Occupancy of the chromophore binding site of opsin activates visual transduction in rod photoreceptors.
(1/2591)
The retinal analogue beta-ionone was used to investigate possible physiological effects of the noncovalent interaction between rod opsin and its chromophore 11-cis retinal. Isolated salamander rod photoreceptors were exposed to bright light that bleached a significant fraction of their pigment, were allowed to recover to a steady state, and then were exposed to beta-ionone. Our experiments show that in bleach-adapted rods beta-ionone causes a decrease in light sensitivity and dark current and an acceleration of the dim flash photoresponse and the rate constants of guanylyl cyclase and cGMP phosphodiesterase. Together, these observations indicate that in bleach-adapted rods beta-ionone activates phototransduction in the dark. Control experiments showed no effect of beta-ionone in either fully dark-adapted or background light-adapted cells, indicating direct interaction of beta-ionone with the free opsin produced by bleaching. We speculate that beta-ionone binds specifically in the chromophore pocket of opsin to produce a complex that is more catalytically potent than free opsin alone. We hypothesize that a similar reaction may occur in the intact retina during pigment regeneration. We propose a model of rod pigment regeneration in which binding of 11-cis retinal to opsin leads to activation of the complex accompanied by a decrease in light sensitivity. The subsequent covalent attachment of retinal to opsin completely inactivates opsin and leads to the recovery of sensitivity. Our findings resolve the conflict between biochemical and physiological data concerning the effect of the occupancy of the chromophore binding site on the catalytic potency of opsin. We show that binding of beta-ionone to rod opsin produces effects opposite to its previously described effects on cone opsin. We propose that this distinction is due to a fundamental difference in the interaction of rod and cone opsins with retinal, which may have implications for the different physiology of the two types of photoreceptors. (+info)
Substrate specificity of lysophospholipase D which produces bioactive lysophosphatidic acids in rat plasma.
(2/2591)
Previously we reported that lysophospholipase D in rat plasma hydrolyzes endogenous unsaturated lysophosphatidylcholines (LPCs) preferentially to saturated LPCs to lysophosphatidic acids with growth factor-like and hormone-like activities. In this study, we examined the possibility that association of LPCs with different proteins in rat plasma has an effect on the preference of lysophospholipase D for unsaturated LPCs. Large portions of various LPCs were found to be recovered in the lipoprotein-poor bottom fraction. Furthermore, the percentages of LPCs associated with albumin isolated from rat plasma were shown not to be consistent with their percentage conversions to lysophosphatidic acids by lysophospholipase D on incubation of rat plasma at 37 degrees C. These results indicate that distinct distributions of LPCs in the plasma protein fractions are not critical factors for the substrate specificity of lysophospholipase D. Experiments with Nagase analbuminemic rats suggested that albumin-LPC complexes are not necessarily required for the hydrolysis by lysophospholipase D; lipoprotein-associate LPCs appeared to be good substrates for the phospholipase. We found that both saturated and unsaturated LPCs are present mainly as 1-acyl isomers in rat plasma. This result indicates that the preference of lysophospholipase D for unsaturated LPCs is not attributable to a difference in position of the acyl group attached to the glycerol backbone of LPC. In addition, lysophospholipase D was also found to attack choline phospholipids with a long chain group and a short chain alkyl group, although their percentage hydrolyses were low. Taken altogether, these results suggest that lysophospholipase D shows higher affinities for free forms of unsaturated acyl type LPCs equilibrated with albumin-bound and lipoprotein-associated forms, than for free forms of saturated acyl type LPCs and analogs of platelet-activating factor. (+info)
Modulation of human airway smooth muscle proliferation by type 3 phosphodiesterase inhibition.
(3/2591)
Elevation in cell cAMP content can inhibit mitogenic signaling in cultured human airway smooth muscle (HASM) cells. We studied the effects of the type 3-selective phosphodiesterase inhibitor siguazodan, the type 4-selective phosphodiesterase inhibitor rolipram, and the nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX) on proliferation of cultured HASM cells. At concentrations selective for the type 3 phosphodiesterase isoform, siguazodan inhibited both [3H]thymidine incorporation (IC50 2 microM) and the increase in cell number (10 microM; 64% reduction) induced by platelet-derived growth factor-BB (20 ng/ml). These effects were mimicked by IBMX. At concentrations selective for type 4 phosphodiesterase inhibition, rolipram was without effect. A 20-min exposure to siguazodan and rolipram did not increase whole cell cAMP levels. However, in HASM cells transfected with a cAMP-responsive luciferase reporter (p6CRE/Luc), increases in cAMP-driven luciferase expression were seen with siguazodan (3.9-fold) and IBMX (16.5-fold). These data suggest that inhibition of the type 3 phosphodiesterase isoform present in airway smooth muscle results in inhibition of mitogenic signaling, possibly through an increase in cAMP-driven gene expression. (+info)
Involvement of phosphodiesterase-cGMP-PKG pathway in intracellular Ca2+ oscillations in pituitary GH3 cells.
