Protein interactome mining defines melatonin MT1 receptors as integral component of presynaptic protein complexes of neurons

In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak levels. Its peripheral and central actions rely either on its intrinsic antioxidant properties or on binding to melatonin MT₁ and MT₂ receptors, belonging to the G protein‐coupled receptor (GPCR) super‐family. Melatonin has been reported to be involved in many functions of the central nervous system such as circadian rhythm regulation, neurotransmission, synaptic plasticity, memory, sleep, and also in Alzheimer's disease and depression. However, little is known about the subcellular localization of melatonin receptors and the molecular aspects involved in neuronal functions of melatonin. Identification of protein complexes associated with GPCRs has been shown to be a valid approach to improve our understanding of their function. By combining proteomic and genomic approaches we built an interactome of MT₁ and MT₂ receptors, which comprises 378 individual proteins. Among the proteins interacting with MT₁, but not with MT₂, we identified several presynaptic proteins, suggesting a potential role of MT₁ in neurotransmission. Presynaptic localization of MT₁ receptors in the hypothalamus, striatum, and cortex was confirmed by subcellular fractionation experiments and immunofluorescence microscopy. MT₁ physically interacts with the voltage‐gated calcium channel Ca_v2.2 and inhibits Ca_v2.2‐promoted Ca²⁺ entry in an agonist‐independent manner. In conclusion, we show that MT₁ is part of the presynaptic protein network and negatively regulates Ca_v2.2 activity, providing a first hint for potential synaptic functions of MT₁.     

Melatonin‐mediated inhibition of Cav3.2 T‐type Ca2+ channels induces sensory neuronal hypoexcitability through the novel protein kinase C‐eta isoform

Recent studies implicate melatonin in the antinociceptive activity of sensory neurons. However, the underlying mechanisms are still largely unknown. Here, we identify a critical role of melatonin in functionally regulating Cav3.2 T‐type Ca²⁺ channels (T‐type channel) in trigeminal ganglion (TG) neurons. Melatonin inhibited T‐type channels in small TG neurons via the melatonin receptor 2 (MT₂ receptor) and a pertussis toxin‐sensitive G‐protein pathway. Immunoprecipitation analyses revealed that the intracellular subunit of the MT₂ receptor coprecipitated with Gα_o. Both shRNA‐mediated knockdown of Gα_o and intracellular application of QEHA peptide abolished the inhibitory effects of melatonin. Protein kinase C (PKC) antagonists abolished the melatonin‐induced T‐type channel response, whereas inhibition of conventional PKC isoforms elicited no effect. Furthermore, application of melatonin increased membrane abundance of PKC‐eta (PKC_η) while antagonism of PKC_η or shRNA targeting PKC_η prevented the melatonin‐mediated effects. In a heterologous expression system, activation of MT₂ receptor strongly inhibited Cav3.2 T‐type channel currents but had no effect on Cav3.1 and Cav3.3 current amplitudes. The selective Cav3.2 response was PKC_η dependent and was accompanied by a negative shift in the steady‐state inactivation curve. Furthermore, melatonin decreased the action potential firing rate of small TG neurons and attenuated the mechanical hypersensitivity in a mouse model of complete Freund's adjuvant‐induced inflammatory pain. These actions were inhibited by T‐type channel blockade. Together, our results demonstrated that melatonin inhibits Cav3.2 T‐type channel activity through the MT₂ receptor coupled to novel G_βγ‐mediated PKC_η signaling, subsequently decreasing the membrane excitability of TG neurons and pain hypersensitivity in mice.     

Dysfunction of serotonergic activity and emotional responses across the light-dark cycle in mice lacking melatonin MT2 receptors

