Expression of Circadian Clock and Melatonin Receptors within Cultured Rat Cardiomyocytes

Melatonin, the pineal gland hormone, provides entrainment of many circadian rhythms to the ambient light/dark cycle. Recently, cardiovascular studies have demostrated melatonin interactions with many physiological processes and diseases, such as hypertension and cardiopathologies. Although membrane melatonin receptors (MT1, MT2) and the transcriptional factor ROR alpha have been reported to be expressed in the heart, there is no evidence of the cell-type expressing receptors as well as the possible role of melatonin on the expression of the circadian clock of cardiomyocytes, which play an important role in cardiac metabolism and function. Therefore, the aim of this study was to evaluate the mRNA and protein expressions of MT1, MT2, and ROR alpha and to determine whether melatonin directly influences expression of circadian clocks within cultured rat cardiomyocytes. Adult rat cardiomyocyte cultures were created, and the cells were stimulated with 1 nM melatonin or vehicle. Gene expressions were assayed by real-time polymerase chain reaction (PCR). The mRNA and protein expressions of membrane melatonin receptors and RORa were established within adult rat cardiomyocytes. Two hours of melatonin stimulation did not alter the expression pattern of the analyzed genes. However, given at the proper time, melatonin kept Rev-erb alpha expression chronically high, specifically 12 h after melatonin treatment, avoiding the rhythmic decline of Rev-erb alpha mRNA. The blockage of MT1 and MT2 by luzindole did not alter the observed melatonin-induced expression of Rev-erb alpha mRNA, suggesting the nonparticipation of MT1 and MT2 on the melatonin effect within cardiomyocytes. It is possible to speculate that melatonin, in adult rat cardiomyocytes, may play an important role in the light signal transduction to peripheral organs, such as the heart, modulating its intrinsic rhythmicity. (Author correspondence: cipolla@icb.usp.br)

Blockage of Angiotensin II type 2 receptor prevents thyroxine-mediated cardiac hypertrophy by blocking Akt activation

Although most of effects of Angiotensin II (Ang II) related to cardiac remodelling can be attributed to type 1 Ang II receptor (AT(1)R), the type 2 receptor (AT(2)R) has been shown to be involved in the development of some cardiac hypertrophy models. In the present study, we investigated whether the thyroid hormone (TH) action leading to cardiac hypertrophy is also mediated by increased Ang II levels or by change on AT(1)R and AT(2)R expression, which could contribute to this effect. In addition, we also evaluated the possible contribution of AT(2)R in the activation of Akt and in the development of TH-induced cardiac hypertrophy. To address these questions, Wistar rats were treated with thyroxine (T(4), 0.1 mg/kg BW/day, i.p.), with or without AT(2)R blocker (PD123319), for 14 days. Cardiac hypertrophy was identified based on heart/body weight ratio and confirmed by analysis of atrial natriuretic factor mRNA expression. Cardiomyocyte cultures were used to exclude the influence of TH-related hemodynamic effects. Our results demonstrate that the cardiac Ang II levels were significantly increased (80%, P < 0.001) as well as the AT(2)R expression (50%, P < 0.05) in TH-induced cardiac hypertrophy. The critical involvement of AT(2)R to the development of this cardiac hypertrophy in vivo was evidenced after administration of AT(2) blocker, which was able to prevent in 40% (P < 0.01) the cardiac mass gain and the Akt activation induced by TH. The role of AT(2)R to the TH-induced cardiomyocyte hypertrophy was also confirmed after using PD123319 in the in vitro studies. These findings improve understanding of the cardiac hypertrophy observed in hyperthyroidism and provide new insights into the generation of future therapeutic strategies.

