Biomedical subjects

D M Cooper

Publications and source records attributed to D M Cooper.

At least 37 records · Page 2Linked to original sources

PubMed2001

Effects of laboratory versus field exercise on leukocyte subsets and cell adhesion molecule expression in children.

In adults, exercise is a powerful and natural stimulator of immune cells and adhesion molecules. Far less is known about these exercise responses during childhood and whether or not exercise in real-life activities of healthy children might influence immune responses. We compared laboratory exercise (10 x 2 min periods of heavy, constant intensity, cycle ergcometer exercise with 1 min rests between exercise in nine subjects, aged 9-15 years) with field exercise (90 min soccer practice in nine different subjects, aged 9-11 years). Blood was sampled before both protocols, 5 min after the 30 min laboratory protocol, and 10-15 min after the 90 min field protocol. Both field and laboratory exercise protocols led to significant (P<0.05) increases in granulocytes, monocytes, and all lymphocyte subpopulations. The mean (SEM) increases were similar for the two protocols except for the significantly greater increase in laboratory compared with field protocols for natural killer cells [142 (39)% vs 12 (16)%, P<0.001] and monocytes [64 (22)% vs 32 (19)%, P<0.001] Both protocols significantly influenced adhesion molecules (such as CD54) which have not been previously studied in children. However, the adhesion molecule CD8+ CD62L increased to a significantly (P < 0.001) greater extent in the laboratory [101 (25)%] versus field [34 (25)%] protocol. Finally, the density of CD632L on lymphocytes significantly decreased with laboratory exercise but showed no change in the field protocol [-20 (3)% vs -3 (3)%, P<0.001]. The rapid and substantial immune response in both laboratory and field protocols suggests that exercise stimulation of the immune system occurs commonly in the real lives of children and may play a role in their overall immune status.

CD3 Complex↗

PubMed2001

Burkholderia cepacia in cystic fibrosis: novel Australian cluster strain without accelerated respiratory deterioration.

OBJECTIVE: To determine whether 4 years of colonization with a novel Australian cluster strain (The 'Hunter' strain) of Burkholderia cepacia (B. cepacia) in cystic fibrosis (CF) patients was associated with more rapid decline in nutritional status and pulmonary function than in non-colonized contemporaries from the same CF clinic. METHODOLOGY: A retrospective review of respiratory function and nutritional data from a single multidisciplinary paediatric CF clinic over 4 years (1993-97). RESULTS: Paired spirometry data for 1993 and 1997 were available in 47 patients without (n = 31) and with B. cepacia (n = 16) colonization (mean (+/- SD) ages in 1993: 12.1 years +/- 4.0 vs 12.6 years +/- 6.5; P = 0.83). Their percentage predicted forced expiratory volume in 1 s (FEV1) (94.2% +/- 16.7 vs 85.9% +/- 21.2; P = 0.19) were not significantly different. The averaged annual fall in FEV1 over 4 years was also not significantly different (3.8% +/- 3.8 vs 3.6% +/- 3.7; P = 0.82). Weight percentile (Wt%), height percentile (Ht%) and percentage age weight for height (%WFH) were not significantly different between groups in 1993. By 1997, Wt% (36.7% +/- 25.1 vs 22.3% +/- 19.6; P = 0.04) and Ht% (42.5% +/- 29.6 vs 17.6% +/- 19.4; P = 0.002) but not %WFH (102% +/- 10.0 vs 106% +/- 11.2; P > 0.10) were lower in subjects with B. cepacia. CONCLUSIONS: In adolescent CF patients, colonization with the Hunter strain of B. cepacia was associated with a deterioration in some nutritional parameters but not with an accelerated decline in FEV1 over 4 years. As varying pathogenicity of B. cepacia strains may account for differing rates of pulmonary decline, further assessment of the consequences of colonization with certain strains of B. cepacia in CF is needed.

Adolescent↗

PubMed2001

Oxidative stress in cystic fibrosis: dietary and metabolic factors.

