Biomedical subjects

D M Cooper

Publications and source records attributed to D M Cooper.

At least 19 recordsLinked to original sources

PubMed2002

Phosphorus in soils and field drainage water in the Thame catchment, UK.

Field drains were sampled at five farms in a catchment in south-central England. The farms were selected to include the main soil types present in the catchment, stagnogleys and pelosols. The phosphorus content of field drainage water was measured on several occasions from 1999 to 2000, under varying flow conditions. The components measured were soluble reactive phosphorus (SRP), total dissolved phosphorus (TDP) and total phosphorus (TP). SRP concentrations in drainage water were lower than in streams in the catchment which had no apparent point source. This indicated that many streams were receiving unidentified point sources of SRP. Measurements of P in field drainage water samples under high flow conditions showed concentrations of particulate phosphorus and SRP up to 1300 and 300 microg l(-1), respectively, these being associated with high suspended sediment concentrations. Comparison of field drain and soil phosphorus contents at the locations sampled did not provide evidence of an inter-relationship. The equilibrium phosphorus content (EPC0) of surface soil was generally higher than the SRP content of drainage water, at one farm by 1 order of magnitude. The variability in measurements suggested a larger-scale and more focussed survey would be required to characterise catchment-scale phosphorus losses from commercial farms by land use and soil type.

Journal Article↗

PubMed2002

The phosphorus budget of the Thame catchment, Oxfordshire: 2. Modelling.

The phosphorus budget of the River Thame was modelled at a daily time scale, using estimates of diffuse and point source contributions of discharge. The model simulated suspended sediment (SS), soluble unreactive phosphorus (SUP), soluble reactive phosphorus (SRP) and particulate phosphorus (PP) concentrations within the main river and major tributaries. Diffuse source estimates of phosphorus loads were based on characteristic losses from identified main landscape classes, with hydrology described by a simple conceptual storage model. In-stream flow was modelled using a kinematic wave equation. Transfer of suspended sediment and phosphorus components was approximated by advection. In-stream sources and sinks included uptake and release of soluble reactive phosphorus by bed sediment, instant equilibration between SRP and the PP concentration on suspended sediment, and flow-related entrainment and deposition of suspended sediment. Simulations at sites within the catchment were compared with measurements made in 1998-1999. Results showed the P budget is dominated by mixing of diffuse and point source water, but some within-river processes have been shown to be capable of significantly influencing SRP concentrations. The development of a sediment entrainment and deposition component of the model has proved particularly valuable in emulating the hysteretic relationship between discharge and suspended sediment concentration in the river. It also provides a measure of available bed sediment.

England↗

PubMed2002

The phosphorus budget of the Thame catchment, Oxfordshire, UK: 1. Mass balance.

The relative magnitudes of annual diffuse and point source loads of phosphorus (P) to the River Thame were estimated from daily and monthly measurements of discharge and concentration. Existing data from gauging and monitoring sites on the river network and at point sources were supplemented by survey data at a range of spatial scales. Results showed that during low flow periods most of the P could be attributed to point sources, while at high flows the figure was less than 10%. The introduction of P stripping at Aylesbury, a major sewage treatment works in the catchment, was estimated to have reduced the annual load of P from the sewage treatment works by approximately 45 t, with a similar reduction in loss from the catchment. This gave a reduction in low flow concentrations of soluble reactive phosphorus (SRP) from 2.5 to 1.7 mg l(-1). Concentrations of SRP in river water remain above eutrophication thresholds because of the influence of other STWs in the catchment and insufficient natural discharge to dilute this.

Environmental Monitoring↗

PubMed2001

A uniform extracellular stimulus triggers distinct cAMP signals in different compartments of a simple cell.

cAMP, the classical second messenger, regulates many diverse cellular functions. The primary effector of cAMP signals, protein kinase A, differentially phosphorylates hundreds of cellular targets. Little is known, however, about the spatial and temporal nature of cAMP signals and their information content. Thus, it is largely unclear how cAMP, in response to different stimuli, orchestrates such a wide variety of cellular responses. Previously, we presented evidence that cAMP is produced in subcellular compartments near the plasma membrane, and that diffusion of cAMP from these compartments to the bulk cytosol is hindered. Here we report that a uniform extracellular stimulus initiates distinct cAMP signals within different cellular compartments. By using cyclic nucleotide-gated ion channels engineered as cAMP biosensors, we found that prostaglandin E(1) stimulation of human embryonic kidney cells caused a transient increase in cAMP concentration near the membrane. Interestingly, in the same time frame, the total cellular cAMP rose to a steady level. The decline in cAMP levels near the membrane was prevented by pretreatment with phosphodiesterase inhibitors. These data demonstrate that spatially and temporally distinct cAMP signals can coexist within simple cells.