Angiotensin type 1 receptor mediates thyroid hormone-induced cardiomyocyte hypertrophy through the Akt/GSK-3 beta/mTOR signaling pathway

Several studies have implicated the renin angiotensin system in the cardiac hypertrophy induced by thyroid hormone. However, whether Angiotensin type 1 receptor (AT(1)R) is critically required to the development of T(3)-induced cardiomyocyte hypertrophy as well as whether the intracellular mechanisms that are triggered by AT(1)R are able to contribute to this hypertrophy model is unknown. To address these questions, we employed a selective small interfering RNA (siRNA, 50 nM) or an AT(1)R blocker (Losartan, 1 mu M) to evaluate the specific role of this receptor in primary cultures of neonatal cardiomyocytes submitted to T(3) (10 nM) treatment. The cardiomyocytes transfected with the AT(1)R siRNA presented reduced mRNA (90%, P < 0.001) and protein (70%, P < 0.001) expression of AT(1)R. The AT(1)R silencing and the AT(1)R blockade totally prevented the T(3)-induced cardiomyocyte hypertrophy, as evidenced by lower mRNA expression of atrial natriuretic factor (66%, P < 0.01) and skeletal alpha-actin (170%, P < 0.01) as well as by reduction in protein synthesis (85%, P < 0.001). The cardiomyocytes treated with T(3) demonstrated a rapid activation of Akt/GSK-3 beta/mTOR signaling pathway, which was completely inhibited by the use of PI3K inhibitors (LY294002, 10 mu M and Wortmannin, 200 nM). In addition, we demonstrated that the AT(1)R mediated the T(3)-induced activation of Akt/GSK-3 beta/mTOR signaling pathway, since the AT(1)R silencing and the AT(1)R blockade attenuated or totally prevented the activation of this signaling pathway. We also reported that local Angiotensin I/II (Ang I/II) levels (120%, P < 0.05) and the AT(1)R expression (180%, P < 0.05) were rapidly increased by T(3) treatment. These data demonstrate for the first time that the AT(1)R is a critical mediator to the T(3)-induced cardiomyocyte hypertrophy as well as to the activation of Akt/GSK-3 beta/mTOR signaling pathway. These results represent a new insight into the mechanism of T(3)-induced cardiomyocyte hypertrophy, indicating that the Ang I/II-AT(1)R-Akt/GSK-3 beta/mTOR pathway corresponds to a potential mediator of the trophic effect exerted by T(3) in cardiomyocytes.

History of hypertension and eplerenone in patients with acute myocardial infarction complicated by heart failure

In the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study ( n = 6632), eplerenone- associated reduction in all- cause mortality was significantly greater in those with a history of hypertension ( Hx- HTN). There were 4007 patients with Hx- HTN ( eplerenone: n = 1983) and 2625 patients without Hx- HTN ( eplerenone: n = 1336). Propensity scores for eplerenone use, separately calculated for patients with and without Hx- HTN, were used to assemble matched cohorts of 1838 and 1176 pairs of patients. In patients with Hx- HTN, all- cause mortality occurred in 18% of patients treated with placebo ( rate, 1430/ 10 000 person- years) and 14% of patients treated with eplerenone ( rate, 1058/ 10 000 person- years) during 2350 and 2457 years of follow- up, respectively ( hazard ratio [ HR]: 0.71; 95% CI: 0.59 to 0.85; P < 0.0001). Composite end point of cardiovascular hospitalization or cardiovascular mortality occurred in 33% of placebo-treated patients ( 3029/ 10 000 person- years) and 28% of eplerenone- treated patients (2438/10 000 person- years) with Hx- HTN ( HR: 0.82; 95% CI: 0.72 to 0.94; P = 0.003). In patients without Hx- HTN, eplerenone reduced heart failure hospitalization ( HR: 73; 95% CI: 0.55 to 0.97; P = 0.028) but had no effect on mortality ( HR: 0.91; 95% CI: 0.72 to 1.15; P = 0.435) or on the composite end point ( HR: 0.91; 95% CI: 0.76 to 1.10; P = 0.331). Eplerenone should, therefore, be prescribed to all of the post - acute myocardial infarction patients with reduced left ventricular ejection fraction and heart failure regardless of Hx- HTN.

