Metabolic fate on 1-hexylcarbamoyl-5-fluorouracil and 5-fluorouracil in mice bearing ascites sarcoma 180.

Patterns of metabolism and disposition in plasma of tumor-bearing mice after oral administration of [6(-14)C]1-hexylcarbamoyl-5-fluorouracil ([6(-14)C]HCFU) resembled those in plasma of normal mice, but elimination of [6(-14)C]HCFU and 5-fluorouracil (FUra) was slower in tumor-bearing mice. The level of 1-(5-hydroxyhexylcarbamoyl)-5-fluorouracil (HHCFU) was lower in tumor-bearing mice. Also detected in plasma were [6(-14)C]HCFU, HHCFU, 1-(3-carboxypropylcarbamoyl)-5-fluorouracil, FUra, 5,6-dihydro-5-fluorouracil, and alpha-fluoro-beta-alanine. FUra originating from [6(-14)C]HCFU was retained over 6 hours, whereas intact FUra after [6(-14)C]FUra administration disappeared within 2 hours. The pattern of metabolism in ascitic fluid was similar to that in plasma after [6(-14)C]HCFU and [6(-14)C]FUra administration, but FUra was retained for a longer period in ascitic fluid. In sarcoma 180 cells, the maximum concentration of total radioactivity was observed 1 or 2 hours after [6(-14)C]FUra or [6(-14)C]HCFU administration, respectively, and the level of intact HCFU was very low. The principal metabolites were nucleotides that were maintained for a long period after administration of both compounds. The pattern of other metabolites after [6(-14)C]HCFU administration was also similar to that after [6(-14)C]FUra administration.

Relationship between antitumor effect and metabolites of 5-fluorouracil in combination treatment with 5-fluorouracil and guanosine in ascites sarcoma 180 tumor system.

The antitumor activity of 5-fluorouracil (FUra) against ascites Sarcoma 180 was significantly enhanced by coadministration of guanosine, and slightly by adenosine, but not by cytidine or uridine. In advanced ascites Sarcoma 180, guanosine also enhanced the action of FUra, but adenosine, uridine, and cytidine did not. The potentiation of antitumor activity by guanosine was reversed by addition of cytidine. The antitumor activity of FUra was significantly potentiated when guanosine was administered either 0 to 15 min before or 5 min after FUra. Changes in metabolites of FUra after potentiation by guanosine were investigated. Total radioactivity in the plasma was significantly decreased 10 min after the combined administration of [6-14C]FUra (3 mg/kg i.p.) and guanosine (100 mg/kg i.p.) in comparison with that of [6-14C]FUra alone and was slightly decreased by coadministration of [6-14C]FUra and adenosine. Conversely, it was significantly increased by uridine or cytidine. The decrease in total radioactivity in the plasma caused by guanosine was completely reversed by addition of cytidine. FUra, 5-fluorouridine, alpha-fluoro-beta-ureidopropionic acid, and alpha-fluoro-beta-alanine were found in the plasma. Intact FUra accounted for about 55% of the total radioactivity. The proportion of metabolites of [6-14C]FUra was not changed by coadministration of [6-14C]FUra and guanosine, adenosine, or cytidine, but the proportion of FUrd was increased by uridine. In the ascitic fluid, the total radioactivity derived from [6-14C]FUra was decreased by its combined administration with guanosine, and it was reversed by addition of cytidine. This pattern was similar to that in the plasma. The main FUra compound was intact FUra itself (90%), and 5-fluorouridine accounted for 1% of the total radioactivity in the ascitic fluid. On the other hand, total radioactivity of [6-14C]FUra in the tumor cells was significantly and slightly increased by guanosine and adenosine, respectively. Total radioactivity after [6-14C]FUra in combination with uridine or cytidine was less than that after [6-14C]FUra alone. Incorporation of [6-14C]FUra into RNA was increased about 3.7 times by its combination with guanosine in comparison with FUra alone, and it was increased 2.0, 0.6, and 0.7 times by adenosine, uridine, and cytidine, respectively. Moreover, FUra-nucleotides were significantly increased by guanosine. The increased radioactivity in RNA and FUra-nucleotides of tumor cells caused by guanosine was completely reversed by cytidine. These changes in incorporation into tumor cells were comparable to those in antitumor activity against ascites Sarcoma 180. The potentiation of antitumor activity of FUra by guanosine was considered to be due to an increase in incorporation of FUra into FUra-nucleotides and RNA in the tumor cells.

