders, in particular sleep dysfunctions, anxiety disorders, schizophrenia and diseases associated with memory deficits.     

Modulation of EEG Rhythms and Changes in Spike Activity of Noradrenergic Neurons of the <Emphasis Type="Italic">Locus Coeruleus</Emphasis> Related to Feedback Sessions by EEG Characteristics

In chronic experiments on awake cats, we studied the dynamics of the spectral power density (SPD) of the α rhythm vs SPD of the θ rhythm ratio and also of the characteristics of impulse activity generated by supposedly noradrenergic (NA) neurons of the locus coeruleus in the course of feedback (FB) sessions by EEG characteristics (EEG-FB). Trainings were performed using a technique analogous to that in EEG-FB sessions for humans. The level of a sound noise signal presented to the animal decreased with increase in the α/θ SPD ratio in the occipital lead. Changes in the level of the sound signal did not depend on EEG modulation in the control series. The animals were trained to correlate changes in the loudness of the sound signal with the power of EEG rhythms and, in such a way, to control the latter. The α/θ SPD ratio in EEG-FB sessions changed mostly due to a significant increase in the α rhythm power. The frequency of the impulse activity of NA neurons increased in a parallel manner with such EEG modulation. Possible mechanisms of the involvement of the cerebral NA system in the formation of the effects of EEG-FB sessions are discussed.     

Evidence for an in vivo and in vitro modulation of endogenous cortical GABA release by α-glycerylphosphorylcholine

The effects of α-glycerylphosphorylcholine (α-GPC) on endogenous cortical GABA release were studied both in vivo and in vitro. In freely moving rats, equipped with epidural cups, α-GPC (30–300 mg/kg i.p.) increased GABA release. This effect was potentiated by atropine, both systematically administered (5 mg/kg i.p.) and locally applied (1.4 μM), but not by mecamylamine (4 mg/kg i.p.). The α-GPC-induced increasein GABA release was abolished in rats pretreated with the α₁ receptor antagonist prazosin (14 μg/kg i.p.). In cortical slices α-GPC (0.4 mM) increased the spontaneous GABA efflux. This effectwas abolished by tetrodotoxin (0.5 μM) and prazosin (1 μM), but not by atropine (0.15 μM) ormecamylamine (2.5μM). These results indicate that the facilitatory response by α-GPC on GABArelease does not depend on a direct activation of either muscarinic or nicotinic receptors, but suggest the involvement of the noradrenergic system.     

Central noradrenergic neurons and the hypertensive effects of intracerebroventricularly administered prostaglandin E2 in anaesthetized rabbits

The participation of central noradrenergic neurons in the pressor responses to intracerebroventricular (i.c.v.) administration of prostaglandin (PG) E₂ was studied in anaesthetized rabbits. The hypertensive effect induced by i.c.v. injection of PGE₂ was inhibited by i.c.v. pretreatment with 6-hydroxydopamine and phentolamine, but not propranolol. These findings suggest that the cerebral noradrenergic neurons may be involved in the development of hypertensive effect of PGE₂ through the adrenergic α-receptors.