Takashi Fujihira

Interleukin-4 as a potent inhibitor of bone resorption

A possible role of interleukin-4 (IL-4) in the regulation of bone turnover was assessed by employing a 45Ca prelabeled-fetal mouse long bone culture system. IL-4 inhibited the bone resorption stimulated by parathyroid hormone (PTH), PTH related protein (PTHrP), 1 alpha, 25, dihydroxy-vitamin D3 [1 alpha, 25 (OH)2 D3], interleukin-1 alpha and - 1 beta (IL-1 alpha, IL-1 beta) and prostaglandin E2 (PGE2). Anti-IL-4 on monoclonal antibody abolished the inhibitory effect of IL-4 on the bone resorption. These results suggest that IL-4 may play an important role on the inhibitory regulation of bone resorption.

Centrally Administered Murine-Leptin Stimulates the Hypothalamus-Pituitary- Adrenal Axis through Arginine-Vasopressin

Starvation induces a decrease in circulating leptin levels and activation of the hypothalamus-pituitary-adrenal (HPA) axis. Leptin inhibits the HPA axis in unfed rodents or genetically leptin-deficient ob/ob mice, whereas it stimulates corticotropin-releasing hormone (CRH) gene expression in the paraventricular nucleus (PVN). However, the interactions between leptin, CRH and the HPA axis are poorly understood and are likely to be complex. We recently demonstrated that central leptin administration caused increases in plasma arginine-vasopressin (AVP) and AVP gene expression of the PVN in nonstressful rats. AVP stimulates the release of adrenocorticotropic hormone (ACTH), but it also potentiates the action of CRH on ACTH release. In this study, we investigated the effects of leptin on plasma ACTH and corticosterone levels, CRH mRNA of the PVN and proopiomelanocortin (POMC) mRNA of the pituitary in nonstrained rats. Intracerebroventricularly administered leptin caused increases in plasma ACTH and corticosterone levels in dose-dependent manners. In Northern blot analyses, the leptin injection induced significant increases in the expression of CRH mRNA in the PVN and POMC mRNA in the pituitary. The increased plasma ACTH and corticosterone levels by leptin were attenuated with intracerebroventricular pretreatment of a V1a receptor antagonist (OPC-21268) or a V1a/V1b receptor antagonist (dP[Tyr(Me)2]AVP), but not with that of a V2 receptor antagonist (OPC-31260). The leptin-induced CRH mRNA expression in the PVN and POMC mRNA expression in the pituitary were also reduced by the pretreatment with OPC-21268 and dP[Tyr(Me)2]AVP. These results suggest that intracerebroventricular leptin administration activates the HPA axis by AVP receptor activation through V1a receptors in the PVN which in turn activates CRH neurons to drive ACTH and corticosterone secretion in concert with AVP in nonstrained rats.

The receptor, metabolism and effects of androgen in osteoblastic MC3T3-E1 cells

We investigated the androgen receptor (AR), metabolism and effects of androgens in osteoblastic MC3T3-E1 cells. AR was proved as a transcript of a 10-kb mRNA and as a 110-kDa protein. An immunocytochemical study showed that AR was located mainly in the nuclei. Specific binding of [3H]DHT was observed in both the nuclear and cytosol fractions. MC3T3-E1 cells possessed approximately 1190 binding sites per cell and most of the sites (1150 sites) situated in the nucleus. The apparent Kd value in the nuclear fraction was 1.35 nM for [3H]DHT binding, and it was similar to that for [3H]testosterone. In the competition analysis, there was not much difference in the displacement of the [3H]DHT binding from AR between the addition of radioinert DHT and testosterone. In studies of 5 alpha-reductase activity and aromatase activity of the cells, both activities were lower than the respective values in classical androgen target tissues. Androgens stimulated the incorporation of [3H]thymidine into the cell, and DHT and testosterone had a similar potency on the cell proliferation. Thus, these results suggest testosterone itself acts mainly on the osteoblasts without conversion to DHT.