Effects of Cobalt Chloride in Junctional Epithelium and Reduced Enamel Epithelium of the Rat Maxillary First Molar

Cobalt is one of the main components of cast metal alloys broadly used in dentistry. It is the constituent of 45 to 70% of numerous prosthetic works. There are evidences that metal elements cause systemic and local toxicity. The purpose of the present study was to evaluate the effects of cobalt on the junctional epithelium and reduced enamel epithelium of the first superior molar in rats, during lactation. To do this, 1-day old rats were used, whose mothers received 300mg of cobalt chloride per liter of distilled water in the drinker, during lactation. After 21 days, the rat pups were killed with an anesthetic overdose. The heads were separated, fixed in ""alfac"", decalcified and embedded in paraffin. Frontal sections stained with hematoxylin and eosin were employed. Karyometric methods allowed to estimate the following parameters: biggest, smallest and mean diameters, D/d ratio, perimeter, area, volume, volume/area ratio, eccentricity, form coefficient and contour index. Stereologic methods allow to evaluate: cytoplasm/nucleus ratio, cell and cytoplasm volume, cell number density, external surface/basal membrane ratio, thickness of the epithelial layers and surface density. All the collected data were subjected to statistic analysis by the non-parametric Wilcoxon-Mann-Whitney test. The nuclei of the studied tissues showed smaller values after karyometry for: diameters; perimeter, area, volume and volume/area ratio. Stereologically, it was observed, in the junctional epithelium and in the reduced enamel epithelium, smaller cells with scarce cytoplasm, reflected in the greater number of cells per mm3 of tissue. In this study, cobalt caused epithelial atrophy, indicating a direct action on the junctional and enamel epithelium.

Effects of Cadmium on the Rat Salivary Glands, During Lactation

Cadmium (Cd) in air, drinking water and food has the potential to affect the health of people, mainly those who live in highly industrialized regions. Cd affects placental function, can cross the placental barrier and directly modify fetal development. Once the organism is particularly susceptible to the exposition to the Cd during the perinatal period, and that this metal can be excreted in the milk, the aim of the present work was to study the effects of the constant exposition to drinkable water containing low levels of Cd during the lactation, on the salivary glands of the rat. Female rats received ad libitum drinking water containing 300mg/l of CdCl2 throughout the whole lactation. Control animals received a similar volume of water without Cd. Lactant rats (21 day old) were killed by lethal dose of anesthetic. The salivary glands were separated, fixed in ""alfac"" solution for 24 h, and serially sectioned. The 6 mu m thick sections were stained with hematoxylin and eosin. Nuclear glandular parameters were estimated, as well as cytoplasm and cell volume, nucleus/cytoplasm ratio, number and surface density, diameters and cell thickness. Mean body weight was 34.86 g for the control group and 18.56 g for the Cd-treated group. Histologically, the glandular acini were significantly smaller, the gland ducts were similar in both groups studied. The connective tissue was more abundant. In conclusion, the salivary glands (submandibular, parotid and sublingual) showed retarded growth after Cd intoxication.

NEONATAL HANDLING AND THE MATERNAL ODOR PREFERENCE IN RAT PUPS: INVOLVEMENT OF MONOAMINES AND CYCLIC AMP RESPONSE ELEMENT-BINDING PROTEIN PATHWAY IN THE OLFACTORY BULB

Early-life environmental events, such as the handling procedure, can induce long-lasting alterations upon several behavioral and neuroendocrine systems. However, the changes within the pups that could be causally related to the effects in adulthood are still poorly understood. In the present study, we analyzed the effects of neonatal handling on behavioral (maternal odor preference) and biochemical (cyclic AMP response element-binding protein (CREB) phosphorylation, noradrenaline (NA), and serotonin (5-HT) levels in the olfactory bulb (OB)) parameters in 7-day-old male and female rat pups. Repeated handling (RH) abolished preference for the maternal odor in female pups compared with nonhandled (NH) and the single-handled (SH) ones, while in RH males the preference was not different than NH and SH groups. In both male and female pups, RH decreased NA activity in the OB, but 5-HT activity increased only in males. Since preference for the maternal odor involves the synergic action of NA and 5-HT in the OB, the maintenance of the behavior in RH males could be related to the increased 5-HT activity, in spite of reduction in the NA activity in the OB. RH did not alter CREB phosphorylation in the OB of both male and females compared with NH pups. The repeated handling procedure can affect the behavior of rat pups in response to the maternal odor and biochemical parameters related to the olfactory learning mechanism. Sex differences were already detected in 7-day-old pups. Although the responsiveness of the hypothalamic-pituitary-adrenal axis to stressors is reduced in the neonatal period, environmental interventions may impact behavioral and biochemical mechanisms relevant to the animal at that early age. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

