Adenosine modulates alpha2-adrenergic receptors through a phospholipase C pathway in brainstem cell culture of rats

Adenosine acts in the nucleus tractus solitarii (NTS), one of the main brain sites related to cardiovascular control. In the present study we show that A(1) adenosine receptor (A(1R)) activation promotes an increase on alpha(2)-adrenoceptor (Alpha(2R)) binding in brainstem cell culture from newborn rats. We investigated the intracellular cascade involved in such modulatory process using different intracellular signaling molecule inhibitors as well as calcium chelators. Phospholipase C, protein kinase Ca(2+)-dependent, IP(3) receptor and intracellular calcium were shown to participate in A(1R)/Alpha(2R) interaction. In conclusion, this result might be important to understand the role of adenosine within the NTS regarding autonomic cardiovascular control. (C) 2009 Elsevier B.V. All rights reserved.

Adenosine Modulatesa alpha(2)-Adrenergic Receptors within Specific Subnuclei of the Nucleus Tractus Solitarius in Normotensive and Spontaneously Hypertensive Rats

Adenosine Is known to modulate neuronal activity within the nucleus tractus solitarius (NTS). The modulatory effect of adenosine A, receptors (A(1R)) on alpha(2)-adrenoceptors (Adr(2R)) was evaluated using quantitative radioautography within NTS subnuclei and using neuronal culture of normotensive (WKY) and spontaneously hypertensive rats (SHR). Radioautography was used in a saturation experiment to measure Adr2R binding parameters (B(max), K(d)) In the presence of 3 different concentrations of N(6)-cyclopentyladenosine (CPA), an A(1R) agonist. Neuronal culture confirmed our radioautographic results. [(3)H]RX821002, an Adr(2R) antagonist, was used as a ligand for both approaches. The dorsomedial/dorsolateral subnucleus of WKY showed an increase in B(max) values (21%) Induced by 10 nmol/L of CPA. However, the subpostremal subnucleus showed a decrease in Kd values (24%) induced by 10 nmol/L of CPA. SHR showed the same pattern of changes as WKY within the same subnuclei; however, the modulatory effect of CPA was induced by I nmol/L (increased B(max), 17%; decreased K(d), 26%). Cell culture confirmed these results, because 10(-5) and 10(-7) mol/L of CPA promoted an Increase in [3H]RX821002 binding of WKY (53%) and SHR cells (48%), respectively. DPCPX, an AIR antagonist, was used to block the modulatory effect promoted by CPA with respect to Adr2R binding. In conclusion, our study shows for the first time an interaction between A(1R) that increases the binding of Adr2R within specific subnuclei of the NTS. This may be important In understanding the complex autonomic response induced by adenosine within the NTS. In addition, changes in interactions between receptors might be relevant to understanding the development of hypertension. (Hypertens Res 2008; 31: 2177-2186)

Sympathetic hyperactivity differentially affects skeletal muscle mass in developing heart failure: role of exercise training