(4/2591)
The present study investigates the potential role of the Ca2+-calmodulin-dependent type I phosphodiesterase (PDE)-cGMP-protein kinase G (PKG) pathway in spontaneous [Ca2+]i oscillations in GH3 cells using fura-2 single cell videoimaging. Vinpocetine (2.5-50 microM), a selective inhibitor of type I PDE, induced a concentration-dependent inhibition of spontaneous [Ca2+]i oscillations in these pituitary cells, and at the same time produced an increase of the intracellular cGMP content. The cell permeable cGMP analog N2,2'-O-dibutyryl-cGMP (dB-cGMP) (1 mM) caused a progressive reduction of the frequency and the amplitude of spontaneous [Ca2+]i oscillations when added to the medium. KT5823 (400 nM), a selective inhibitor of cGMP-dependent protein kinase (PKG), produced an increase of baseline [Ca2+]i and the disappearance of spontaneous [Ca2+]i oscillations. When KT5823 was added before vinpocetine, the PKG inhibitor counteracted the [Ca2+]i lowering effect of the cGMP catabolism inhibitor. Finally, the removal of extracellular Ca2+ or the blockade of L-type voltage-sensitive calcium channels (VSCC) by nimodipine produced a decrease of cytosolic cGMP levels. Collectively, the results of the present study suggest that spontaneous [Ca2+]i oscillations in GH3 cells may be regulated by the activity of type I PDE-cGMP-PKG pathway. (+info)
Mutational analysis of the phoD promoter in Bacillus subtilis: implications for PhoP binding and promoter activation of Pho regulon promoters.
(5/2591)
The PhoP-PhoR two-component regulatory system controls the phosphate deficiency response in B. subtilis. A number of Pho regulon genes which require PhoP approximately P for activation or repression have been identified. The studies reported here were initiated to understand the PhoP-DNA interaction necessary for Pho promoter regulation. The regulatory region of phoD was characterized in detail using oligo-directed mutagenesis, DNase I footprinting, and in vivo transcription assays. These data reveal basic principles of PhoP binding relevant to PhoP's interaction with other Pho regulon promoters. Our results show that: (i) a dimer of PhoP approximately P is able to bind two consensus repeats in a stable fashion; (ii) PhoP binding is highly cooperative within the core promoter region, which is located from -66 to -17 on the coding strand and contains four TT(A/T/C)ACA-like repeats; (iii) specific bases comprising the TT(A/T/C)ACA consensus are essential for transcriptional activation, but the specific base pairs of the intervening sequences separating the consensus repeats are not important for either PhoP binding or promoter activation; (iv) the spacing between two consensus repeats within a putative dimer binding site in the core region is important for both PhoP binding and promoter activation; (v) the exact spacing between two dimer binding sites within the core region is important for promoter activation but less so for PhoP binding affinity, as long as the repeats are on the same face of the helix; and (vi) the 5' secondary binding region is important for coordinated PhoP binding to the core binding region, making it nearly essential for promoter activation. (+info)
SVPD-post-labeling detection of oxidative damage negates the problem of adventitious oxidative effects during 32P-labeling.
(6/2591)
The exploitation of oxidative DNA lesions as biomarkers of oxidative stress in vivo requires techniques that allow for the precise and valid measurement of oxidative damage to DNA. Previously, endogenous levels of the oxidative lesion 8-hydroxy-2'-deoxyguanosine (8-HO-dG) in rat tissues determined by a micrococcal nuclease/calf spleen phosphodiesterase-based 32P-post-labeling protocol were found to be at least 10-fold higher than those determined by HPLC with electrochemical detection. This was attributed to the adventitious oxidation of the normal nucleotides (dGp) occurring during the labeling stage of the postlabeling protocol, which could only be prevented by the introduction of additional chromatographic steps to remove the unmodified species prior to labeling. In the present study we report that an alternative snake venom phosphodiesterase-based 32P-post-labeling procedure (SVPD-postlabeling) negates the problem of adventitious oxidative damage during labeling by virtue of a unique digestion strategy. In SVPD-post-labeling, digestion yields certain lesions (thymine glycols, phosphoglycolates and abasic sites) as damage-containing dimer species which are ready substrates for labeling. In contrast, the undamaged DNA is recovered as mononucleoside species (dN) which are not substrates for labeling and so remain undetected. Furthermore, even if the mononucleosides are oxidized during labeling, they will not contribute to the level of damage detected. Indeed, we demonstrate that neither the external gamma-irradiation of the digested DNA samples nor increasing the incubation time of the labeling reaction alters the levels of damage detected by SVPD-post-labeling. The negation of adventitious oxidative effects during labeling deems that an optimized SVPD-post-labeling procedure should be well-suited for the biomonitoring of endogenous oxidative stress in vivo. (+info)
The metabotropic receptor mGluR6 may signal through G(o), but not phosphodiesterase, in retinal bipolar cells.