Melatonin (MLT) levels fluctuate according to the external light/dark cycle in both diurnal and nocturnal mammals. We previously demonstrated that melatonin MT₂ receptor knockout (MT₂^−/−) mice show a decreased nonrapid eye movement sleep over 24 hours and increased wakefulness during the inactive (light) phase. Here, we investigated the role of MT₂ receptors in physiological light/dark cycle fluctuations in the activity of dorsal raphe nucleus (DRN) serotonin (5-HT) neurons and anxiety- and depression-like behavior. We found that the 5-HT burst-firing activity was tonically reduced across the whole 24 hours in MT₂^−/− mice compared with MT₂^+/+ mice.  Importantly, the physiological changes in the spontaneous firing activity of DRN 5-HT neurons during the light/dark cycle were nullified in MT₂^−/− mice, with a higher DRN 5-HT neural firing activity during the light phase in MT₂^−/− than in MT₂^+/+ mice. The role of MT₂ receptors over DRN 5-HT neurons was confirmed by acute pharmacological studies in which the selective MT₂ receptors agonist UCM1014 dose dependently inhibited DRN 5-HT activity, mostly during the dark phase. Compared with MT₂^+/+, MT₂^−/− mice displayed an anxiety-like phenotype in the novelty-suppressed feeding and in the light/dark box tests; while anxiety levels in the light/dark box test were lower during the dark than during the light phase in MT₂^+/+ mice, the opposite was seen in MT₂^−/− mice. No differences between MT₂^+/+ and MT₂^−/− mice were observed for depression-like behavior in the forced swim and in the sucrose preference tests. These results suggest that MT₂ receptor genetic inactivation impacts 5-HT neurotransmission and interferes with anxiety levels by perturbing the physiologic light/dark pattern.     

Nociceptive responses in melatonin MT2 receptor knockout mice compared to MT1 and double MT1/MT2 receptor knockout mice

Melatonin, a neurohormone that binds to two G protein-coupled receptors MT₁ and MT_2, is involved in pain regulation, but the distinct role of each receptor has yet to be defined. We characterized the nociceptive responses of mice with genetic inactivation of melatonin MT₁ (MT₁^−/−), or MT₂ (MT₂^−/−), or both MT₁/MT₂ (MT₁^−/−/MT₂^−/−) receptors in the hot plate test (HPT), and the formalin test (FT). In HPT and FT, MT₁^−/− display no differences compared to their wild-type littermates (CTL), whereas both MT₂^−/− and MT₁^−/−/MT₂^−/− mice showed a reduced thermal sensitivity and a decreased tonic nocifensive behavior during phase 2 of the FT in the light phase. The MT₂ partial agonist UCM924 induced an antinociceptive effect in MT₁^−/− but not in MT₂^−/− and MT₁^−/−/MT₂^−/− mice. Also, the competitive opioid antagonist naloxone had no effects in CTL, whereas it induced a decrease of nociceptive thresholds in MT₂^−/− mice. Our results show that the genetic inactivation of melatonin MT₂, but not MT₁ receptors, produces a distinct effect on nociceptive threshold, suggesting that the melatonin MT₂ receptor subtype is selectively involved in the regulation of pain responses.     

Functional MT1 and MT2 melatonin receptors in mammals

Melatonin, dubbed the hormone of darkness, is known to regulate a wide variety of physiological processes in mammals. This review describes well-defined functional responses mediated through activation of high-affinity MT₁ and MT₂ proteinteoupled receptors viewed as potential targets for drug discovery. MT₁ melatonin receptors modulate neuronal firing, arterial vasoconstriction, cell proliferation in cancer cells, and reproductive and metabolic functions. Ativation of MT₂ melatonin receptors phase shift circadian rhythms of neuronal firing in the suprachiasmatic nucleus, inhibit dopamine release in retina, induce vasodilation and inhibition of leukocyte rolling in arterial beds, and enhance immune responses. The melatonin-mediated responses elicited by activation of MT₁ and MT₂ native melatonin receptors are dependent on circadian time, duration and mode of exposure to endogenous or exogenous melatonin, and functional receptor sensitivity. Together, these studies underscore the importance of carefully linking each melatonin receptor type to specific functional responses in target tissues to facilitate the design and development of novel therapeutic agent.     

Membrane depolarization causes a direct activation of G protein-coupled receptors leading to local Ca2+ release in smooth muscle

Membrane depolarization activates voltage-dependent Ca²⁺ channels (VDCCs) inducing Ca²⁺ release via ryanodine receptors (RyRs), which is obligatory for skeletal and cardiac muscle contraction and other physiological responses. However, depolarization-induced Ca²⁺ release and its functional importance as well as underlying signaling mechanisms in smooth muscle cells (SMCs) are largely unknown. Here we report that membrane depolarization can induce RyR-mediated local Ca²⁺ release, leading to a significant increase in the activity of Ca²⁺ sparks and contraction in airway SMCs. The increased Ca²⁺ sparks are independent of VDCCs and the associated extracellular Ca²⁺ influx. This format of local Ca²⁺ release results from a direct activation of G protein-coupled, M₃ muscarinic receptors in the absence of exogenous agonists, which causes activation of Gq proteins and phospholipase C, and generation of inositol 1,4,5-triphosphate (IP₃), inducing initial Ca²⁺ release through IP₃ receptors and then further Ca²⁺ release via RyR2 due to a local Ca²⁺-induced Ca²⁺ release process. These findings demonstrate an important mechanism for Ca²⁺ signaling and attendant physiological function in SMCs.     

Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain

The involvement of melatonin in mammalian brain pathophysiology has received growing interest, but information about the anatomical distribution of its two G‐protein‐coupled receptors, MT₁ and MT₂, remains elusive. In this study, using specific antibodies, we examined the precise distribution of both melatonin receptors immunoreactivity across the adult rat brain using light, confocal, and electron microscopy. Our results demonstrate a selective MT₁ and MT₂ localization on neuronal cell bodies and dendrites in numerous regions of the rat telencephalon, diencephalon, and mesencephalon. Confocal and ultrastructural examination confirmed the somatodendritic nature of MT₁ and MT₂ receptors, both being localized on neuronal membranes. Overall, striking differences were observed in the anatomical distribution pattern of MT₁ and MT₂ proteins, and the labeling often appeared complementary in regions displaying both receptors. Somadendrites labeled for MT₁ were observed for instance in the retrosplenial cortex, the dentate gyrus of the hippocampus, the islands of Calleja, the medial habenula, the suprachiasmatic nucleus, the superior colliculus, the substantia nigra pars compacta, the dorsal raphe nucleus, and the pars tuberalis of the pituitary gland. Somadendrites endowed with MT₂ receptors were mostly observed in the CA3 field of the hippocampus, the reticular thalamic nucleus, the supraoptic nucleus, the inferior colliculus, the substantia nigra pars reticulata, and the ventrolateral periaqueductal gray. Together, these data provide the first detailed neurocytological mapping of melatonin receptors in the adult rat brain, an essential prerequisite for a better understanding of melatonin distinct receptor function and neurophysiology.     

Melatonin induces calcium release from CCK‐8‐ and thapsigargin‐sensitive cytosolic stores in pancreatic AR42J cells

Abstract: Melatonin is produced following circadian rhythm with high levels being released at night and has been implicated in the regulation of physiological processes in major tissues, including the pancreas. The aim of our study was to examine the effects of melatonin on intracellular free Ca²⁺ concentration ([Ca²⁺]_c) in AR42J pancreatic cells. Our results show that stimulation of cells with 1 nm cholecystokinin (CCK)‐8 led to a transient increase in [Ca²⁺]_c followed by a decrease towards a value close to the prestimulation level. Melatonin (at the concentrations 1, 10, 100 μm and 1 mm ) induced changes in [Ca²⁺]_c that consisted of single or short lasting spikes in the form of oscillations or slow transient increases followed by a slow reduction towards a value close to the resting level. Depletion of intracellular Ca²⁺ stores by stimulation of cells with 1 nm CCK‐8 or 1 μm thapsigargin (Tps) blocked Ca²⁺ responses evoked by melatonin in the majority of cells. Conversely, prior stimulation of cells with 1 mm melatonin in the absence of extracellular Ca²⁺ inhibited Ca²⁺ mobilization in response to a secondary application of CCK‐8 or Tps. In summary, our results show that melatonin releases Ca²⁺ from intracellular stores and can therefore modulate the responses of the pancreas to CCK‐8. The source for Ca²⁺ mobilization most probably is the endoplasmic reticulum. These data raise the possibility that melatonin also involves Ca²⁺ signalling, in addition to other intracellular messengers, to modulate cellular function.     