Bacurau AV, Jardim MA, Ferreira JC, Bechara LR, Bueno CR Jr, Alba-Loureiro TC, Negrao CE, Casarini DE, Curi R, Ramires PR, Moriscot AS, Brum PC. Sympathetic hyperactivity differentially affects skeletal muscle mass in developing heart failure: role of exercise training. J Appl Physiol 106: 1631-1640, 2009. First published January 29, 2009; doi:10.1152/japplphysiol.91067.2008.-Sympathetic hyperactivity (SH) is a hallmark of heart failure (HF), and several lines of evidence suggest that SH contributes to HF-induced skeletal myopathy. However, little is known about the influence of SH on skeletal muscle morphology and metabolism in a setting of developing HF, taking into consideration muscles with different fiber compositions. The contribution of SH on exercise tolerance and skeletal muscle morphology and biochemistry was investigated in 3- and 7-mo-old mice lacking both alpha(2A)- and alpha(2C)-adrenergic receptor subtypes (alpha(2A)/alpha(2C)ARKO mice) that present SH with evidence of HF by 7 mo. To verify whether exercise training (ET) would prevent skeletal muscle myopathy in advanced-stage HF, alpha(2A)/alpha(2C)ARKO mice were exercised from 5 to 7 mo of age. At 3 mo, alpha(2A)/alpha(2C)ARKO mice showed no signs of HF and preserved exercise tolerance and muscular norepinephrine with no changes in soleus morphology. In contrast, plantaris muscle of alpha(2A)/alpha(2C)ARKO mice displayed hypertrophy and fiber type shift (IIA -> IIX) paralleled by capillary rarefaction, increased hexokinase activity, and oxidative stress. At 7 mo, alpha(2A)/alpha(2C)ARKO mice displayed exercise intolerance and increased muscular norepinephrine, muscular atrophy, capillary rarefaction, and increased oxidative stress. ET reestablished alpha(2A)/alpha(2C)ARKO mouse exercise tolerance to 7-mo-old wild-type levels and prevented muscular atrophy and capillary rarefaction associated with reduced oxidative stress. Collectively, these data provide direct evidence that SH is a major factor contributing to skeletal muscle morphological changes in a setting of developing HF. ET prevented skeletal muscle myopathy in alpha(2A)/alpha(2C)ARKO mice, which highlights its importance as a therapeutic tool for HF.

Ethanol consumption and pineal melatonin daily profile in rats

It is well known that melatonin participates in the regulation of many important physiological functions such as sleep-wakefulness cycle, motor coordination and neural plasticity, and cognition. However, as there are contradictory results regarding the melatonin production diurnal profile under alcohol consumption, the aim of this paper was to study the phenomenology and mechanisms of the putative modifications on the daily profile of melatonin production in rats submitted to chronic alcohol intake. The present results show that rats receiving 10% ethanol in drinking water for 35 days display an altered daily profile of melatonin production, with a phase delay and a reduction in the nocturnal peak. This can be partially explained by a loss of the daily rhythm and the 25% reduction in tryptophan hydroxylase activity and, mainly, by a phase delay in arylalkylamine N-acetyltransferase gene expression and a 70% reduction in its peak activity. Upstream in the melatonin synthesis pathway, the results showed that noradrenergic signaling is impaired as well, with a decrease in beta 1 and alpha 1 adrenergic receptors` mRNA contents and in vitro sustained loss of noradrenergic-stimulated melatonin production by glands from alcohol-treated rats. Together, these results confirm the alterations in the daily melatonin profile of alcoholic rats and suggest the possible mechanisms for the observed melatonin synthesis modification.

Matrix metalloproteinases cleave the beta(2)-adrenergic receptor in spontaneously hypertensive rats

Rodrigues SF, Tran ED, Fortes ZB, Schmid-Schonbein GW. Matrix metalloproteinases cleave the beta(2)-adrenergic receptor in spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 299: H25-H35, 2010. First published April 9, 2010; doi:10.1152/ajpheart.00620.2009.-We recently observed the enhanced serine and matrix metalloproteinase (MMP) activity in the spontaneously hypertensive rat (SHR) compared with its normotensive Wistar-Kyoto (WKY) rat and the cleavage of membrane receptors in the SHR by MMPs. We demonstrate in vivo that MMP-7 and MMP-9 injection leads to a vasoconstrictor response in microvessels of rats that is blocked by a specific MMP inhibitor (GM-6001, 1 mu M). Multiple pathways may be responsible. Since the beta(2)-adrenergic receptor (beta(2)-AR) is susceptible to the action of endogenous MMPs, we hypothesize that MMPs in the plasma of SHRs are able to cleave the extracellular domain of the beta(2)-AR. SHR arterioles respond in an attenuated fashion to beta(2)-AR agonists and antagonists. Aorta and heart muscle of control Wistar rats were exposed for 24 h (37 C) to fresh plasma of male Wistar and WKY rats and SHRs with and without doxycycline (30 mu M) and EDTA (10 mM) to reduce MMP activity. The density of extracellular and intracellular domains of beta(2)-AR was determined by immunohistochemistry. The density of the extracellular domain of beta(2)-AR is reduced in aortic endothelial cells and cardiac microvessels of SHRs compared with that of WKY or Wistar rats. Treatment of the aorta and the heart of control Wistar rats with plasma from SHRs, but not from WKY rats, reduced the number of extracellular domains, but not intracellular domains, of beta(2)-AR in aortic endothelial cells and cardiac microvessels. MMP inhibitors (EDTA and doxycycline) prevented the cleavage of the extracellular domain. Thus MMPs may contribute to the reduced density of the extracellular domain of beta(2)-AR in blood vessels and to the increased arteriolar tone of SHRs compared with normotensive rats.

