Akiko Katoh

Oxytocin: Crossing the Bridge between Basic Science and Pharmacotherapy

Is oxytocin the hormone of happiness? Probably not. However, this small nine amino acid peptide is involved in a wide variety of physiological and pathological functions such as sexual activity, penile erection, ejaculation, pregnancy, uterus contraction, milk ejection, maternal behavior, osteoporosis, diabetes, cancer, social bonding, and stress, which makes oxytocin and its receptor potential candidates as targets for drug therapy. In this review, we address the issues of drug design and specificity and focus our discussion on recent findings on oxytocin and its heterotrimeric G protein‐coupled receptor OTR. In this regard, we will highlight the following topics: (i) the role of oxytocin in behavior and affectivity, (ii) the relationship between oxytocin and stress with emphasis on the hypothalamo–pituitary–adrenal axis, (iii) the involvement of oxytocin in pain regulation and nociception, (iv) the specific action mechanisms of oxytocin on intracellular Ca2+ in the hypothalamo neurohypophysial system (HNS) cell bodies, (v) newly generated transgenic rats tagged by a visible fluorescent protein to study the physiology of vasopressin and oxytocin, and (vi) the action of the neurohypophysial hormone outside the central nervous system, including the myometrium, heart and peripheral nervous system. As a short nine amino acid peptide, closely related to its partner peptide vasopressin, oxytocin appears to be ideal for the design of agonists and antagonists of its receptor. In addition, not only the hormone itself and its binding to OTR, but also its synthesis, storage and release can be endogenously and exogenously regulated to counteract pathophysiological states. Understanding the fundamental physiopharmacology of the effects of oxytocin is an important and necessary approach for developing a potential pharmacotherapy.

Highly Visible Expression of an Oxytocin-Monomeric Red Fluorescent Protein 1 Fusion Gene in the Hypothalamus and Posterior Pituitary of Transgenic Rats

We have generated rats bearing an oxytocin (OXT)-monomeric red fluorescent protein 1 (mRFP1) fusion transgene. The mRFP1 fluorescence was highly visible in ventral part of the supraoptic nucleus (SON) and the posterior pituitary in a whole mount. mRFP1 fluorescence in hypothalamic sections was also observed in the SON, the paraventricular nucleus (PVN), and the internal layer of the median eminence. Salt loading for 5 d caused a marked increase in mRFP1 fluorescence in the SON, the PVN, the median eminence, and the posterior pituitary. In situ hybridization histochemistry revealed that the expression of the mRNA encoding the OXT-mRFP1 fusion gene was observed in the SON and the PVN of euhydrated rats and increased dramatically after chronic salt loading. The expression of the endogenous OXT and the arginine vasopressin (AVP) genes were significantly increased in the SON and the PVN after chronic salt loading in both nontransgenic and transgenic rats. These responses were not different between male and female rats. Compared with nontransgenic rats, euhydrated and salt-loaded male and female transgenic rats showed no significant differences in plasma osmolality, sodium concentration, OXT, and AVP levels. Finally, we succeeded in generating a double-transgenic rat that expresses both the OXT-mRFP1 fusion gene and the AVP-enhanced green fluorescent protein fusion gene. Our new transgenic rats are valuable new tools to study the physiology of the hypothalamo-neurohypophysial system.

Exaggerated Response of a Vasopressin–Enhanced Green Fluorescent Protein Transgene to Nociceptive Stimulation in the Rat

Nociceptive stimulation elicits neuroendocrine responses such as arginine vasopressin (AVP) release as well as activation of the hypothalamo-pituitary-adrenal axis. We have generated novel transgenic rats expressing an AVP–enhanced green fluorescent protein (eGFP) fusion gene, and we examined the effects of nociceptive stimulation on transgene expression in the hypothalamus after subcutaneous injection of saline or formalin into the bilateral hindpaws in these rats. We have assessed (1) AVP levels in plasma and the changes of eGFP mRNA and AVP heteronuclear RNA (hnRNA) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) using in situ hybridization histochemistry, (2) gene expression changes in distinct magnocellular and parvocellular divisions of the PVN, (3) eGFP fluorescence in the SON, the PVN, the median eminence (ME), and the posterior pituitary gland (PP). Plasma AVP levels were significantly increased 15 min after formalin injection. In the same time period, the AVP hnRNA levels in the PVN were increased, especially in the parvocellular division of the PVN in formalin-injected rats. In the same region, eGFP mRNA levels after formalin injection were also significantly increased to a much greater extent than those of AVP hnRNA. The eGFP fluorescence in the SON, the PVN, the ME, and the PP was markedly increased in formalin-injected rats and especially increased in the parvocellular divisions of the PVN. Together, our results demonstrate robust and rapid changes in the expression of the AVP-eGFP transgene in the rat hypothalamus after acute nociceptive stimulation.