Pancreatic protease activation by alcohol metabolite depends on Ca2+ release via acid store IP3 receptors

Toxic alcohol effects on pancreatic acinar cells, causing the often fatal human disease acute pancreatitis, are principally mediated by fatty acid ethyl esters (non-oxidative products of alcohol and fatty acids), emptying internal stores of Ca²⁺. This excessive Ca²⁺ liberation induces Ca²⁺-dependent necrosis due to intracellular trypsin activation. Our aim was to identify the specific source of the Ca²⁺ release linked to the fatal intracellular protease activation. In 2-photon permeabilized mouse pancreatic acinar cells, we monitored changes in the Ca²⁺ concentration in the thapsigargin-sensitive endoplasmic reticulum (ER) as well as in a bafilomycin-sensitive acid compartment, localized exclusively in the apical granular pole. We also assessed trypsin activity in the apical granular region. Palmitoleic acid ethyl ester (POAEE) elicited Ca²⁺ release from both the ER as well as the acid pool, but trypsin activation depended predominantly on Ca²⁺ release from the acid pool, that was mainly mediated by functional inositol 1,4,5- trisphosphate receptors (IP₃Rs) of types 2 and 3. POAEE evoked very little Ca²⁺ release and trypsin activation when IP₃Rs of both types 2 and 3 were knocked out. Antibodies against IP₃Rs of types 2 and 3, but not type 1, markedly inhibited POAEE-elicited Ca²⁺ release and trypsin activation. We conclude that Ca²⁺ release through IP₃Rs of types 2 and 3 in the acid granular Ca²⁺ store induces intracellular protease activation, and propose that this is a critical process in the initiation of alcohol-related acute pancreatitis.     

Melatonin‐mediated inhibition of Purkinje neuron P‐type Ca2+ channels in vitro induces neuronal hyperexcitability through the phosphatidylinositol 3‐kinase‐dependent protein kinase C delta pathway

Although melatonin receptors are widely expressed in the mammalian central nervous system and peripheral tissues, there are limited data regarding the functions of melatonin in cerebellar Purkinje cells. Here, we identified a novel functional role of melatonin in modulating P‐type Ca²⁺ channels and action‐potential firing in rat Purkinje neurons. Melatonin at 0.1 μm reversibly decreased peak currents (I_Ba) by 32.9%. This effect was melatonin receptor 1 (MT_R1) dependent and was associated with a hyperpolarizing shift in the voltage dependence of inactivation. Pertussis toxin pretreatment, intracellular application of QEHA peptide, and a selective antibody raised against the G_β subunit prevented the inhibitory effects of melatonin. Pretreatment with phosphatidylinositol 3‐kinase (PI3K) inhibitors abolished the melatonin‐induced decrease in I_Ba. Surprisingly, melatonin responses were not regulated by Akt, a common downstream target of PI3K. Melatonin treatment significantly increased protein kinase C (PKC) activity 2.1‐fold. Antagonists of PKC, but not of protein kinase A, abolished the melatonin‐induced decrease in I_Ba. Melatonin application increased the membrane abundance of PKC_δ, and PKC_δ inhibition (either pharmacologically or genetically) abolished the melatonin‐induced I_Ba response. Functionally, melatonin increased spontaneous action‐potential firing by 53.0%; knockdown of MT_R1 and blockade of P‐type channels abolished this effect. Thus, our results suggest that melatonin inhibits P‐type channels through MT_R1 activation, which is coupled sequentially to the βγ subunits of G_i/o‐protein and to downstream PI3K‐dependent PKC_δ signaling. This likely contributes to its physiological functions, including spontaneous firing of cerebellar Purkinje neurons.     

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non‐24‐hour sleep‐wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT₁ and MT₂, belonging to the G protein‐coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT₁ and MT₂. Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT₁ and MT₂ by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT₁ or MT₂. None of the mABs cross‐reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR‐KO mice as control. MT₂ expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta‐cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.     

Intracellular calcium release mediated by noradrenaline and acetylcholine in mammalian pineal cells

Abstract: The effects of noradrenergic and cholinergic receptor agonists on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in single dissociated rat pineal cells were investigated by microfluorimetric measurements in Fura-2 acetoxymethyl ester (Fura-2/AM) loaded cells. Noradrenaline (NA) evoked characteristic biphasic increments of intracellular Ca²⁺ consisting of one or more leading spikes followed by a plateau, resulting from the release of Ca²⁺ from intracellular stores and from the influx of Ca²⁺ from the external medium, respectively. This response was reproduced by the α₁ adrenoceptor agonist, phenylephrine (PE), in the presence of the β-adrenoceptor antagonist, propranolol, and was abolished when NA or PE was applied in conjunction with the α₁-adrenoceptor antagonist, prazosin. The curve relating the peak amplitude of the Ca²⁺ increments to different PE concentrations (0.5–10 μM) showed a half-maximum response at 0.6 μM PE, and saturation at concentrations greater than 2 μM. Acetylcholine (ACh) also elicited transient Ca²⁺ increments consisting of an abrupt rise to a maximum value which decayed exponentially to the basal Ca²⁺ level. A half-maximum response was achieved at 59 μM ACh. The muscarinic cholinergic receptor agonist, carbachol (CCh), similarly activated Ca²⁺ increments while the muscarinic antagonist, atropine, abolished them. In the absence of extracellular Ca²⁺, repetitive stimuli with either α₁-adrenergic and muscarinic agonists produced a progressive decrement in the amplitude of the Ca²⁺ signals because of the depletion of intracellular stores. However, extinction of the response to muscarinic agonists did not preclude a response to adrenergic agonists, while the contrary was not true. These results suggest that these agonists liberate Ca²⁺ from two functionally distinct, caffeine-insensitive, Ca²⁺ intracellular stores.     