Pyk2 mediates increased adrenergic contractile responses in arteries from DOCA-salt mice - Vasoactive Peptide Symposium

The calcium-dependent proline-rich tyrosine kinase (Pyk2), a nonreceptor protein activated by tyrosine phosphorylation, links G protein-coupled receptors to vascular responses. We tested the hypothesis that enhanced vascular reactivity in deoxycorticosterone acetate (DOCA)-salt hypertensive mice is due to increased activation of Pyk2. Aorta and small mesenteric arteries from DOCA-salt and uninephrectomized (UNI) male C57B1/6 mice were used. Systolic blood pressure (mm Hg) was higher in DOCA (126 +/- 3) vs. UNI (100 +/- 4) mice. Vascular responses to phenylephrine (1 nM to 100 mu M) were greater both in aorta and small mesenteric arteries from DOCA-salt than UNI, but treatment with Tyrphostin A-9 (0.1 mu M, Pyk2 inhibitor) abolished the difference among the groups. Pyk2 levels, as well as phospho-Pyk2(Tyr402), paxillin, and phospho-paxillin(Tyr118) were increased in DOCA-salt aorta. Incubation of vessels with Tyrphostin A-9 restored phosphorylation of Pyk2 and paxillin. Increased activation of Pyk2 contributes to increased vascular contractile responses in DOCA-salt mice. J Am Soc Hypertens 2008;2(6): 431-438. (C) 2008 American Society of Hypertension. All rights reserved.

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.

Acquisition of Pavlovian Fear Conditioning Using beta-Adrenoceptor Activation of the Dorsal Premammillary Nucleus as an Unconditioned Stimulus to Mimic Live Predator-Threat Exposure

In the present work, we sought to mimic the internal state changes in response to a predator threat by pharmacologically stimulating the brain circuit involved in mediating predator fear responses, and explored whether this stimulation would be a valuable unconditioned stimulus (US) in an olfactory fear conditioning paradigm (OFC). The dorsal premammillary nucleus (PMd) is a key brain structure in the neural processing of anti-predatory defensive behavior and has also been shown to mediate the acquisition and expression of anti-predatory contextual conditioning fear responses. Rats were conditioned by pairing the US, which was an intra-PMd microinjection of isoproterenol (ISO; beta-adrenoceptor agonist), with amyl acetate odor-the conditioned stimulus (CS). ISO (10 and 40 nmol) induced the acquisition of the OFC and the second-order association by activation of beta-1 receptors in the PMd. Furthermore, similar to what had been found for contextual conditioning to a predator threat, atenolol (beta-1 receptor antagonist) in the PMd also impaired the acquisition and expression of OFC promoted by ISO. Considering the strong glutamatergic projections from the PMd to the dorsal periaqueductal gray (dPAG), we tested how the glutamatergic blockade of the dPAG would interfere with the OFC induced by ISO. Accordingly, microinjections of NMDA receptor antagonist (AP5, 6 nmol) into the dPAG were able to block both the acquisition, and partially, the expression of the OFC. In conclusion, we have found that PMd beta-1 adrenergic stimulation is a good model to mimic predatory threat-induced internal state changes, and works as a US able to mobilize the same systems involved in the acquisition and expression of predator-related contextual conditioning. Neuropsychopharmacology (2011) 36, 926-939; doi:10.1038/npp.2010.231; published online 5 January 2011

