P2X2 receptors supply extracellular choline as a substrate for acetylcholine synthesis.

Acetylcholine (ACh), an excitatory neurotransmitter, is biosynthesized from choline in cholinergic neurons. Import from the extracellular space to the intracellular environment through the high-affinity choline transporter is currently regarded to be the only source of choline for ACh synthesis. We recently demonstrated that the P2X2 receptor, through which large cations permeate, functions as an alternative pathway for choline transport in the mouse retina. In the present study, we investigated whether choline entering cells through P2X2 receptors is used for ACh synthesis using a recombinant system. When P2X2 receptors expressed on HEK293 cell lines were stimulated with ATP, intracellular ACh concentrations increased. These results suggest that P2X2 receptors function in a novel pathway that supplies choline for ACh synthesis.

Non-neuronal cardiac acetylcholine system playing indispensable roles in cardiac homeostasis confers resiliency to the heart.

BACKGROUND: We previously established that the non-neuronal cardiac cholinergic system (NNCCS) is equipped with cardiomyocytes synthesizes acetylcholine (ACh), which is an indispensable endogenous system, sustaining cardiac homeostasis and regulating an inflammatory status, by transgenic mice overexpressing choline acetyltransferase (ChAT) gene in the heart. However, whole body biological significances of NNCCS remain to be fully elucidated. METHODS AND RESULTS: To consolidate the features, we developed heart-specific ChAT knockdown (ChATKD) mice using 3 ChAT-specific siRNAs. The mice developed cardiac dysfunction. Factors causing it included the downregulation of cardiac glucose metabolism along with decreased signal transduction of Akt/HIF-1alpha/GLUT4, leading to poor glucose utilization, impairment of glycolytic metabolites entering the tricarboxylic (TCA) cycle, the upregulation of reactive oxygen species (ROS) production with an attenuated scavenging potency, and the downregulated nitric oxide (NO) production via NOS1. ChATKD mice revealed a decreased vagus nerve activity, accelerated aggression, more accentuated blood basal corticosterone levels with depression-like phenotypes, several features of which were accompanied by cardiac dysfunction. CONCLUSION: The NNCCS plays a crucial role in cardiac homeostasis by regulating the glucose metabolism, ROS synthesis, NO levels, and the cardiac vagus nerve activity. Thus, the NNCCS is suggested a fundamentally crucial system of the heart.

S-Nitroso-N-Pivaloyl-D-Penicillamine, a novel non-neuronal ACh system activator, modulates cardiac diastolic function to increase cardiac performance under pathophysiological conditions.

We have previously reported the development of a novel chemical compound, S-Nitroso-N-Pivaloyl-D-Penicillamine (SNPiP), for the upregulation of the non-neuronal cardiac cholinergic system (NNCCS), a cardiac acetylcholine (ACh) synthesis system, which is different from the vagus nerve releasing of ACh as a neurotransmitter. However, it remains unclear how SNPiP could influence cardiac function positively, and whether SNPiP could improve cardiac function under various pathological conditions. SNPiP-injected control mice demonstrated a gradual upregulation in diastolic function without changes in heart rate. In contrast to some parameters in cardiac function that were influenced by SNPiP 24 h or 48 h after a single intraperitoneal (IP) injection, 72 h later, end-systolic pressure, cardiac output, end-diastolic volume, stroke volume, and ejection fraction increased. IP SNPiP injection also improved impaired cardiac function, which is a characteristic feature of the db/db heart, in a delayed fashion, including diastolic and systolic function, following either several consecutive injections or a single injection. SNPiP, a novel NNCCS activator, could be applied as a therapeutic agent for the upregulation of NNCCS and as a unique tool for modulating cardiac function via improvement in diastolic function.

Potentiating a non-neuronal cardiac cholinergic system reinforces the functional integrity of the blood brain barrier associated with systemic anti-inflammatory responses.