OBJECTIVE: To examine oxidative stress in CF by measuring 8-iso-PGF2alpha and antioxidant defenses, in relation to dietary intake, immune function and clinical status. METHODS: We measured total plasma concentrations of 8-iso-PGF2alpha and dietary antioxidants (vitamin E, vitamin C, beta-carotene), erythrocyte antioxidant enzyme activities (glutathione peroxidase and superoxide dismutase), lung function and dietary intake in 21 CF subjects and 21 healthy age- and gender-matched controls. RESULTS: Total plasma 8-iso-PGF2alpha concentration (median [quartile 1-quartile 3]) was significantly higher in CF subjects compared to controls (214 pg/mL (155-331) vs. 135 pg/mL (101-168), p = 0.001). Neutrophil, monocyte and total white cell counts were elevated in the CF group and these correlated with 8-iso-PGF2alpha concentration. Despite similar dietary intake, lower plasma antioxidant concentrations were observed in the CF group (vitamin E, p < 0.001, vitamin C, p = 0.004, beta-carotene, p = 0.001). 8-iso-PGF2alpha correlated negatively with plasma vitamin E, C and beta-carotene concentrations. CONCLUSION: Oxidative stress is increased in CF patients, despite normal dietary antioxidant intake. The immune response appears to be a key factor causing oxidative stress. Antioxidant intervention aimed at reducing oxidative stress in CF needs to be assessed.

Adolescent↗

PubMed2001

Training, muscle volume, and energy expenditure in nonobese American girls.

Little is known about the relationship among training, energy expenditure, muscle volume, and fitness in prepubertal girls. Because physical activity is high in prepubertal children, we hypothesized that there would be no effect of training. Forty pre- and early pubertal (mean age 9.1 +/- 0.1 yr) nonobese girls enrolled in a 5 day/wk summer school program for 5 wk and were randomized to control (n = 20) or training groups (n = 20; 1.5 h/day, endurance-type exercise). Total energy expenditure (TEE) was measured using doubly labeled water, thigh muscle volume using magnetic resonance imaging, and peak O(2) uptake (VO(2 peak)) using cycle ergometry. TEE was significantly greater (17%, P < 0.02) in the training girls. Training increased thigh muscle volume (+4.3 +/- 0.9%, P < 0.005) and VO(2 peak) (+9.5 +/- 6%, P < 0.05), effects surprisingly similar to those observed in adolescent girls using the same protocol (Eliakim A, Barstow TJ, Brasel JA, Ajie H, Lee W-NP, Renslo R, Berman N, and Cooper DM, J Pediatr 129: 537-543, 1996). We further compared these two sample populations: thigh muscle volume per weight was much lower in adolescent compared with prepubertal girls (17.0 +/- 0.3 vs. 27.8 +/- 0.6 ml/kg body mass; P < 0.001), and allometric analysis revealed remarkably low scaling factors relating muscle volume (0.34 +/- 0.05, P < 0.0001), TEE (0.24 +/- 0. 06, P < 0.0004), and VO(2 peak) (0.28 +/- 0.07, P < 0.0001) to body mass in all subjects. Muscle and cardiorespiratory functions were quite responsive to brief training in prepubertal girls. Moreover, a retrospective, cross-sectional analysis suggests that increases in muscle mass and VO(2 peak) may be depressed in nonobese American girls as they mature.

Body Height↗

PubMed2001

Flow-independent nitric oxide exchange parameters in healthy adults.

