Processing of daily and seasonal light information in the mammalian circadian clock

It is necessary for an organism's survival that many physiological functions and behaviours demonstrate daily and seasonal variations. A crucial component for the temporal control in mammals is the circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Neurons in the SCN generate a rhythm in electrical activity with a period of about 24h. The SCN receives photic information from photoreceptive ganglion cells in the retina and processes the information, detecting dawn and dusk as well as encoding day-length. Information processing by the SCN is optimized to extract relevant irradiance information and reduce interferences. Neuronal coupling pathways, including GABAergic signalling, are employed to distribute information and synchronize SCN subregions to form a uniform timing signal. Encoding of day-length is manifested in SCN neuronal activity patterns and may be the product of network interactions rather than being based on the single cell.     

Effects of melatonin on water metabolism and renal function in male Syrian hamsters (Mesocricetus auratus)

The pineal indoleamine, melatonin, has been shown to influence many physiological systems within the mammalian body. Few studies, however, have examined the influence of melatonin on renal function. This study investigated the effects of melatonin on water metabolism and renal function. Young adult male Syrian hamsters were maintained on a long photoperiod (LD 14:10) in metabolic cages. The animals received daily (1700) injections of either control vehicle or 25 micrograms of melatonin for 85 consecutive days. Melatonin administration resulted in significant increases in water consumption and urine production. Water budgets were also significantly influenced by melatonin, as were urinary osmolality, urinary sodium, and potassium concentrations, but urinary calcium concentrations were essentially unaltered. When excretion rates for sodium, potassium, and calcium were calculated, no differences were observed between the vehicle control and melatonin-treated groups. Injections of melatonin also significantly decreased plasma antidiuretic hormone (ADH). These results demonstrate that afternoon injections of melatonin can alter renal function, which may involve direct (i.e., on ADH secretion and/or thirst mechanisms) or indirect (i.e., behavioral) effects.     

Coexpression of MT1 and RORalpha1 melatonin receptors in the Syrian hamster Harderian gland

Melatonin acts through several specific receptors, including membrane receptors (MT(1) and MT(2)) and members of the RZR/ROR nuclear receptors family, which have been identified in a large variety of mammalian and nonmammalian cells types. Both membrane and nuclear melatonin receptors have been partially characterized in Harderian gland of the Syrian hamster. Nevertheless, the identities of these receptors were unknown until this study, where the coexistence of MT(1) and RORalpha(1) in this gland was determined by nested RT-PCR followed by amplicon sequencing and Western-blot. Furthermore, the cellular localization of both receptors was determined by immunohistochemistry. Thus, MT(1) receptor was localized exclusively at the basal side of the cell acini, supporting the hypothesis that this receptor is activated by the pineal-synthesized melatonin. On the contrary, although a RORalpha(1)-immunoreactivity was observed in nuclei of epithelial cells of both sexes, an extranuclear specific staining, which was more frequently among those cells of males, was also seen. The implication of this possible nuclear exclusion of RORalpha(1) on the role of this indoleamine against oxidative stress is discussed.     

Neural pathways involved in the photoperiodic control of reproductive physiology and behavior in female hamsters (Mesocricetus auratus)

Female hamsters received horizontal knife cuts to investigate the role of hypothalamic connections in the photoperiodic control of female reproductive functions. Knife cuts placed ventral to or through the paraventricular nucleus (PVN), but dorsal to the suprachiasmatic nucleus (SCN), prevented photoperiod-induced acyclicity and uterine regression in animals maintained under a nonstimulatory photoperiod for 10 weeks. The animals were then ovariectomized and tested for lordosis behavior following subcutaneous injections of ovarian hormones to investigate the photoperiodic modulation of female sexual behavior. Animals exposed to a nonstimulatory photoperiod were less behaviorally sensitive to treatment with estradiol benzoate (EB) alone, but did not differ from animals maintained under a stimulatory photoperiod when EB was combined with progesterone. The effect of photoperiod and behavioral sensitivity to hormone replacement was independent of the surgical condition. The results are consistent with the hypothesis that dorsal projections from the SCN to the PVN mediate gonadal responses to short photoperiods. They also indicate that photoperiod-induced changes in behavioral sensitivity to gonadal steroids may be mediated by neural pathways distinct from those that mediate the gonadal changes.     

