Aclidinium inhibits cholinergic and tobacco smoke-induced MUC5AC in human airways.

Mucus hypersecretion and mucin MUC5AC overexpression are pathological features of chronic obstructive pulmonary disease (COPD). This study examines the inhibitory effect of aclidinium, a new long-acting muscarinic antagonist, on MUC5AC expression in human airway epithelial cells. MUC5AC mRNA (RT-PCR) and protein expression (ELISA and immunohistochemistry) were studied in human bronchial tissue and differentiated human airway epithelial cells activated with carbachol (100 µM) or cigarette smoke extract in the absence or presence of aclidinium. Carbachol increased MUC5AC mRNA and protein expression in human bronchus and cultured epithelial cells. Aclidinium inhibited the carbachol-induced MUC5AC mRNA and protein expression with potency (half maximal inhibitory concentration) ~1 nM in human bronchus and cultured airway epithelial cells. AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, inhibited carbachol-induced MUC5AC responses, indicating EGFR transactivation. Aclidinium inhibited carbachol-induced phospho-EGFR and phospho-p44/42 MAPK expression. In cultured airway epithelial cells transfected with small interfering (si)RNA against muscarinic receptor subtypes, siRNA-M3 but not siRNA-M2 blocked carbachol-induced MUC5AC expression. Cigarette smoke-induced MUC5AC upregulation in cultured airway epithelial cells was suppressed by aclidinium. In conclusion, aclidinium decreases carbachol and tobacco smoke-induced MUC5AC overexpression in human airway epithelial cells. This effect may contribute to the clinical efficacy of aclidinium in mucus hypersecretory diseases including COPD.

The human area postrema and other nuclei related to the emetic reflex express cAMP phosphodiesterases 4B and 4D.

Phosphodiesterase 4 (PDE4) inhibitors, i.e. rolipram, are being extensively investigated as therapeutic agents in several diseases. Emesis is one of the most common side effects of PDE4 inhibitors. Given the fact that the area postrema is considered the chemoreceptor trigger zone for vomiting, the present study investigates the regional distribution and cellular localization of the four gene transcripts of the PDE4 subfamily (PDE4A, PDE4B, PDE4C and PDE4D) in human brainstem. In situ hybridization histochemistry was used to locate the mRNA distribution of the four PDE4 subfamilies in the area postrema and related nuclei of human postmortem brainstem. We have found that in the brainstem PDE4B and PDE4D mRNA expression is abundant and distributed not only in neuronal cells, but also in glial cells, and on blood vessels. The hybridization signals for PDE4B and PDE4D mRNAs in the area postrema were stronger than those in any other nuclei in the brainstem. They were also found in vomiting-related nuclei such as the nucleus of the solitary tract and the dorsal vagal motor nucleus. These findings suggest that cAMP signaling modification in the area postrema could mediate the emetic effects of PDE4 inhibitors in human brainstem.

Necrotic cell death induced by photodynamic treatment of human lung adenocarcinoma A-549 cells with palladium(II)-tetraphenylporphycene.

In this study we describe photodamaging and photokilling effects of palladium(II)-tetraphenylporphycene (PdTPPo) (previously incorporated into dipalmitoylphosphatidylcholine liposomes) on the human lung adenocarcinoma A-549 cell line. No dark cytotoxicity was found when the drug was applied at 10(-6) M or 5 x 10(-7) M for 1 or 18 h, respectively. After 1-h treatment with 10(-7) M or 5 x 10(-7) M PdTPPo followed by red light irradiation for variable times, dose-dependent lethal effects were observed in A-549 cells. Apoptosis was not found after the above photodynamic treatments or under even milder sublethal conditions. In contrast to HeLa cells subjected to PdTPPo photosensitization where either apoptosis or necrosis were induced, morphological analysis and electrophoretical DNA pattern of A-549 cells always revealed a clearly necrotic death mechanism. However, A-549 cells died by apoptosis after serum and L-glutamine deprivation, indicating that only the photodynamically induced apoptosis was inhibited. Immunofluorescent labeling revealed that microtubules and actin microfilaments were immediately and strongly damaged by photodynamic treatments with PdTPPo. No metaphase arrest and/or mitotic alterations were observed after phototreatments. Present results show that the cell type plays a fundamental role in relation to the apoptotic or necrotic response to photosensitization, and that cytoskeletal components are important targets implicated in cell death processes.

