Cardiac myocyte exosomes: stability, HSP60, and proteomics.

Exosomes, which are 50- to 100-nm-diameter lipid vesicles, have been implicated in intercellular communication, including transmitting malignancy, and as a way for viral particles to evade detection while spreading to new cells. Previously, we demonstrated that adult cardiac myocytes release heat shock protein (HSP)60 in exosomes. Extracellular HSP60, when not in exosomes, causes cardiac myocyte apoptosis via the activation of Toll-like receptor 4. Thus, release of HSP60 from exosomes would be damaging to the surrounding cardiac myocytes. We hypothesized that 1) pathological changes in the environment, such as fever, change in pH, or ethanol consumption, would increase exosome permeability; 2) different exosome inducers would result in different exosomal protein content; 3) ethanol at "physiological" concentrations would cause exosome release; and 4) ROS production is an underlying mechanism of increased exosome production. We found the following: first, exosomes retained their protein cargo under different physiological/pathological conditions, based on Western blot analyses. Second, mass spectrometry demonstrated that the protein content of cardiac exosomes differed significantly from other types of exosomes in the literature and contained cytosolic, sarcomeric, and mitochondrial proteins. Third, ethanol did not affect exosome stability but greatly increased the production of exosomes by cardiac myocytes. Fourth, ethanol- and hypoxia/reoxygenation-derived exosomes had different protein content. Finally, ROS inhibition reduced exosome production but did not completely inhibit it. In conclusion, exosomal protein content is influenced by the cell source and stimulus for exosome formation. ROS stimulate exosome production. The functions of exosomes remain to be fully elucidated.

Sidestream smoke exposure enhances rapidly adapting receptor responses to substance P in young guinea pigs.

We determined the effect of sidestream tobacco smoke (SS) exposure on responses of lung rapidly adapting receptors (RARs), peak tracheal pressure (Ptr), and arterial blood pressure (ABP) to substance P in young guinea pigs. Guinea pigs were exposed to SS or filtered air from day 8 to days 41-45 of life. They were then anesthetized and given three doses of intravenous substance P (1.56-4.94 nmol/kg). SS exposure augmented substance P-evoked increases in RAR activity (P = 0.029 by analysis of variance) but not substance P-evoked increases in peak Ptr or decreases in ABP. Neurokinin 1-receptor blockade (CP-96345, 400 nmol/kg) attenuated substance P-evoked increases in RAR activity (P = 0.001) and ABP (P = 0.009) but not in peak Ptr (P = 0.06). This chronic exposure to SS in young guinea pigs exaggerates RAR responsiveness to substance P. The findings may help explain the increased incidence of airway hyperresponsiveness and cough in children chronically exposed to environmental tobacco smoke.

Exposing guinea pigs to ozone for 1 wk enhances responsiveness of rapidly adapting receptors.

Acute exposure to ozone causes changes in breathing pattern and lung function which may be caused in part by stimulation of rapidly adapting receptors (RARs). The consequences of repeated daily ozone exposure on RAR responsiveness are unknown, although ozone-induced changes in pulmonary function diminish with repeated exposure. Accordingly, we investigated whether repeated daily ozone exposure diminishes the general responsiveness of RARs. Guinea pigs (n = 30) were exposed to 0.5 parts/million ozone or filtered air (8 h/day for 7 days). The animals were then anesthetized, and RAR impulse activity, dynamic compliance (Cdyn), and lung resistance were recorded at baseline and in response to four stimuli: substance P, methacholine, hyperinflation, and removal of positive end-expiratory pressure. Repeated daily ozone exposure exaggerated RAR responses to substance P, methacholine, and hyperinflation without causing physiologically relevant effects on baseline or substance P- and methacholine-induced changes in Cdyn and lung resistance. Because agonist-evoked changes in RAR activity preceded Cdyn changes, the data suggest that repeated daily ozone exposure enhances RAR responsiveness via a mechanism other than changes in Cdyn.

Chronic exposure to sidestream tobacco smoke augments lung C-fiber responsiveness in young guinea pigs.

