Juan Pablo Prestifilippo

The rapid release of corticosterone from the adrenal induced by ACTH is mediated by nitric oxide acting by prostaglandin E2

The adrenal cortex is a major stress organ in mammals that reacts rapidly to a multitude of external and internal stressors. Adrenocorticotropin (ACTH) is the main stimulator of the adrenal cortex, activating corticosteroid synthesis and secretion. We evaluated the mechanism of action of ACTH on adrenals of male rats, preserving the architecture of the gland in vitro. We demonstrated that both sodium nitroprusside (NP), a nitric oxide (NO) donor, and ACTH stimulate corticosterone release. NO mediated the acute response to ACTH because Nω-nitro-l-arginine methyl ester, a NO synthase inhibitor, and hemoglobin, a NO scavenger, blocked the stimulation of corticosterone release induced by ACTH. NP stimulated prostaglandin E release, which in turn stimulated corticosterone release from the adrenal. Additionally, indomethacin, which inhibits cyclooxygenase, and thereby, prostaglandin release, prevented corticosterone release from the adrenal induced by both NP and ACTH, demonstrating that prostaglandins mediate acute corticosterone release. Corticosterone content in adrenals after incubation with ACTH or NP was lower than in control glands, indicating that any de novo synthesis of corticosterone during this period was not sufficient to keep up with the release of the stored hormone. The release induced by ACTH or NP depleted the corticosterone content in the adrenal by ≈40% compared with the content of glands incubated in buffer. The mechanism of rapid release is as follows: NO produced by NO synthase activation by ACTH activates cyclooxygenase, which generates PGE2, which in turn releases corticosterone stored in microvesicles and other organelles.

Inhibition of Salivary Secretion by Activation of Cannabinoid Receptors

It is known that marijuana use decreases saliva secretion. Therefore, we hypothesized that cannabinoid receptors (CBs) are located in salivary glands to mediate that effect. In these experiments, we used the submandibular gland (SMG) of male rats, which is one of the major salivary glands. Mammalian tissues contain at least two types of CBs, CB1 and CB2, mainly located in the nervous system and peripheral tissues, respectively. Both receptors are coupled to Gi protein and respond by inhibiting the activity of adenylyl cyclase. We demonstrated that both CB1 and CB2 are present in the SMG, each showing specific localizations. The best-known endocannabinoid is anandamide (AEA), which binds with high affinity to CB1 and CB2. We showed that AEA markedly reduced forskolin-induced increase of cAMP content in vitro. This effect was blocked by AM251 and AM630 (CB1 and CB2 antagonists, respectively), indicating that both receptors are implicated in SMG physiology. In addition, we showed that AEA injected intraglandularly to anesthetized rats Inhibited norepinephrine (NE)– and methacholine (MC)–stimulated saliva secretion in vivo and that both AM251 or AM630 prevented the inhibitory action of AEA. Also, the intraglandular injection of AM251 increased saliva secretion induced by lower doses of NE or MC. This increase was synergized after coinjection with AM630. Therefore, we concluded that AEA decreases saliva secretion in the SMG acting through CB1 and CB2 receptors.