Inhibition of corticosteroid-binding globulin caused by a severe stressor is apparently mediated by the adrenal but not by glucocorticoid receptors.

The effect of stress on serum corticosteroid-binding globulin (CBG) was studied in adult male Sprague-Dawley rats. CBG was measured either by a homologous radioimmunoassay (RIA) or by a binding assay (BA) using 3H-corticosterone. Exposure of adult male rats to a severe stressor such as immobilization (IMO) for 1 h did not alter serum CBG levels, but a significant decrease was found after 6 and especially 24 h IMO. This decrease was not observed after 24 h exposure to a milder treatment such as food and water deprivation. The effect of different periods of exposure to two stressors, IMO or restraint, was also studied. The following results were obtained:serum CBG levels were reduced by IMO, but only by restraint; IMO-induced reduction of CBG levels was always observed 24 h after starting exposure to IMO, independently of the actual period of exposure to the stressor; and IMO-induced inhibition of CBG was proportional to the hours of exposure to the stressor. Although IMO-induced inhibition of CBG was prevented by adrenalectomy, a role for glucocorticoid acting through their classical type II receptors is unclear as far as treatment of rats with the glucocorticoid receptor antagonist RU486 (100 mg/kg) did not prevent the inhibition caused by IMO. The present data clearly indicate that acute exposure to a stressor is able to decrease CBG levels provided that duration of exposure to the stressor and its intensity are high and that the effect is tested at least 6 h after the onset of stress. The effect appears to be mediated by some adrenal factor(s) other than glucocorticoids.

Acute stress attenuates but does not abolish circadian rhythmicity of serum thyrotrophin and growth hormone in the rat.

The effects of acute immobilization (IMO) on daily rhythms of corticosterone, thyroid-stimulating hormone (TSH) and growth hormone (GH) were studied in adult male rats. Two hours of IMO increased serum corticosterone, this increase still being observed 3 h after finishing stress exposure. In the dark period corticosterone levels did not differ in control and IMO rats, but higher levels were observed again in the morning of the day after. Immobilization lowered serum GH and TSH levels throughout the 24-h period that followed exposure to the stressor. Such an effect was more marked in GH than in TSH. In addition, GH, but not TSH, levels were found to be reduced significantly by IMO at 08.30 h of the next day. None the less, daily rhythms of GH and TSH were still persistent and roughly similar to those of control rats. The daily rhythm of food intake was measured in a separate experiment and it was observed, as expected, that IMO reduced food intake only in the dark period of the lighting cycle. It appears therefore unlikely that IMO-induced anorexia was the major factor responsible for the inhibition of GH and TSH caused by IMO at 11.00 and 19.00 h, considering that the amount of food intake was very low and similar in control and IMO rats during this period. However, anorexia might have contributed to inhibition of GH and TSH secretion afterwards. Thus, in a third experiment we studied the contribution of IMO-induced anorexia to the changes in hormone levels observed 24 h after stress by introducing a group of pair-fed rats. It was found that IMO, but not pair-feeding, reduced TSH levels, whereas a similar reduction of GH was found in the two conditions. It might be concluded that acute stress transiently altered corticosterone secretion, the only long-lasting effect being a slight increase in its morning levels on the following stress. Immobilization also causes an inhibition of GH and TSH secretion in the rat that persists for several hours after finalization of exposure to the stressor, but daily rhythms were still apparent. It appears that the contribution of stress-induced anorexia is different in GH than in TSH. In conclusion, an acute severe stressor such as IMO, although modifying circulating levels of some hormones, particularly in the hours following exposure to the stressor, did not appear to interfere greatly with the expression of circadian rhythms of anterior pituitary hormones.

Comparison of the behavioural and endocrine response to forced swimming stress in five inbred strains of rats.