Antagonistic effect of polyinosinic-polycytidylic acid on the cell lethality produced by 5-fluorouracil in human colon carcinoma cells in vitro.

The effect of polyinosinic X polycytidylic acid [poly(I) X poly(C)] on the cell lethality produced by 5-fluorouracil [FUra] and 5-fluorouridine [FUrd] was examined in human colon carcinoma cell line HT-29. Pretreatment of cells with poly(I) X poly(C) as well as during exposure to FUra or FUrd resulted in antagonism of the cell lethality generated by the fluoropyrimidines. Antagonism of FUra cytotoxicity was also produced by the 2'-O-methylated analogue, polyinosinic X poly-2'-O-methylcytidylic acid, but not by the individual single-stranded polynucleotides or by the component mononucleotides, cytidine 5'-phosphate and inosine 5'-phosphate. In contrast, cytidine 5'-phosphate blocked the toxicity of FUrd. The antagonism by poly(I) X poly(C) of FUra and FUrd cytotoxicity was related to the inhibition of their metabolism to fluorouridine triphosphate and their incorporation into RNA and not to inhibition of the synthesis of RNA. Antibodies to leukocyte and fibroblast interferons did not reverse the antagonistic activity of poly(I) X poly(C). These results indicate that poly(I) X poly(C) may be interfering with the transport and/or initial metabolism of FUra and FUrd to fluorouridine monophosphate which is independent of the ability of the double-stranded RNA to induce interferon.

Synergistic antitumor effect of fluoropyrimidines and polyinosinic-polycytidylic acid against L1210 leukemia.

The effect of polyriboinosinic X polyribocytidylic acid [poly(I) X poly(C)] on the antitumor activity of 5-fluorouracil (FUra) and 5-fluorouridine (FUrd) was evaluated in mice bearing L1210 leukemia. Coadministration intravenously of poly(I) X poly(C) and either FUra or FUrd on days 1,5, and 9 to mice bearing L1210 leukemia implanted subcutaneously resulted in a 40% greater increase in life span at the optimal antitumor dose versus FUra and FUrd alone. This effect appeared to result from greater host tolerance of a dose of FUra or FUrd which would otherwise be cytotoxic. The protective effect of poly(I) X poly(C) was also evident in non-tumor-bearing mice, as well as following administration of drug intraperitoneally to mice bearing the tumor implanted intraperitoneally. FUrd incorporation into RNA in the spleen, bone marrow, and small intestine revealed little or no changes after coadministration of poly(I) X poly(C). (2', 5')Oligo(A) synthetase activity, an indication of interferon activity, was markedly depressed in the spleen and bone marrow following treatment with FUrd; however, poly(I) X poly(C) administered together with FUrd returned (2', 5')oligo(A) synthetase activity to normal levels. These data indicate that poly(I) X poly(C) ameliorates the host toxicity of fluoropyrimidines, possibly via an interferon-mediated effect, and thereby results in enhanced therapeutic efficacy of the antimetabolite as an antitumor agent.

In vivo antitumor activity of multiple injections of recombinant interleukin 2, alone and in combination with three different types of recombinant interferon, on various syngeneic murine tumors.

We have used several transplantable experimental murine tumors to evaluate the potentiation of antitumor activity by a combination of human recombinant interleukin 2 (rHIL2) and recombinant interferons (rIFNs). The combination of rHIL2 and either human hybrid recombinant alpha-interferon A/D (rIFN-alpha A/D) or mouse recombinant beta-interferon (rIFN-beta) induced the s.c. adenocarcinoma 755, which had been established for 8 days, to regress, although rHIL2 or the rIFNs alone hardly inhibited the tumor's growth. Eight injections of the rHIL2-rIFN-alpha A/D combination cured 38% of the tumor-bearing mice. The rHIL2-rIFN-beta combination achieved a complete cure only when given in more than 13 injections. The administration of rHIL2 and mouse recombinant gamma-interferon (rIFN-gamma) markedly inhibited tumor growth of the s.c. established adenocarcinoma 755, but did not cure any of the mice. Other tumors, B16-F10 melanoma, and colon tumors 38 and 26 responded almost as well to a rHIL2-rIFN-alpha A/D or -beta combination, but not to a rHIL2-rIFN-gamma combination. The growth of Lewis lung carcinoma was inhibited to a lesser extent by all combinations, for which there were no long-term survivors. The combination therapy of rHIL2 and rIFN-beta produced a marked regression of the tumor in beige mice which have low natural killer activity, suggesting the activated natural killer cells not to be responsible for the therapeutic effect. And T-cell immunity may be important in the regression of s.c. established tumors, because of the lesser potentiation of antitumor activity in athymic mice. These results demonstrate that combination therapies of rHIL2 and rIFN-alpha A/D or -beta can function synergistically in the various s.c. established murine tumor systems and give further evidence in support of their clinical potential.