Centrally administered relaxin-3 induces Fos expression in the osmosensitive areas in rat brain and facilitates water intake.

The expression of the relaxin-3 gene, detected as a new member of the insulin superfamily using human genomic databases, is abundantly present in the brain and testis. Intracerebroventricularly (icv) administered relaxin-3 stimulates food intake. Icv administered relaxin (identical to relaxin-2 in humans) affects the secretion of vasopressin and drinking behavior. Relaxin-3 partly binds relaxin family peptide receptor 1, which is a specific receptor to relaxin. Thus, we hypothesized that relaxin-3 would have physiological effects in the body fluid balance. However, the effects of relaxin-3 in the body fluid balance remain unknown. In the present study, we revealed that icv administered relaxin-3 induced dense Fos-like immunoreactivity (Fos-LI) in the rat hypothalamus and circumventricular organs including the organum vasculosum of the lamina terminalis, the median preoptic nucleus, supraoptic nucleus (SON), the subfornical organ (SFO) and the paraventricular nucleus (PVN), that are related to the central regulation of body fluid balance. Icv administered relaxin-3 (54, 180 and 540 pmol/rat) also induced a significant increase in c-fos gene expression in a dose-dependent manner in the SON, SFO and PVN. Further, icv administered relaxin-3 (180 pmol/rat) significantly increased water intake, and the effect was as strong as that of relaxin-2 (180 pmol/rat). These results suggest that icv administered relaxin-3 activates osmosensitive areas in the brain and plays an important role in the regulation of body fluid balance.

Robust Up-Regulation of Nuclear Red Fluorescent-Tagged Fos Marks Neuronal Activation in Green Fluorescent Vasopressin Neurons after Osmotic Stimulation in a Double-Transgenic Rat

The up-regulation in the expression of mRNA or protein encoded by the c-fos gene is widely used as a marker of neuronal activation elicited by various stimuli. To facilitate the detection of activated neurons, we generated transgenic rats expressing a fusion gene consisting of c-fos coding sequences in frame with monomeric red fluorescent protein 1 (mRFP1) under the control of c-fos gene regulatory sequences (c-fos-mRFP1 rats). In c-fos-mRFP1 transgenic rats, 90 min after hypertonic saline ip administration, nuclear mRFP1 fluorescence was observed abundantly in brain regions known to be osmosensitive, namely the median preoptic nucleus, organum vasculosum lamina terminalis, supraoptic nucleus, paraventricular nucleus, and subfornical organ. Immunohistochemistry for Fos protein confirmed that the distribution of Fos-like immunoreactivity in nontransgenic rats was similar to those of mRFP1 fluorescence after ip administration of hypertonic saline in the transgenic rats. Several double-transgenic rats were obtained from matings between transgenic rats expressing an arginine vasopressin-enhanced green fluorescent protein fusion gene (AVP-eGFP rats) and c-fos-mRFP1 rats. In these double-transgenic rats, almost all eGFP neurons in the supraoptic nucleus and PVN expressed nuclear mRFP1 fluorescence 90 min after hypertonic saline administration. The c-fos-mRFP1 rats are a powerful tool that enables the facile identification of activated neurons in the nervous system. Furthermore, when combined with transgenes expressing another fluorophore under the control of cell-specific regulatory sequences, activation of specific neuronal cell types in response to physiological cues can be readily detected.

Efficacy of intratympanic steroid administration on idiopathic sudden sensorineural hearing loss in comparison with hyperbaric oxygen therapy

The efficacy of intratympanic steroid administration was examined in comparison with hyperbaric oxygen (HBO) therapy in patients with idiopathic sudden sensorineural hearing loss (ISSNHL).