Long-term daily melatonin infusion induces a large increase in N-acetyltransferase activity, hydroxyindole-O-methyltransferase activity, and melatonin content in the Harderian gland and eye of pinealectomized male Siberian hamsters (Phodopus sungorus)

The effects of long-term daily melatonin infusions on the melatonin synthetic pathway in the Harderian glands and eyes of male Siberian hamsters were studied. Hamsters were pinealectomized (PX) and infused daily for 8 hr with either melatonin (6 microg/hr) or vehicle for 7 days in short photoperiod (SP, 10L:14D), followed by 14 wk in either SP (SP group) or in constant darkness (DD group). After the infusion period (15 wk), the infusion was stopped and animals were transferred into SP for 3 wk. The hamsters were then killed at midday or midnight. Exogenous melatonin infusion caused an increase in the Harderian gland weight, which was still evident 3 wk after the end of the treatment. In addition, exogenous melatonin increased endogenous melatonin concentrations (4-fold) and hydroxyindole-O-methyltransferase (HIOMT) activity (2-fold). N-acetyltransferase (NAT) activity, however, was not increased, and no day/night difference in melatonin content and HIOMT activity was observed in the Harderian glands. In the eye, melatonin infusions significantly increased day and night-time melatonin levels (up to 3-fold) and both NAT and HIOMT activities (up to 3.5-fold). This effect of melatonin treatment was observed in both SP and DD groups. These observations demonstrate that exogenously-infused melatonin at relatively high doses activates the synthesis of endogenous melatonin in the Harderian gland and eye of the Siberian hamster. Circulating levels of melatonin were also markedly increased, indicating that in these conditions melatonin may be released from extra-pineal sites.     

C-terminal domains within human MT1 and MT2 melatonin receptors are involved in internalization processes

Abstract:  Melatonin, a molecule implicated in a variety of diseases, including cancer, often exerts its effects through G-protein-coupled melatonin receptors, MT₁ and MT₂. In this study, we sought to understand further the domains involved in the function and desensitization patterns of these receptors through site-directed mutagenesis. Two mutations were constructed in the cytoplasmic C-terminal tail of each receptor subtype: (i) a cysteine residue in the C-terminal tail was mutated to alanine, thus removing a putative palmitoylation site, and a site possibly required for normal receptor function (MT₁C7.72A and MT₂C7.77A) and (ii) the C-terminal tail in the MT₁ and MT₂ receptors was truncated, removing the putative phosphorylation and β-arrestin binding sites (MT₁Y7.64 and MT₂Y7.64). These mutations did not alter the affinity of 2-[¹²⁵I]-iodomelatonin binding to the MT₁ or MT₂ receptors. Using confocal microscopy, it was determined that the putative palmitoylation site (cysteine residue) did not play a role in receptor internalization; however, this residue was essential for receptor function, as determined by 3',5'-cyclic adenosine monophosphate (cAMP) accumulation assays. Truncation of the C-terminal tail of both receptors (MT₁Y7.64 and MT₂Y7.64) inhibited internalization as well as the cAMP response, suggesting the importance of the C-terminal tail in these receptor functions.     