Children chronically exposed to environmental tobacco smoke (ETS) have more coughs, wheezes, and airway obstruction, which may result in part from stimulation of lung C fibers. We examined the effect of chronic exposure to sidestream tobacco smoke (SS, a surrogate for ETS) on lung C-fiber responsiveness in guinea pigs, in which dynamic compliance (Cdyn), lung resistance, tracheal pressure, arterial blood pressure, and heart rate were also monitored. Guinea pigs were exposed to SS (1 mg/mm(3) total suspended particulates) or filtered air 5 days/wk from 1 to 6 wk of age. They were then anesthetized, and lung C fibers (n = 55), identified by a conduction velocity of <2.0 m/s, were tested for responsiveness to chemical and mechanical stimuli. SS exposure doubled C-fiber responsiveness to left atrial capsaicin (P = 0.02) and lung hyperinflation (P = 0.03) but had no effect on responsiveness to inhaled capsaicin or bradykinin or on baseline activity. The data indicate that chronically exposing young guinea pigs to SS enhances C-fiber sensitivity to certain stimuli and may help explain respiratory symptoms in children exposed to ETS.

Exposing young guinea pigs to sidestream tobacco smoke decreases rapidly adapting receptor responsiveness.

We exposed 21 young guinea pigs to 5 wk of either sidestream tobacco smoke (SS) or filtered air (FA). The exposure started on day 8 of life and ended at 41-45 days of life. The animals were then anesthetized, and lung rapidly adapting receptor (RAR) and slowly adapting receptor (SAR) activities and peak tracheal pressure (TP) were examined in response to mainstream smoke. SS exposure did not alter baseline RAR activity. Low-nicotine smoke increased RAR activity in the FA but not in the SS group. High-nicotine smoke increased RAR activity in both groups but more so in the FA than in the SS group. Baseline TP was lower in the SS group. Both low- and high-nicotine smoke increased TP but more so in the FA than in the SS group. The increase in RAR activity preceded the increase in TP. SS exposure increased baseline SAR activity but did not affect the variable responses of SARs to low- and high-nicotine smoke. We suggest that exposing guinea pigs to SS during development diminishes the responsiveness of RARs to acute inhalation of mainstream smoke.

Effects of extended sidestream smoke exposure on components of the C-fiber axon reflex.

We have previously shown that young guinea pigs repeatedly exposed to sidestream cigarette smoke (SS) develop decreased airway reactivity of the C-fiber system without changing reactivity to one of its neurotransmitters, substance P (SP). This study was designed to determine whether the decreased reactivity was due to decreased responsiveness to another neurotransmitter, neurokinin A (NKA), decreased lung SP content, decreased affinity or number of NK1 receptors, and/or decreased number of C-fibers. Duncan Hartley guinea pigs were exposed to filtered air (FA) or to SS for 6 h/day, 5 days/week for 5 weeks starting at 1 week of age. SS exposure did not change, (1) airway reactivity to NKA injected into the pulmonary artery of their isolated perfused lungs (n = 6-7 each group), (2) lung SP content as measured by enzyme immunoassay (n = 12 each group), (3) NK1 receptor number or affinity as measured by radioligand binding (n = 7 each group), or (4) SP-immunoreactive nerve profiles of the terminal bronchioles or small airways (n = 6 each group). Thus, SS exposure does not decrease C-fiber system by reducing NKA responsiveness, decreasing SP content, changing NK1 receptors, or decreasing the number of C-fibers.

Photoperiod and pinealectomy do not affect cold-induced deposition of brown adipose tissue in the Long-Evans rat.

Brown adipose tissue is a major thermogenic effector of cold-induced nonshivering thermogenesis. Previous studies indicate that melatonin and/or short photoperiod are involved in the increase in brown fat deposition seen in certain cold-acclimated rodents. The present study was undertaken, in part, to determine whether the pineal is a necessary component in the cold-induced increase in thermogenic capacity characteristic of the cold-acclimated laboratory rat. Under a 12L:12D light cycle, pinealectomized rats did not differ from sham-operated rats in their ability to increase brown fat deposition in the cold (5 degrees C). Moreover, in a subsequent set of experiments performed at 9 degrees C, intact rats maintained at a short photoperiod (9L:15D) exhibited the same degree of brown fat hypertrophy/hyperplasia as did those kept at a long photoperiod (15L:9D). These data thus indicate that: (a) the intact pineal is not necessary for the cold-induced increase in brown adipose tissue occurring in the cold-acclimated rat; and (b) photoperiod does not significantly modulate the magnitude of this increase.