Currently accepted techniques utilize the plateau concentration of nitric oxide (NO) at a constant exhalation flow rate to characterize NO exchange, which cannot sufficiently distinguish airway and alveolar sources. Using nonlinear least squares regression and a two-compartment model, we recently described a new technique (Tsoukias et al. J Appl Physiol 91: 477-487, 2001), which utilizes a preexpiratory breath hold followed by a decreasing flow rate maneuver, to estimate three flow-independent NO parameters: maximum flux of NO from the airways (J(NO,max), pl/s), diffusing capacity of NO in the airways (D(NO,air), pl x s(-1) x ppb(-1)), and steady-state alveolar concentration (C(alv,ss), ppb). In healthy adults (n = 10), the optimal breath-hold time was 20 s, and the mean (95% intramaneuver, intrasubject, and intrapopulation confidence interval) J(NO,max), D(NO,air), and C(alv,ss) are 640 (26, 20, and 15%) pl/s, 4.2 (168, 87, and 37%) pl x s(-1) x ppb(-1), and 2.5 (81, 59, and 21%) ppb, respectively. J(NO,max) can be estimated with the greatest certainty, and the variability of all the parameters within the population of healthy adults is significant. There is no correlation between the flow-independent NO parameters and forced vital capacity or the ratio of forced expiratory volume in 1 s to forced vital capacity. With the use of these parameters, the two-compartment model can accurately predict experimentally measured plateau NO concentrations at a constant flow rate. We conclude that this new technique is simple to perform and can simultaneously characterize airway and alveolar NO exchange in healthy adults with the use of a single breathing maneuver.

Adult↗

PubMed2001

Fitness, training, and the growth hormone-->insulin-like growth factor I axis in prepubertal girls.

We recently demonstrated that a brief endurance type training program led to increases in thigh muscle mass and peak oxygen uptake (VO(2)) in prepubertal girls. In this study, we examined the effect of training on the GH-->insulin-like growth factor I (GH-->IGF-I) axis, a system known to be involved both in the process of growth and development and in the response to exercise. Healthy girls (mean age 9.17 +/- 0.10 yr old) volunteered for the study and were randomized to control (n = 20) and training groups (n = 19) for 5 weeks. Peak VO(2), thigh muscle volume, and blood samples [for IGF-I, IGF-binding proteins (IGFBP)-1 to -6, and GHBP] were measured. At baseline, IGF-I was significantly correlated with both peak VO(2) (r = 0.44, P < 0.02) and muscle volume (r = 0.58, P < 0.004). IGFBP-1 was negatively correlated with muscle volume (r = -0.71, P < 0.0001), as was IGFBP-2. IGFBP-4 and -5 were significantly correlated with muscle volume. We found a threshold value of body mass index percentile (by age) of about 71, above which systematic changes in GHBP, IGFBP-1, and peak VO(2) per kilogram were noted, suggesting decreases in the following: 1) GH function, 2) insulin sensitivity, and 3) fitness. Following the training intervention, IGF-I increased in control (19.4 +/- 9.6%, P < 0.05) but not trained subjects, and both IGFBP-3 and GHBP decreased in the training group (-4.2 +/- 3.1% and -9.9 +/- 3.8%, respectively, P < 0.05). Fitness in prepubertal girls is associated with an activated GH-->IGF-I axis, but, paradoxically, early in a training program, children first pass through what appears to be a neuroendocrine state more consistent with catabolism.

Body Height↗

PubMed2001

Plastic bronchitis and the role of bronchoscopy in the acute chest syndrome of sickle cell disease.

STUDY OBJECTIVES: To review the prevalence, clinical features, and role of bronchoscopy in patients with plastic bronchitis during the acute chest syndrome (ACS) of sickle cell disease (SCD). DESIGN: Eight-year review of clinical experience. SETTING: Tertiary referral children's hospital. PATIENTS: Twenty-six pediatric inpatients with 29 ACS episodes requiring diagnostic bronchoscopy. RESULTS: Of the pediatric inpatients with ACS who underwent bronchoscopy, plastic bronchitis was diagnosed in 21 of 29 episodes (72%). There was no difference in clinical features between the patients with and without plastic bronchitis. Bronchoscopy was an essential diagnostic tool, but its therapeutic benefits were doubtful. CONCLUSIONS: This is the first report of the prevalence of plastic bronchitis in patients with ACS of SCD. In our patient population, this condition was found to be common. The role of diagnostic bronchoscopy is essential. A large series, multicenter study is required to determine whether bronchoscopy and BAL are therapeutically beneficial when added to currently practiced supportive care.

Acute Disease↗

PubMed2001

Beta(2)-adrenoceptor polymorphism and body mass index are associated with adult-onset asthma in sedentary but not active women.

