cAMP and human neutrophil chemotaxis. Elevation of cAMP differentially affects chemotactic responsiveness.

Neutrophils (PMN) treated with cAMP elevating agents were evaluated for their chemotactic responsiveness to FMLP and leukotriene B4 (LTB4). PGE1 and isoproterenol, increased PMN cyclic AMP production and inhibited chemotaxis to both FMLP and LTB4. In contrast, forskolin, which activates adenylate cyclase directly, inhibited chemotaxis to FMLP but not to LTB4. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was required for inhibition of PMN chemotaxis to FMLP by forskolin, PGE1, and isoproterenol. Isoproterenol and PGE1 inhibited PMN chemotaxis to LTB4 in the absence of IBMX and chemotaxis was further inhibited in the presence of IBMX. PMN cAMP levels were stimulated 2- to 3-fold with isoproterenol, 6- to 10-fold with PGE1, and 5- to 7-fold with forskolin over basal levels in the presence of IBMX. These observations demonstrate that total cellular cAMP concentration is not correlated with inhibition of PMN chemotaxis to all stimuli; forskolin, which increased cyclic AMP 5- to 7-fold over basal levels, did not inhibit chemotaxis to LTB4, whereas isoproterenol, which increased cyclic AMP only 2- to 3-fold over basal levels, inhibited chemotaxis to LTB4. PMN cAMP extrusion was determined under basal conditions and in the presence of PGE1, isoproterenol, or forskolin. PMN extruded cAMP under all conditions examined.     

Axonal Transport of the Ca2+-Dependent Protein Modulator of 3'':5''-Cyclic-AMP Phosphodiesterase in the Rabbit Visual System

Water-soluble proteins were extracted from individual retinas, optic nerves, combined optic tracts and lateral geniculate bodies, and superior colliculi of rabbits at 1, 3, and 18 days after injection of [3H]leucine into the right eye. The Ca2+-dependent protein modulator of 3':5'-cyclic-AMP phosphodiesterase (calmodulin) was isolated from these samples by a two-step polyacrylamide gel electrophoresis procedure. An analysis of the radioactivity incorporated into the total soluble proteins and the calmodulin revealed that most of the calmodulin was axonally transported at a slow rate (2--4 mm/day) and represented about 0.45% of the total transported soluble protein.     

Severe Painful Vaso-Occlusive Crises and Mortality in a Contemporary Adult Sickle Cell Anemia Cohort Study

Background

Frequent painful vaso-occlusive crises (VOCs) were associated with mortality in the Cooperative Study of Sickle Cell Disease (CSSCD) over twenty years ago. Modern therapies for sickle cell anemia (SCA) like hydroxyurea are believed to have improved overall patient survival. The current study sought to determine the relevance of the association between more frequent VOCs and death and its relative impact upon overall mortality compared to other known risk factors in a contemporary adult SCA cohort.

Methods

Two hundred sixty four SCA adults were assigned into two groups based on patient reported outcomes for emergency department (ED) visits or hospitalizations for painful VOC treatment during the 12 months prior to evaluation.

Results

Higher baseline hematocrit (p = 0.0008), ferritin (p = 0.005), and HDL cholesterol (p = 0.01) were independently associated with 1 or more painful VOCs requiring an ED visit or hospitalization for acute pain. During a median follow-up of 5 years, mortality was higher in the ED visit/hospitalization group (relative risk [RR] 2.68, 95% CI 1.1-6.5, p = 0.03). Higher tricuspid regurgitatant jet velocity (TRV) (RR 2.41, 95% CI 1.5-3.9, p < 0.0001), elevated ferritin (RR 4.00, 95% CI 1.8-9.0, p = 0.001) and lower glomerular filtration rate (RR=2.73, 95% CI 1.6-4.6, p < 0.0001) were also independent risk factors for mortality.

Conclusions

Severe painful VOCs remain a marker for SCA disease severity and premature mortality in a modern cohort along with other known risk factors for death including high TRV, high ferritin and lower renal function. The number of patient reported pain crises requiring healthcare utilization is an easily obtained outcome that could help to identify high risk patients for disease modifying therapies.

Trial Registration

ClinicalTrials.gov NCT00011648 http://clinicaltrials.gov/     

Activation of Cyclic AMP-Generating Systems in Brain Membranes and Slices by the Diterpene Forskolin: Augmentation of Receptor-Mediated Responses

The diterpene forskolin markedly activates adenylate cyclase in membranes from various rat brain regions and elicits marked accumulations of radioactive cyclic AMP in adenine-labeled slices from cerebral cortex, cerebellum, hippocampus, striatum, superior colliculi, hypothalamus, thalamus, and medulla-pons. In cerebral cortical slices, forskolin has half-maximal effects at 20-30 microM on cyclic AMP levels, both alone and in the presence of the phosphodiesterase inhibitor ZK 62771. The presence of a very low dose of forskolin (1 microM) can augment the response of brain cyclic AMP-generating systems to norepinephrine, isoproterenol, histamine, serotonin, dopamine, adenosine, prostaglandin E2, and vasoactive intestinal peptide. Forskolin does not augment responses to combinations of histamine-norepinephrine adenosine-norepinephrine, or histamine-adenosine. For norepinephrine and isoproterenol in rat cerebral cortical slices and for histamine in guinea pig cerebral cortical slices, the presence of 1 microM-forskolin augments the apparent efficacy of the amine, whereas for adenosine, prostaglandin E2, and vasoactive intestinal peptide, the major effect of 1 microM-forskolin is to increase the apparent potency of the stimulatory agent. In rat striatal slices, forskolin reveals a significant response of cyclic AMP systems to dopamine and augments the dopamine-elicited activation of adenylate cyclase in rat striatal membranes. The activation of cyclic AMP systems by forskolin is rapid and reversible, and appears to involve both direct activation of adenylate cyclase and facilitation and/or enhancement of receptor-mediated activation of the enzyme.     