1-Methyl-3-isobutylxanthine↗

PubMed2001

Fitness, acute exercise, and anabolic and catabolic mediators in cystic fibrosis.

Exercise can stimulate catabolic inflammatory cytokines even in healthy children. For patients with cystic fibrosis (CF), this may be problematic because CF is characterized by increased inflammation and suppressed growth. We examined fitness and the response to brief exercise of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), insulinlike growth factor-I (IGF-I), and IGF binding protein-1 (IGFBP-1) in 14 subjects with CF (10.5 +/- 0.8 yr of age), 9 of whom were treated with ibuprofen, and 14 healthy control subjects (11.6 +/- 0.5 yr of age, NS). Subjects performed brief intermittent, constant work rate protocol (scaled to each individual's exercise capacity) with blood and urine sampling. Peak V O(2) was correlated with IGF-I (r = 0.68, p < 0.01) in control subjects but not in subjects with CF. In subjects with CF, baseline IL-6 was 79% greater (p < 0.05) and IGF-I was 47% lower than in control subjects (p < 0.05). Post hoc analysis revealed a progressive increase in the IL-6 response to exercise, with the lowest increase observed in control subjects (11.8 +/- 4.6 pg/L/kJ), higher increases in patients with CF treated with ibuprofen (23.4 +/- 7.7 pg/L/kJ), and highest in subjects with CF not receiving ibuprofen (29.2 +/- 7.5 pg/L/kJ). Qualitatively similar results were observed for TNF-alpha. Exercise also significantly increased IGFBP-1 in both control subjects and subjects with CF. Brief exercise can increase even chronically elevated inflammatory mediators in CF, and this response may be attenuated by ibuprofen.

Adolescent↗

PubMed2001

Adenovirus encoded cyclic nucleotide-gated channels: a new methodology for monitoring cAMP in living cells.

The current, static methodologies for measuring cyclic AMP (cAMP) may underestimate its regulatory properties. Here, we have exploited the Ca2+-conducting properties of cyclic nucleotide-gated (CNG) channels to measure cAMP in live cells, in response to various stimuli. We placed a mutated CNG channel with high sensitivity to cAMP in adenovirus to maximize and render facile its expression in numerous cell types. The ready, continuous nature of the readout contrasted with the traditional approach, which yielded similar static information, but lacked any continuous or interactive qualities. It seems fair to predict that this readily adopted approach will broaden the perception of cAMP signaling.

Adenoviridae↗

PubMed2001

Persistent interactions between the two transmembrane clusters dictate the targeting and functional assembly of adenylyl cyclase.

Adenylyl cyclases possess complex structures like those of the ATP binding cassette (ABC) transporter family, which includes the cystic fibrosis transmembrane regulator, the P-glycoprotein, and ATP-sensitive K(+) channels [1-4]. These structures comprise a cytosolic N terminus followed by two tandem six-transmembrane cassettes, each associated with a highly homologous (ATP binding) cytosolic loop [5-8]. The catalytic domains, which are located in the two large cytoplasmic loops, are highly conserved and well studied. The crystal structure of these domains has even been described recently [9, 10]. However, nothing is known of the function or organization of the 12 transmembrane segments. In the present study we adopted a range of strategies including live-cell fluorescence resonance energy transfer (FRET) microscopy, coimmunoprecipitation, and functional assays of various truncated and substituted, fluorescently-tagged molecules to analyze the trafficking and activity of this molecule. When expressed as individual peptides, the two transmembrane domains - largely independently of any cytosolic region - formed a tight complex that was delivered to the plasma membrane. This cooperation between the two intact transmembrane domains was essential and sufficient to target the enzyme to the plasma membrane of the cell. The extracellular loop between the ninth and tenth transmembrane segments, which contains an N-glycosylation site, was also necessary. Furthermore, the interaction between the two transmembrane clusters played a critical role in bringing together the cytosolic catalytic domains to express functional adenylyl cyclase activity in the intact cell.

Adenylyl Cyclases↗

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↗