Parathyroid Hormone-Related Peptide-(1–34) [PTHrP- (1–34)] Induces Vasopressin Release from the Rat Supraoptic Nucleus in Vitro through a Novel Receptor Distinct from a Type I or Type II PTH/PTHrP Receptor

PTH and PTH-related peptide (PTHrP) bind to a type I PTH/PTHrP receptor expressed in bone and kidney or a type II receptor in nonclassical target tissue with equal affinity and similar bioactivities. PTHrP is abundant in the central nervous system, but its physiological role remains unknown. Herein, we examined the role of PTHrP-(1–34) on arginine vasopressin (AVP) release from the rat supraoptic nucleus (SON). Application of PTHrP-(1–34) to SON slices caused an increase in AVP release in a concentration-dependent manner. Neither PTHrP-(7–34) nor PTH-(1–34) had any effect on AVP release from the SON. PTHrP-(1–34)-induced AVP release was antagonized by a large excess of PTHrP-(7–34) and by H89, an inhibitor of cAMP-dependent protein kinase (A kinase), but not by PTH-(1–34) or PTH-(13–34). PTHrP-(1–34), but not PTH-(1–34), also dose-dependently increased the levels of cAMP in the SON. 125I-Labeled PTHrP-(1–34) bound specifically to crude membranes isolated from the SON. Scatchard analysis showed a single cl...

Centrally Administered Murine Leptin Stimulates Plasma Arginine-Vasopressin Secretion and Increases the Level of mRNA Expression in the Supraoptic Nucleus of Conscious Rats

The product of the ob gene protein, leptin, has been suggested to function as an endogenous mediator of the cardiovascular system via sympathetic nerve activity. Moreover, extensive distribution of leptin receptor-like immunoreactivity has been demonstrated in the choroid plexus, cerebral cortex, hippocampus, thalamus and hypothalamus, especially in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). In this study, we have investigated the in vivo effects of leptin on plasma arginine-vasopressin (AVP) secretion and the level of AVP messenger ribonucleotic acid (AVP mRNA) in the SON of conscious rats. Intracerebroventricularly administered leptin increased plasma AVP concentration in a dose-dependent manner (0–400 pmol/rat). The maximal effect was obtained at 15 min after the administration of leptin. Furthermore, in Northern blot analyses, the levels of AVP mRNa in the SON increased approximately 2-fold from the basal level after the administration of leptin. AVP mRNA expression in the PVN was also increased by leptin. However, leptin had no effects on plasma oxytocin (OXT) secretion and OXT gene expression in the SON. In conclusion, leptin is involved in AVP secretion via the central nervous system, however, its physiological role is unknown.

Stimulatory Effect of Interleukin‐1α on Proliferation through a Ca2+/Calmodulindependent Pathway of a Human Thyroid Carcinoma Cell Line, NIM 1

NIM 1 cells, a human thyroid cell line established from a patient with thyroid papillary adenocarcinoma, produce cytokines such as interleukin‐1α (IL‐1α) and granulocyte‐colony stimulating factor. In the present study, we investigated the signal transduction pathway in the proliferation of NIM 1 cells evoked by IL‐1α. Incubation of NIM 1 cells with IL‐1α for 48 h increased the incorporation of 3H‐thymidine (3H‐TdR). The stimulatory effect of IL‐1α was evident at 0.01 ng/ml and the maximal effect was seen at 10 ng/ml. IL‐1α evoked an influx of 45Ca into NIM 1 cells within 3 min in a concentration‐dependent manner (0.01–1 ng/ml). These stimulatory effects of IL‐1α on both 3H‐TdR incorporation and 45Ca influx were similarly inhibited by nicardipine, an inhibitor of voltage‐dependent Ca2+ channels, in a concentration‐dependent manner (10–1000 nM). The stimulatory effect of IL‐1α on 3H‐TdR incorporation was inhibited by N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7), an antagonist of calmodulin, but not by 1‐(5‐isoquinoline sulfonyl)‐2‐methylpiperazine (H‐7), an inhibitor of protein kinase C. While the culture medium initially contained 0.75 mM Ca2+, inhibition of 3H‐TdR incorporation by nicardipine and W‐7 under these baseline conditions was also recognized. These results suggest that IL‐1α stimulates cell proliferation through a Ca2+/calmodulin‐dependent pathway in NIM 1 cells.