Intraovarian expression of GnRH-1 and gonadotropin mRNA and protein levels in Siberian hamsters during the estrus cycle and photoperiod induced regression/recrudescence

The hypothalamic-pituitary-gonadal (HPG) axis is the key reproductive regulator in vertebrates. While gonadotropin releasing hormone (GnRH), follicle stimulating (FSH), and luteinizing (LH) hormones are primarily produced in the hypothalamus and pituitary, they can be synthesized in the gonads, suggesting an intraovarian GnRH-gonadotropin axis. Because these hormones are critical for follicle maturation and steroidogenesis, we hypothesized that this intraovarian axis may be important in photoperiod-induced ovarian regression/recrudescence in seasonal breeders. Thus, we investigated GnRH-1 and gonadotropin mRNA and protein expression in Siberian hamster ovaries during (1) the estrous cycle; where ovaries from cycling long day hamsters (LD;16L:8D) were collected at proestrus, estrus, diestrus I, and diestrus II and (2) during photoperiod induced regression/recrudescence; where ovaries were collected from hamsters exposed to 14 weeks of LD, short days (SD;8L:16D), or 8 weeks post-transfer to LD after 14 weeks SD (PT). GnRH-1, LHβ, FSHβ, and common α subunit mRNA expression was observed in cycling ovaries. GnRH-1 expression peaked at diestrus I compared to other stages (p < 0.05). FSHβ and LHβ mRNA levels peaked at proestrus and diestrus I (p < 0.05), with no change in the α subunit across the cycle (p > 0.05). SD exposure decreased ovarian mass and plasma estradiol concentrations (p < 0.05) and increased GnRH-1, LHβ, FSHβ, and α subunit mRNA expression as compared to LD and, except for LH, compared to PT (p < 0.05). GnRH and gonadotropin protein was also dynamically expressed across the estrous cycle and photoperiod exposure. The presence of cycling intraovarian GnRH-1 and gonadotropin mRNA suggests that these hormones may be locally involved in ovarian maintenance during SD regression and/or could potentially serve to prime ovaries for rapid recrudescence.     

Maternal transfer of photoperiodic information in Siberian hamsters. vi. effects of time-dependent 1-hr melatonin infusions in the mother on photoperiod-induced testicular development of her offspring

Abstract: We tested in Siberian hamsters the nature of the maternal signal that relays photoperiodic information to the developing fetuses. As previous investigations have identified maternal hormonal and circadian components in this process, the specific goal of this presentation is to determine quality of the signal that connotes daylength when it is imparted to the fetus. Does the function of the signal received by the fetus best support the coincidence or duration hypotheses of photoperiodic induction? Pregnant hamsters received 1 or 8 hr melatonin or vehicle infusions everyday. Juveniles of intact mothers gestated on 16 hr of light per day (16L) experienced maximal suppression of testicular development when reared on 14L. However, when intact mothers gestated on 10L received a 1‐hr melatonin infusion daily at 20:00–21:00 hr, their young responded to 14L with greatly accelerated testicular development. In the absence of the maternal pineal gland (and, therefore, the maternal melatonin signal), the effects of maternal melatonin infusions were reversed. Here, only the juveniles of 16L‐gestated females infused at 20:00–21:00 hr daily responded to 14L with enhanced testicular development. All other groups showed the same extent of gonadal development, independent of the time or type of infusion their mothers received. Testicular development on 14L of all juveniles from pinealectomized mothers gestated on 10L was of the same magnitude, regardless of the type and time of infusion their mothers received during pregnancy. The results suggest that the maternal signal transferred to the fetuses during gestation consists not only of the daily melatonin signal, but also some circadian‐based component that greatly affects the effect of the former. The timing, and not the duration, of the maternal melatonin signal with respect to the animals’ (mother and fetus) circadian day is of crucial importance in the transfer of photoperiodic information from mother to fetus.     

Circadian rhythm of mt1 melatonin receptor expression in the suprachiasmatic nucleus of the C3H/HeN mouse