We previously reported that the heart-specific choline acetyltransferase (ChAT) gene overexpressing mice (ChAT tg) show specific phenotypes including ischemic tolerance and the CNS stress tolerance. In the current study, we focused on molecular mechanisms responsible for systemic and localized anti-inflammatory phenotypes of ChAT tg. ChAT tg were resistant to systemic inflammation induced by lipopolysaccharides due to an attenuated cytokine response. In addition, ChAT tg, originally equipped with less reactive Kupffer cells, were refractory to brain cold injury, with decreased blood brain barrier (BBB) permeability and reduced inflammation. This is because ChAT tg brain endothelial cells expressed more claudin-5, and their astrocytes were less reactive, causing decreased hypertrophy. Moreover, reconstruction of the BBB integrity in vitro confirmed the consolidation of ChAT tg. ChAT tg were also resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neuronal toxicity due to lower mortality rate and neuronal loss of substantia nigra. Additionally, ChAT tg subjected to MPTP showed attenuated BBB disruption, as evident from reduced sodium fluorescein levels in the brain parenchyma. The activated central cholinergic pathway of ChAT tg lead to anti-convulsive effects like vagus nerve stimulation. However, DSP-4, a noradrenergic neuron-selective neurotoxin against the CNS including the locus ceruleus, abrogated the beneficial phenotype and vagotomy attenuated expression of claudin-5, suggesting the link between the cholinergic pathway and BBB function. Altogether, these findings indicate that ChAT tg possess an anti-inflammatory response potential, associated with upregulated claudin-5, leading to the consolidation of BBB integrity. These characteristics protect ChAT tg against systemic and localized inflammatory pathological disorders, which targets the CNS.

A Novel Nitric Oxide Donor, S-Nitroso-NPivaloyl-D-Penicillamine, Activates a Non-Neuronal Cardiac Cholinergic System to Synthesize Acetylcholine and Augments Cardiac Function.

BACKGROUND/AIMS: In a previous study, we reported that cardiomyocytes were equipped with non-neuronal cardiac cholinergic system (NNCCS) to synthesize acetylcholine (ACh), which is indispensable for maintaining the basic physiological cardiac functions. The aim of this study was to identify and characterize a pharmacological inducer of NNCCS. METHODS: To identify a pharmacological inducer of NNCCS, we screened several chemical compounds with chemical structures similar to the structure of S-nitroso-N-acetyl-DL-penicillamine (SNAP). Preliminary investigation revealed that SNAP is an inducer of non-neuronal ACh synthesis. We screened potential pharmacological inducers in H9c2 and HEK293 cells using western blot analysis, luciferase assay, and measurements of intracellular cGMP, NO2 and ACh levels. The effects of the screened compound on cardiac function of male C57BL6 mice were also evaluated using cardiac catheter system. RESULTS: Among the tested compounds, we selected S-nitroso-Npivaloyl-D-penicillamine (SNPiP), which gradually elevated the intracellular cGMP levels and nitric oxide (NO) levels in H9c2 and HEK293 cells. These elevated levels resulted in the gradual transactivation and translation of the choline acetyltransferase gene. Additionally, in vitro and in vivo SNPiP treatment elevated ACh levels for 72 h. SNPiP-treated mice upregulated their cardiac function without tachycardia but with enhanced diastolic function resulting in improved cardiac output. The effect of SNPiP was dependent on SNPiP nitroso group as verified by the ineffectiveness of N-pivaloyl-D-penicillamine (PiP), which lacks the nitroso group. CONCLUSION: SNPiP is identified to be one of the important pharmacological candidates for induction of NNCCS.

Diethylstilbestrol administration inhibits theca cell androgen and granulosa cell estrogen production in immature rat ovary.

Diethylstilbestrol (DES), a strong estrogenic compound, is well-known to affect the reproductive system. In this study, we investigated the effects of DES administration on gonadotropin levels and ovarian steroidogenesis in prepubertal rats. DES treatment acutely reduced serum LH levels, followed by a reduction in the expression of various steroidogenesis-related genes in theca cells. Serum FSH levels were almost unaffected by DES-treatment, even though Cyp19a1 expression was markedly reduced. Serum progesterone, testosterone and estradiol levels were also declined at this time. LH levels recovered from 12 h after DES-treatment and gradually increased until 96 h with a reduction of ERα expression observed in the pituitary. Steroidogenesis-related genes were also up-regulated during this time, except for Cyp17a1 and Cyp19a1. Consistent with observed gene expression pattern, serum testosterone and estradiol concentrations were maintained at lower levels, even though progesterone levels recovered. DES-treatment induced the inducible nitric oxide synthase (iNOS) in granulosa cells, and a nitric oxide generator markedly repressed Cyp19a1 expression in cultured granulosa cells. These results indicate that DES inhibits thecal androgen production via suppression of pituitary LH secretion and ovarian Cyp17a1 expression. In addition, DES represses Cyp19a1 expression by inducing iNOS gene expression for continuous inhibition of estrogen production in granulosa cells.

