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.     

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.     

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.     

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.     

Melatonin MT1 and MT2 receptor ERK signaling is differentially dependent on Gi/o and Gq/11 proteins

G protein-coupled receptors (GPCRs) transmit extracellular signals into cells by activating G protein- and β-arrestin-dependent pathways. Extracellular signal-regulated kinases (ERKs) play a central role in integrating these different linear inputs coming from a variety of GPCRs to regulate cellular functions. Here, we investigated human melatonin MT₁ and MT₂ receptors signaling through the ERK1/2 cascade by employing different biochemical techniques together with pharmacological inhibitors and siRNA molecules. We show that ERK1/2 activation by both receptors is exclusively G protein-dependent, without any participation of β-arrestin1/2 in HEK293 cells. ERK1/2 activation by MT₁ is only mediated though G_i/o proteins, while MT₂ is dependent on the cooperative activation of G_i/o and G_q/11 proteins. In the absence of G_q/11 proteins, however, MT₂-induced ERK1/2 activation switches to a β-arrestin1/2-dependent mode. The signaling cascade downstream of G proteins is the same for both receptors and involves activation of the PI3K/PKCζ/c-Raf/MEK/ERK cascade. The differential G protein dependency of MT₁- and MT₂-mediated ERK activation was confirmed at the level of EGR1 and FOS gene expression, two ERK1/2 target genes. G_i/o/G_q/11 cooperativity was also observed in Neuroscreen-1 cells expressing endogenous MT₂, whereas in the mouse retina, where MT₂ is engaged into MT₁/MT₂ heterodimers, ERK1/2 signaling is exclusively G_i/o-dependent. Collectively, our data reveal differential signaling modes of MT₁ and MT₂ in terms of ERK1/2 activation, with an unexpected G_i/o/G_q/11 cooperativity exclusively for MT₂. The plasticity of ERK activation by MT₂ is highlighted by the switch to a β-arrestin1/2-dependent mode in the absence of G_q/11 proteins and by the switch to a G_i/o mode when engaged into MT₁/MT₂ heterodimers, revealing a new mechanism underlying tissue-specific responses to melatonin.     

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.     

Effect of MT1 melatonin receptor deletion on melatonin-mediated phase shift of circadian rhythms in the C57BL/6 mouse

In the mouse suprachiasmatic nucleus (SCN), melatonin activates MT1 and MT2 G-protein coupled receptors, which are involved primarily in inhibition of neuronal firing and phase shift of circadian rhythms. This study investigated the ability of melatonin to phase shift circadian rhythms in wild type (WT) and MT1 melatonin receptor knockout (KO) C57BL/6 mice. In WT mice, melatonin (90 microg/mouse, s.c.) administered at circadian time 10 (CT10; CT12 onset of activity) significantly phase advanced the onset of the circadian activity rhythm (0.60 +/- 0.09 hr, n = 41) when compared with vehicle treated controls (-0.02 +/- 0.07 hr, n = 28) (P < 0.001). In contrast, C57 MT1KO mice treated with melatonin did not phase shift circadian activity rhythms (-0.10 +/- 0.12 hr, n = 42) when compared with vehicle treated mice (-0.12 +/- 0.07 hr, n = 43). Similarly, in the C57 MT1KO mouse melatonin did not accelerate re-entrainment to a new dark onset after an abrupt advance of the dark cycle. In contrast, melatonin (3 and 10 pm) significantly phase advanced circadian rhythm of neuronal firing in SCN brain slices independent of genotype with an identical maximal shift at 10 pm (C57 WT: 3.61 +/- 0.38 hr, n = 3; C57 MT(1)KO: 3.45 +/- 0.11 hr, n = 4). Taken together, these results suggest that melatonin-mediated phase advances of circadian rhythms of neuronal firing in the SCN in vitro may involve activation of the MT2 receptor while in vivo activation of the MT1 and possibly the MT2 receptor may be necessary for the expression of melatonin-mediated phase shifts of overt circadian activity rhythms.     

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.     