This report studied the diurnal and circadian rhythms of mt1 melatonin receptor expression in the SCN of C3H/HeN mice maintained in either a light:dark (LD) cycle or in constant dark for a minimum of 6 wk. Diurnal times (ZT) were assessed with reference to the onset of the light period (ZT0) and circadian times (CT) were established by determining the phase of wheel running activity of each mouse before sacrifice. 2-[125I]-Iodomelatonin binding in the SCN revealed low amplitude diurnal and circadian rhythms with highest levels of binding 2 hr after lights on (41.3+/-1.7 fmol/mg protein, n = 5, at ZT2) or at the beginning of the subjective day (48.6+/-2.1 fmol/mg protein, n = 6, CT2), respectively. The expression of mt1 mRNA, determined by in situ hybridization with a 35S-labeled mouse mt1 riboprobe, showed robust diurnal and circadian rhythms. In animals housed under a LD cycle, low levels of expression were observed during the day, with a rapid rise in mt1 melatonin receptor expression at the beginning of the dark period (ZT14), coincident with an abrupt increase in levels of circulating melatonin measured by radioimmunoassay. In animals housed under constant dark conditions, a robust peak of mt1 mRNA expression occurred in the middle of the subjective night (CT18), 8 hr before the peak of protein expression, while the lowest levels of mt1 mRNA expression were observed during the day (CTI10). Results suggest that mt1 melatonin receptor rhythm in the C3H/HeN mouse SCN is regulated both by light and by the biological clock as distinct rhythms of both mRNA and protein are differentially expressed under a LD cycle and constant dark conditions.     

A test of the coincidence and duration models of melatonin action in Siberian hamsters. II. The effects of 4- and 8-hr melatonin infusions on testicular development of pinealectomized juvenile Siberian hamsters (Phodopus sungorus)

In a previous paper we demonstrated that properly timed 1-hr infusions of 50 ng melatonin effectively suppressed testicular development in juvenile Siberian hamsters. Only melatonin infused between 20:00 and 21:00 hr was effective in animals exposed to 16L (lights off 20:00 hr). In this paper we further investigate the importance of the coincidence and duration hypotheses of daily exposure of melatonin. Prepubertal Siberian hamsters received either 4- or 8-hr melatonin infusions at various times either on long photoperiod (LD 16:8 = 16L) or on short photoperiod (LD 10:14 = 10L). Daily 8-hr melatonin infusions suppressed testicular development in both photoperiods. Daily 4-hr, 50 ng/hr, melatonin infusions at 17:00-21:00 hr inhibited testicular growth in 16L and daily 4-hr melatonin infusions (either 50 ng/h or 50 ng/day) inhibited testicular growth at 17:00-21:00 hr in 10L. We also tested the efficacy of an interrupted melatonin infusion of long duration (8 hr). Pinealectomized prepubertal male Siberian hamsters, born on 16L, were infused with two signals of 4 hr separated by an interval of 2 hr. Melatonin-infused groups had significantly inhibited testicular growth compared to vehicle-infused animals. Testicular development was maximally inhibited only in those groups in which the period of melatonin sensitivity identified in the previous paper (20:00-21:00 hr) overlapped or immediately followed a period of melatonin infusion. Considering the restrictions of the experimental design employed in these studies, the results are best explained by the hypothesis that the photoperiodic gonadal response in juvenile Siberian hamsters is regulated by the coincidence in time of exogenously administered melatonin with an intrinsic rhythm of sensitivity to melatonin, which occurred at 20:00-21:00 hr. The duration of the melatonin signal alone can not explain the results.     

Melatonin rhythm and other outputs of the master circadian clock in the desert goat (Capra hircus) are entrained by daily cycles of ambient temperature

In desert areas, mammals such as camel and goat are exposed to harsh environmental conditions. The ambient temperature (Ta) cycles have been shown to entrain the circadian clock in the camel. In the present work, we assumed that, in the goat living in a desert biotope, Ta cycles would have the same synchronizing effect on the central clock. Therefore, the effects of Ta cycles on body temperature (Tb), locomotor activity (LA) and melatonin (Mel) rhythms as outputs of the master circadian clock have been studied. The study was performed on bucks kept first under constant conditions of total darkness (DD) and constant Ta, then maintained under DD conditions but exposed to Ta cycles with heat period during subjective day and cold period during subjective night. Finally, the Ta cycles were reversed with highest temperatures during the subjective night and the lowest temperatures during the subjective day. Under constant conditions, the circadian rhythms of Tb and LA were free running with an endogenous period of 25.3 and 25.0 hours, respectively. Ta cycles entrained the rhythms of Tb and LA to a period of exactly 24.0 hours; while when reversed, the Ta cycles led to an inversion of Tb and LA rhythms. Similarly, Ta cycles were also able to entrain Mel rhythm, by adjusting its secretion to the cooling phase before and after Ta cycles inversion. All together, these results show that the Ta cycles entrain the master circadian clock in the goat.     