Ocular melatonin rhythms in the goldfish, Carassius auratus.

Ocular melatonin rhythms in the goldfish were studied and compared to those in the pineal organ and plasma. Under light:dark (LD) of 12 h light:12 h dark, melatonin contents in the eye as well as the pineal organ and plasma exhibited clear day-night changes with higher levels at mid-dark than at mid-light. However, melatonin contents in the eye at mid-light and mid-dark were approximately 100 and 9 times greater than those in the pineal organ, respectively. Day-night changes of ocular melatonin persisted after pinealectomy, which abolished those in plasma melatonin under LD 12:12. Ocular melatonin contents in the pinealectomized fish at mid-light were significantly higher than those in the sham-operated control. Under constant darkness (DD), circadian melatonin rhythms were observed in the eye but damped on the 3rd day, whereas plasma melatonin rhythms generated by the pineal organ persisted for at least 3 days. Under constant light, ocular melatonin contents exhibited a significant fluctuation with a smaller amplitude than that under DD, whereas plasma melatonin remained at low levels. These results indicate the involvement of LD cycles, a circadian clock, and the pineal organ in the regulation of ocular melatonin rhythms in the goldfish.

Melatonin signal transduction in the goldfish, Carassius auratus.

Generation and reception of melatonin signals in the goldfish, Carassius auratus, are reviewed. The photoreceptive pineal gland of the goldfish generates circulating melatonin rhythms according to a given photoperiod under light-dark cycles and in a circadian manner under continuous dark conditions. Melatonin is also produced in the retina in a similar fashion. Melatonin produced in the pineal gland and retina is considered to act as internal zeitgeber in the brain and retina, respectively, controlling various physiological events via specific melatonin binding sites that are coupled with G protein. The goldfish exhibit clear diurnal locomotor activity rhythms under light-dark cycles and free-running rhythms under constant conditions. However, the relationship between melatonin and locomotor activity rhythms in the goldfish remains unclear. Further studies should be required to demonstrate the roles of melatonin in the circadian system in this species.

Increase in ubiquitin-immunoreactive nuclei in rat pituitary luteinizing hormone cells after castration.

Immunocytochemical detection of ubiquitin in the nucleus of rat LH cells and the effects of castration and testosterone replacement on the occurrence of immunoreactive ubiquitin in the nucleus were investigated. Immunoreactive ubiquitin occurred in certain nuclei, mostly belonging to identified LH cells. The concentration of testosterone in blood was altered by castration and implantation of testosterone into castrated rats, and the occurrence of ubiquitin was examined weekly for the following 4 weeks. In castrated rats, the proportion of LH cells with ubiquitin-immunoreactive nuclei was high throughout the experiment. In castrated rats implanted with testosterone, on the contrary, the proportion remained significantly lower. Ubiquitin may be involved in the cellular activity of LH cells in the rat pituitary.

Characterization of extrathymic T cells induced by recombinant murine interferon-beta in the peritoneal cavity of nude mice.

The effects of recombinant murine interferon-beta (rMuIFN-beta) on extrathymic lymphocyte formation in the peritoneal cavity of BALB/c athymic nude mice and tumor-bearing nude mice were examined for comparison with BALB/c normal euthymic mice. The 7 days of administration of 1 x 10(5) IU rMuIFN-beta caused the number of these cells to increase remarkably in the peritoneal cavity as a unique subset of asialoGM1+CD4+CD8-TcR alpha beta+ T cells. The asialoGM1+CD4+ T cells produced IL-2 and IFN-gamma in primary culture without stimulant but did not proliferate. Thus, extrathymic T cells can be induced easily in the peritoneal cavity in addition to the thymus for host defense systems.

Enhanced therapeutic effects of anti-tumour agents against growth and metastasis of colon carcinoma 26 when given in combination with interferon and interleukin-2.