Nitric Oxide at the Crossroad of Immunoneuroendocrine Interactions

Nitric oxide (NO) was initially described as a mediator of endothelial relaxation, and now its participation is recognized in numerous physiological and pathological processes. It was demonstrated that lipopolysaccharide‐stimulated corticotropin‐releasing factor release involves NO production. Furthermore, it has been shown that interleukin (IL)‐1, tumor necrosis factor (TNF)‐α, IL‐6, and IL‐2 can stimulate adrenocorticotropic hormone release from anterior pituitary via NO. Also, we found that NO released from hypothalamic NOergic neurons in response to norepinephrine diffuses to luteinizing hormone‐releasing hormone (LHRH) neurons that activate cyclooxygenase and guanylate cyclase. This activation results in an increase in prostaglandin E2 and cyclic guanosine monophosphate, respectively, which leads to the exocytosis of LHRH granules. During pathological conditions, such as manganese intoxication, NO production is increased, leading to an increase in LHRH secretion that can advance puberty. In another study we demonstrated that NO reduces oxytocin as well as vasopressin secretion from the posterior pituitary, suggesting it has a modulatory role during dehydration. An increase in NO synthase (NOS) activity and protein in the hippocampus and cerebellum was found in offspring of rats that were subjected to prenatal stress, and this was correlated with behavioral changes in adults. Also NO participates in signal transduction pathways in peripheral tissue in physiological processes, such as in corticosterone release from the adrenal gland. Pathological conditions, such as tumors of the head and neck, that are treated with radiation are followed by xerostomy. In a rat model, radiation diminished NOS activity in the submandibulary gland, and this was followed by inhibition in salivary secretion. In summary, this review describes the wide participation of NO in the cross‐talk between neuroendocrine and neuroimmune systems in physiological and pathological processes.

Histamine prevents functional and morphological alterations of submandibular glands induced by ionising radiation.

Purpose: Xerostomia is a common, disturbing side-effect among patients treated with radiotherapy for head-and-neck cancer. The aim of the present work was to investigate whether histamine could prevent salivary gland dysfunction and histological alterations exerted by ionising radiation. Materials and methods: Forty-eight rats were divided into four groups. Histamine and histamine-5 Gy groups received a daily subcutaneous histamine injection (0.1 mg/kg) starting 24 h before irradiation. Histamine-5 Gy and untreated-5 Gy groups were irradiated with a single dose of whole-body Cesium-137 irradiation. Control and untreated-5 Gy groups were given daily saline injections. Three days post irradiation metacholine-induced salivary secretion was measured or animals were sacrificed and submandibular gland (SMG) removed, stained and histological characteristics were evaluated. Proliferation and apoptosis markers were studied by immunohistochemistry. Results: Radiation decreased salivary secretion by 40% in comparison to untreated rats, which was associated with loss of SMG mass, alteration of epithelial architecture, partial loss of secretor granular material, diminished proliferation and a remarkable apoptotic response. In contrast, histamine completely reversed the reduced salivation induced by radiation, conserved glandular mass with normal appearance and preserved the structural organisation of secretor granules. Radiation-induced toxicity is prevented by histamine essentially by suppressing apoptosis of ductal and acinar cells, reducing the number of apoptotic cells per field (19.0 ± 3.8 vs. 106.0 ± 12.0 in untreated animals, P < 0.001), and also by preventing the radiation-induced decrease in cell proliferation. Conclusions: Histamine prevents morphological and functional radiation-induced damage on SMG, representing a potential radioprotector for treatment of patients undergoing radiotherapy for head and neck malignancies.

Hippocampal mitochondrial dysfunction with decreased mtNOS activity in prehepatic portal hypertensive rats.

Portal hypertension is a major complication of human cirrhosis that frequently leads to central nervous system dysfunction. In our study, rats with prehepatic portal hypertension developed hippocampal mitochondrial dysfunction as indicated by decreased respiratory rates, respiratory control and mitochondrial nitric oxide synthase (mtNOS) activity in mitochondria isolated from the whole hippocampus. Succinate-dependent respiratory rates decreased by 29% in controlled state 4 and by 42% in active state 3, and respiratory control diminished by 20%. Portal hypertensive rats showed a decreased mtNOS activity of 46%. Hippocampal mitochondrial dysfunction was associated with ultrastructural damage in the mitochondria of hippocampal astrocytes and endothelial cells. Swollen mitochondria, loss of cristae and rupture of outer and inner membrane was observed in astrocytes and endothelial cells of the blood-brain barrier in parallel with the ammonia gradient. It is concluded that the moderate increase in plasma ammonia that followed portal hypertension was the potential primary cause of the observed alterations.