STUDY OBJECTIVE: Beta(2)-adrenoceptor Gly16 polymorphism has been associated with asthma severity and beta(2)-adrenoceptor receptor downregulation, but not with the diagnosis of asthma. Glu27 polymorphism may limit beta(2)-adrenoceptor downregulation and predict body mass index (BMI), particularly among sedentary persons. In addition, BMI predicts asthma. We hypothesized that these DNA sequence variants predict adult-onset asthma only in sedentary women. DESIGN: Nested case-control study. SETTING: Nurses' Health Study, a large, prospective cohort study with participants throughout the United States. PARTICIPANTS: Among lifelong nonsmokers, 171 women with adult-onset, medication-requiring asthma and 137 age-matched control subjects. MEASUREMENTS: Physical activity and BMI were self-reported by previously validated questionnaire items. Genomic DNA was obtained from buccal brushings collected via first-class mail. RESULTS: Of 76 sedentary women, the adjusted odds ratios of Gly16 allele were 7.4 (p = 0.047) for asthma and 13.8 (p = 0.02) for steroid-requiring asthma. No similar associations were observed among 232 active women (p = 0.91). Sedentary individuals with both Gly16 and Glu27 alleles had a less elevated risk for asthma. BMI was associated with asthma and Glu27 allele among sedentary women. CONCLUSION: This exploratory analysis suggests an important gene/environment interaction for asthma involving physical activity level. Further study in larger populations is warranted to confirm if sedentary lifestyle unmasks a genetic risk for asthma.

Adult↗

PubMed2001

Antinociceptive activity of and clinical experience with buprenorphine in swine.

We performed antinociceptive testing on swine receiving buprenorphine. Intravenous access was achieved, and animals were allowed to recover for 24 h. Baseline skin-twitch latency to a focused light source was determined for each animal. Animals received intravenous (i.v.) buprenorphine at 0.08 (n =1), 0.16 (n = 1), 0.005 (n = 5), 0.01 (n = 5), or 0.02 mg/kg (n = 6). Skin-twitch latency was determined 15, 30, 60, 120, 180, 240, 300, 360, 420, 480, 540, and 600 min after buprenorphine administration. Analgesic activity as measured by a significant increase in latency time over baseline values occurred at all time points except 480 min in animals that received 0.02 mg/kg buprenorphine i.v. Analgesic activity to 420 min was demonstrated in animals that received 0.01 mg/kg buprenorphine i.v. Analgesic activity was not demonstrated at any time point in animals that received 0.005 mg/kg buprenorphine i.v. A retrospective analysis of postoperative care records was performed to determine whether 0.01 mg/kg buprenorphine i.v. or intramuscularly (i.m.) postoperatively to swine provided clinically relevant analgesia. Records of swine receiving buprenorphine from 1997 to 2000 were reviewed for indications of treatment failure, such as pain or a change in analgesic regimen from that used routinely. Treatment failure occurred in 18 of 416 (4.3%) cases treated with buprenorphine. This failure occurred in 17% of cases with problems categorized as inflammatory in nature and in 15.5% of those with systemic problems or organ failure. We concluded that antinociceptive testing predicted that buprenorphine administered at 0.01 mg/kg i.v. in swine likely would provide analgesic efficacy for 6 h and when administered at 0.02 mg/kg i.v. likely would provide 10 h analgesia. Clinical signs of pain in animals recovering from surgery were not observed in the majority of cases when buprenorphine was administered twice or thrice daily at 0.01 mg/kg i.m. or i.v. However, buprenorphine was less effective at treating signs of pain associated with inflammation, organ failure, or systemic disease than at ameliorating pain associated with surgical incisions and orthopedic, dental, and ophthalmic procedures.

Analgesics, Opioid↗

PubMed2000

Regulation of a Ca2+-sensitive adenylyl cyclase in an excitable cell. Role of voltage-gated versus capacitative Ca2+ entry.