Effect of adenosine receptor ligands on cAMP content in human airways and peripheral lung.

Adenosine causes airway obstruction in asthmatics and smokers. Theophylline and cromolyn, drugs used to treat these patients, bind to human lung adenosine receptors (ARs). This study investigated whether A1ARs and/or A2ARs are functionally present in human lung and airways, and whether theophylline and/or cromolyn antagonize their function. Peripheral lung or airway fragments from 21 people were incubated for 15 min with (1) an A1AR agonist, N6-cyclopentyladenosine (CPA, 5 to 1,000 nM), or (2) an A2AR agonist, either 5'-N-ethylcarboxamido adenosine (NECA, 0.5 to 20 microM) or 2-[p-(2-carboxyethyl)-phenethyl amino]-5'-N-ethylcarboxamido adenosine (CGS 21680, 0.5 to 28 microM), in the presence of the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine (50 nM) and/or (3) theophylline (1 mM) and/or (4) cromolyn (500 microM). Adenosine deaminase (2 U/ml) and the phosphodiesterase inhibitor Ro 20-1724 (2 mM) were present in all incubations. Cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. In peripheral lung, CPA did not change baseline or isoproterenol-stimulated cAMP content. However, both NECA (20 microM) and CGS 21680 (28 microM) significantly (P < 0.05) increased cAMP content 220 +/- 4% and 201 +/- 32%, respectively (mean +/- SEM). In airways, 20 microM NECA increased cAMP content 129 +/- 34%, and 28 microM CGS 21680 increased it 52 +/- 20% (both P < 0.05). In both peripheral lung and airway tissue, NECA-induced increase in cAMP was antagonized by theophylline (P < 0.05) but not cromolyn. The lungs of younger, nonsmokers had lower baseline cAMP content but did not respond differentially to A2AR stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

GDP binding to hamster brown fat mitochondria is reduced during hibernation.

Preparation for hibernation is accompanied by increased thermogenic capacity of brown fat (BAT), an important site of thermogenesis during arousal from hibernation. This study examined whether that thermogenic capacity is reduced in hibernation and reactivated during arousal. In one set of experiments, Syrian hamsters were exposed to short photoperiod (10:14 light-dark) and cold (7 degrees C). Those not hibernating at death (n = 10) served as controls for those that were (n = 9). A third group (n = 10) was killed 80-90 min after arousal was initiated by manual perturbation. Mitochondrial GDP binding (nmol/mg mitochondrial protein) was used to estimate thermogenic capacity. In a second experimental series, BAT citrate (si)-synthase and 3-hydroxyacyl-CoA dehydrogenase activities were measured in hibernating and nonhibernating hamsters. Although there were no differences in the maximum activities of these enzymes, GDP binding was markedly lower in the hibernators relative to the nonhibernators (0.214 +/- 0.031 vs. 0.535 +/- 0.039). However, in the partially aroused hamsters, GDP binding had doubled (0.438 +/- 0.04). Thus hibernation is accompanied by a substantial reduction of BAT thermogenic capacity (as manifested by GDP binding), which is reversed during arousal. The rapidity of this reversal indicates that it does not involve the synthesis of new GDP binding sites.

Decreased testosterone levels do not mediate short-photoperiod-induced brown fat changes.

Previous studies showed that short photoperiod increased brown fat (BAT) mass and reduced gonadal size and gonadal hormone secretion in hamsters. The present study investigated the possibility that the effects on BAT were dependent on reduced levels of gonadal hormones. BAT from male Syrian hamsters exposed to short photoperiod for 10 wk was significantly greater in mass, protein content, and total maximal citrate synthase and beta-hydroxyacyl-CoA dehydrogenase activities than was BAT from long-photoperiod hamsters, These differences between short- and long-photoperiod exposure were observed in hamsters housed at 21 as well as at 8 degrees C. Short photoperiod also increased the total recovered mitochondrial GDP binding, a finding consistent with increased BAT thermogenic capacity. These short-photoperiod effects were neither mimicked by castration of long-photoperiod hamsters nor prevented by high levels of testosterone administered to short-photoperiod animals. Castration did attenuate the effects of short photoperiod on BAT growth if, after surgery and prior to short-photoperiod exposure, the animals were housed at a long photoperiod for 2-3 wk. In contrast, in hamsters immediately placed at short photoperiod after surgery, castration did not inhibit short-photoperiod effects. The present three experiments demonstrate that, in addition to increasing BAT mass, short photoperiod elevates the thermogenic capacity of BAT, and this elevation does not require the absence or a much reduced testosterone level.