Melatonin recovers sleep phase delayed by MK-801 through the melatonin MT2 receptor- Ca2+-CaMKII-CREB pathway in the ventrolateral preoptic nucleus

Melatonin (MLT) is widely used to treat sleep disorders although the underlying mechanism is still elusive. In mice, using wheel-running detection, we found that exogenous MLT could completely recover the period length prolonged by N-methyl-D-aspartate receptor (NMDAR) impairment due to the injection of the NMDAR antagonist MK-801, a preclinical model of psychosis. The analysis of the possible underlying mechanisms indicated that MLT could regulate the homeostatic state in the ventrolateral preoptic nucleus (VLPO) instead of the circadian process in the suprachiasmatic nucleus (SCN). In addition, our data showed that MK-801 decreased Ca²⁺-related CaMKII expression and CREB phosphorylation levels in the VLPO, and MLT could rescue these intracellular impairments but not NMDAR expression levels. Accordingly, Gcamp6 AAV virus was injected in-vivo to further monitor intracellular Ca²⁺ levels in the VLPO, and MLT demonstrated a unique ability to increase Ca²⁺ fluorescence compared with MK-801-injected mice. Additionally, using the selective melatonin MT₂ receptor antagonist 4-phenyl-2-propionamidotetralin (4P-PDOT), we discovered that the pharmacological effects of MLT upon NMDAR impairments were mediated by melatonin MT₂ receptors. Using electroencephalography/electromyography (EEG/EMG) recordings, we observed that the latency to the first nonrapid eye movement (NREM) sleep episode was delayed by MK-801, and MLT was able to recover this delay. In conclusion, exogenous MLT by acting upon melatonin MT₂ receptors rescues sleep phase delayed by NMDAR impairment via increasing intracellular Ca²⁺ signaling in the VLPO, suggesting a regulatory role of the neurohormone on the homeostatic system.     

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.     

Melatonin enhances the human mesenchymal stem cells motility via melatonin receptor 2 coupling with Gαq in skin wound healing

Melatonin, a circadian rhythm–promoting molecule, has a variety of biological functions, but the functional role of melatonin in the motility of mesenchymal stem cells (MSCs) has yet to be studied. In a mouse skin excisional wound model, we found that transplantation of umbilical cord blood (UCB)‐MSCs pretreated with melatonin enhanced wound closure, granulation, and re‐epithelialization at mouse skin wound sites, where relatively more UCB‐MSCs which were engrafted onto the wound site were detected. Thus, we identified the signaling pathway of melatonin, which affects the motility of UCB‐MSCs. Melatonin (1 μm ) significantly increased the motility of UCB‐MSCs, which had been inhibited by the knockdown of melatonin receptor 2 (MT₂). We found that Gαq coupled with MT₂ and that the binding of Gαq to MT₂ uniquely stimulated an atypical PKC isoform, PKCζ. Melatonin induced the phosphorylation of FAK and paxillin, which were concurrently downregulated by blocking of the PKC activity. Melatonin increased the levels of active Cdc42 and Arp2/3, and it has the ability to stimulate cytoskeletal reorganization‐related proteins such as profilin‐1, cofilin‐1, and F‐actin in UCB‐MSCs. Finally, a lack of MT₂ expression in UCB‐MSCs during a mouse skin transplantation experiment resulted in impaired wound healing and less engraftment of stem cells at the wound site. These results demonstrate that melatonin signaling via MT₂ triggers FAK/paxillin phosphorylation to stimulate reorganization of the actin cytoskeleton, which is responsible for Cdc42/Arp2/3 activation to promote UCB‐MSCs motility.     

Melatonin receptor structures shed new light on melatonin research

The tryptophan derivative melatonin is an evolutionary old molecule that is involved in a pleiotropy of physiological functions. In humans, age‐related decline of circulating melatonin levels and/or dysregulation of its circadian synthesis pattern have been associated with several disorders and disease states. Several molecular targets have been proposed for melatonin since its discovery, in 1959. Among them, melatonin MT₁ and MT₂ receptors are the best characterized melatonin targets, mediating melatonin effects in a variety of tissues. They belong to the superfamily of G protein‐coupled receptors. Two back‐to‐back articles published in the “Nature” Journal earlier this year present the first crystal structures of the human MT₁ and MT₂ in its inactive states. Here, we will briefly outline the discovery path of melatonin receptors until their structural elucidation and discuss how these new findings will guide future research toward a better understanding of their function and rational drug design.     