Hyalinizing clear cell carcinoma arising from the base of the tongue

Hyalinizing clear cell carcinoma is a low-grade indolent and rare salivary gland tumor originally described by Milchgrub et al. in 1994. We herein report a case of this tumor of the base of the tongue. A 66-year-old Japanese woman presented with a large painless mass in the throat. Computed tomography and magnetic resonance imaging revealed a 40×30-mm well-defined ovoid tumor arising from the base of the tongue. She underwent tracheostomy followed by a resection of the tumor via the transmandibular approach combined with a right-sided supra-omohyoid neck dissection. Because the tumor invasion of the surrounding tissue was limited, the surgical defect at the base of the tongue was relatively small, and no reconstructive procedure needed to be performed. The tumor was histopathologically diagnosed as hyalinizing clear cell carcinoma of the minor salivary gland. Her postoperative clinical course was uneventful. No aspiration or difficulty upon deglutition was recognized when she started transoral ingestion on the eighth postoperative day. The patient is currently free from disease 21 months after surgery. The pathology, clinical characteristics, and treatment of hyalinizing clear cell carcinoma are bibliographically reviewed.

Specific expression of an oxytocin-enhanced cyan fluorescent protein fusion transgene in the rat hypothalamus and posterior pituitary

We have generated rats bearing an oxytocin (OXT)-enhanced cyan fluorescent protein (eCFP) fusion transgene designed from a murine construct previously shown to be faithfully expressed in transgenic mice. In situ hybridisation histochemistry revealed that the Oxt-eCfp fusion gene was expressed in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) in these rats. The fluorescence emanating from eCFP was observed only in the SON, the PVN, the internal layer of the median eminence and the posterior pituitary (PP). In in vitro preparations, freshly dissociated cells from the SON and axon terminals showed clear eCFP fluorescence. Immunohistochemistry for OXT and arginine vasopressin (AVP) revealed that the eCFP fluorescence co-localises with OXT immunofluorescence, but not with AVP immunofluorescence in the SON and the PVN. Although the expression levels of the Oxt-eCfp fusion gene in the SON and the PVN showed a wide range of variations in transgenic rats, eCFP fluorescence was markedly increased in the SON and the PVN, but decreased in the PP after chronic salt loading. The expression of the Oxt gene was significantly increased in the SON and the PVN after chronic salt loading in both non-transgenic and transgenic rats. Compared with wild-type animals, euhydrated and salt-loaded male and female transgenic rats showed no significant differences in plasma osmolality, sodium concentration and OXT and AVP levels, suggesting that the fusion gene expression did not disturb any physiological processes. These results suggest that our new transgenic rats are a valuable new tool to identify OXT-producing neurones and their terminals.

Fluorescent visualisation of the hypothalamic oxytocin neurones activated by cholecystokinin-8 in rats expressing c-fos-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 fusion transgenes.

The up‐regulation of c‐fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c‐fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c‐fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c‐fos gene regulatory sequences that drive the expression of a c‐fos‐eGFP fusion protein. In c‐fos‐eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)‐8, which selectively activates oxytocin (OXT)‐secreting neurones in the hypothalamus. In double transgenic rats that express c‐fos‐eGFP and an OXT‐monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1‐positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK‐8. It is possible that not only a plane image, but also three‐dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.

Possible contribution of pannexin channel to ATP-induced currents in vitro in vasopressin neurons isolated from the rat supraoptic nucleus

Release of arginine vasopressin (AVP) from magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) is controlled by the electrical activity of these neurons. ATP plays a crucial role in the regulation of SON MNCs by activating the purinergic P2X and P2Y receptors. Recent reports of interaction between P2X receptors and pannexin channels have provided new insights into the physiology of the central nervous system; however, the function of pannexin channels has not been assessed in AVP neurons. In the present study, we examined the possible contribution of the pannexin channel in ATP-induced responses in SON AVP neurons. We used the whole-cell patch-clamp technique in isolated rat SON MNCs that express an AVP-enhanced green fluorescent protein transgene. The ATP-induced current was inhibited in a concentration-dependent manner by pannexin channel blockers carbenoxolone and mefloquine, whereas the connexin channel blockers flufenamic acid and lanthanum had no effect. Multi-cell reverse transcriptase-polymerase chain reaction experiments confirmed the existence of pannexin-1 mRNA in AVP neurons. The involvement of the ATP-activated transient receptor potential vanilloid and acid-sensing ion channels was excluded. These results suggest that pannexin channels in SON AVP neurons are involved in the regulatory mechanisms of neuronal activity.