In nonexcitable cells, we had previously established that Ca(2+)-sensitive adenylyl cyclases, whether expressed endogenously or heterologously, were regulated exclusively by capacitative Ca(2+) entry (Fagan, K. A., Mahey, R. and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438-12444; Fagan, K. A., Mons, N., and Cooper, D. M. F. (1998) J. Biol. Chem. 273, 9297-9305). Relatively little is known about how these enzymes are regulated by Ca(2+) in excitable cells, where they predominate. Furthermore, no effort has been made to determine whether the prominent voltage-gated Ca(2+) entry, which typifies excitable cells, overwhelms the effect of any capacitative Ca(2+) entry that may occur. In the present study, we placed the Ca(2+)-stimulable, adenylyl cyclase type VIII in an adenovirus vector to optimize its expression in the pituitary-derived GH(4)C(1) cell line. In these cells, a modest degree of capacitative Ca(2+) entry could be discerned in the face of a dramatic voltage-gated Ca(2+) entry. Nevertheless, both modes of Ca(2+) entry were equally efficacious at stimulating adenylyl cyclase. A striking release of Ca(2+) from intracellular stores, triggered either by ionophore or thyrotrophin-releasing hormone, was incapable of stimulating the adenylyl cyclase. It thus appears as though the intimate colocalization of adenylyl cyclase with capacitative Ca(2+) entry channels is an intrinsic property of these molecules, regardless of whether they are expressed in excitable or nonexcitable cells.

Adenylyl Cyclases↗

PubMed2000

Regulation of the Ca2+-inhibitable adenylyl cyclase type VI by capacitative Ca2+ entry requires localization in cholesterol-rich domains.

The endogenous Ca(2+)-inhibitable adenylyl cyclase type VI of C6-2B glioma cells is regulated only by capacitative Ca(2+) entry and not by a substantial elevation of [Ca(2+)](i) from either intracellular stores or via ionophore-mediated Ca(2+) entry (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F. (1995) J. Biol. Chem. 270, 1149-1155; Fagan, K. A., Mons, N., and Cooper, D. M. F. (1998) J. Biol. Chem. 273, 9297-9305). The present studies explored the role of cholesterol-rich domains in maintaining this functional association. The cholesterol-binding agent, filipin, profoundly inhibited adenylyl cyclase activity. Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin did not affect forskolin-stimulated adenylyl cyclase activity and did not affect capacitative Ca(2+) entry. However, cholesterol depletion completely ablated the regulation of adenylyl cyclase by capacitative Ca(2+) entry. Repletion of cholesterol restored the sensitivity of adenylyl cyclase to capacitative Ca(2+) entry. Adenylyl cyclase catalytic activity and immunoreactivity were extracted into buoyant caveolar fractions with Triton X-100. The presence of adenylyl cyclase in such structures was eliminated by depletion of plasma membrane cholesterol. Altogether, these data lead us to conclude that adenylyl cyclase must occur in cholesterol-rich domains to be susceptible to regulation by capacitative Ca(2+) entry. These findings are the first indication of regulatory significance for the localization of adenylyl cyclase in caveolae.

Adenylyl Cyclase Inhibitors↗

PubMed2000

Sustained endothelial nitric-oxide synthase activation requires capacitative Ca2+ entry.

Endothelial nitric-oxide synthase (eNOS), a Ca(2+)/calmodulin-dependent enzyme, is critical for vascular homeostasis. While eNOS is membrane-associated through its N-myristoylation, the significance of membrane association in locating eNOS near sources of Ca(2+) entry is uncertain. To assess the Ca(2+) source required for eNOS activation, chimera containing the full-length eNOS cDNA and HA-tagged aequorin sequence (EHA), and MHA (myristoylation-deficient EHA) were generated and transfected into COS-7 cells. The EHA chimera was primarily targeted to the plasma membrane while MHA was located intracellularly. Both constructs retained enzymatic eNOS activity and aequorin-mediated Ca(2+) sensitivity. The plasma membrane-associated EHA and intracellular MHA were compared in their ability to sense changes in local Ca(2+) concentration, demonstrating preferential sensitivity to Ca(2+) originating from intracellular pools (MHA) or from capacitative Ca(2+) entry (EHA). Measurements of eNOS activation in intact cells revealed that the eNOS enzymatic activity of EHA was more sensitive to Ca(2+) influx via capacitative Ca(2+) entry than intracellular release, whereas MHA eNOS activity was more responsive to intracellular Ca(2+) release. When eNOS activation by CCE was compared with that generated by an equal rise in [Ca(2+)](i) due to the Ca(2+) ionophore ionomycin, a 10-fold greater increase in NO production was found in the former condition. These results demonstrate that EHA and MHA chimera are properly targeted and retain full functions of eNOS and aequorin, and that capacitative Ca(2+) influx is the principle stimulus for sustained activation of eNOS on the plasma membrane in intact cells.