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.     

Melatonin promotes regeneration of injured motor axons via MT1 receptors

Melatonin is an ancient multi‐tasking molecule produced by the pineal gland and by several extrapineal tissues. A variety of activities has been ascribed to this hormone in different physiological and pathological contexts, but little is known about its role in peripheral neuroregeneration. Here, we have exploited two different types of injury to test the capability of melatonin to stimulate regeneration of motor axons: (a) the acute and reversible presynaptic degeneration induced by the spider neurotoxin α‐Latrotoxin and (b) the compression/transection of the sciatic nerve. We found that in both cases melatonin administration accelerates the process of nerve repair. This pro‐regenerative action is MT₁‐mediated, and at least in part due to a sustained activation of the ERK1/2 pathway. These findings reveal a receptor‐mediated, pro‐regenerative action of melatonin in vivo that holds important clinical implications, as it posits melatonin as a safe candidate molecule for the treatment of a number of peripheral neurodegenerative conditions.     

Enhancement of high glucose‐induced PINK1 expression by melatonin stimulates neuronal cell survival: Involvement of MT2/Akt/NF‐κB pathway

Hyperglycemia is a representative hallmark and risk factor for diabetes mellitus (DM) and is closely linked to DM‐associated neuronal cell death. Previous investigators reported on a genome‐wide association study and showed relationships between DM and melatonin receptor (MT), highlighting the role of MT signaling by assessing melatonin in DM. However, the role of MT signaling in DM pathogenesis is unclear. Therefore, we investigated the role of mitophagy regulators in high glucose‐induced neuronal cell death and the effect of melatonin against high glucose‐induced mitophagy regulators in neuronal cells. In our results, high glucose significantly increased PTEN‐induced putative kinase 1 (PINK1) and LC‐3B expressions; as well it decreased cytochrome c oxidase subunit 4 expression and Mitotracker? fluorescence intensity. Silencing of PINK1 induced mitochondrial reactive oxygen species (ROS) accumulation and mitochondrial membrane potential impairment, increased expressions of cleaved caspases, and increased the number of annexin V‐positive cells. In addition, high glucose‐stimulated melatonin receptor 1B (MTNR1B) mRNA and PINK1 expressions were reversed by ROS scavenger N‐acetyl cysteine pretreatment. Upregulation of PINK1 expression in neuronal cells is suppressed by pretreatment with MT₂ receptor‐specific inhibitor 4‐P‐PDOT. We further showed melatonin stimulated Akt phosphorylation, which was followed by nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) phosphorylation and nuclear translocation. Silencing of PINK1 expression abolished melatonin‐regulated mitochondrial ROS production, cleaved caspase‐3 and caspase‐9 expressions, and the number of annexin V‐positive cells. In conclusion, we have demonstrated the melatonin stimulates PINK1 expression via an MT₂/Akt/NF‐κB pathway, and such stimulation is important for the prevention of neuronal cell apoptosis under high glucose conditions.     

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.     

Renal Angiotensin II Receptors,Hyperinsulinemia, and Obesity

Angiotensin II, via activation of AT₁ receptors in the kidney regulates sodium/fluid homeostasis and blood pressure. An exaggerated action of angiotensin II mediated via activation of AT₁ receptors has been implicated in the increased renal sodium retention and the resetting of the pressure natriuresis in obesity related hypertension. Treatment of obese Zucker rats with AT₁ receptor blockers reduces blood pressure to a greater extent and produces greater natriuresis. Also, there is an increased membranal AT₁ receptor numbers and angiotensin II produces greater activation of sodium transporters in the isolated tubules from obese Zucker rats. Interestingly, AT₂ receptors, which are believed to be beneficial to the renal and cardiovascular function in terms of their action on kidney and blood vessels, are greatly increased in proximal tubular membranes of obese Zucker rats. Whole animal and in vitro studies indicate that higher plasma insulin level, generally associated with obesity, is responsible for the up‐regulation of both AT₁ and AT₂ receptors in the kidney. Determining the consequence of selective blocking of AT₁ receptors and/or activation of the AT₂ receptors on renal and cardiovascular function, and the effect of lowering insulin on these receptors present an important area of further investigation in obesity.