The local C-fiber contribution to ozone-induced effects on the isolated guinea pig lung.

We studied the local role of C-fibers, in the absence of systemic effects and blood components of inflammation, on lung responses to ozone. Guinea pigs were pretreated with capsaicin to deplete C-fibers or with vehicle. One week later their isolated, buffer-perfused lungs were exposed to 0.8 ppm ozone or air for 2 hr. In some lungs (9 or 10 each group), increasing doses of methacholine followed by capsaicin were injected into the pulmonary artery. In separate lungs (n = 5, each group), lung Substance P content by EIA and NK1 receptor characteristics by radioligand binding were measured. Analyses were performed by two-way ANOVA with a significant interaction indicative of C-fibers playing a role in ozone responses. Ozone increased R(L) and decreased C(dyn), effects which were apparently not dependent on C-fibers. Ozone increased responsiveness to methacholine, an effect which was reduced by depletion of C-fibers. Ozone increased and C-fiber depletion decreased lung responses to capsaicin. C-fiber depletion but not ozone decreased lung substance P content. C-fiber depletion increased the affinity but did not change the number of NK1 receptors, while ozone had no effect. We conclude that the ozone, in the absence of systemic effects and the blood components of inflammation, increased muscarinic reactivity in part via the local effects of C-fibers.

Nitric oxide contributes to substance P-induced increases in lung rapidly adapting receptor activity in guinea-pigs.

1. Substance P induces fluid flux via nitric oxide, and fluid flux stimulates lung rapidly adapting receptors (RARs). We therefore proposed that nitric oxide contributes to substance P-evoked increases in RAR activity. Since substance P decreases dynamic compliance (Cdyn), which can stimulate RARs, we also determined whether nitric oxide contributed to substance P-induced effects on pulmonary function. 2. In anaesthetized guinea-pigs, the effects of substance P on RAR activity, Cdyn, pulmonary resistance (RL), and arterial blood pressure were measured before and after i.v. infusion of NG-methyl-L-arginine (L-NMMA; a nitric oxide synthase inhibitor), or L-NMMA followed by L-arginine (a nitric oxide precursor which reverses the effects of L-NMMA). 3. Substance P-evoked increases in RAR activity were blunted by L-NMMA (P = 0.006) but not by L-NMMA-L-arginine (P = 0.42). 4. Substance P-evoked decreases in Cdyn were slightly inhibited by L-NMMA (P = 0.02) and slightly enhanced by L-NMMA-L-arginine (P = 0.004). However, at the time at which L-NMMA maximally reduced substance P-induced RAR stimulation (the first 30 s), it did not change substance P-induced decreases in Cdyn. 5. Substance P-evoked increases in RL were not changed by L-NMMA (P = 0.10) and were enhanced by L-NMMA-L-arginine (P = 0.03). 6. L-NMMA-evoked increases in mean arterial blood pressure were reversed by L-arginine. Substance P-evoked decreases in mean arterial blood pressure were not changed by L-NMMA or by L-NMMA-L-arginine. 7. We conclude that nitric oxide contributes to substance P-evoked increases in RAR activity and that the increases are most probably independent of decreases in Cdyn.

Hyperprolactinemia prevents short photoperiod-induced changes in brown fat.

Previous studies demonstrated that short photoperiod exposure significantly decreases circulating prolactin levels. The present study investigated the possibility that concomitant changes in brown fat tissue mass, protein content, thermogenic capacity, and carcass composition are dependent on this change in prolactin levels. Male golden (Syrian) hamsters were sham operated and exposed to a short (10L:14D) or long (14L:10D) photoperiod. A third group was implanted with exogenous pituitaries under the right kidney capsule and exposed to a short photoperiod. In experiment I, 4 wk of short- vs. long-photoperiod exposure did not result in significant changes in circulating prolactin levels, nor was there an increase in brown fat mass, protein content, or thermogenic capacity. Four weeks of short-photoperiod exposure did significantly increase carcass lipid content. However, this increase did not occur in hamsters exposed to 4 wk of short photoperiod but made hyperprolactinemic (implanted with two exogenous pituitaries). Ten weeks of short photoperiod significantly reduced circulating prolactin levels. Concomitantly, brown fat mass, protein content, and thermogenic capacity, as well as carcass fat, were increased. These short-photoperiod-induced changes were not observed in similarly exposed hamsters that were made hyperprolactinemic via two implanted pituitaries. In experiment II, similar changes in brown fat and body composition occurred in sham-operated hamsters exposed to 10 wk of short photoperiod. These changes were prevented in hamsters exposed to 10 wk of short photoperiod but made hyperprolactinemic via only one implanted pituitary. These results suggest that decreased prolactin is a necessary condition for the increased brown fat mass, protein content, and thermogenic capacity that occurs when golden hamsters are exposed to short photoperiod.