PROTECTION OF RADIATION-INDUCED DAMAGE TO THE HEMATOPOIETIC SYSTEM, SMALL INTESTINE AND SALIVARY GLANDS IN RATS BY JNJ7777120 COMPOUND, A HISTAMINE H4 LIGAND

Based on previous data on the histamine radioprotective effect on highly radiosensitive tissues, in the present work we aimed at investigating the radioprotective potential of the H4R ligand, JNJ7777120, on ionizing radiation-induced injury and genotoxic damage in small intestine, salivary glands and hematopoietic tissue. For that purpose, rats were divided into 4 groups. JNJ7777120 and JNJ7777120-irradiated groups received a daily subcutaneous JNJ7777120 injection (10 mg/kg) starting 24 h before irradiation. Irradiated groups received a single dose of 5 Gy on whole-body using Cesium-137 source and were sacrificed 3 or 30 days after irradiation. Tissues were removed, fixed, stained with hematoxylin and eosin or PAS staining and histological characteristics were evaluated. Proliferative and apoptotic markers were studied by immunohistochemistry, while micronucleus assay was performed to evaluate DNA damage. Submandibular gland (SMG) function was evaluated by methacholine-induced salivation. Results indicate that JNJ7777120 treatment diminished mucosal atrophy and preserved villi and the number of crypts after radiation exposure (240±8 vs. 165±10, P<0.01). This effect was associated to a reduced apoptosis and DNA damage in intestinal crypts. JNJ7777120 reduced radiation-induced aplasia, preserving medullar components and reducing formation of micronucleus and also it accelerated bone marrow repopulation. Furthermore, it reduced micronucleus frequency in peripheral blood (27±8 vs. 149±22, in 1,000 erythrocytes, P<0.01). JNJ7777120 completely reversed radiation-induced reduced salivation, conserving glandular mass with normal histological appearance and reducing apoptosis and atrophy of SMG. JNJ7777120 exhibits radioprotective effects against radiation-induced cytotoxic and genotoxic damages in small intestine, SMG and hematopoietic tissues and, thus, could be of clinical value for patients undergoing radiotherapy.

Role of the endocannabinoid system in ethanol-induced inhibition of salivary secretion.

AIM The aim of the present study was to determine whether the endocannabinoid system could be involved in the ethanol-induced inhibition of salivation in adult male Wistar rats. METHODS Salivary secretion induced by different concentrations of methacholine, a cholinergic agonist, and the endocannabinoid arachidonoyl ethanolamide (anandamide, AEA) production in the submandibular gland (SMG) were determined in rats after ethanol (3 g/kg) administration by gastric gavage. To study the participation of cannabinod receptors in ethanol action, we evaluated methacholine-induced salivary secretion after ethanol administration when CB1 or CB2 receptors were blocked by intra-SMG injections of their selective antagonists AM251 and AM630, respectively. Additionally, we evaluated the in vitro effect of ethanol (0.1 M) on SMG production of cAMP, alone or combined with AM251 or AM630. RESULTS Acute ethanol administration increased AEA production in SMG and also inhibited the methacholine-induced saliva secretion that was partially restored by intraglandular injection of AM251 or AM630. In addition, ethanol significantly reduced the forskolin-induced increase in cAMP content in SMG in vitro while treatment with AM251 blocked this response. CONCLUSION We conclude that the inhibitory effect produced by ethanol on submandibular gland salivary secretion is mediated, at least in part, by the endocannabinoid system.

Participation of the endocannabinoid system in lipopolysaccharide-induced inhibition of salivary secretion