Novel channel-mediated choline transport in cholinergic neurons of the mouse retina.

Choline uptake into the presynaptic terminal of cholinergic neurons is mediated by the high-affinity choline transporter and is essential for acetylcholine synthesis. In a previous study, we reported that P2X2 purinoceptors are selectively expressed in OFF-cholinergic amacrine cells of the mouse retina. Under specific conditions, P2X2 purinoceptors acquire permeability to large cations, such as N-methyl-d-glucamine, and therefore potentially could act as a noncanonical pathway for choline entry into neurons. We tested this hypothesis in OFF-cholinergic amacrine cells of the mouse retina. ATP-induced choline currents were observed in OFF-cholinergic amacrine cells, but not in ON-cholinergic amacrine cells, in mouse retinal slice preparations. High-affinity choline transporters are expressed at higher levels in ON-cholinergic amacrine cells than in OFF-cholinergic amacrine cells. In dissociated preparations of cholinergic amacrine cells, ATP-activated cation currents arose from permeation of extracellular choline. We also examined the pharmacological properties of choline currents. Pharmacologically, α,ß-methylene ATP did not produce a cation current, whereas ATPγS and benzoyl-benzoyl-ATP (BzATP) activated choline currents. However, the amplitude of the choline current activated by BzATP was very small. The choline current activated by ATP was strongly inhibited by pyridoxalphosphate-6-azophenyl-2',4'-sulfonic acid. Accordingly, P2X2 purinoceptors expressed in HEK-293T cells were permeable to choline and similarly functioned as a choline uptake pathway. Our physiological and pharmacological findings support the hypothesis that P2 purinoceptors, including P2X2 purinoceptors, function as a novel choline transport pathway and may provide a new regulatory mechanism for cholinergic signaling transmission at synapses in OFF-cholinergic amacrine cells of the mouse retina.NEW & NOTEWORTHY Choline transport across the membrane is exerted by both the high-affinity and low-affinity choline transporters. We found that choline can permeate P2 purinergic receptors, including P2X2 purinoceptors, in cholinergic neurons of the retina. Our findings show the presence of a novel choline transport pathway in cholinergic neurons. Our findings also indicate that the permeability of P2X2 purinergic receptors to choline observed in the heterologous expression system may have a physiological relevance in vivo.

Various Regulatory Modes for Circadian Rhythmicity and Sexual Dimorphism in the Non-Neuronal Cardiac Cholinergic System.

Cardiomyocytes possess a non-neuronal cardiac cholinergic system (NNCCS) regulated by a positive feedback system; however, its other regulatory mechanisms remain to be elucidated, which include the epigenetic control or regulation by the female sex steroid, estrogen. Here, the NNCCS was shown to possess a circadian rhythm; its activity was upregulated in the light-off phase via histone acetyltransferase (HAT) activity and downregulated in the light-on phase. Disrupting the circadian rhythm altered the physiological choline acetyltransferase (ChAT) expression pattern. The NNCCS circadian rhythm may be regulated by miR-345, independently of HAT, causing decreased cardiac ChAT expression. Murine cardiac ChAT expression and ACh contents were increased more in female hearts than in male hearts. This upregulation was downregulated by treatment with the estrogen receptor antagonist tamoxifen, and in contrast, estrogen reciprocally regulated cardiac miR-345 expression. These results suggest that the NNCCS is regulated by the circadian rhythm and is affected by sexual dimorphism.

Non-neuronal cardiac cholinergic system influences CNS via the vagus nerve to acquire a stress-refractory propensity.