Chemoimmunotherapy of pulmonary metastases was investigated in a protocol of combined anti-tumour agents and interferon-beta and/or interleukin-2. The combination of interferon-beta and interleukin-2 after treatment with etoposide or cisplatin exerted profound therapeutic effects in an experimental model (lung colonization) using colon carcinoma 26, which was resistant to interferon-beta or to interleukin-2 alone. Cured mice treated with anti-tumour agents and cytokines rejected re-implanted tumours. Moreover, this approach also had profound effects on spontaneous pulmonary metastases, together with the effect on primary tumours. However, this combination of cytokines did not enhance the anti-tumour activity of etoposide in athymic mice with pulmonary metastases. Injections of tumour-bearing BALB/c mice with a combination of etoposide and these cytokines resulted in a marked increase in CD8+, asialo-GM1+ cells. Thus the combined treatment with interferon-beta and interleukin-2 after administration of cytotoxic drugs may induce specific anti-tumour immunity, and such combinations may offer a new approach to the development of effective therapy for cancer metastases.

Inhibitory effects of bovine lactoferrin on colon carcinoma 26 lung metastasis in mice.

In order to determine the effects of the multifunctional iron-binding glycoprotein, lactoferrin (LF), and related compounds on tumor growth and metastasis, bovine LF (bLF), and bLF hydrolysate and lactoferricin (bLFcin), active products generated by acid-pepsin hydrolysis were administered orally to BALB/c mice bearing subcutaneous (s.c.) implants of the highly metastatic colon carcinoma 26 (Co 26Lu). bLF and the bLF hydrolysate demonstrated significant inhibition of lung metastatic colony formation from s.c. implanted tumors without appreciable effects on tumor growth. bLFcin displayed a tendency for inhibition of lung metastasis. On the other hand, bLF did not exert marked anti-metastatic activity in athymic nude mice bearing Co 26Lu, though bLF had a tendency to inhibit the lung metastatic colony formation associated with anti-asialoGM1 antibody (Ab) treatment. AsialoGM1+ and CD8+ cells in white blood cells were increased after treatment with bLF. In vitro, the viability of Co 26Lu-F55 cells was markedly decreased when co-cultured with white blood cells from mice administrated bLF p.o., but recovered on treatment with anti-asialoGM1 Ab or anti-CD8 mAb and complement. The results suggest bLF and related compounds might find application as tools in the control of metastasis and that asialoGM1+ and CD8+ cells in the blood are important for their inhibitory effects.

Effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine on life-span and 5-fluorouracil metabolism in mice with hepatic metastases.

5-Fluorouracil (5FU) is rapidly metabolised in the liver by dihydrouracil dehydrogenase. Bromovinyluracil is formed in the liver from (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) by pyrimidine nucleoside phosphorylase and is a potent inhibitor of dihydrouracil dehydrogenase. The co-administration of 5FU (intravenously) and BVDU (orally) was investigated in normal BDF1 mice and in those bearing liver metastases of Lewis lung carcinoma. 5FU alone rapidly disappeared from plasma and liver within 60 min of dosing. Administered with BVDU, 5FU persisted in plasma and liver for 60-180 min. The combination also significantly enhanced the life-span of tumour-bearing mice. 5FU plus BVDU may have therapeutic potential in the treatment of primary and secondary liver tumours.

Antitumor activity of 1,2-diaminocyclohexane--platinum complexes against sarcoma-180 ascites form.

Platinum complexes derived from three isomers of 1,2-diaminocyclohexane have been synthesized and their antitumor activities were evaluated against ascites Sarcoma-180. All the platinum complexes had high antitumor activity. Platinum complexes derived from cis-1,2-diaminocyclohexane were more effective than those derived from trans-l-and trans-d-1,2-diaminocyclohexane. Among the platinum complexes tested, oxalato(cis-1,2-diminocyclohexane)platinum had a remarkably high therapeutic index. Modification of the nonleaving group as well as that of the leaving group is important in order to find better antitumor platinum complexes.

Inhibitory effects of bovine lactoferrin on intestinal polyposis in the Apc(Min) mouse.

