Serum endocannabinoids and N-acyl ethanolamines and the influence of simulated solar UVR exposure in humans in vivo.

Solar ultraviolet radiation (UVR) exposure of human skin has beneficial and harmful effects on health, including impact on immune function, inflammation and reportedly mood, but these are not fully elucidated. Since the endocannabinoid system is implicated in many activities including mood alteration, our objective was to (i) determine and quantify circulating levels of a wide range of endocannabinoid and N-acyl ethanolamine (NAE) species (ii) evaluate whether these are modulated by cutaneous UVR exposures, as attained through repeated low level summer sunlight exposure. Wearing goggles to prevent eye exposure, 16 healthy volunteers (23-59 y; 10 light skin, phototype II, and 6 dark skin, phototype V) received the same UVR exposures (1.3 SED, 95% UVA/5% UVB) thrice weekly for 6 weeks, whilst casually dressed to expose ∼35% skin surface area. Blood samples were taken at baseline, days 1, 3 and 5 of week one, then at weekly intervals, and analysed by LC-MS/MS. Eleven endocannabinoids and NAEs were detected and quantified at baseline, with N-palmitoyl ethanolamine the most abundant (30% of total). Levels did not vary according to phototype (p > 0.05), except for the NAE docosapentaenoyl ethanolamide, which was higher in phototype II than V (p = 0.0002). Level of the endocannabinoid, 2-AG, was elevated during the UVR exposure course (p < 0.05 vs. baseline for all subjects; p < 0.01 for each phototype group), with maximum levels reached by week 2-3, while NAE species did not significantly alter. These findings suggest differential involvement of the cutaneous endocannabinoid system in low dose solar UVR responses in humans.

Alterations in calcium signaling and a decrease in Bcl-2 expression: possible correlation with apoptosis in aged striatum.

Aging is a multifaceted process associated with various functional and structural deficits that might be evolved in degenerative diseases. It has been shown that neurodegenerative disorders are associated with alterations in Ca(2+) homeostasis. Thus, in the present work, we have investigated Ca(2+) signaling and apoptosis in aged striatum. Our results show that glutamate and NMDA evoke a greater Ca(2+) rise in striatum slices from aged animals. However, this difference is not present when glutamate is tested in the absence of external Ca(2+). Immunostaining of glutamate receptors shows that only NMDA receptors (NR1) are increased in the striatum of aged rats. Increases in mitochondrial Ca(2+) content and in the reactive oxygen species levels were also observed in aged animals, which could be associated with tissue vulnerability. In addition, a decrease in the Bcl-2 protein expression and an enhancement in apoptosis were also present in aged striatum. Together the results indicate that, in aged animals, alterations in Ca(2+) handling coupled to an increase in ROS accumulation and a decrease in the prosurvival protein Bcl-2 may contribute to apoptosis induction and cell death in rat striatum.

Long-term nitric oxide deficiency causes muscarinic supersensitivity and reduces beta(3)-adrenoceptor-mediated relaxation, causing rat detrusor overactivity.

BACKGROUND AND PURPOSE: Overactive bladder is a complex and widely prevalent condition, but little is known about its physiopathology. We have carried out morphological, biochemical and functional assays to investigate the effects of long-term nitric oxide (NO) deficiency on muscarinic receptor and beta-adrenoceptor modulation leading to overactivity of rat detrusor muscle. EXPERIMENTAL APPROACH: Male Wistar rats received N(omega)-nitro-L-arginine methyl ester (L-NAME) in drinking water for 7-30 days. Functional responses to muscarinic and beta-adrenoceptor agonists were measured in detrusor smooth muscle (DSM) strips in Krebs-Henseleit solution. Measurements of [(3)H]inositol phosphate, NO synthase (NOS) activity, [(3)H]quinuclidinyl benzilate ([(3)H]QNB) binding and bladder morphology were also performed. KEY RESULTS: Long-term L-NAME treatment significantly increased carbachol-induced DSM contractile responses after 15 and 30 days; relaxing responses to the beta(3)-adrenoceptor agonist BRL 37-344 were significantly reduced at 30 days. Constitutive NOS activity in bladder was reduced by 86% after 7 days and maintained up to 30 days of L-NAME treatment. Carbachol increased sixfold the [(3)H]inositol phosphate in bladder tissue from rats treated with L-NAME. [(3)H]QNB was bound with an apparent K(D) twofold higher in bladder membranes after L-NAME treatment compared with that in control. No morphological alterations in DSM were found. CONCLUSIONS AND IMPLICATIONS: Long-term NO deficiency increased rat DSM contractile responses to a muscarinic agonist, accompanied by significantly enhanced K(D) values for muscarinic receptors and [(3)H]inositol phosphate accumulation in bladder. This supersensitivity for muscarinic agonists along with reductions of beta(3)-adrenoceptor-mediated relaxations indicated that overactive DSM resulted from chronic NO deficiency.