PKC stimulated by glucagon decreases UT-A1 urea transporter expression in rat IMCD

It is well-known that glucagon increases fractional excretion of urea in rats after a protein intravenous infusion. This effect was investigated by using: (a) in vitro microperfusion technique to measure [(14)C]-urea permeability (Pu x 10(-5) cm/s) in inner medullary collecting ducts (IMCD) from normal rats in the presence of 10(-7) M of glucagon and in the absence of vasopressin and (b) immunoblot techniques to determine urea transporter expression in tubule suspension incubated with the same glucagon concentration. Seven groups of IMCDs (n = 47) were studied. Our results revealed that: (a) glucagon decreased urea reabsorption dose-dependently; (b) the glucagon antagonist des-His(1)-[Glu(9)], blocked the glucagon action but not vasopressin action; (c) the phorbol myristate acetate, decreased urea reabsorption but (d) staurosporin, restored its effect; e) staurosporin decreased glucagon action, and finally, (f) glucagon decreased UT-A1 expression. We can conclude that glucagon reduces UT-A1 expression via a glucagon receptor by stimulating PKC.

Aquaporin 2 expression increased by glucagon in normal rat inner medullary collecting ducts

Yano Y, Cesar KR, Araujo M, Rodrigues Jr. AC, Andrade LC, Magaldi AJ. Aquaporin 2 expression increased by glucagon in normal rat inner medullary collecting ducts. Am J Physiol Renal Physiol 296: F54-F59, 2009. First published October 1, 2008; doi: 10.1152/ajprenal.90367.2008.-It is well known that Glucagon (Gl) is released after a high protein diet and participates in water excretion by the kidney, principally after a protein meal. To study this effect in in vitro perfused inner medullary collecting ducts (IMCD), the osmotic water permeability (Pf; mu m/s) at 37 degrees C and pH 7.4 in normal rat IMCDs (n = 36) perfused with Ringer/HCO(3) was determined. Gl (10(-7) M) in absence of Vasopressin (AVP) enhanced the Pf from 4.38 +/- 1.40 to 11.16 +/- 1.44 mu m/s (P < 0.01). Adding 10(-8), 10(-7), and 10(-6) M Gl, the Pf responded in a dose-dependent manner. The protein kinase A inhibitor H8 blocked the Gl effect. The specific Gl inhibitor, des-His(1)-[Glu(9)] glucagon (10(-7) M), blocked the Gl-stimulated Pf but not the AVP-stimulated Pf. There occurred a partial additional effect between Gl and AVP. The cAMP level was enhanced from the control 1.24 +/- 0.39 to 59.70 +/- 15.18 fm/mg prot after Gl 10(-7) M in an IMCD cell suspension. The immunoblotting studies indicated an increase in AQP2 protein abundance of 27% (cont 100.0 +/- 3.9 vs. Gl 127.53; P = 0.0035) in membrane fractions extracted from IMCD tubule suspension, incubated with 10(-6) M Gl. Our data showed that 1) Gl increased water absorption in a dose-dependent manner; 2) the anti-Gl blocked the action of Gl but not the action of AVP; 3) Gl stimulated the cAMP generation; 4) Gl increased the AQP2 water channel protein expression, leading us to conclude that Gl controls water absorption by utilizing a Gl receptor, rather than a AVP receptor, increasing the AQP2 protein expression.