Aequorin↗

PubMed2000

Ca(2+), Sr(2+), and Ba(2+) identify distinct regulatory sites on adenylyl cyclase (AC) types VI and VIII and consolidate the apposition of capacitative cation entry channels and Ca(2+)-sensitive ACs.

Ca(2+)-sensitive adenylyl cyclases may act as early integrators of the two major second messenger-signaling pathways mediated by Ca(2+) and cAMP. Ca(2+) stimulation of adenylyl cyclase type I (ACI) and adenylyl cyclase type VIII (ACVIII) is mediated by calmodulin and the site on these adenylyl cyclases that interacts with calmodulin has been defined. By contrast, the mechanism whereby Ca(2+) inhibits adenylyl cyclase type V (ACV) and adenylyl cyclase type VI (ACVI) is unknown. In this study, Ca(2+), Sr(2+), and Ba(2+) were compared to probe the involvement of E-F hand-like domains in both Ca(2+) stimulation and inhibition of ACVIII and ACVI, respectively. HEK 293 cells transfected with ACVIII cDNA and C6-2B glioma cells (where the endogenous adenylyl cyclases is predominantly ACVI) were used to compare the effects of these three cations in in vitro and in vivo measurements. The in vitro data identified two Ca(2+) regulatory sites for both ACVIII and ACVI. Strikingly different potency series for these cations at mediating high affinity stimulation and inhibition of ACVIII and ACVI, respectively, effectively rule out the possibility that calmodulin or proteins utilizing similar Ca(2+)-binding motifs mediate inhibition of ACVI. On the other hand, the low affinity inhibition that is common to both ACVIII and ACVI showed virtually identical potency profiles for the IIa cation series, indicating a common site of action. Remarkably, whereas Sr(2+) was rather ineffective at regulating these cyclases (particularly ACVI) in vitro, adequate concentrations accumulated in the vicinity of these enzymes as a consequence of capacitative cation entry to partially regulate both of these activities in vivo. This latter finding consolidates earlier observations that Ca(2+)-sensitive adenylyl cyclases detect and respond to capacitative cation entry rather than global cytosolic cation concentrations.

Adenylyl Cyclase Inhibitors↗

PubMed2000

Lipid peroxidation as determined by plasma isoprostanes is related to disease severity in mild asthma.

Oxidlative stress is believed to play an important role in the pathophysiology of asthma. Recently discovered F2-isoprostanes, of which 8-iso-PGF2alpha is the most well-known isomer, have emerged as the most reliable marker of in vivo oxidative stress. The aim of this study was to examine 8-iso-PGF2alpha as a biomarker of oxidative stress in mild asthma in relation to endogenous and dietary antioxidant protection. Total (free and esterified) plasma 8-iso-PGF2alpha, plasma dietary antioxidants (vitamins E and C, Beta-carotene, Zn, and Se), and erythrocyte antioxidant enzyme activities (glutathione peroxidase and superoxide dismutase) were measured in 15 mild asthmatics and 15 age-and sex-matched controls. Total plasma 8-iso-PGF2alpha levels [median (quartile 1 - quartile 3)] were significantly increased in the asthmatics [213 pg/mL (122-455) vs. 139 pg/mL (109-174), P= 0.042]. The 8-iso PGF2alpha levels were found to be associated with clinical asthma severity (P = 0.044) and inhaled corticosteroid use (P = 0.027) in asthmatics. No differences were observed in the plasma dietary antioxidant vitamins. The asthmatics had significantly lower plasma levels of Zn (P = 0.027) and Se (P = 0.006). Plasma Se correlated negatively with 8-iso-PGF2alpha (r = -0.725, P= 0.002). No differences between the groups were observed for glutathione peroxidase or superoxide dismutase, however, superoxide dismutase activity was negatively associated with asthma severity (P = 0.042). In conclusion, oxidative stress is increased in mildly asthmatics, as reflected by increased plasma levels of 8-iso-PGF2alpha and a deficiency in plasma Zn and Se. The isoprostane 8-iso-PGF2alpha may provide a useful tool in intervention studies aimed at improving clinical status in asthma.