Coincident light and clock regulation of pseudoresponse regulator protein 37 (PRR37) controls photoperiodic flowering in sorghum

Optimal flowering time is critical to the success of modern agriculture. Sorghum is a short-day tropical species that exhibits substantial photoperiod sensitivity and delayed flowering in long days. Genotypes with reduced photoperiod sensitivity enabled sorghum's utilization as a grain crop in temperate zones worldwide. In the present study, Ma(1), the major repressor of sorghum flowering in long days, was identified as the pseudoresponse regulator protein 37 (PRR37) through positional cloning and analysis of SbPRR37 alleles that modulate flowering time in grain and energy sorghum. Several allelic variants of SbPRR37 were identified in early flowering grain sorghum germplasm that contain unique loss-of-function mutations. We show that in long days SbPRR37 activates expression of the floral inhibitor CONSTANS and represses expression of the floral activators Early Heading Date 1, FLOWERING LOCUS T, Zea mays CENTRORADIALIS 8, and floral induction. Expression of SbPRR37 is light dependent and regulated by the circadian clock, with peaks of RNA abundance in the morning and evening in long days. In short days, the evening-phase expression of SbPRR37 does not occur due to darkness, allowing sorghum to flower in this photoperiod. This study provides insight into an external coincidence mechanism of photoperiodic regulation of flowering time mediated by PRR37 in the short-day grass sorghum and identifies important alleles of SbPRR37 that are critical for the utilization of this tropical grass in temperate zone grain and bioenergy production.     

Effects on bone by the light/dark cycle and chronic treatment with melatonin and/or hormone replacement therapy in intact female mice

Abstract: In this study, the effects of the light/dark cycle, hormone replacement therapy (HRT), and nocturnal melatonin supplementation on osteogenic markers and serum melatonin levels were examined in a blind mouse model (MMTV‐Neu transgenic mice). Melatonin levels in this mouse strain (FVB/N) with retinal degeneration (rd?/?) fluctuate in a diurnal manner, suggesting that these mice, although blind, still perceive light. Real‐time RT‐PCR analyses demonstrated that Runx2, Bmp2, Bmp6, Bglap, and Per2 mRNA levels coincide with melatonin levels. The effect of chronic HRT (0.5 mg 17β‐estradiol + 50 mg progesterone in 1800 kcal of diet) alone and in combination with melatonin (15 mg/L drinking water) on bone quality and density was also assessed by histomorphometry and microcomputed tomography, respectively. Bone density was significantly increased (P < 0.05) after 1 yr of treatment with the individual therapies, HRT (22% increase) and nocturnal melatonin (20% increase) compared to control. Hormone replacement therapy alone also increased surface bone, decreased trabecular space, and decreased the number of osteoclasts without affecting osteoblast numbers compared to the control group (P < 0.05). Chronic HRT + melatonin therapy did not significantly increase bone density, even though this combination significantly increased Bglap mRNA levels. These data suggest that the endogenous melatonin rhythm modulates markers important to bone physiology. Hormone replacement therapy with or without nocturnal melatonin in cycling mice produces unique effects on bone markers and bone density. The effects of these therapies alone and combined may improve bone health in women in perimenopause and with low nocturnal melatonin levels from too little sleep, too much light, or age.     