Calmodulin stimulation of adenylate cyclase in rat brain membranes does not require GTP

Calcium stimulates adenylate cyclase activity in rat cerebral cortical membranes with either ATP or AppNHp as substrate. In contrast, isoproterenol stimulates the cerebral cortical enzyme with ATP as substrate but not with AppNHp as substrate unless exogenous GTP is added. In rat striatal membranes, calcium or dopamine stimulate adenylate cyclase activity with ATP as substrate, but not with AppNHp as substrate. GTP restores the dopamine but not the calcium response. The inhibitory guanine nucleotide GDP-βS antagonizes dopamine and GppNHp stimulation of the brain adenylate cyclases, but not stimulation by calcium of either rat cerebral cortical or striatal enzymes. Results indicate that GTP is not requisite to calcium-calmodulin activation of adenylate cyclases in brain membranes. In addition, calcium-calmodulin cannot activate striatal adenylate cyclases with a nonphosphorylating nucleotide, AppNHp, as substrate.     

Frequent,low‐amplitude disturbances drive high tree turnover rates on a remote,cyclone‐prone Polynesian island

Aim How important are frequent, low‐intensity disturbances to tree community dynamics of a cyclone‐prone forest? We tested the following hypotheses concerning the ‘inter‐cataclysm’ period on a remote Polynesian island: (1) tree turnover would be high and recruitment rates would be significantly higher than mortality; (2) low‐intensity disturbance would result in a marginal increase in tree mortality in the short term; (3) turnover would vary among species and would be associated with plant traits linked to differences in life history; and (4) mortality and recruitment events would be spatially non‐random. Location Tutuila, a volcanic island in the Samoan Archipelago, Polynesia. Methods We censused the tree (stem diameter ≥ 10 cm) community in 3.9 ha of tropical forest three times over a 10‐year period, 1998–2008. We calculated annual mortality, recruitment and turnover rates for 36 tree species. We tested for non‐random spatial patterns and predictors of mortality, and non‐random spatial patterns of tree recruitment. A 2004 cyclone passing within 400 km allowed us to measure the effects of a non‐cataclysmic disturbance on vital rates. Results Annual turnover was 2.8% and annual recruitment was 3.6%; these are some of the highest rates in the tropics, and likely to be a response to a cyclone that passed < 50 km from Tutuila in 1991. Species turnover rates over 10 years were negatively correlated with wood specific gravity, and positively correlated with annual stem diameter increment. Mortality was spatially aggregated, and a function of site, species and an individual’s growth rate. Recruitment was highest on ground with low slope. The low‐magnitude cyclone disturbance in 2004 defoliated 29% of all trees, but killed only 1.8% of trees immediately and increased annual mortality over 5 years by 0.7%. Main conclusions The inter‐cataclysm period on Tutuila is characterized by frequent, low‐amplitude disturbances that promote high rates of tree recruitment and create a dynamic, non‐equilibrium or disturbed island disequilibrium tree community. Species with low wood density and fast growth rates have enhanced opportunities for recruitment between cataclysms, but also higher probabilities of dying. Our results suggest that increases in the frequency of cyclone activity could shift relative abundances towards disturbance‐specialist species and new forest turnover rates.     

Interaction of forskolin with the P-glycoprotein multidrug transporter.

Forskolin and 1,9-dideoxyforskolin, an analogue that does not activate adenylyl cyclase, were tested for their ability to enhance the cytotoxic effects of adriamycin in human ovarian carcinoma cells, SKOV3, which are sensitive to adriamycin and express low levels of P-glycoprotein, and a variant cell line, SKVLB, which overexpresses the P-glycoprotein and has the multidrug resistance (MDR) phenotype. Forskolin and 1,9-dideoxyforskolin both increased the cytotoxic effects of adriamycin in SKVLB cells, yet had no effect on SKOV3 cells. Two photoactive derivatives of forskolin have been synthesized, 7-O-[[2-[3-(4-azido-3- [125I]iodophenyl)propionamido]ethyl] carbamyl]-7-deacetylforskolin, 125I-7-AIPP-Fsk, and 6-O-[[2-[3-(4-azido-3- [125I]iodophenyl)propionamido]ethyl]carbamyl]forskolin, 125I-6-AIPP-Fsk, which exhibit specificity for labeling the glucose transporter and adenylyl cyclase, respectively (Morris et al., 1991). Both photolabels identified a 140-kDa protein in membranes from SKVLB cells whose labeling was inhibited by forskolin and 1,9-dideoxyforskolin. There was no specific labeling of proteins in membranes from the SKOV3 cells. The overexpressed 140-kDa protein in SKVLB membranes was identified as the P-glycoprotein by immunoblot analysis and immunoprecipitation using anti-P-glycoprotein antiserum. Total inhibition of photolabeling of the P-glycoprotein was observed with verapamil, nifedipine, diltiazem, and vinbalastine, and partial inhibition was observed with colchicine and cytochalasin B. Forskolin was less effective at inhibiting the photolabeling of the P-glycoprotein than 1,9-dideoxyforskolin or a lipophilic derivative of forskolin. The data are consistent with forskolin binding to the P-glycoprotein analogous to that of other chemosensitizing drugs that have been shown to partially reverse MDR.(ABSTRACT TRUNCATED AT 250 WORDS)