Molecular and cellular pharmacological properties of 5‐methoxycarbonylamino‐N‐acetyltryptamine (MCA‐NAT): a nonspecific MT3 ligand

Abstract: 5‐Methoxycarbonylamino‐N‐acetyltryptamine (MCA‐NAT) has been initially described as a ligand at non MT₁, non MT₂ melatonin binding site (MT3) selective versus MT₁ and MT₂, two membrane melatonin receptors. MCA‐NAT activity has been reported by others in different models, in vivo, particularly in the intra‐ocular pressure (IOP) models in rabbits and monkeys. Its activity was systematically linked to either MT3 or to a new, yet unknown, melatonin receptor. In this article, the melatonin receptor pharmacology of MCA‐NAT is described. MCA‐NAT has micromolar range affinities at the melatonin receptors MT₁ and MT₂, while in functional studies, MCA‐NAT proved to be a powerful MT₁/MT₂ partial agonist in the sub‐micromolar range. These data strongly suggest that MCA‐NAT actions might be mediated by these receptors in vivo. Finally, as described by others, we show that MCA‐NAT is unable to elicit any type of receptor‐like functional responses from Chinese hamster ovary cells over‐expressing quinone reductase 2, the MT3.     

Melatonin ameliorates acute necrotizing pancreatitis by the regulation of cytosolic Ca2+ homeostasis

ObjectivesThis study aims to investigate the relationship between the protective effects of melatonin in pancreas and the expression of sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and Na⁺/Ca²⁺ exchanger (NCX) in rats with acute necrotizing pancreatitis (ANP), to verify whether melatonin ameliorates ANP by alleviating calcium overload.MethodsNinety-six male Sprague-Dawley rats were randomly divided into four groups (sham operation group, ANP group, melatonin treatment group, melatonin contrast group). ANP was induced by the retrograde injection of 4% taurocholate (1 ml/kg body weight) into the biliopancreatic duct. Melatonin (50 mg/kg body weight) was administered 30 min before the induction of ANP in the melatonin treatment group. Rats in each group were euthanized at 1, 4, and 8 h after ANP induction. Pancreatic tissues were removed to measure SERCA and NCX levels and cytosolic calcium ion (Ca²⁺) concentration ([Ca²⁺]_i).ResultsAt each time point, SERCA and NCX levels in the melatonin treatment group were significantly higher than that in the ANP group, and lower than that in the sham group and the melatonin contrast group. These levels did not differ between the 4- and 8-h time points in the ANP group. [Ca²⁺]_i in pancreatic acinar cells was higher in the melatonin treatment group than in the sham group and the melatonin contrast group, but lower than in the ANP group, at each time point.ConclusionMelatonin can reduce pancreatic damage via the up-regulation of SERCA and NCX expression, which can alleviate calcium overload in pancreatic acinar cells.     

Adenosine 5'-triphosphate (ATP) receptors induce intracellular calcium changes in mouse leydig cells