Adolescent↗

PubMed2000

Characteristics of the Ca(2+)-dependent inhibition of cyclic AMP accumulation by histamine and thapsigargin in human U373 MG astrocytoma cells.

1. Histamine, acting on H(1)-receptors, caused a Ca(2+)-dependent inhibition of forskolin- and isoprenaline-induced cyclic AMP accumulation in monolayers of human U373 MG cells (IC(50) 1.3+/-0.3 microM, maximum inhibition 66+/-3%). The inhibition was not reversed by the protein kinase inhibitor K-252A. 2. Thapsigargin also inhibited cyclic AMP accumulation (IC(50) 6.0+/-0.3 nM, maximum inhibition 72+/-1%). In the absence of extracellular Ca(2+) 5 microM thapsigargin caused only a 12+/-2% inhibition of cyclic AMP accumulation. 3. The inhibitory effect of 100 nM thapsigargin on forskolin-stimulated cyclic AMP accumulation was blocked by La(3+) (best-fit maximum inhibition 81+/-4%, IC(50) 125+/-8 nM). In contrast, the inhibitory action of 10 microM histamine was much less sensitive to reversal by 1 microM La(3+) (33+/-5% reversal, compared with 78+/-6% reversal of the inhibition by thapsigargin measured concurrently). However, in the presence of both thapsigargin and histamine the inhibition of cyclic AMP accumulation was reversed by 1 microM La(3+) to the same extent as the inhibition by thapsigargin alone. 4.++Thapsigargin (5 microM)+1 microM La(3+) caused only a 20+/-1% inhibition of histamine-stimulated phosphoinositide hydrolysis. 5. There was no indication from measurement of intracellular Ca(2+) of any persistent La(3+)-insensitive Ca(2+) entry component activated by histamine. 6. The results provide evidence that Ca(2+) entry is required for the inhibition by histamine and thapsigargin of drug-induced cyclic AMP accumulation in U373 MG astrocytoma cells. The differential sensitivity of the inhibitory action of the two agents to block by La(3+) suggests that more than one pathway of Ca(2+) entry is involved.

Adenylyl Cyclase Inhibitors↗

PubMed2000

Adiposity, lipid levels, and brief endurance training in nonobese adolescent males.

Physical activity during childhood and adolescence may influence the development of childhood obesity and cardiovascular disease later in life. Research focused prospectively on the effects of training on lipid levels in nonobese subjects, and studies using noninvasive measurements of subcutaneous and intraabdominal fat are lacking. It was hypothesized in nonobese sedentary adolescent males that a brief endurance-type exercise training intervention would reduce body fat and improve lipid profiles. Thirty-eight healthy, nonobese sedentary adolescent males (mean age 16 +/- 0.7 years old; 18 controls, 20 trained) completed a 5-week prospective, randomized, controlled study. Adiposity was measured using magnetic resonance images of the thigh and abdomen (subcutaneous abdominal adipose tissue [SAAT] and intraabdominal adipose tissue [IAAT]). Lipid measurements included serum triglycerides (TG), total cholesterol (TC), HDL and LDL cholesterol. There was no change in body weight in either control or training groups. Training led to small but significant reductions in thigh fat (-4.6 +/- 1.5%, p < 0.03) and SAAT% (1.7 +/- 0.8%, p < 0.02). There was no change in IAAT%. Unexpectedly in the control group there were significant increases in thigh fat (5.2 +/- 1.7%, p<0.01), SAAT% (1.8 +/- 0.6, p < 0.007) and IAAT% (4.5 +/- 1.1, p < 0.0007). Training-induced changes in adiposity were not accompanied by changes in circulating lipids. In nonobese adolescent males a brief period of endurance training led to reductions in body fat depots without weight change while body fat increased rapidly in the control group. Exercise training did not change lipid levels, the latter may require more sustained alterations in patterns of physical activity.

