Oxidative stress has a critical role in the pathogenesis of Age-related Macular Degeneration (AMD), a multifactorial disease that includes age, gene variants of complement regulatory proteins and smoking as the main risk factors. Stress-induced premature cellular senescence (SIPS) is postulated to contribute to this condition. In this study, we hypothesized that oxidative damage, promoted by endogenous or exogenous sources, could elicit a senescence response in RPE cells, which would in turn dysregulate the expression of major players in AMD pathogenic mechanisms. We showed that exposure of a human RPE cell line (ARPE-19) to a cigarette smoke concentrate (CSC), not only enhanced Reactive Oxygen Species (ROS) levels, but also induced 8-Hydroxydeoxyguanosine-immunoreactive (8-OHdG) DNA lesions and phosphorylated-Histone 2AX-immunoreactive (p-H2AX) nuclear foci. CSC-nuclear damage was followed by premature senescence as shown by positive senescence associated-β-galactosidase (SA-β-Gal) staining, and p16INK4a and p21Waf-Cip1 protein upregulation. N-acetylcysteine (NAC) treatment, a ROS scavenger, decreased senescence markers, thus supporting the role of oxidative damage in CSC-induced senescence activation. ARPE-19 senescent cultures were also established by exposure to hydrogen peroxide (H2O2), which is an endogenous stress source produced in the retina under photo-oxidation conditions. Senescent cells upregulated the proinflammatory cytokines IL-6 and IL-8, the main markers of the senescence-associated secretory phenotype (SASP). Most important, we show for the first time that senescent ARPE-19 cells upregulated vascular endothelial growth factor (VEGF) and simultaneously downregulated complement factor H (CFH) expression. Since both phenomena are involved in AMD pathogenesis, our results support the hypothesis that SIPS could be a principal player in the induction and progression of AMD. Moreover, they would also explain the striking association of this disease with cigarette smoking.

Nitric oxide (NO) is generated from L-arginine by NO synthases. Localization of the brain enzyme has been carried out in the rat; however, despite data suggesting that NO is a major regulator of vascular and neural functions in man, there is no information about the localization of NO synthase in human tissues. Rabbit antisera to NO synthase purified from rat brain (antisera A and B) were raised, tested by Western blotting, affinity purification and enzyme immunoprecipitation assay, and used to investigate the distribution of the enzyme in a variety of human tissues by immunohistochemistry. Antisera to two synthetic peptides from cloned neural NO synthase were used to aid specificity testing. Anti-sera A and B reacted with a approximately 160-kDa protein in Western blots of human brain extracts, gave immunostaining of nerves, and precipitated enzyme activity from rat brain homogenates. Antiserum B to NO synthase also reacted with proteins of M(r) between 125 and 140 kDa in extracts of well-vascularised tissues, and immunostained vascular endothelium; the neural and vascular immunoreactivity persisted after affinity purification of antiserum B with the approximately 160 kDa protein. Endothelial staining with antiserum B was seen in respiratory tract, liver, skin and umbilicus; syncytial trophoblasts stained in the placenta. Neural staining with antiserum A and B was seen in the myenteric and submucous plexus, and in nerve fibres in smooth muscle of the gut and in many areas of the central nervous system, particularly cortex, hippocampus, hypothalamus, cerebellum, brain stem and spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

No abstract

Since nitric oxide has been found to control the function of many organs of the body by the non-adrenergic, non-cholinergic branch of the autonomic nervous system, we hypothesized that it might play a role in salivary secretion. Therefore, we investigated the distribution of nitric oxide synthase (NOS) throughout the submaxillary gland and also studied the ability of inhibitors of NOS to interfere with salivation induced by a cholinergic agonist, metacholine, and by a polypeptide, substance P. The secretory responses were determined in rats anesthetized with chlorolose following intravenous injection of the various pharmacological agents. There was no basal flow of saliva and dose-response curves were obtained by sequential intravenous injection of increasing doses of the drugs. Then, in the same animal, the same dose-response curves were performed in the presence of NOS inhibitors. L-Nitro-arginine-methyl-ester (L-NAME; 20 mg/kg) produced an over 50% inhibition of the dose-related salivation induced by metacholine. Similar results were produced with L-N^G-monomethyl-L-arginine (L-NMMA; 5 mg/kg). The salivation induced by much lower molar doses of substance P was dramatically greater than that obtained with metacholine. The response to substance P was almost completely inhibited by L-NMMA at the lowest dose (0.3 mg/kg), but at higher doses (1 mg/kg), the inhibition was only around 60% and at the highest dose (3 mg/kg) only about 20%. In control rats, there were roughly equal amounts of calcium-dependent and calcium-independent NOS in the gland at this time. At the end of the experiment, the effect of the inhibitor of NOS, L-NMMA, on the NOS activity in the submandibular gland was determined. At this time, the Ca²⁺-dependent NOS was decreased and the Ca²⁺-independent NO was increased. The prior injection of L-NMMA reduced calcium-dependent NOS activity by approximately 70% but calcium-independent activity by only 30%. These results indicate that, at least at the end of the experiment, the blockade of NOS imposed by NMMA was incomplete. This could account in part for the failure of the inhibitors to block completely the stimulatory effect of the two secretagogues. Analysis of the distribution of NOS in the salivary gland revealed that it was not present in the acinar cells, but in neural terminals within the gland and also in the ductile system which contained neural (n) NOS in the apical membrane of the excretory and striated ducts, the cytoplasm of granular convoluted tubules and, to a lesser extent, in the cytoplasm of excretory and striated ducts. Macrophage (inducible) NOS was also found not only in the macrophages, but also in the tubules and ducts. Since drugs were used that would act on the receptors in the gland, the role of NO in our conditions is probably mediated by nNOS and iNOS in the ductile and tubular structures. Since iNOS would already be active, it is unlikely to play a role in this acute secretory activity. Rather the nNOS in these non-neural cells is probably ac...