Asuka Mano-Otagiri

Our previous studies, as well as other investigations, demonstrated that non-neuronal acetylcholine (ACh) produced by cardiomyocytes—that is, the non-neuronal cardiac cholinergic system (NNCCS)—is indispensable for sustaining the physiological functions and structural integrity of cardiomyocytes and for protecting the heart from ischemic/hypoxic insults, hypertrophic stress, and hypersympathetic conditions. These findings were supported by pharmacologically manipulated models in non-neuronal ACh systems and by gain- or loss-of-function models in the NNCCS. Nevertheless, the mechanisms underlying this phenomenon (i.e., sustention and protection) and the target of the NNCCS in cardiomyocytes remain to be fully elucidated. Our conditional murine model with heart-specific deletion of the choline acetyltransferase (ChAT) gene in the heart (hChAT KO mice) revealed cardiac dysfunction associated with heart failure symptoms. The representative culprit targets were the mitochondria with a disorganized appearance and dysfunction, accompanied by a reduction in mitochondrial DNA, membrane potential, and ATP production. Alternatively, malfunctioning mitochondria impaired cardiac energy metabolism and nicotinic receptor-mediated calcium responses in the mitochondria and down-regulated the mitochondrial calcium uniporter (MCU), leading to poor calcium handling by the mitochondria. The impaired cardiac function in hChAT KO mice induced systemic inflammatory responses and attenuated blood–brain barrier function, further influencing higher brain functions, including the aggravation of depression-like phenomenon. These specifically characteristic phenotypes indicate that the NNCCS principally plays a crucial role in sustaining mitochondrial functions through nicotinic receptors in the mitochondria and that the signal is indispensable for maintaining mitochondrial functions and integrity.

Corticotropin‐releasing factor (CRF) plays roles in stress‐related responses through its type 1 (CRF ₁ ) and type 2 receptors. Both CRF and CRF ₁ are expressed in the rat colon. Peripheral CRF administration and various stressors increase colonic motility and defecation. Stress induces CRF release in the colon, suggesting CRF may mediate stress‐related responses of the colon. The vagal nodose ganglion (NG) transduces visceral information, including colonic sensation, to the brain. However, it remains unclear whether the CRF/CRF ₁ system is involved in vagal afferent functions. This study, therefore, aimed to clarify the involvement of the CRF/CRF ₁ system in relaying visceral sensory information to the brain and the effect of stress exposure on vagal nerve function. The experiments were conducted in male rats. First, CRF ₁ ‐like immunoreactivity (CRF ₁ ‐LI) was characterized in the NG. Second, the effects of vagotomy on CRF ₁ ‐LI in the NG, intraperitoneally administered CRF‐induced fecal output, and c‐Fos expression in the nucleus tractus solitarius (NTS) were evaluated. Subsequently, a fast blue retrograde tracer was microinjected into the proximal colon. Finally, we analyzed CRF‐ or stress‐induced phosphorylation of cyclic AMP‐response element‐binding protein (pCREB) in the NG. CRF ₁ mRNA and CRF ₁ ‐LI were detected, and CRF ₁ ‐LI accumulated on the proximal side of the ligated region of the nerve trunk, and CRF ₁ ‐LI was detected in most cholinergic neurons. CRF ₁ siRNA suppressed the expression of CRF ₁ ‐LI in the NG. Subdiaphragmatic vagotomy decreased the number of CRF ₁ ‐positive cells in the NG while it did not affect CRF‐induced fecal output. CRF‐induced c‐Fos expression in the NTS was suppressed by vagotomy. A neuronal tracing study showed that approximately half of CRF ₁ ‐positive cells expressed fast blue in the NG. Intraperitoneal CRF, a selective CRF ₁ agonist, or immobilization stress induced pCREB expression and increases in CRF ₁ ‐positive cells in the NG. In contrast, a CRF ₁ antagonist reduced the immobilization‐induced increase in the expression of pCREB in the NG. These results suggest that the CRF/CRF ₁ system is involved in the signal transduction of colonic sensory information to the central nervous system via the NG.