Cytoplasmic calcium ([Ca²⁺]_i) changes evoked by adenosine 5¹-triphosphate (ATP) were recorded in cultured individual Leydig cells within 10–18 h after cell dispersion. [Ca²⁺]_i was monitored using Fura-2AM loaded cells with a digital ratio imaging system. Five micromolars ATP induced biphasic [Ca²⁺]_i responses in most cells (94%,n=100), characterized by a fast increase from a basal level (126±5 nMSE,n=60 cells) to a peak (5–7 times above basal levels) within seconds, followed by a slow decrease toward a plateau level (2–3 times above basal) within 5 min. The peak phase of the [Ca²⁺]_i response increased with ATP concentrations (1–100 μM ATP) in a dose-dependent manner with an IC₅₀ of 5.9±1.2 μM, and it desensitized in a reversible manner with repeated application of 5 μM ATP at <5-min intervals. The [Ca²⁺]_i peak response was dependent on Ca²⁺ release from an intracellular pool, whereas the plateau phase was dependent on extracellular [Ca²⁺]. ATP did not appear to induce formation of nonspecific membrane pores, since stimulation for 10 min with ATP (10–100 μM) in the presence of extracellular Lucifer yellow (LY) (5 mg/mL) did not result in dye loading of the cells. [Ca²⁺]_i transients were elicited by other adenosine nucleotides with an order of potencies (ATP>Adenosine diphosphate [ADP]>Adenosine> Adenosine monophosphate [AMP]) that was compatible with the expression of P₂ receptors. [Ca²⁺]_i responses were suppressed by the purinergic P₂ receptor antagonist, suramin. These results provide functional evidence for the expression of purinergic P₂ receptors in Leydig cells.     

Acyl derivatives of coenzyme A inhibit platelet function via antagonism at P2Y1 and P2Y12 receptors: A new finding that may influence the design of anti-thrombotic agents

We have performed a detailed investigation of the effects on platelet function of coenzyme A (CoA) and several acyl-CoAs. Platelet aggregation was measured by turbidimetry and by platelet counting; platelet shape change was measured using light scattering; P-selectin, Ca²⁺ mobilization and vasodilator-stimulated phosphoprotein (VASP) phosphorylation were measured by flow cytometry. The compounds investigated inhibited ADP-induced platelet aggregation; those with saturated acyl groups containing 16-18 carbons were most effective. The effects of palmitoyl-CoA (16:0) were studied in depth. It inhibited platelet shape change and Ca²⁺ mobilization brought about by ADP (but not other agonists) indicating antagonism at P2Y₁ receptors, and also inhibited ADP-induced P-selectin expression. Effects of palmitoyl-CoA on the platelet aggregation and Ca²⁺ mobilization induced by several different agonists and agonist combinations were compared with those of MRS 2179 (a P2Y₁ antagonist) and AR-C69931 (a P2Y₁₂ antagonist), and were consistent with palmitoyl-CoA acting mainly at P2Y₁ but also with partial antagonism at P2Y₁₂ receptors. Antagonism at P2Y₁₂ receptors was confirmed in studies of VASP-phosphorylation. Palmitoyl-CoA did not act as an antagonist at P2X₁ receptors. The results are discussed in relation to the possibility that acyl-CoAs may contribute as endogenous modulators of platelet function and might serve as lead compounds for the design of novel antithrombotics.     

Genetic deletion of MT1 and MT2 melatonin receptors differentially abrogates the development and expression of methamphetamine‐induced locomotor sensitization during the day and the night in C3H/HeN mice

Abstract: This study explored the role of the melatonin receptors in methamphetamine (METH)‐induced locomotor sensitization during the light and dark phases in C3H/HeN mice with genetic deletion of the MT₁ and/or MT₂ melatonin receptors. Six daily treatments with METH (1.2 mg/kg, i.p.) in a novel environment during the light phase led to the development of locomotor sensitization in wild‐type (WT), MT₁KO and MT₂KO mice. Following four full days of abstinence, METH challenge (1.2 mg/kg, i.p.) triggered the expression of locomotor sensitization in METH‐pretreated but not in vehicle (VEH)‐pretreated mice. In MT₁/MT₂KO mice, the development of sensitization during the light phase was significantly reduced and the expression of sensitization was completely abrogated upon METH challenge. During the dark phase the development of locomotor sensitization in METH‐pretreated WT, MT₁KO and MT₂KO mice was statistically different from VEH‐treated controls. However, WT and MT₂KO, but not MT₁KO mice receiving repeated VEH pretreatments during the dark phase expressed a sensitized response to METH challenge that is of an identical magnitude to that observed upon 6 days of METH pretreatment. We conclude that exposure to a novel environment during the dark phase, but not during the light phase, facilitated the expression of sensitization to a METH challenge in a manner dependent on MT₁ melatonin receptor activation by endogenous melatonin. We suggest that MT₁ and MT₂ melatonin receptors are potential targets for pharmacotherapeutic intervention in METH abusers.     