Adipose Tissue↗

PubMed2000

Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion.

Cyclic AMP is a ubiquitous second messenger that coordinates diverse cellular functions. Current methods for measuring cAMP lack both temporal and spatial resolution, leading to the pervasive notion that, unlike Ca(2+), cAMP signals are simple and contain little information. Here we show the development of adenovirus-expressed cyclic nucleotide-gated channels as sensors for cAMP. Homomultimeric channels composed of the olfactory alpha subunit responded rapidly to jumps in cAMP concentration, and their cAMP sensitivity was measured to calibrate the sensor for intracellular measurements. We used these channels to detect cAMP, produced by either heterologously expressed or endogenous adenylyl cyclase, in both single cells and cell populations. After forskolin stimulation, the endogenous adenylyl cyclase in C6-2B glioma cells produced high concentrations of cAMP near the channels, yet the global cAMP concentration remained low. We found that rapid exchange of the bulk cytoplasm in whole-cell patch clamp experiments did not prevent the buildup of significant levels of cAMP near the channels in human embryonic kidney 293 (HEK-293) cells expressing an exogenous adenylyl cyclase. These results can be explained quantitatively by a cell compartment model in which cyclic nucleotide-gated channels colocalize with adenylyl cyclase in microdomains, and diffusion of cAMP between these domains and the bulk cytosol is significantly hindered. In agreement with the model, we measured a slow rate of cAMP diffusion from the whole-cell patch pipette to the channels (90% exchange in 194 s, compared with 22-56 s for substances that monitor exchange with the cytosol). Without a microdomain and restricted diffusional access to the cytosol, we are unable to account for all of the results. It is worth noting that in models of unrestricted diffusion, even in extreme proximity to adenylyl cyclase, cAMP does not reach high enough concentrations to substantially activate PKA or cyclic nucleotide-gated channels, unless the entire cell fills with cAMP. Thus, the microdomains should facilitate rapid and efficient activation of both PKA and cyclic nucleotide-gated channels, and allow for local feedback control of adenylyl cyclase. Localized cAMP signals should also facilitate the differential regulation of cellular targets.

Adenoviridae↗

PubMed2000

Effect of single wrist exercise on fibroblast growth factor-2, insulin-like growth factor, and growth hormone.

Anabolic effects of exercise are mediated, in part, by fibroblast growth factor-2 (FGF-2), insulin-like growth factor-I (IGF-I), and growth hormone (GH). To identify local vs. systemic modification of these mediators, 10 male subjects performed 10 min of unilateral wrist-flexion exercise. Blood was sampled from catheters placed in basilic veins of both arms. Lactate was significantly increased only in the exercising arm. FGF-2 decreased dramatically (P < 0.01) in both the resting (from 1.49 +/- 0.32 to nadir at 0.11 +/- 0.11 pg/ml) and exercising arm (1.80 +/- 0.60 to 0.29 +/- 0.14 pg/ml). Small but significant increases were found in both the resting and exercising arm for IGF-I and IGF binding protein-3 (IGFBP-3). GH was elevated in blood sampled from both the resting (from 1.04 +/- 0.68 to a peak of 2.57 +/- 0.53 ng/ml) and exercising arm (1.04 +/- 0.66 to 2.43 +/- 0.42 ng/ml, P < 0.05). Unilateral wrist exercise was not sufficiently intense to increase circulating lactate or heart rate, but it led to systemic changes in GH, IGF-I, IGFBP-3, and FGF-2. Low-intensity exercise involving small muscle groups can influence the circulating levels of growth factors.

Adult↗