Corticotrophin‐releasing factor (CRF) regulates the hypothalamic‐pituitary‐adrenal axis response to stress through its type 1 receptor (CRF₁) in the corticotrophs of the anterior pituitary. Although CRF₁ mRNA expression has been confirmed in the rat pituitary, the distribution pattern of CRF₁ protein in the pituitary has not been reported. Therefore, we generated an antiserum against the amino acid fragment corresponding to the 177–188 sequence of the first extracellular loop of the rat CRF₁. Using the antiserum, CRF₁‐like immunoreactivity (CRF₁‐LI) was detected in the anterior lobe cells of the rat pituitary where some of them expressed intense signals. CRF₁‐LI also appeared in the intermediate lobe cells and on the fibre‐like elements of the posterior lobe of the pituitary. Dual immunofluorescence labelling showed that corticotrophs exhibited the highest percentage of CRF₁ (male: 27.1 ± 3.0%, female: 18.0 ± 3.0%), followed by lactotrophs (male: 6.7 ± 3.0%, female: 12.1 ± 1.3%), gonadotrophs (male: 2.6 ± 1.0%, female: 7.5 ± 0.5%), thyrotrophs (male: 2.9 ± 0.1%, female: 5.3 ± 1.2%) and somatotrophs (male: 1.1 ± 0.3%, female: 1.2 ± 0.5%). The percentage of CRF₁‐LI‐positive cells that were corticotrophs was significantly higher in male rats than in female rats, whereas CRF₁‐LI‐positive lactotrophs and gonadotrophs were significantly higher in female rats than in male rats. Almost all of the melanotrophs were positive for CRF₁ in the intermediate lobe (98.9 ± 0.2%). CRF₁‐LI and the percentage of CRF₁‐LI in corticotrophs were decreased in the anterior pituitary, and the distribution patterns were altered from a diffuse to punctate one by adrenalectomy; the changes were restored by treatment with dexamethasone (100 μg/kg bw). These results suggest that CRF₁ is involved in the modulation of the functions of the pituitary; moreover, protein expression and the distribution patterns of CRF₁ are regulated by glucocorticoids in the rat anterior pituitary.

To clarify the role of ghrelin in the regulatory mechanism of energy metabolism, we analyzed the effects of centrally and peripherally administered ghrelin on noradrenaline release in the brown adipose tissue (BAT) of rats using a microdialysis system. I.c.v. administration of ghrelin at a dose of 500 pmol suppressed noradrenaline release in BAT, and microinjection of ghrelin (50 pmol) into the paraventricular nucleus (PVN) or arcuate nucleus (ARC) of the hypothalamus also suppressed noradrenaline release in BAT. In addition, i.v. administered ghrelin (30 nmol) suppressed noradrenaline release in BAT, and this suppression was blocked by a vagotomy. Neither i.c.v. nor i.v. administration of des-acyl ghrelin, which does not bind to GH secretagogue receptor type 1a (GHS-R1a), affected noradrenaline release in BAT. These results indicate that ghrelin increases energy storage by suppressing the activity of the sympathetic nerve innervating BAT. It seems that the PVN and ARC, which express GHS-R1a, are the sites of action of ghrelin in the brain and that the action of peripheral ghrelin on the sympathetic nerve activity innervating BAT is mediated by the vagal nerve, which also expresses GHS-R1a.

GH secretagogue (GHS)/ghrelin stimulates GH secretion by binding mainly to its receptor (GHS-R) on GHRH neurons in the arcuate nucleus (Arc) of the hypothalamus. GHRH, somatostatin, and neuropeptide Y (NPY) in the hypothalamus are involved in the regulatory mechanism of GH secretion. We previously created transgenic (Tg) rats whose GHS-R expression is reduced in the Arc, showing lower body weight and shorter nose-tail length. GH secretion is decreased in female Tg rats. To clarify how GHS-R affects GHRH expression in the Arc, we compared the numbers of GHS-R-positive, GHRH, and NPY neurons between Tg and wild-type rats. Immunohistochemical analysis showed that the numbers of GHS-R-positive neurons, GHRH neurons, and GHS-R-positive GHRH neurons were reduced in Tg rats, whereas the numbers of NPY neurons and GHS-R-positive NPY neurons did not differ between the two groups. The numbers of Fos-positive neurons and Fos-positive GHRH neurons in response to KP-102 were decreased in Tg rats. Competitive RT-PCR analysis of GHRH mRNA expression in the cultured hypothalamic neurons showed that KP-102 increased NPY mRNA expression level and that NPY decreased GHRH mRNA expression level. KP-102 increased GHRH mRNA expression level in the presence of anti-NPY IgG. GH increased somatostatin mRNA expression. Furthermore, GH and somatostatin decreased GHRH mRNA expression, whereas KP-102 showed no significant effect on somatostatin mRNA expression. These results suggest that GHS-R is involved in the up-regulation of GHRH and NPY expression and that NPY, somatostatin, and GH suppress GHRH expression. It is also suggested that the reduction of GHRH neurons of Tg rats is induced by a decrease in GHS-R expression.

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