We previously developed cardiac ventricle-specific choline acetyltransferase (ChAT) gene-overexpressing transgenic mice (ChAT tgm), i.e. an in vivo model of the cardiac non-neuronal acetylcholine (NNA) system or non-neuronal cardiac cholinergic system (NNCCS). By using this murine model, we determined that this system was responsible for characteristics of resistance to ischaemia, or hypoxia, via the modulation of cellular energy metabolism and angiogenesis. In line with our previous study, neuronal ChAT-immunoreactivity in the ChAT tgm brains was not altered from that in the wild-type (WT) mice brains; in contrast, the ChAT tgm hearts were the organs with the highest expression of the ChAT transgene. ChAT tgm showed specific traits in a central nervous system (CNS) phenotype, including decreased response to restraint stress, less depressive-like and anxiety-like behaviours and anti-convulsive effects, all of which may benefit the heart. These phenotypes, induced by the activation of cardiac NNCCS, were dependent on the vagus nerve, because vagus nerve stimulation (VS) in WT mice also evoked phenotypes similar to those of ChAT tgm, which display higher vagus nerve discharge frequency; in contrast, lateral vagotomy attenuated these traits in ChAT tgm to levels observed in WT mice. Furthermore, ChAT tgm induced several biomarkers of VS responsible for anti-convulsive and anti-depressive-like effects. These results suggest that the augmentation of the NNCCS transduces an effective and beneficial signal to the afferent pathway, which mimics VS. Therefore, the present study supports our hypothesis that activation of the NNCCS modifies CNS to a more stress-resistant state through vagus nerve activity.

Remote ischemic preconditioning with a specialized protocol activates the non-neuronal cardiac cholinergic system and increases ATP content in the heart.

Ischemic preconditioning (IPC) renders the targeted organ resistant to prolonged ischemic insults, leading to organoprotection. Among several means to achieve IPC, we reported that remote ischemic preconditioning (RIPC) activates the non-neuronal cardiac cholinergic system (NNCCS) to accelerate de novo ACh synthesis in cardiomyocytes. In the current study, we aimed to optimize a specific protocol to most efficiently activate NNCCS using RIPC. In this study, we elucidated that the protocol with 3 min of ischemia repeated three times increased cardiac ChAT expression (139.2 ± 0.4%; P < 0.05) as well as ACh (14.2 ± 2.0× 10(-8) M; P< 0.05) and ATP content (2.13 ± 0.19 µmol/g tissue; P < 0.05) in the heart. Moreover, in the specific protocol, several characteristic responses against energy starvation and for obtaining adequate energy were observed; therefore, it is suggested that RIPC evokes a robust response by the heart to activate NNCCS through the modification of energy metabolism.

Nicotinic receptor-dependent and -independent effects of galantamine, an acetylcholinesterase inhibitor, on the non-neuronal acetylcholine system in C2C12 cells.

We previously reported that satellite cells possess the ability to produce angiogenic factors, including fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) in vivo. However, whether C2C12 cells possess a non-neuronal cholinergic system (NNCS) or non-neuronal ACh (NNA) remains to be studied; therefore, we investigated the system using C2C12 cells and its regulatory mechanisms. C2C12 cells synthesized ACh, the level of which was comparable with that of cardiomyocytes, and the synthesis was augmented by the acetylcholinesterase inhibitor galantamine. The ChAT promoter activity was upregulated by nicotine or galantamine, partly through nicotinic receptors for both agents as well as through a non-nicotinic receptor pathway for galantamine. Further, VEGF secretion by C2C12 cells was also increased by nicotine or galantamine through nicotinic receptors as well as partly through non-nicotinic pathways in the case of galantamine. These results suggest that C2C12 cells are equipped with NNCS or NNA, which is positively regulated through nicotinic or non-nicotinic pathways, particularly in the case of galantamine. These results provide a novel concept that myogenic cells expressing NNA can be a therapeutic target for regulating angiogenic factor synthesis.

miR-449a contributes to glucocorticoid-induced CRF-R1 downregulation in the pituitary during stress.