(7/2591)
Bipolar cells are retinal interneurons that receive synaptic input from photoreceptors. Glutamate, the photoreceptor transmitter, hyperpolarizes On bipolar cells by closing nonselective cation channels, an effect mediated by the metabotropic receptor mGluR6. Previous studies of mGluR6 transduction have suggested that the receptor couples to a phosphodiesterase (PDE) that preferentially hydrolyzes cGMP, and that cGMP directly gates the nonselective cation channel. This hypothesis was tested by dialyzing On bipolar cells with nonhydrolyzable analogs of cGMP. Whole-cell recordings were obtained from On bipolar cells in slices of larval tiger salamander retina. Surprisingly, On bipolar cells dialyzed with 8-(4-chlorophenylthio)-cyclic GMP (8-pCPT-cGMP), or 8-bromo-cyclic GMP (8-Br-cGMP) responded normally to glutamate or L-2-amino-4-phosphonobutyrate (L-APB). Response amplitudes and kinetics were not significantly altered compared with cells dialyzed with cGMP alone. Comparable results were obtained with the PDE inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) or with 8-pCPT-cGMP and IBMX together, indicating that PDE is not required for mGluR6 signal transduction. Addition of the G-protein subunit G(o)alpha to the pipette solution suppressed the cation current and occluded the glutamate response, whereas dialysis with G(i)alpha or with transducin Gbetagamma had no significant effect on either the cation current or the response. Dialysis of an antibody directed against G(o)alpha also reduced the glutamate response, indicating a functional role for endogenous G(o)alpha. These results indicate that mGluR6 may signal through G(o), rather than a transducin-like G-protein. (+info)
Membrane topology and cellular location of the Treponema pallidum glycerophosphodiester phosphodiesterase (GlpQ) ortholog.
(8/2591)
Recent reports that isolated Treponema pallidum outer membranes contain an ortholog for glycerophosphodiester phosphodiesterase (GlpQ) (D. V. Shevchenko, D. R. Akins, E. J. Robinson, M. Li, O. V. Shevchenko, and J. D. Radolf, Infect. Immun. 65:4179-4189, 1997) and that this protein is a potential opsonic target for T. pallidum (C. E. Stebeck, J. M. Shaffer, T. W. Arroll, S. A. Lukehart, and W. C. Van Voorhis, FEMS Microbiol. Lett. 154:303-310, 1997) prompted a more detailed investigation of its physicochemical properties and cellular location. [14C]palmitate radiolabeling studies of a GlpQ-alkaline phosphatase fusion expressed in Escherichia coli confirmed the prediction from DNA sequencing that the protein is lipid modified. Studies using Triton X-114 phase partitioning revealed that the protein's amphiphilicity is due to lipid modification and that a substantial portion of the polypeptide is associated with the T. pallidum peptidoglycan sacculus. Three different approaches, i.e., (i) proteinase K treatment of intact treponemes, (ii) indirect immunofluorescence analysis of treponemes encapsulated in agarose beads, and (iii) opsonophagocytosis of treponemes incubated with antiserum against recombinant GlpQ by rabbit peritoneal macrophages, confirmed that GlpQ is entirely subsurface in T. pallidum. Moreover, rabbits hyperimmunized with GlpQ were not protected against intradermal challenge with virulent treponemes. Circular dichroism spectroscopy confirmed that the recombinant form of the polypeptide lacked discernible evidence of denaturation. Finally, GlpQ was not radiolabeled when T. pallidum outer membranes were incubated with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazarene, a photoactivatable, lipophilic probe which promiscuously labels both proteins and lipids within phospholipid bilayers. Taken as a whole, these studies indicate that the T. pallidum GlpQ ortholog is a periplasmic protein associated predominantly with the spirochete's peptidoglycan-cytoplasmic membrane complex. (+info)