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)

New Perspectives on beta-Adrenergic Mediation of Innate and Learned Fear Responses to Predator Odor

In the present study, we investigated the role of noradrenergic transmission in unconditioned and conditioned responses to predatory threats. First, we examined the effects of systemically injected beta-blockers on unconditioned and contextual conditioned response to cat odor. The centrally acting beta-blocker (propranolol) was able to impair unconditioned responses, as well as the acquisition of the contextual fear to cat odor; however, the peripherally acting (nadolol) was not effective. Next, we examined the neural substrate underlying the noradrenergic modulation of the defensive response to cat odor and focused on the dorsal premammillary nucleus (PMd), because it represents the hypothalamic site most responsive to predatory threats and, at the same time, presents a dense plexus of noradrenergic fibers. We were able to see that propranolol significantly reduced PMd-Fos expression in response to cat odor and that beta-adrenoceptor blockade in the PMd, before cat odor exposure, reduced defensive responses to the cat odor and to the cat odor-related environment. We have also shown that beta-adrenoceptor blockade in the PMd, before the exposure to cat odor-related context, impaired the contextual conditioned responses. Overall, the present results provide convincing evidence suggesting that central noradrenergic mediation is critical for the expression of unconditioned and contextual conditioned antipredatory responses. We have further shown that the PMd appears to be an important locus to mediate these beta-adrenoceptor effects.

Crotalphine induces potent antinociception in neuropathic pain by acting at peripheral opioid receptors

Neuropathic pain is an important clinical problem and it is usually resistant to the current therapy. We have recently characterized a novel analgesic peptide, crotalphine, from the venom of the South American rattlesnake Crotalus durissus terrificus. In the present work, the antinociceptive effect of crotalphine was evaluated in an experimental model of neuropathic pain induced in rats by chronic constriction, of sciatic nerve. The effect of the peptide was compared to that induced by the crude venom, which confirmed that crotalphine is responsible for the antinociceptive effect of the crotalid venom on neuropathic pain. For characterization of neuropathic pain, the presence of hyperalgesia, allodynia and spontaneous pain was assessed at different times after nerve constriction. These phenomena were detected 24 h after surgery and persisted at least for 14 days. The pharmacological treatments were performed on day 14 after surgery. Crotalphine (0.2-5 mu g/kg) and the crude venom (400-1600 mu g/kg) administered p.o. inhibited hyperalgesia, allodynia and spontaneous pain induced by nerve constriction. The antinociceptive effect of the peptide and crude venom was long lasting, since it was detected up to 3 days after treatment. Intraplantar injection of naloxone (1 mu g/paw) blocked the antinociceptive effect, indicating the involvement of opioid receptors in this phenomenon. Gabapentin (200 mg/kg, p.o.), and morphine (5 mg/kg, s.c.), used as positive controls, blocked hyperalgesia and partially inhibited allodynia induced by nerve constriction. These data indicate that crotalphine induces a potent and long lasting opioid antinociceptive effect in neuropathic pain that surpasses that observed with standard analgesic drugs. (C) 2008 Elsevier B.V. All rights reserved.

TRPV1 receptors modulate retinal development

We investigated the possible participation of TRPV1 channels in retinal apoptosis and overall development. Retinas from newborn, male albino rats were treated in vitro with capsazepine, a TRPV1 antagonist. The expression of cell cycle markers was not changed after TRPV1 blockade, whereas capsazepine reduced the number of apoptotic cells throughout the retina,increased ERK1/2 and p38 phosphorylation and slightly reduced JNK phosphorylation. The expression of BAD, Bcl-2, as well as integral and cleaved capsase-3 were similar in all experimental conditions. Newborn rats were kept for 2 months after receiving high doses of capsazepine. In their retinas, calbindin and parvalbumin protein levels were upregulated, but only the number of amacrine-like, parvalbumin-positive cells was increased. The numbers of calretinin, calbindin, ChAT, vimentin, PKC-alpha and GABA-positive cells were similar in both conditions. Protein expression of synapsin Ib was also increased in the retinas of capsazepine-treated rats. Calretinin, vimentin, GFAP, synapsin Ia, synaptophysin and light neurofilament protein levels were not changed when compared to control values. Our results indicate that TRPV1 channels play a role in the control of the early apoptosis that occur during retinal development, which might be dependent on MAPK signaling. Moreover, it seems that TRPV1 function might be important for neuronal and synaptic maturation in the retina. (C) 2011 ISDN. Published by Elsevier Ltd. All rights reserved.