DAILY VARIATION OF CONSTITUTIVELY ACTIVATED NUCLEAR FACTOR KAPPA B (NFKB) IN RAT PINEAL GLAND

In mammals, the production of melatonin by the pineal gland is mainly controlled by the suprachiasmatic nuclei (SCN), the master clock of the circadian system. We have previously shown that agents involved in inflammatory responses, such as cytokines and corticosterone, modulate pineal melatonin synthesis. The nuclear transcription factor NFKB, detected by our group in the rat pineal gland, modulates this effect. Here, we evaluated a putative constitutive role for the pineal gland NFKB pathway. Male rats were kept under 12 h: 12 h light-dark (LD) cycle or under constant darkness (DD) condition. Nuclear NFKB was quantified by electrophoretic mobility shift assay on pineal glands obtained from animals killed throughout the day at different times. Nuclear content of NFKB presented a daily rhythm only in LD-entrained animals. During the light phase, the amount of NFKB increased continuously, and a sharp drop occurred when lights were turned off. Animals maintained in a constant light environment until ZT 18 showed diurnal levels of nuclear NFKB at ZT15 and ZT18. Propranolol (20 mg/kg, i.p., ZT 11) treatment, which inhibits nocturnal sympathetic input, impaired nocturnal decrease of NFKB only at ZT18. A similar effect was observed in free-running animals, which secreted less nocturnal melatonin. Because melatonin reduces constitutive NFKB activation in cultured pineal glands, we propose that this indolamine regulates this transcription factor pathway in the rat pineal gland, but not at the LD transition. The controversial results regarding the inhibition of pineal function by constant light or blocking sympathetic neurotransmission are discussed according to the hypothesis that the prompt effect of lights-off is not mediated by noradrenaline, which otherwise contributes to maintaining low levels of nuclear NFKB at night. In summary, we report here a novel transcription factor in the pineal gland, which exhibits a constitutive rhythm dependent on environmental photic information. (Author correspondence: rpmarkus@usp.br)

Dissociation of circadian and light inhibition of melatonin release through forced desynchronization in the rat

Pineal melatonin release exhibits a circadian rhythm with a tight nocturnal pattern. Melatonin synthesis is regulated by the master circadian clock within the hypothalamic suprachiasmatic nucleus (SCN) and is also directly inhibited by light. The SCN is necessary for both circadian regulation and light inhibition of melatonin synthesis and thus it has been difficult to isolate these two regulatory limbs to define the output pathways by which the SCN conveys circadian and light phase information to the pineal. A 22-h light-dark (LD) cycle forced desynchrony protocol leads to the stable dissociation of rhythmic clock gene expression within the ventrolateral SCN (vlSCN) and the dorsomedial SCN (dmSCN). In the present study, we have used this protocol to assess the pattern of melatonin release under forced desynchronization of these SCN subregions. In light of our reported patterns of clock gene expression in the forced desynchronized rat, we propose that the vlSCN oscillator entrains to the 22-h LD cycle whereas the dmSCN shows relative coordination to the light-entrained vlSCN, and that this dual-oscillator configuration accounts for the pattern of melatonin release. We present a simple mathematical model in which the relative coordination of a single oscillator within the dmSCN to a single light-entrained oscillator within the vlSCN faithfully portrays the circadian phase, duration and amplitude of melatonin release under forced desynchronization. Our results underscore the importance of the SCN`s subregional organization to both photic input processing and rhythmic output control.

Melatonin inhibits LPS-induced NO production in rat endothelial cells

Endothelial cells produce NO by activation of constitutive nitric oxide synthase (NOS) and transcription of inducible NOS (iNOS). We have previously shown that melatonin, in the nanomolar range, inhibits activation of constitutive NOS, and in the present paper, we evaluated whether it could interfere with the expression of iNOS, which is activated by lipopolysaccharide (LPS), a major component of gram-negative bacteria cell walls. Primary cultures of rat endothelial cells were loaded with fluorescent probe for NO detection. Nuclear factor kappa B (NF-kappa B) translocation in endothelial cells elicited by LPS was measured by electromobility shift assay, and the vasodilation of aortic rings was accessed by recording isometric contraction. Melatonin in a micromolar but not in a nanomolar range inhibits the NO production induced by LPS. This effect is not dependent on the activation of G protein-coupled melatonin receptors. The nuclear NF-kappa B translocation is a process necessary for iNOS transcription, and melatonin also inhibits its translocation. LPS induced vasodilation only in endothelium-intact aortic rings, and melatonin (10 mu m) inhibits the vasodilation. Here, we show that concentrations compatible with nocturnal melatonin surge (nm) did not interfere with the activity of iNOS. Considering that micromolar melatonin concentrations could be locally achieved through production by activated immune competent cells, extra-pineal melatonin could have a protective effect against tissue injury. We propose that melatonin blocked the LPS-induced vasodilation by inhibiting the NF-kappa B pathway. Finally, we propose that the effect of melatonin on vascular reactivity is one of the mechanisms that underlies the protective effect of this indolamine against LPS.