N-methyl-D-aspartate receptor responses are differentially modulated by noncompetitive receptor antagonists and ethanol in inbred long-sleep and short-sleep mice: behavior and electrophysiology

BACKGROUND: Short-sleep (SS) mice exhibit higher locomotor activity than do long-sleep (LS) mice when injected with low doses of ethanol or the noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist MK-801 (dizocilpine). SS mice also have higher densities of brain NMDARs. However, two strains of LS X SS recombinant inbred (RI) mice also show differential activation to ethanol and MK-801, but have similar numbers of NMDARs. Here we used inbred LS (ILS) and SS (ISS) mice to investigate further the relationship between NMDARs and sensitivity to the stimulant effects of low doses of ethanol. METHODS: Open field activity and spontaneous alternations were measured after saline or drug injection. [3H]MK-801 binding parameters were determined in hippocampus, cortex, striatum, and nucleus accumbens. Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region of hippocampal slices. RESULTS: Systemic injection of either ethanol or MK-801 increased locomotor activity to a greater extent in ISS mice than in ILS mice. The competitive NMDAR antagonist 2-carboxypiperazin-4-yl-propyl-1-lphosphonic acid (+/- CPP) depressed activity of ILS, but not ISS, mice. No strain differences were observed in spontaneous alternations or in the number or affinity of NMDARs in the brain regions examined. Likewise, the magnitudes of hippocampal NMDAR-mediated fEPSPs were similar in ILS and ISS mice and were inhibited to the same extent by a competitive NMDAR antagonist. However, both ethanol and the NMDAR NR2B receptor antagonist ifenprodil inhibited the late component of hippocampal NMDAR fEPSPs to a greater extent in ISS, than in ILS, mice. CONCLUSIONS: Differential ethanol- and MK-801-induced behavioral activation in ILS and ISS mice was not associated with differences in NMDAR number. Nonetheless, pharmacological differences in hippocampal NMDAR responsiveness suggest that ISS mice express NMDARs that have a greater sensitivity to noncompetitive, but not competitive, NMDAR antagonists. These differences, which may reflect differences in NMDAR subunit composition, could underlie the differential responsiveness to low doses of ethanol in ILS and ISS mice.     

Acute hypoxia activates store-operated Ca2+ entry and increases intracellular Ca2+ concentration in rat distal pulmonary venous smooth muscle cells

Rationale

Exposure to acute hypoxia causes vasoconstriction in both pulmonary arteries (PA) and pulmonary veins (PV). The mechanisms on the arterial side have been studied extensively. However, bare attention has been paid to the venous side.

Objectives

To investigate if acute hypoxia caused the increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i), and Ca²⁺ influx through store-operated calcium channels (SOCC) in pulmonary venous smooth muscle cells (PVSMCs).

Methods

Fluorescent microscopy and fura-2 were used to measure effects of 4% O₂ on [Ca²⁺]_i and store-operated Ca²⁺ entry (SOCE) in isolated rat distal PVSMCs.

Measurements and main results

In PVSMCs perfused with Ca²⁺-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca²⁺ stores in the sarcoplasmic reticulum (SR) and nifedipine to prevent Ca²⁺ entry through L-type voltage-depended Ca²⁺ channels (VDCC), hypoxia markedly enhanced both the increase in [Ca²⁺]_i caused by restoration of extracellular [Ca²⁺] and the rate at which extracellular Mn²⁺ quenched fura-2 fluorescence. Moreover, the increased [Ca²⁺]_i in PVSMCs perfused with normal salt solution was completely blocked by SOCC antagonists SKF-96365 and NiCl₂ at concentrations that SOCE >85% was inhibited but [Ca²⁺]_i responses to 60 mM KCl were not altered. On the contrary, L-type VDCC antagonist nifedipine inhibited increase in [Ca²⁺]_i to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca²⁺]_i caused by hypoxia was completely abolished by perfusion with Ca²⁺-free KRBS.

Conclusions

These results suggest that acute hypoxia enhances SOCE via activating SOCCs, leading to increased [Ca²⁺]_i in distal PVSMCs.KEYWORDS : Calcium signaling, pulmonary venous smooth muscle (PVSM), store-operated Ca²⁺ entry (SOCE), intracellular Ca²⁺ concentration ([Ca²⁺]_i)