Biomedical subjects

John A Allen

Publications and source records attributed to John A Allen.

7 recordsLinked to original sources

PubMed2006

Lipid raft microdomains and neurotransmitter signalling.

Lipid rafts are specialized structures on the plasma membrane that have an altered lipid composition as well as links to the cytoskeleton. It has been proposed that these structures are membrane domains in which neurotransmitter signalling might occur through a clustering of receptors and components of receptor-activated signalling cascades. The localization of these proteins in lipid rafts, which is affected by the cytoskeleton, also influences the potency and efficacy of neurotransmitter receptors and transporters. The effect of lipid rafts on neurotransmitter signalling has also been implicated in neurological and psychiatric diseases.

Animals↗

PubMed2006

Energized, polarized, and actively respiring mitochondria are required for acute Leydig cell steroidogenesis.

The first and rate-limiting step in the biosynthesis of steroid hormones is the transfer of cholesterol into mitochondria, which is facilitated by the steroidogenic acute regulatory (StAR) protein. Recent study of Leydig cell function has focused on the mechanisms regulating steroidogenesis; however, few investigations have examined the importance of mitochondria in this process. The purpose of this investigation was to determine which aspects of mitochondrial function are necessary for acute cAMP-stimulated Leydig cell steroidogenesis. MA-10 cells were treated with 8-bromoadenosine 3',5'-cyclic monophosphate (cAMP) and different site-specific agents that disrupt mitochondrial function, and the effects on acute cAMP-stimulated progesterone synthesis, StAR mRNA and protein, mitochondrial membrane potential (Deltapsim), and ATP synthesis were determined. cAMP treatment of MA-10 cells resulted in significant increases in both cellular respiration and Deltapsim. Dissipating Deltapsim with carbonyl cyanide m-chlorophenyl hydrazone resulted in a profound reduction in progesterone synthesis, even in the presence of newly synthesized StAR protein. Preventing electron transport in mitochondria with antimycin A significantly reduced cellular ATP, potently inhibited steroidogenesis, and reduced StAR protein levels. Inhibiting mitochondrial ATP synthesis with oligomycin reduced cellular ATP, inhibited progesterone synthesis and StAR protein, but had no effect on Deltapsim. Disruption of intramitochondrial pH with nigericin significantly reduced progesterone production and StAR protein but had minimal effects on Deltapsim. 22(R)-hydroxycholesterol-stimulated progesterone synthesis was not inhibited by any of the mitochondrial reagents, indicating that neither P450 side-chain cleavage nor 3beta-hydroxysteroid dehydrogenase activity was inhibited. These results indicate that Deltapsim, mitochondrial ATP synthesis, and mitochondrial pH are all required for acute steroid biosynthesis. These results suggest that mitochondria must be energized, polarized, and actively respiring to support Leydig cell steroidogenesis, and alterations in the state of mitochondria may be involved in regulating steroid biosynthesis.

Adenosine Triphosphatases↗

PubMed2005

Beta-adrenergic receptor stimulation promotes G alpha s internalization through lipid rafts: a study in living cells.

Upon binding hormones or drugs, many G protein-coupled receptors are internalized, leading to receptor recycling, receptor desensitization, and down-regulation. Much less understood is whether heterotrimeric G proteins also undergo agonist-induced endocytosis. To investigate the intracellular trafficking of G alpha s, we developed a functional G alpha s-green fluorescent protein (GFP) fusion protein that can be visualized in living cells during signal transduction. C6 and MCF-7 cells expressing G alpha s-GFP were treated with 10 microM isoproterenol, and trafficking was assessed with fluorescence microscopy. Upon isoproterenol stimulation, G alpha s-GFP was removed from the plasma membrane and internalized into vesicles. Vesicles containing G alpha s-GFP did not colocalize with markers for early endosomes or late endosomes/lysosomes, revealing that G alpha s does not traffic through common endocytic pathways. Furthermore, G alpha s-GFP did not colocalize with internalized beta2-adrenergic receptors, suggesting that G alpha s and receptors are removed from the plasma membrane by distinct endocytic pathways. Nonetheless, activated G alpha s-GFP did colocalize in vesicles labeled with fluorescent cholera toxin B, a lipid raft marker. Agonist significantly increased G alpha s protein in Triton X-100 -insoluble membrane fractions, suggesting that G alpha s moves into lipid rafts/caveolae after activation. Disruption of rafts/caveolae by treatment with cyclodextrin prevented agonist-induced internalization of G alpha s-GFP, as did overexpression of a dominant-negative dynamin. Taken together, these results suggest that receptor-activated G alpha s moves into lipid rafts and is internalized from these membrane microdomains. It is suggested that agonist-induced internalization of G alpha s plays a specific role in G protein-coupled receptor-mediated signaling and could enable G alpha s to traffic into the cellular interior to regulate effectors at multiple cellular sites.