Kallikrein-kinin system in the plasma of the snake Bothrops jararaca.

1. Bothrops jararaca venom (BJV) caused a fall in the carotid artery blood pressure of the anaesthetized snake. This effect was tachyphylactic and was potentiated by captopril, a kininase II inhibitor; it was partially antagonized by promethazine plus cimetidine and was not affected by atropine. 2. Similar hypotensive effects were obtained by administration of trypsin or a partially purified BJV kininogenase to the snake. 3. Incubation of Bothrops jararaca plasma (BJP) with trypsin released a substance (or substances) that produced hypotension in the snake but not in the rat; this hypotensive effect was also potentiated by captopril. 4. The trypsinised plasma contracted Bothrops jararaca isolated uterus, a pharmacological preparation weakly sensitive to bradykinin. Trypsinised plasma was inactive on pigeon oviduct and rat uterus and displayed a weak action on the guinea-pig ileum. Similar effects were observed with incubates of a fraction of BJP, containing globulins, with a partially purified BJV kininogenase. 5. Like mammalian kinins, the substance(s) was(were) dialysable, thermostable in acid but not in alkaline pH, and inactivated by chymotrypsin but not by trypsin. Its(their) inactivation by BJP or BJP kininase II was inhibited by captopril. 6. These findings strongly suggest that, besides releasing histamine, BJV or trypsin release a kininlike substance (or substances) from the snake plasma. 7. Since BJV and other kininogenases active on mammalian plasma were shown to be unable to release kinins from BJP, in experiments conducted on pharmacological preparations suitable for the assay of mammalian kinins, these data also suggest that the snake Bothrops jararaca, like birds, may have developed its own kallikrein-kinin system.

Effect of estrogen on intracellular signaling pathways linked to activation of M(2)- and M(3)-muscarinic acetylcholine receptors in the rat myometrium.

The estrogen treatment of adult female rats induces an increase in myometrium sensitivity to cholinergic agonists and in this tissue the presence of M(2)- and M(3)-muscarinic acetylcholine (mACh) receptor was shown. We now report the effect of estrogen on intracellular signaling pathways linked to activation of M(2)- and M(3)-mACh receptor subtypes. The intracellular cyclic AMP accumulation and [3H]-inositol phosphates content were measured in myometrium strips from rats in estrus (control) and estradiol-treated rats (12.5 microg/100 g body weight, sc, 24 h before experiments) (the plasma estradiol level was 30.9+/-3.5 pg/ml and 119.3+/-14.1 pg/ml from control and estrogen-treated rats, respectively). Estrogen treatment increased 2.5-fold the intracellular cyclic AMP accumulation induced by 10 microM forskolin. The effects of muscarinic agonist and antagonists on cyclic AMP accumulation were tested. Carbachol reduced the forskolin-induced intracellular cyclic AMP content, 3.0 and 10.5-fold, in myometrium from control and estradiol-treated rats, respectively. This inhibitory effect failed to occur when carbachol was incubated in the presence of methoctramine. Carbachol also induced increase on total [3H]-inositol phosphates accumulation in myometrium from estradiol-treated rats when compared with control rats. This effect was reversed by pfHHSiD. These studies suggest the modulation by estrogen of intracellular signaling pathways linked to activation of M(2)- and M(3)-mACh receptors in the rat myometrium.

Effect of chymotrypsin on rat blood pressure.

1. Chymotrypsin (Cht) administration (14 mg/kg, i.v.) to rats always caused hypertension; hypotension preceded this effect in 64% of the observations (n = 11). 2. A 68% reduction of circulating kininogen but not of angiotensinogen was observed after Cht administration. 3. Cht effects were not affected by captopril, [Sar1-Leu8]-angiotensin II and alpha-adrenoceptor antagonists. In 70% of the observations (n = 10) hypotension was abolished by a mixture of histamine H1- and H2-antagonists. Therefore histamine release may explain hypotension. 4. Cht released in vitro from rat plasma, a substance producing hypertension in the rat and contraction of the guinea-pig ileum. Both effects were antagonized by [Sar1-Leu8]-angiotensin II. 5. In spite of this angiotensin release in vitro, the hypertensive component of the in vivo response to Cht seems to be due to some other substance.