Pharmacological properties of purinergic receptors and their effects on proliferation and induction of neuronal differentiation of P19 embryonal carcinoma cells

We have used P19 embryonal carcinoma cells as in vitro model for early neurogenesis to study ionotropic P2X and metabotropic P2Y receptor-induced Ca2+ transients and their participation in induction of proliferation and differentiation. In embryonic P19 cells, P2Y(1), P2Y(2) and P2X(4) receptors or P2X-heteromultimers with similar P2X4 pharmacology were responsible for ATP and ATP analogue-induced Ca2+ transients. In neuronal-differentiated cells, P2Y(2), P2Y(6), P2X(2) and possibly P2X(2)/P2X(6) heteromeric receptors were the major mediators of the elevations in intracellular free calcium concentration [Ca2+](i). We have collected evidence for the involvement of metabotropic purinergic receptors in proliferation induction of undifferentiated and neural progenitor cells by using a BrdU-incorporation assay. ATP-, UTP-, ADP-, 2-MeS-ATP- and ADP-beta S-induced proliferation in P19 cells was mediated by P2Y, and P2Y2 receptors as judged from pharmacological profiles of receptor responses. ATP-provoked acceleration of neuronal differentiation, determined by analysis of nestin and neuron-specific enolase gene and protein expression, also resulted from P2Y, and P2Y2 receptor activation. Proliferation- and differentiation-induction involved the activation of inositol-trisphosphate sensitive intracellular Ca2+ stores. (C) 2008 ISDN. Published by Elsevier Ltd. All rights reserved.

Retinal removal up-regulates cannabinoid CB1 receptors in the chick optic tectum

The endocannabinoid system has been implicated in several neurobiological processes, including neurodegeneration and neuro protection. The aim of this study was to evaluate the effects of unilateral retinal ablation on the expression of the cannabinoid receptor subtype 1 (CB1) at both protein and mRNA levels in the optic tectum of the adult chick brain. After different survival times postlesion (2-30 days), the chick brains were subjected to immunohistochemical, immunoblotting, and real-time PCR procedures to evaluate CB1 expression. TUNEL and Fluoro-Jade B were used to verify the possible occurrence of cell death, and immunostaining for the microtubule-associated protein MAP-2 was performed to verify possible dendritic remodeling after lesions. No cell death could be observed in the deafferented tectum, at least up to 30 days postlesion, although Fluoro-Jade B could reveal degenerating axons and terminals. Retinal ablation seems to generate an increase of CB1 protein in the optic tectum and other retinorecipient visual areas, which paralleled an increase in MAP-2 staining. On the other hand, CB, mRNA levels were not changed after retinal ablation. Our results reveal that CB, expression in visual structures of the adult chick brain may be negatively regulated by the retinal innervation. The increase of CB1 receptor expression observed after retinal removal indicates that these receptors are not presynaptic in retinal axons projecting to the tectum and suggests a role of the cannabinoid system in plasticity processes ensuing after lesions. (c) 2008 Wiley-Liss, Inc.

Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca2+](i)) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of G alpha(q/11)-coupled M-1, M-3 and M-5 receptors and intracellular calcium stores, whereas G alpha(i/o)-protein coupled M-2 receptor activity mediated neuronal differentiation. (C) 2008 Elsevier Inc. All rights reserved.