Small mammals of the mid-Araguaia River in central Brazil, with the description of a new species of climbing rat

The mid-Araguaia River basin in central Brazil is considered a priority area for biodiversity conservation, and Parque Estadual do Cantao (PEC) is one of the most important protected areas in this ecotone between Cerrado and Amazonia. This area suffers an intensive human pressure with high rates of deforestation, and still remains poorly studied in terms of biodiversity. From June 2007 to November 2008 we sampled small mammals from both banks of the mid-Araguaia River, in the states of Tocantins and Para. Data are given about morphological traits, geographic distribution and natural history of 22 species of small non-volant mammals (eight marsupials and 14 rodents) surveyed at PEC and its surroundings. We also present mitochondrial phylogenetic analyses that allow species identification within the genera: Oecomys, Oligoryzomys and Rhipidomys, and delineate an undescribed species of Thrichomys. Based on morphologic and molecular data, we describe a new species of Rhipidomys previously assigned to R. nitela, which is apparently endemic to the Araguaia-Tocantins basin in the Cerrado. Additionally, our phylogenetic analyses provide support for the role played by the Araguaia River as an important geographic barrier for two sister species of Rhipidomys.

TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway

Nuclear factor-kappa B (NFKB), a pivotal player in inflammatory responses, is constitutively expressed in the pineal gland. Corticosterone inhibits pineal NFKB leading to an enhancement of melatonin production, while tumor necrosis factor (TNF) leads to inhibition of Aa-nat transcription and the production of N-acetylserotonin in cultured glands. The reduction in nocturnal melatonin surge favors the mounting of the inflammatory response. Despite these data, there is no clear evidence of the ability of the pineal gland to recognize molecules that signal infection. This study investigated whether the rat pineal gland expresses receptors for lipopolysaccharide (LPS), the endotoxin from the membranes of Gram-negative bacteria, and to establish the mechanism of action of LPS. Here, we show that pineal glands possess both CD14 and toll-like receptor 4 (TLR4), membrane proteins that bind LPS and trigger the NFKB pathway. LPS induced the nuclear translocation of p50/p50 and p50/RELA dimers and the synthesis of TNF. The maximal expression of TNF in cultured glands coincides with an increase in the expression of TNF receptor 1 (TNFR1) in isolated pinealocytes. In addition, LPS inhibited the synthesis of N-acetylserotonin and melatonin. Therefore, the pineal gland transduces Gram-negative endotoxin stimulation by producing TNF and inhibiting melatonin synthesis. Here, we provide evidence to reinforce the idea of an immune-pineal axis, showing that the pineal gland is a constitutive player in the innate immune response.

EXPRESSION OF COCAINE- AND AMPHETAMINE-REGULATED TRANSCRIPT IN THE RAT FOREBRAIN DURING POSTNATAL DEVELOPMENT

Cocaine- and amphetamine-regulated transcript (CART) is widespread in the rodent brain. CART has been implicated in many different functions including reward, feeding, stress responses, sensory processing, learning and memory formation. Recent studies have suggested that CART may also play a role in neural development. Therefore, in the present study we compared the distribution pattern and levels of CART mRNA expression in the forebrain of male and female rats at different stages of postnatal development: P06, P26 and P66. At 6 days of age (P06), male and female rats showed increased CART expression in the somatosensory and piriform cortices, indusium griseum, dentate gyrus, nucleus accumbens, and ventral premammillary nucleus. Interestingly, we found a striking expression of CART mRNA in the ventral posteromedial and ventral posterolateral thalamic nuclei. This thalamic expression was absent at P26 and P66. Contrastingly, at P06 CART mRNA expression was decreased in the arcuate nucleus. Comparing sexes, we found increased CART mRNA expression in the anteroventral periventricular nucleus of adult females. In other regions including the CA1, the lateral hypothalamic area and the dorsomedial nucleus of the hypothalamus, CART expression was not different comparing postnatal ages and sexes. Our findings indicate that CART gene expression is induced in a distinct temporal and spatial manner in forebrain sites of male and female rats. They also suggest that CART peptide participate in the development of neural pathways related to selective functions including sensory processing, reward and memory formation. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.