Animals↗

PubMed2005

Mitochondrial function in Leydig cell steroidogenesis.

The first and rate-limiting step in the biosynthesis of steroid hormones is the transfer of cholesterol into mitochondria, which is facilitated by the steroidogenic acute regulatory (StAR) protein. Recent studies of Leydig cell function have focused on the molecular events controlling steroidogenesis; however, few studies have examined the importance of the mitochondria. The purpose of this investigation was to determine which aspects of mitochondrial function are necessary for Leydig cell steroidogenesis. MA-10 tumor Leydig cells were treated with 8-bromo-cAMP (cAMP) and site-specific mitochondrial disrupters, pro-oxidants, and their effects on progesterone synthesis, StAR expression, mitochondrial membrane potential (delta psi(m)) and ATP synthesis were determined. Dissipating delta psi(m) with CCCP inhibited progesterone synthesis, even in the presence of newly synthesized StAR protein. The electron transport inhibitor antimycin A significantly reduced cellular ATP, inhibited steroidogenesis, and reduced StAR protein expression. The F0/F1 ATPase inhibitor oligomycin reduced cellular ATP and inhibited progesterone synthesis and StAR protein expression, but had no effect on delta psi(m). Disruption of pH with nigericin significantly reduced progesterone production and StAR protein, but had minimal effects on delta psi(m). Sodium arsenite at low concentrations inhibited StAR protein but not mRNA expression and inhibited progesterone without disrupting delta psi(m). The mitochondrial Ca2+ inhibitor Ru360 also inhibited StAR protein expression. These results demonstrate that delta psi(m), ATP synthesis, delta pH and [Ca2+]mt are all required for steroid biosynthesis, and that mitochondria are sensitive to oxidative stress. These results suggest that mitochondria must be energized, polarized, and actively respiring to support Leydig cell steroidogenesis and alterations in the state of mitochondria may be involved in regulating steroid biosynthesis.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2004

A source-sink hypothesis for abyssal biodiversity.

Bathymetric gradients of biodiversity in the deep-sea benthos constitute a major class of large-scale biogeographic phenomena. They are typically portrayed and interpreted as variation in alpha diversity (the number of species recovered in individual samples) along depth transects. Here, we examine the depth ranges of deep-sea gastropods and bivalves in the eastern and western North Atlantic. This approach shows that the abyssal molluscan fauna largely represents deeper range extensions for a subset of bathyal species. Most abyssal species have larval dispersal, and adults live at densities that appear to be too low for successful reproduction. These patterns suggest a new explanation for abyssal biodiversity. For many species, bathyal and abyssal populations may form a source-sink system in which abyssal populations are regulated by a balance between chronic extinction arising from vulnerabilities to Allee effects and immigration from bathyal sources. An increased significance of source-sink dynamics with depth may be driven by the exponential decrease in organic carbon flux to the benthos with increasing depth and distance from productive coastal systems. The abyss, which is the largest marine benthic environment, may afford more limited ecological and evolutionary opportunity than the bathyal zone.

Animal Migration↗

PubMed2004

Bacterial endotoxin lipopolysaccharide and reactive oxygen species inhibit Leydig cell steroidogenesis via perturbation of mitochondria.