Chemopreventive effects of bovine lactoferrin (bLF), previously shown to strongly inhibit intestinal carcinogenesis in rats (K. Sekine, E. Watanabe, J. Nakamura, N. Takasuka, D.J. Kim, M. Asamoto, V. Krutovskikh, T.H. Baba, T. Ota, M.A. Moore, M. Masuda, H. Sugimoto, H. Nishino, T. Kakizoe, H. Tsuda, Inhibition of azoxymethane-initiated colon tumor by bovine lactoferrin administration in F344 rats, Jpn. J. Cancer Res. 88 (1997) 523-526; K. Sekine, Y. Ushida, T. Kuhara, M. Iigo, H. Baba-Toriyama, M.A. Moore, M. Murakoshi, Y. Satomi, H. Nishino, T. Kakizoe, H. Tsuda, Inhibition of initiation and early stage development of aberrant crypt foci and enhanced natural killer activity in male rats administered bovine lactoferrin concomitantly with azoxymethane, Cancer Lett. 121 (1997) 211-216), on spontaneous intestinal polyp development were assessed in the ApcMin mouse, a model for both familial adenomatous polyposis and sporadic colon cancers. In the experiment, 54 mice at 6 weeks of age were given 2% bLF (15 mice), 0.2% bLF (15 mice) and AIN-93G (24 mice) as basal diet ad libitum for 8 weeks. An overall tendency for a reduction in the total number of polyps in the small intestine was evident in the bLF-treated animals, along with significant suppression in the jejunum at the 2% dose (P < 0.05, 68% of the control). In addition, body growth suppression, presumed to be due to anemia and/or intussusception as a consequence of numerous polyps in the intestine, was alleviated. No toxic effects were observed in the intestinal epithelium. Although not as obvious as observed for the rat case, the data suggest that bLF may be a chemopreventor of intestinal polyposis.

Inhibition of initiation and early stage development of aberrant crypt foci and enhanced natural killer activity in male rats administered bovine lactoferrin concomitantly with azoxymethane.

The influence of concomitant administration of bovine lactoferrin (bLF) on induction of aberrant crypt foci (ACF) by azoxymethane was investigated in male F344 rats. Two percent bLF and 3% Bifidobacterium longum (B. longum), as a positive control, significantly decreased the numbers of ACF as well as the total numbers of aberrant crypts reproducibly in three independent studies (2% bLF, P < 0.01; 3% B. longum, P < 0.05). Most importantly large size foci composed of four or more crypts were always significantly decreased by 2% bLF (P < 0.05). Additional investigation of the natural killer activity of spleen cells demonstrated enhancement by bLF (P < 0.01) and B. longum (P < 0.01) in line with the levels of influence on foci induction, indicating a possible role for elevated immune cytotoxicity in the observed inhibition.

Enhancing effect of bromovinyldeoxyuridine on antitumor activity of 5-fluorouracil against adenocarcinoma 755 in mice. Increased therapeutic index and correlation with increased plasma 5-fluorouracil levels.

A marked inhibition of the growth of solid tumor adenocarcinoma 755 was achieved by the combination of 5-fluorouracil (5-FU) with bromovinyldeoxyuridine (BVDU). The therapeutic index (LD50/ED50) for the combination of BVDU plus 5-FU was 8.1 and 3.9 upon intraperitoneal (i.p.) or oral (p.o.) administration, respectively. The therapeutic index of i.p. 5-FU given alone was 2.3, whereas for p.o. 5-FU given alone no therapeutic index could be established because of insufficient activity of the compound. Thus, the therapeutic index of 5-FU increased significantly when combined with BVDU. Pharmacokinetic studies revealed that upon i.p. or p.o. 5-FU administration plasma 5-FU levels rapidly declined, but that, in the combination with BVDU, the plasma clearance of 5-FU, especially following p.o. administration, was slowed down considerably. Antitumor activity of 5-FU correlated with AUC (area under the concentration x time curve), within the plasma 5-FU concentration range from 0.02 to 0.4 microgram/ml.

Enhancing effect of bromovinyldeoxyuridine on antitumor activity of 5'-deoxy-5-fluorouridine against adenocarcinoma 755 in mice. Correlation with pharmacokinetics of plasma 5-fluorouracil levels.

5'-Deoxy-5-fluorouridine (DFUR), whether or not combined with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was pursued in BDF1 mice from both a pharmacokinetic viewpoint, following a single oral dose administration, and an anticancer viewpoint, following 5 daily oral doses in mice inoculated subcutaneously with adenocarcinoma 755 tumor cells. Half-life (t1/2) values for the elimination of DFUR and 5-fluorouracil (5-FU) from plasma following DFUR (100 mg/kg) administration were about 0.80 and 0.39 hr, respectively. Plasma 5-FU AUC (area under the curve) values following oral DFUR (100 mg/kg) was 0.224 micrograms.hr/ml. If DFUR (100 mg/kg) was combined with BVDU (10 mg/kg) the t1/2 and AUC values for 5-FU increased from 0.39 to 1.24 hr, and from 0.224 to 1.699 micrograms.hr/ml, respectively. Thus, BVDU significantly increased the plasma levels of 5-FU. It had no effect on the plasma levels of DFUR. At 100 mg/kg, DFUR did not show a significant antitumor activity. At 500 mg/kg it effected a 90% inhibition in tumor growth. When combined with BVDU (10 mg/kg), DFUR at 100, 200 and 300 mg/kg reduced tumor growth by 96, 100 and 100%, respectively. The antitumor activity achieved by DFUR, in the presence or absence of BVDU, correlated highly significantly with the AUC values for plasma 5-FU.