Evidence that membrane-bound G protein-coupled melatonin receptors MT1 and MT2 are not involved in the neuroprotective effects of melatonin in focal cerebral ischemia

Melatonin is synthesized and released by the pineal gland in a circadian rhythm, and many of its peripheral actions are mediated via membrane MT1 and MT2 receptors. Apart from its metabolic functions, melatonin is a potent neuroprotective molecule owing to its antioxidative actions. The roles of MT1 and MT2 in the neuroprotective effects of melatonin and cell signaling after cerebral ischemia remain unknown. With the use of MT1 and MT2 knockout (mt1/2(-/-) ) mice treated with melatonin, we evaluated brain injury, edema formation, inducible nitric oxide synthase (iNOS) activity, and signaling pathways, including CREB, ATF-1, p21, Jun kinase (JNK)1/2, p38 phosphorylation, resulting from ischemia/reperfusion injury. We show that the infarct volume and brain edema do not differ between mt1/2(-/-) and wild-type (WT) animals, but melatonin treatment decreases infarct volume in both groups and brain edema in WT animals after middle cerebral artery occlusion. Notably, melatonin's neuroprotective effect was even more pronounced in mt1/2(-/-) animals compared to that in WT animals. We also demonstrate that melatonin treatment decreased CREB, ATF-1, and p38 phosphorylation in both mt1/2(-/-) and WT mice, while p21 and JNK1/2 were reduced only in melatonin-treated WT animals in the ischemic hemisphere. Furthermore, melatonin treatment lowered iNOS activity only in WT animals. We provide evidence that the absence of MT1 and MT2 has no unfavorable effect on ischemic brain injury. In addition, the neuroprotective effects of melatonin appear to be mediated through a mechanism independent of its membrane receptors. The underlying mechanism(s) should be further studied using selective melatonin receptor agonists and antagonists.     

Functional expression of MT2 (Mel1b) melatonin receptors in human PAZ6 adipocytes

Several reports have demonstrated that the pineal hormone, melatonin, plays an important role in body mass regulation in mammals. To date, however, the target tissues and relevant biochemical mechanisms involved remain uncharacterized. As adipose tissue is the principal site of energy storage in the body, we investigated whether melatonin could also act on this tissue. Semiquantitative RT-PCR analysis revealed the expression of MT1 and MT2 melatonin receptor mRNAs in the human brown adipose cell line, PAZ6, as well as in human brown and white adipose tissue. Binding analysis with 2-[(125)I]iodomelatonin ((125)I-Mel) revealed the presence of a single, high affinity binding site in PAZ6 adipocytes with a binding capacity of 7.46 +/- 1.58 fmol/mg protein and a K(d) of 457 +/- 5 pM. Both melatonin and the MT2 receptor-selective antagonist, 4-phenyl-2-propionamidotetraline, competed with 2-[(125)I]iodomelatonin binding, with respective K(i) values of 3 x 10(-11) and 1.5 x 10(-11) M. Functional expression of melatonin receptors in PAZ6 adipocytes was indicated by the melatonin-induced, dose-dependent inhibition of forskolin-stimulated cAMP levels and basal cGMP levels with IC(50) values of 2 x 10(-9) and 3 x 10(-10) M, respectively. Modulation of the cGMP pathway by melatonin further supports functional expression of MT2 receptors, as this pathway was shown to be specific for that subtype in humans. In addition, long-term melatonin treatment of PAZ6 adipocytes was found to decrease the expression of the glucose transporter Glut4 and glucose uptake, an important parameter of adipocyte metabolism. These results suggest that melatonin may act directly at MT2 receptors on human brown adipocytes to regulate adipocyte physiology.     

Apelin secretion and expression of apelin receptors in 3T3-L1 adipocytes are differentially regulated by angiotensin type 1 and type 2 receptors

Adipocytes play pivotal roles in regulating metabolism through secretion of a variety of adipokines, which in turn is regulated by other metabolic factors (e.g., insulin). Understanding the regulations of adipokine secretion is important because adipokines are implicated with metabolic disorders, such as, obesity and diabetes mellitus. Here, we investigated the regulatory roles of angiotensin II (AngII) on the secretion of apelin in 3T3-L1 adipocytes, and distinct signaling pathways mediated by AngII receptor type 1 (AT₁) and type 2 (AT₂) were revealed. It was found that activation of AT₁ receptors stimulates apelin secretion in Ca²⁺, protein kinase C, and MAPK kinase dependent ways while activation of AT₂ receptors inhibits apelin secretion through cAMP and cGMP dependent pathways. Furthermore, we demonstrate that the expression of apelin receptor (APJ) is also similarly regulated by AT₁ and AT₂ receptors. Finally, a detailed AngII signaling map is proposed.     