The hypothalamic-pituitary-adrenal axis is controlled by the feedback of glucocorticoids on the hypothalamus and pituitary. Stress increases CRF, ACTH, and glucocorticoid secretion. The expression of not only CRF mRNA in the hypothalamus and proopiomelanocortin mRNA in corticotrophs, but also CRF type 1 receptor (CRF-R1) mRNA and protein on corticotrophs are downregulated through glucocorticoids. However, the mechanisms underlying the glucocorticoid-induced CRF-R1 downregulation are not fully understood. Short RNA molecules, called microRNAs (miRNAs), are posttranscriptional regulators that usually induce translational repression or gene silencing via binding to complementary sequences within target mRNAs. We hypothesized that glucocorticoids may induce the expression of miRNAs in the pituitary, which are involved in glucocorticoid-induced downregulation of CRF-R1. We found 3 miRNAs with sequences predicted to bind to the CRF-R1 3' untranslated region (3'-UTR) by database search. Expression of 1 of these miRNAs (miR-449a) was significantly higher in the anterior pituitary of restrained rats than in that of unrestrained control rats. Expression of miR-449a was evident in many anterior pituitary cells, including corticotrophs. Although overexpression of miR-449a decreased CRF-R1 mRNA and CRF-R1 protein expression, knockdown of miR-449a attenuated dexamethasone-induced suppression of CRF-R1 mRNA and CRF-R1 protein expression in the monolayer-cultured pituitary cells. Notably, luciferase activity was significantly lower in cells cotransfected with a luciferase vector containing the CRF-R1 3'-UTR and a miR-449a vector. miR-449a expression was significantly increased by dexamethasone. Adrenalectomy attenuated restraint-induced increase in miR-449a expression in the pituitary. These results indicated that miR-449a plays an important role in stress-induced, glucocorticoid-mediated downregulation of CRF-R1 expression.

Increased expression of miR-325-3p by urocortin 2 and its involvement in stress-induced suppression of LH secretion in rat pituitary.

Urocortin 2 (Ucn2) is a member of the corticotropin releasing factor (CRF) peptide family, which binds to CRF type 2 receptor. We previously reported on expression of Ucn2 in proopiomelanocortin cells of rat pituitary and its inhibitory action on LH secretion. We also demonstrated that Ucn2 is involved in the mechanism underlying immobilization-induced suppression of LH secretion; the details remain unclear. Here, we found that Ucn2 increased the expression of miR-325-3p, one of three microRNAs with predicted sequence for binding to LH ß-subunit 3'-untranslated region (3'-UTR) in monolayer cultured rat anterior pituitary cells, and that miR-325-3p was expressed in LH cells of the anterior pituitary. Immobilization also increased miR-325-3p expression in the anterior pituitary, and its increase was blocked by pretreatment with anti-Ucn2 IgG. Overexpression of miR-325-3p in cultured pituitary cells significantly suppressed intracellular contents and secretion of LH, while miR-325-3p knockdown blocked Ucn2-induced suppression of intracellular contents and secretion of LH. Coexpression of miR-325-3p with LH ß-subunit 3'-UTR-fused luciferase vector significantly suppressed luciferase activity compared with that of mock transfectants. These results suggest that miR-325-3p is involved in immobilization-induced suppression of LH translation and secretion and that Ucn2 plays a role in the increase in miR-325-3p expression.

The effects of ghrelin/GHSs on AVP mRNA expression and release in cultured hypothalamic cells in rats.

Ghrelin, the endogenous ligand for growth hormone secretagogues (GHSs) receptor (GHS-R), increases adrenocorticotropin (ACTH) and cortisol (corticosterone) as well as GH secretion in humans and animals. However, the site of GHSs action to induce ACTH secretion is not fully understood. To clarify the mechanisms of the action of ghrelin/GHSs on ACTH secretion, we analyzed the effects of KP-102 and ghrelin on the mRNA expression and release of corticotropin releasing factor (CRF) and arginine vasopressin (AVP), ACTH secretagogues, in monolayer-cultured hypothalamic cells of rats. Incubation of cells with KP-102 for 4h and 8h and with ghrelin for 4h significantly increased AVP mRNA expression and release without changing CRF mRNA expression. CRF levels in culture media were undetectable. Suppression of GHS-R expression by siRNA blocked ghrelin- and KP-102-induced AVP mRNA expression and release. NPY significantly increased AVP mRNA expression and release. Furthermore, treatment of cells with anti-NPY IgG blocked KP-102-induced AVP mRNA expression and release. We previously reported that KP-102 significantly increases NPY mRNA expression in cultured hypothalamic cells. Taken together, these results suggest that ACTH secretion by ghrelin/GHSs is induced mainly through hypothalamic AVP, and that NPY mediates the action of ghrelin/GHSs.