Differential effects of 2,2'-anhydro-5-ethyluridine, a uridine phosphorylase inhibitor, on the antitumor activity of 5-fluorouridine and 5-fluoro-2'-deoxyuridine.

2,2'-Anhydro-5-ethyluridine (ANEUR), a potent inhibitor of uridine phosphorylase, markedly potentiated the antitumor activity of fluorouridine (FUR) against murine mammary adenocarcinoma 755 in BDF1 mice and human colon adenocarcinoma LS174T in athymic-nude mice. Whereas ANEUR annihilated the antitumor activity of 5-fluoro-2'-deoxyuridine (FUdR) and 5'-deoxy-5-fluorouridine (DFUR) in the murine adenocarcinoma 755 system, it did not alter the antitumor activity of FUdR in the human adenocarcinoma LS174T system. In vitro, ANEUR proved inhibitory to the phosphorolytic cleavage of both FUR and FUdR by uridine phosphorylase, and this could explain why in vivo conversion of FUR and FUdR to 5-fluorouracil was suppressed. FUR can be held directly responsible for the antitumor effects observed in the murine adenocarcinoma 755 system, whereas in the activity against human adenocarcinoma LS174T may be mediated by both FUR and FUdR.

A possible role of the pineal gland in acute immobilization-related suppression of naloxone-induced LH release in ovariectomized estrogen-primed rats.

It has been recently reported that acute immobilization stress almost completely suppresses the luteinizing hormone (LH) release induced by naloxone, a mu-opioid antagonist, in ovariectomized estrogen-primed rats. The present study examined the possible involvement of the pineal gland in the acute immobilization-related suppression of the naloxone-induced LH release. An intraventricular (ICV) injection of 15 microg naloxone produced an abrupt increase in circulating LH concentrations in non-stressed rats. The naloxone-induced LH release was completely eliminated when tested 60 min after the end of a 30 min session of acute immobilization. The same stress conditions did not affect LH-releasing hormone (LHRH)-induced LH release, suggesting that the stress-related suppression of the naloxone-induced LH release was a suprapituitary event. In chronically-pinealectomized rats, but not in sham-pinealectomized rats, naloxone injected 60 min after the end of the stress session evoked a significant increase in serum LH concentrations. However, naloxone injected ICV during the acute immobilization did not elicit LH release in either pinealectomized or sham-operated rats. Under non-stressed conditions, the LH secretory response to naloxone was similar in pinealectomized and sham-operated animals. The same stress (30 min immobilization) significantly increased pineal melatonin content as well as plasma melatonin concentrations in rats bearing intact pineal glands, indicating that stress actually affected the pineal function. These results provide evidence for a role of the pineal in the suppression of the LH response to naloxone after stress, but not during stress.

Circadian rhythm of melatonin release from the photoreceptive pineal organ of a teleost, ayu (Plecoglossus altivelis) in flow-thorough culture.

In the present study, we tested whether the pineal organ of ayu (Plecoglossus altivelis), an osmerid teleost close relative of salmonids, harbours a circadian oscillator regulating rhythmic melatonin release using flow-through culture. The pineal organ maintained under light/dark cycles released melatonin in a rhythmic fashion with high levels during the dark phase. A circadian rhythm of melatonin release persisted in constant darkness for at least four cycles. Characteristics of the circadian rhythm (free-running period, phase and amplitude) exhibited small variations among cultures when the data was normalized, indicating that this system is sufficient for the analysis of the circadian rhythm both at qualitative and quantitative levels. Six-hour extension of the light phase from the normal onset time of the dark phase or exposure to constant light for 36 or 48 h before transfer to constant darkness significantly inhibited melatonin release. Phase shifts in the circadian rhythm of melatonin release were also observed. Thus, the ayu pineal organ contains all the three essential components of the circadian system (a circadian clock, the photoreceptor responsible for photic entrainment of the clock, and melatonin generating system as an output pathway). This system should provide a useful model for analysing the physiological and molecular basis of the vertebrate circadian system. In addition, further comparative studies using salmonids and related species including ayu will provide some insight into the evolution of the roles of the pineal organ in the vertebrate circadian system.