Renal cortical cyclooxygenase 2 expression is differentially regulated by angiotensin II AT(1) and AT(2) receptors

Macula densa cyclooxygenase 2 (COX-2)-derived prostaglandins serve as important modulators of the renin-angiotensin system, and cross-talk exists between these two systems. Cortical COX-2 induction by angiotensin-converting enzyme (ACE) inhibitors or AT(1) receptor blockers (ARBs) suggests that angiotensin II may inhibit cortical COX-2 by stimulating the AT(1) receptor pathway. In the present studies we determined that chronic infusion of either hypertensive or nonhypertensive concentrations of angiotensin II attenuated cortical COX-2. Angiotensin II infusion reversed cortical COX-2 elevation induced by ACE inhibitors. However, we found that angiotensin II infusion further stimulated cortical COX-2 elevation induced by ARBs, suggesting a potential role for an AT(2) receptor-mediated pathway when the AT(1) receptor was inhibited. Both WT and AT(2) receptor knockout mice were treated for 7 days with either ACE inhibitors or ARBs. Cortical COX-2 increased to similar levels in response to ACE inhibition in both knockout and WT mice. In WT mice ARBs increased cortical COX-2 more than ACE inhibitors, and this stimulation was attenuated by the AT(2) receptor antagonist PD123319. In the knockout mice ARBs led to significantly less cortical COX-2 elevation, which was not attenuated by PD123319. PCR confirmed AT(1a) and AT(2) receptor expression in the cultured macula densa cell line MMDD1. Angiotensin II inhibited MMDD1 COX-2, and CGP42112A, an AT(2) receptor agonist, stimulated MMDD1 COX-2. In summary, these results demonstrate that macula densa COX-2 expression is oppositely regulated by AT(1) and AT(2) receptors and suggest that AT(2) receptor-mediated cortical COX-2 elevation may mediate physiologic effects that modulate AT(1)-mediated responses.     

Variations of mt1 melatonin receptor density in the rat uterus during decidualization,the estrous cycle and in response to exogenous steroid treatment

The expression of mt1 receptor protein in the rat uterus was investigated using an anti-mt1 polyclonal antibody against the rat mt1 receptor. A melatonin receptor protein of 37 kDa was detectable by Western blotting in the rat uterine membrane preparations. Autoradiography with the melatonin ligand, 2-[125I]iodomelatonin, was used to localize melatonin receptors in the uterus of the estrous rats and to study the changes of melatonin receptors in pregnancy. Melatonin receptors were found to be localized in the estrous rat uterine antimesometrial stroma. As decidualization of the uterine stroma progressed during pregnancy, the melatonin binding sites were progressively reduced and became confined to the antimesometrial non-decidualized outer stroma. 2-[125I]Iodomelatonin binding sites were not seen in the mesometrial stromal cells during pregnancy. The role of ovarian hormones in the regulation of uterine melatonin receptors was examined by studying the binding at various phases of the estrous cycle, after ovariectomy with and without follow-on treatment of estradiol (E2), progesterone (P4) or both. 2-[125I]Iodomelatonin binding in the rat uterus fluctuated during the estrous cycle, being lowest during metestrus. Ovariectomy caused an almost 70% reduction of 2-[125I]iodomelatonin binding compared with the control. Injections of ovariectomized (OVX) rats with E2 or P4 alone or in combination for 11 days induced a partial restoration of 2-[125I]iodomelatonin binding in the OVX rats. The results show that mt1 melatonin receptors in the rat antimesometrial stroma are regulated by ovarian hormones.