Chronic inflammatory disease and acute infection are well known to inhibit gonadal steroidogenesis. Previous studies have demonstrated that immune activation in response to lipopolysaccharide (LPS) results in reductions in serum testosterone, and this is a direct effect on the Leydig cell. We hypothesize that during the early onset of LPS endotoxemia in vivo, testicular macrophages produce reactive oxygen species (ROS) leading to perturbation of Leydig cell mitochondria and an inhibition in steroidogenesis. To investigate the mechanism of LPS inhibition of Leydig cell steroidogenesis, alterations in mitochondria and markers of oxi-dative stress were assessed in vivo and in Leydig cell pri- mary culture. After a single injection of mice with LPS, serum testosterone was significantly decreased within 2 h. LPS injection of mice resulted in significant reductions in steroidogenic acute regulatory protein (StAR) and 3beta-hydroxysteroid dehydogenase-Delta4-Delta5 isomerase (3beta-HSD) proteins. LPS significantly increased lipid peroxidation of Leydig cell membranes, indicating that LPS results in oxidative damage in vivo. Mitochondria in Leydig cells isolated from LPS-injected mice were disrupted and showed a marked reduction in the mitochondrial membrane potential (DeltaPsim). Similar to the effects of LPS, treatment of Leydig cells with hydrogen peroxide acutely inhibited steroidogenesis, reduced StAR and 3beta-HSD protein levels, and disrupted DeltaPsim. These results suggest that LPS acutely inhibits Leydig cell function by ROS-mediated disruption of Leydig cell mitochondria. Taken together, these results demonstrate the necessity of having respiring mitochondria with an intact DeltaPsim to facilitate StAR function and Leydig cell steroidogenesis. The acute effects of LPS demonstrate how sensitive Leydig cell mitochondrial steroidogenesis is to inflammation-induced oxidative stress.

Animals↗

PubMed2003

Reactive oxygen disrupts mitochondria in MA-10 tumor Leydig cells and inhibits steroidogenic acute regulatory (StAR) protein and steroidogenesis.

Reactive oxygen species (ROS) are involved in a variety of pathophysiological conditions of the testis, and oxidative stress is known to inhibit ovarian and testicular steroidogenesis. The site of ROS-mediated inhibition of steroidogenesis in the corpus luteum and MA-10 tumor Leydig cells was shown to be the hormone-sensitive mitochondrial cholesterol transfer step. The purpose of this study was to examine the effects of ROS on steroidogenic acute regulatory (StAR) protein in MA-10 cells and determine the extent to which MA-10 cell mitochondria are sensitive to oxidative stress. cAMP-stimulated progesterone production was inhibited in a dose-dependent manner in MA-10 cells exposed to H(2)O(2). StAR protein, but not mRNA levels, was decreased in parallel to changes in progesterone production. Even at the highest concentrations of H(2)O(2) tested, there was no effect on P450 side-chain cleavage enzyme protein levels. Oxidative stress from exposure to exogenous xanthine oxidase and xanthine resulted in the inhibition of both progesterone production and StAR protein expression. The mature 30- and 32-kDa intramitochondrial forms of StAR were decreased relative to the 37-kDa extramitochondrial precursor form of StAR, indicating that the ROS-mediated inhibition of StAR protein was due, in part, to the inhibition of mitochondrial import and processing. Vital staining with the fluorescent dye tetramethylrhodamine ethyl ester was used to visualize changes in the mitochondrial electrochemical gradient-dependent membrane potential (Deltapsim). ROS caused a significant dissipation of Deltapsi(m) and time-dependent loss of tetramethylrhodamine ethyl ester fluorescence. The inhibitory effects of H(2)O(2) were transient. There was no evidence for ROS-induced cell death, and following H(2)O(2) removal in the presence of continuous treatment with 8-bromo-cAMP, StAR protein levels and progesterone production were restored. In addition, there was no loss of cell viability following treatment with H(2)O(2) or xanthine/xanthine oxidase as determined by trypan blue exclusion. H(2)O(2) did not cause a significant decrease in total cellular ATP levels. These data indicate that oxidative stress-mediated perturbation of the mitochondria and dissipation of Deltapsi(m) results in the inhibition of StAR protein expression and its import, processing, and cholesterol transfer activity. These findings confirm earlier studies demonstrating the requirement for maintenance of an intact Deltapsi(m) for StAR protein function in cholesterol transport. The significant reduction in the 32- to 30-kDa mature forms of StAR, cessation of cholesterol transport, and loss of Deltapsi(m) are consistent with mitochondrial perturbation because of oxidative stress. This mechanism likely contributes to a host of pathophysiological events evident in testicular disorders such as infection, reperfusion injury, aging, cryptorchidism, and varicocele.

Adenosine Triphosphate↗