OBJECTIVE The aim of the present paper was to assess whether lipopolysaccharide (LPS)-induced inhibition of salivary secretion involves the activation of the endocannabinoid system and the participation of tumor necrosis factor (TNF)alpha in the submandibular gland. DESIGN Pharmacological approaches were performed by using CB1 and/or CB2 cannabinoid receptor antagonists, AM251 and AM630, respectively, injected into the submandibular gland, to study the participation of the endocannabinoid system in LPS inhibitory effects on metacholine-induced salivary secretion. To assess the participation of TNFalpha on LPS inhibitory effects, salivary secretion was studied in LPS treated rats after the intraglandular injection of etanercept, a soluble form of TNF receptor which blocks TNFalpha action. Finally, to evaluate the possible interplay between endocannabinoids and TNFalpha on the submandibular gland function reduced during LPS challenge, the salivary secretion was studied after the intraglandular injection of this cytokine alone or concomitantly with AM251 and AM630. RESULTS AM251 and AM630, injected separately or concomitantly, partially prevented LPS-induced inhibition of salivation. Also, anandamide synthase activity was increased in submandibular glands extracted from rats 3h after LPS injection, suggesting that the endocannabinoid system was activated in response to this challenge. On the other hand, etanercept, prevented the inhibitory effect of LPS on salivary secretion and moreover, TNFalpha injected intraglandularly inhibited salivary secretion, being this effect prevented by AM251 and AM630 injected concomitantly. CONCLUSION The present results demonstrate the participation of the endocannabinoid system and TNFalpha on salivary responses during systemic inflammation induced by LPS.

Functional and morphological alterations induced by uranyl nitrate in rat submandibular gland

Exposure to uranium (U) is an occupational hazard to workers who continually handle it and an environmental risk to the population at large. Adverse effects of U on different tissues, particularly kidneys, have been reported. The aim of the present study was to investigate whether U might produce damage to the rat submandibular gland (SMG). Uranyl nitrate (UN) was used to evaluate the secretory responses to norepinephrine (NE) or to the parasympathomimetic agent, methacholine (MC), along with some morphological and histological parameters. In addition, the presence of U in saliva was determined by atomic absorption spectrometric techniques. Results indicate that intraperitoneal (i.p.) injection of a single dose of UN (2 or 4 mg kg−1) induced a functional decrease in the induced secretory responses in the rat SMG, demonstrating for the first time that U interferes with salivary secretion. Moreover, salivary responses to MC as well as to NE were decreased after UN administration, in time- and dose-dependent manner, displaying a higher diminution after 7 days post injection. In agreement with the functional studies, the injection of UN produced morphological alterations of SMG, consisting in a reduction of mean acinar area and a marked vacuolization. Data indicate that UN produced an adverse effect in a dose- and time-dependent manner on SMG function and morphology. Furthermore, it was shown that U was incorporated in saliva and therefore, these findings may contribute to create a useful, noninvasive method to detect the exposure to U.

Endocannabinoids mediate hyposalivation induced by inflammogens in the submandibular glands and hypothalamus

OBJECTIVE The aim of this study was to investigate the factors that could participate on salivary glands hypofunction during inflammation and the participation of endocannabinoids in hyposalivation induced by the presence of inflammogens in the submandibular gland (SMG) or in the brain. DESIGN Salivary secretion was assessed in the presence of inflammogens and/or the cannabinoid receptor antagonist AM251 in the SMG or in the brain of rats. At the end of the experiments, some systemic and glandular inflammatory markers were measured and histopathological analysis was performed. RESULTS The inhibitory effect observed 1h after lipopolysaccharide (LPS, 50μg/50μl) injection into the SMG (ig) was completely prevented by the injection of AM251 (5μg/50μl) by the same route (P<0.05). The LPS (ig)-induced increase in PGE2 content was not altered by AM251 (ig), while the glandular production of TNFα induced by the endotoxin (P<0.001) was partially blocked by it. Also, LPS injection produced no significant changes in the wet weight of the SMG neither damage to lipid membranes of its cells, nor significant microscopic changes in them, after hispopathological analysis, compared to controls. Finally, TNFα (100ng/5μl) injected intracerebro-ventricularly (icv) inhibited methacholine-induced salivary secretion evaluated 30min after (P<0.01), but the previous injection of AM251 (500ng/5μl, icv) prevented completely that effect. CONCLUSION We conclude that endocannabinoids mediate the hyposialia induced by inflammogens in the SMG and in the brain. The hypofunction would be due to changes on signalling pathway produced by inflammatory compounds since anatomical changes were not observed.