In utero and postnatal effects of sidestream cigarette smoke exposure on lung function, hyperresponsiveness, and neuroendocrine cells in rats.

We evaluated whether sidestream smoke (SS) exposure in utero and/or postnatally causes airway obstruction and hyperresponsiveness, and whether the effect is associated with neuroendocrine cell hyperplasia. Pregnant Sprague-Dawley rats were exposed to filtered air (FA) or to SS (total suspended particulate concentration, 1.00 +/- 0.07 mg/m3, CO, 4.9 +/- 0.7 ppm; nicotine, 344 +/- 85 micrograms/m3; mean +/- SD) for 4 hr/day, 7 days/week from Day 3 of gestation until birth and then their female pups were exposed to either FA or SS for 7-10 weeks postnatally. This resulted in four exposure conditions: in utero FA followed by postnatal FA (FA/FA), in utero FA followed by postnatal SS (FA/SS), in utero SS followed by postnatal FA (SS/FA), and in utero SS followed by postnatal SS (SS/SS). The lungs from the pups (n = 6-8 of each exposure combination) were then placed in an isolated buffer-perfused system where transpulmonary pressure, airflow, and pulmonary artery pressure (Ppa) were measured while increasing doses of methacholine were injected into the pulmonary artery. Three lungs from each group were then fixed in 1% paraformaldehyde and neuroendocrine cells were identified immunohistochemically using antibodies to neuron-specific enolase. As compared to lungs from FA/FA-exposed rats, lungs from SS/SS-exposed rats exhibited 24% lower Cdyn (p = 0.0006, ANOVA), greater reactivity to methacholine (p = 0.0001, repeated measures ANOVA), and more neuroendocrine cells per centimeter basal lamina (p = 0.0006, ANOVA). Lungs from SS/FA- or FA/SS-exposed rats were not different from lungs from FA/FA-exposed rats in any of these parameters. We conclude that exposure to SS both pre- and postnatally (but not only pre- or only postnatally) results in lungs which are less compliant, more reactive to methacholine, and have a greater number of neuroendocrine cells.

Substance P contributes to rapidly adapting receptor responses to pulmonary venous congestion in rabbits.

1. This study tested the hypothesis that substance P stimulates rapidly adapting receptors (RARs), contributes to the increase in RAR activity produced by mild pulmonary congestion, and evokes an augmented response from RARs when combined with near-threshold levels of pulmonary congestion. 2. RAR activity, peak tracheal pressure, arterial blood pressure and left atrial pressure were measured in paralysed, anaesthetized and ventilated rabbits. Substance P was given i.v. in one-half log incremental doses to a maximum of 3 micrograms kg-1. Mild pulmonary congestion was produced by inflating a balloon in the left atrium to increase left atrial pressure by 5 mmHg. Near-threshold levels of pulmonary congestion were produced by increasing left atrial pressure by 2 mmHg. 3. Substance P produced dose-dependent increases in RAR activity. The highest dose given increased the activity from 1.3 +/- 0.5 to 11.0 +/- 3.1 impulses bin-1. Increases in left atrial pressure of 5 mmHg increased RAR activity from 3.8 +/- 1.4 to 14.7 +/- 3.9 impulses bin-1. Blockade of NK1 receptors with CP 96345 significantly attenuated RAR responses to substance P and to mild pulmonary congestion. 4. Doses of substance P, which alone had no effect, stimulated the RARs when delivered during near-threshold levels of pulmonary congestion. 5. The findings suggest that substance P augments the stimulatory effect of mild pulmonary congestion on RAR activity, most probably by enhancing hydraulically induced microvascular leak.