Kallikrein-kinin system in the plasma of snakes.

Using pharmacological preparations suitable for assay of mammalian kinins, it was shown that Bothrops jararaca (Bj) venom and other kininogenases were unable to release kinins from snake plasma. The kallikrein-kinin system presents species-specificity in birds. In order to detect such a specificity in snakes, the effects of Bj venom on snake blood pressure and the effect of incubates of snake plasma with trypsin, on snake blood pressure and snake uterus, were studied. The possibility of activating snake plasma kallikrein with ellagic acid, glass beads or kaolin was also investigated. Whereas plasma of the snakes Waglerophis merremii (Wm) and Crotalus durissus (Cd), were shown to contain factor XII, prekallikrein, kininogen, kininases and to present a low but definite activation rate of the kinin system, the plasmas of Bj, Bothrops mojeni (Bm) and Oxyrophus trigeminus (Ot), yielded only kininogen and kininases. Activation of the system was not even detected by the sensitive substrate Ac-Phe-Arg-Nan (acetyl-phenylalanyl-arginyl-4nitro-anilide), indicating that the plasma of these species does not possess either factor XII and/or prekallikrein. Snake plasma may constitute an interesting model for the study of blood clotting, fibrinolytic and complement systems.

Reproductive cycle of the Neotropical Crotalus durissus terrificus: I. Seasonal levels and interplay between steroid hormones and vasotocinase.

Crotaline snakes present delayed fertilization and sperm storage because secondary vitellogenesis is not completed by the time of mating. The release of vitellogenesis and synchrony between ovulation and fertilization suggest a steroidal modulation. We investigated changes of sexual steroid levels during reproduction in the Neotropical rattlesnake Crotalus durissus terrificus, analyzing macroscopical variations of reproductive condition (vitellogenesis, pregnancy, and post-partum) and plasma levels of estradiol, progesterone, and vasotocinase cystine aminopeptidase (CAP) activity over 2 years. Data showed 44.4% non-reproductive snakes (40.1% primary vitellogenesis and 4.3% post-partum) and 55.6% reproductive (36.8% secondary vitellogenesis and 18.8% pregnant). Estradiol was low in spring and summer, increasing in autumn till it peaked in winter. Estradiol in secondary vitellogenesis was significantly higher than in primary vitellogenesis, or in pregnant and post-partum females, Progesterone dropped significantly in autumn compared to summer, winter, and spring. Pregnant females showed the highest levels of progesterone compared to primary or secondary vitellogenesis, or post-partum females. CAP activity showed lowest values in reproductive females in autumn and greatest levels in post-partum females. A significant negative linear relationship was obtained between CAP activity and estradiol. The combination of morphological observations, levels of steroids and CAP activity allowed us to suggest a similar morphological reproductive pattern between temperate and tropical rattlesnakes, and to infer the role of estradiol, progesterone and CAP activity on vitellogenesis, gestation and sperm storage, respectively.

Reproductive cycle of the Neotropical Crotalus durissus terrificus: II. Establishment and maintenance of the uterine muscular twisting, a strategy for long-term sperm storage.

Crotaline snakes store sperm by means of a uterine musculature twisting (UMT). We investigated the influence of plasma levels of estradiol and progesterone and vasotocinase cystine aminopeptidase (CAP) activity on UMT formation and maintenance, and the in vitro uterine reactivity for AVT in Crotalus durissus terrificus in primary or secondary vitellogenesis with or without UMT. Frequency of females in secondary vitellogenesis with UMT is significantly higher than in primary one. Estradiol levels did not vary in all conditions studied, however, significantly low levels of progesterone were found in snakes in secondary vitellogenesis with UMT compared to those without it. UMT is always observed when high levels of estradiol and low levels of progesterone are detected. CAP activity did not change in the presence of UMT. AVT produced concentration-response contractions of the isolated uterus of snakes in all stages analysed and the pD2 value and maximum contractile response were significantly higher in primary vitellogenesis without UMT than in other reproductive conditions, indicating that uterus of those snakes presents a higher contractile capacity which may favour UMT establishment. In conclusion, we show a relationship of UMT and estradiol/progesterone balance and a possible participation of AVT in UMT formation and maintenance in the Neotropical rattlesnake.