Some inbred strains of rats showed behavioural differences in the forced swimming test, which is considered a putative animal model of depression. In the present work, the behavioural and physiological responses to forced swimming were studied in male and female rats of five inbred strains of rats: Brown-Norway (BN), Fischer 344 (FIS), Lewis (LEW), Spontaneously Hypertensive (SHR) and Wistar-Kyoto (WKY). Physiological measures were aimed at characterizing emotional reactivity, a very important issue which has usually been approached by studying a single endocrine system, and its relationship to the forced swimming behaviour. The four indices of reactivity to stress used were serum glucose, ACTH, corticosterone and prolactin. No behavioural differences between sexes were observed in the forced swimming test. In addition, BN and WKY rats showed passive behaviour compared with the other three strains, the FIS strain being the most active. Whereas only minor differences were found in the resting levels of the variables studied with regard to either sex or strain, pituitary-adrenal (PA) and glucose responses to 15 min forced swimming differed among sexes and strains. Stress-induced hyperglycaemia was lowest in WKY and highest in SHR, being lower in females than in males. The lowest ACTH and corticosterone responses to forced swimming were observed in LEW and the highest in FIS. Female rats showed a clearly higher PA response to stress in all strains. Prolactin response to stress was very similar between sexes and strains. It might thus be concluded that: (i) there are important inter-strain differences in the forced swimming behaviour, with no differences between sexes; (ii) the various physiological indices of emotional reactivity follow a different trend and no warranted conclusion on differences in emotional reactivity should be based upon a single endocrine system or even only upon physiological measures; (iii) we cannot be sure, therefore, whether or not there are differences in emotionality between the strains studied in spite of well-established inter-strains differences in the forced swimming behaviour.

Direct evidence for chronic stress-induced facilitation of the adrenocorticotropin response to a novel acute stressor.

The ACTH response to CRF and the role of glucocorticoids on the pituitary-adrenal responsiveness to acute stressors after a period of chronic stress were assessed in Sprague-Dawley rats. After chronic immobilization (IMO) an enhanced ACTH response to CRF administration was observed. In another experiment, control and chronic IMO rats were adrenalectomized (ADX) or sham-adrenalectomized (SHAM) and 2 days later killed in resting conditions or after having been subjected to acute IMO or tail-shock for 30 min. Chronic IMO increased basal corticosterone but not adrenocorticotropin (ACTH) levels in SHAM rats. As expected, ADX increased ACTH levels in all conditions. In response to the novel acute stressor (shock), ACTH levels were drastically dependent on the presence of corticosterone: thus, whereas in SHAM rats chronic IMO reduced the ACTH response to shock, in ADX rats a clear enhancement of the ACTH response to shock was observed in chronic IMO rats. In order to demonstrate that, in our experimental conditions, chronic stress also induces down-regulation of glucocorticoid receptors in the hippocampus, an additional experiment was done: rats subjected chronically to IMO or administered 5 mg corticosterone (B) were adrenalectomized and killed 20 h later under basal conditions. Both chronic IMO and chronic B administration decreased glucocorticoid type II binding in the hippocampus. From these results, it is concluded that chronic IMO induces facilitation of the ACTH response to novel acute stressors which is uncovered after corticosterone removal.

The effect of acute and chronic ACTH administration on pituitary-adrenal response to acute immobilization stress. Relationship to changes in corticosteroid-binding globulin.

The effect of single and chronic ACTH administration on serum levels of the corticosteroid-binding globulin (CBG) and pituitary-adrenal (PA) responsiveness to acute immobilization (IMO) stress was studied in adult Sprague-Dawley rats. Single ACTH administration significantly reduced CBG levels but did not alter PA response to acute IMO. Chronic ACTH administration caused a greater fall in CBG than single ACTH administration and blunted PA response to IMO. The effect of chronic ACTH administration on CGB levels recovered 2 days after the last administration, but the ACTH response to IMO was normal only by day 7 after the last ACTH injection. The present data indicate that ACTH administration to rats reduced CBG levels and impaired PA response to acute stress, but impaired PA responsiveness cannot be solely attributed to changes in CBG.

Chronic stress affects in vivo hypothalamic somatostatin release but not in vitro GH responsiveness to somatostatin in rats.

One week after stereotaxical implantation of a push-pull cannula into the median eminence (ME), rats were stressed by immobilization for 2 h daily for 7 days. Thereafter, ME was perfused for 1 h in basal, stress and recovery conditions, respectively, and somatostatin (SRIH) was measured in perfusate fractions. Pituitaries were in vitro perifused to assess GH responsiveness to SRIH. In the stressed group, basal SRIH release was significantly higher than in the control group and stress caused a significant sharp peak in neurohormone release. GH responsiveness to SRIH was not affected in pituitaries obtained from stressed donors. High SRIH levels secreted under chronic stress thus did not impair the GH pituitary response to SRIH.

Chronic but not acute exposure to stress is associated with hypothalamic vasoactive intestinal polypeptide (VIP) release into median eminence.

The influence of stress on hypothalamic VIP release into the pituitary portal blood has not been assessed at present despite the fact that this peptide has been implicated in the control of several pituitary hormones and especially in the release of prolactin (PRL) caused by stress. In the present work the effect of stress on the in vivo release of VIP into the pituitary portal blood of male rats was evaluated by means of push-pull perfusion (PPP) of median eminence (EM). VIP content in the PPP liquid was successfully measured and their levels agree well with pituitary portal blood levels measured by other authors. Whereas plasma PRL levels strongly increased during acute immobilization (IMO), no changes in VIP secretion into the ME were observed. VIP release into the ME was also unaffected by exposure to ether. In contrast, in chronically immobilized rats a significant increase in VIP release into the ME was obtained in response to acute IMO. The present data argue against a major role of hypothalamic VIP in PRL release caused by acute stress and show that chronic stress qualitatively alters the signal of hypothalamic VIP to the pituitary.

Effects of chronic immobilization stress on GH and TSH secretion in the rat: response to hypothalamic regulatory factors.

The effect of chronic immobilization (2 h/day) for 13 days on basal and stress levels of GH and TSH, and their response to various hypothalamic regulatory factors was studied in male Sprague-Dawley rats. Chronic immobilization (IMO) resulted in reduced serum TSH levels in stress situations but not in resting conditions. GH secretion was inhibited both in resting and stress situations. Chronic IMO impaired both GH and TSH responses to GRH and TRH, respectively, but also to another peptide (VIP) stimulatory for the two hormones. Whereas somatostatin administration inhibited GH secretion in control but not in chronic IMO rats, its inhibitory effect on TSH was slight and similar in the two experimental groups. The present results suggest that chronic exposure to a severe stressor such as IMO alters GH and TSH secretion, at least in part by changes in the response of the pituitary to the hypothalamic regulatory factors. The actual influence of chronic IMO on the release of these peptides into the median eminence remains to be studied.

Role of somatostatin in the acute immobilization stress-induced GH decrease in rat.

In the present work we have investigated to what extent somatostatin (SRIF) release from median eminence (ME) is affected by stress immobilization (IMO) in unanesthetized rats stereotaxically implanted with a push-pull cannula (PPC). One week after implantation, the ME was perfused with artificial cerebrospinal fluid for 1 hour in basal, stress and recovery conditions respectively. Samples were collected every 15 min and SRIF was measured by RIA. In another group of animals, a jugular cannula was inserted the day before and plasma samples were taken off simultaneously with the ME perfusate for GH and SRIF analysis respectively. SRIF release from the ME is rapidly (15 min) and significantly increased (58 +/- 11 vs 28 +/- 5 pg/15 min; n = 7; P < 0.01) in rats bearing only PPC. Intriguingly, animals bearing a jugular catheter plus a PPC showed no increase in SRIF release during the first 15 min of IMO in spite of a striking decrease of plasma GH (27.2 +/- 3.8 vs 3.6 +/- 1.3 ng/ml; n = 6; P < 0.001) observed at this time. However, in spite that the animals responded with a significant increase in SRIF, the response was later and more reduced than in animals without jugular cannula. Since our two rat groups--as result of jugular cannula surgery 24 hours before--showed differences such as a food intake, body weight gain, plasma GH levels and basal SRIF release, we think that these differences could explain the modifications in the regulatory mechanisms involved in GH control under acute stress.

Chronic stress induced changes in LH secretion: the contribution of anorexia associated to stress.

The effects of chronic intermittent immobilization (IMO) on serum LH levels of adult male rats were studied. Chronic IMO (2 h daily for 13 days) did not alter basal LH levels, but abolished the LH response to acute stressors (IMO and tailshock). The inhibition of LH caused by acute exposure to IMO for 4 or 18 h was similar in control and chronic IMO rats. Also the LH response to exogenous LHRH administration was normal in chronically stressed rats. When a group of rats eating the same amount of food as that eaten by immobilized rats was introduced (pair-fed), an inhibition of LH response to acute stressors quite similar to that found in chronic IMO rats was observed. These data indicate that chronic stress-induced inhibition of LH release caused by short-term exposure to acute stressors was located above the pituitary and was mainly due to anorexia accompanying daily exposure to the stressor.

Evidence for the involvement of serotonin in acute stress-induced release of luteinizing hormone in the male rat.

The influence of serotonin on luteinizing hormone (LH) release caused by exposure to two acute stressors differing in their intensity (restraint in tubes and immobilization in woodboards) was studied in adult male rats. Inhibition of serotonin synthesis with p-chlorophenylalanine (PCPA) significantly abolished LH release caused by immobilization (IMO). Administration of the serotonin antagonists mianserine and methiothepin also eliminated LH release caused by IMO without altering basal LH levels. These data represent the first evidence that a classical neurotransmitter (serotonin) is involved in LH release caused by stress in the rat.

Effect of regularity of exposure to chronic immobilization stress on the circadian pattern of pituitary adrenal hormones, growth hormone, and thyroid stimulating hormone in the adult male rat.

Circadian variation of serum levels of adrenocorticotropin hormone (ACTH), corticosterone, growth hormone (GH), and thyroid-stimulating hormone (TSH) were studied in three groups of adult male rats exposed to chronic intermittent immobilization stress (IMO) for 2 hr daily under different schedules. IMO resulted in reduced food intake, body weight loss, and increased adrenal weight. ACTH levels were not affected but corticosterone levels were increased in all IMO rats as compared to control ones during the diurnal phase of the circadian cycle. IMO decreased serum GH and TSH levels but the circadian pattern of secretion was influenced in a complex way depending on the specific pattern of daily exposure to IMO. Differences observed between the IMO groups were not caused by differences in food intake because its circadian rhythm was very similar in all IMO groups. These results suggest that regularity of exposure to immobilization alters in a complex fashion circadian GH and TSH rhythms.

Negative feedback of corticosterone on the pituitary-adrenal axis is maintained after inhibition of serotonin synthesis with parachlorophenylalanine.

The role of the serotonergic transmission on the negative feedback of corticosterone on the pituitary-adrenal (PA) axis was studied in adult male rats. Animals were given p-chlorophenylalanine (PCPA) and 24 h later were administered corticosterone, 2 h before being subjected to 20 min of noise stress. The main results were as follows: First, PCPA administration increased resting levels of both corticotropin (ACTH) and corticosterone but did not alter PA response to noise stress; second, ACTH response to stress was eliminated in rats given PCPA; third, corticosterone reduced ACTH levels in nonstressed rats only and this effect was maintained after PCPA administration. Taken together, the present results suggest a mediator role for serotonin in the noise-stress-induced PA hormone release but no role in the negative feedback of corticosterone on the PA axis.

Blockade of opioid receptors with naltrexone inhibits thyrotropin increase after noise stress but does not prevent the decrease caused by immobilization.

The influence of naltrexone-induced opioid receptor blockade on the response of thyrotropin to two different acute stressors was studied in adult male rats. Naltrexone slightly but significantly reduced basal thyrotropin levels and abolished the increase in serum thyrotropin caused by acute noise stress. In contrast, the opioid antagonist did not prevent the decrease in serum thyrotropin caused by another much more severe stressor such as immobilization. The present data offer the first evidence that endogenous opioids could play a stimulatory role in the control of thyrotropin secretion in a presumably physiological condition such as the response to a mild stressor. In addition, factors other than opioids could be involved in the inhibition of thyrotropin secretion under severe stress.

Forced swimming test in rats: effect of desipramine administration and the period of exposure to the test on struggling behavior, swimming, immobility and defecation rate.

The effect of desipramine administration and the duration of the daily exposure to forced swimming on some variables has been studied in adult male rats. Desipramine administration (15 mg/kg) significantly increased struggling behavior in the first and second 5-min periods of a single exposure to forced swimming. Swimming was reduced in the first 5 min and remained unchanged thereafter. Immobility was decreased in the second and the third 5-min periods. After a pre-exposure to forced swimming for 15 min the day before, the drug was effective in increasing struggling behavior and reducing immobility during a subsequent 5-min test. Swimming was not modified. Daily exposure to forced swimming for 3 days caused a decline in struggling behavior and swimming, while increasing immobility and the defecation rate. The duration of daily exposure to forced swimming did not alter the changes in the variables measured. The present results indicate that a one-day test can be used to discriminate between saline- and desipramine-treated rats, and that struggling behavior could be a reliable measure of the positive action of antidepressants. The finding that behavioral changes over the 3 days were independent of the duration of exposure to swimming argues against the interpretation of the results which suggest that the responses are caused by the appearance of a behavioral despair state, and suggests that these behaviors might be trait-markers in the rat. In addition, the changes in struggling behavior and immobility over the 3 days cannot be attributed to a behavioral adaptation to the test because the defecation rate increased rather than decreased during successive forced swimming tests.