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• W1993813725 abstract "The fatty acid (FA) docosahexaenoic acid (DHA, 22: 6n-3) is highly enriched in membrane phospholipids of the central nervous system and retina. Loss of DHA because of n-3 FA deficiency leads to suboptimal function in learning, memory, olfactory-based discrimination, spatial learning, and visual acuity. G protein-coupled receptor (GPCR) signal transduction is a common signaling motif in these neuronal pathways. Here we investigated the effect of n-3 FA deficiency on GPCR signaling in retinal rod outer segment (ROS) membranes isolated from rats raised on n-3-adequate or -deficient diets. ROS membranes of second generation n-3 FA-deficient rats had ∼80% less DHA than n-3-adequate rats. DHA was replaced by docosapentaenoic acid (22:5n-6), an n-6 FA. This replacement correlated with desensitization of visual signaling in n-3 FA-deficient ROS, as evidenced by reduced rhodopsin activation, rhodopsin-transducin (Gt) coupling, cGMP phosphodiesterase activity, and slower formation of metarhodopsin II (MII) and the MII-Gt complex relative to n-3 FA-adequate ROS. ROS membranes from n-3 FA-deficient rats exhibited a higher degree of phospholipid acyl chain order relative to n-3 FA-adequate rats. These findings reported here provide an explanation for the reduced amplitude and delayed response of the electroretinogram a-wave observed in n-3 FA deficiency in rodents and nonhuman primates. Because members of the GPCR family are widespread in signaling pathways in the nervous system, the effect of reduced GPCR signaling due to the loss of membrane DHA may serve as an explanation for the suboptimal neural signaling observed in n-3 FA deficiency. The fatty acid (FA) docosahexaenoic acid (DHA, 22: 6n-3) is highly enriched in membrane phospholipids of the central nervous system and retina. Loss of DHA because of n-3 FA deficiency leads to suboptimal function in learning, memory, olfactory-based discrimination, spatial learning, and visual acuity. G protein-coupled receptor (GPCR) signal transduction is a common signaling motif in these neuronal pathways. Here we investigated the effect of n-3 FA deficiency on GPCR signaling in retinal rod outer segment (ROS) membranes isolated from rats raised on n-3-adequate or -deficient diets. ROS membranes of second generation n-3 FA-deficient rats had ∼80% less DHA than n-3-adequate rats. DHA was replaced by docosapentaenoic acid (22:5n-6), an n-6 FA. This replacement correlated with desensitization of visual signaling in n-3 FA-deficient ROS, as evidenced by reduced rhodopsin activation, rhodopsin-transducin (Gt) coupling, cGMP phosphodiesterase activity, and slower formation of metarhodopsin II (MII) and the MII-Gt complex relative to n-3 FA-adequate ROS. ROS membranes from n-3 FA-deficient rats exhibited a higher degree of phospholipid acyl chain order relative to n-3 FA-adequate rats. These findings reported here provide an explanation for the reduced amplitude and delayed response of the electroretinogram a-wave observed in n-3 FA deficiency in rodents and nonhuman primates. Because members of the GPCR family are widespread in signaling pathways in the nervous system, the effect of reduced GPCR signaling due to the loss of membrane DHA may serve as an explanation for the suboptimal neural signaling observed in n-3 FA deficiency. G protein-coupled receptor (GPCR) 1The abbreviations used are: GPCR, G protein-coupled receptor; DHA or 22:6n-3, docosahexaenoic acid; DPA or 22:5n-6, docosapentaenoic acid; FA, fatty acid; MI and MII, metarhodopsin I and II, respectively; Gt, transducin; PDE, phosphodiesterase; ROS, rod outer segment(s); ERG, electroretinogram; HPLC, high pressure liquid chromatography; DPH, diphenylhexatriene; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; PLE, plasmalogen PE; TBS, Tris-buffered saline; DTPA, diethylenetriaminepentaacetic acid. signaling is ubiquitous in the retina, brain, and nervous system and includes vision, taste, odor, and many neurotransmitter and channel signaling pathways leading to cognitive function (1Salem Jr., N. Litman B. Kim H.Y. Gawrisch K. Lipids. 2001; 36: 945-959Google Scholar). Docosahexaenoic acid (DHA, 22:6n-3), a long chain polyunsaturated fatty acid (FA) of the n-3 series, is highly enriched in the membrane phospholipids of the brain, neuronal tissue, and retina (2Salem N. Spiller G.A. Scala J. Current Topics in Nutrition and Disease: New Protective Roles for Selected Nutrients. Alan R. Liss Inc., New York1989: 109-228Google Scholar). In n-3 FA deficiency, membrane phospholipid DHA is replaced in both the retina and brain by docosapentaenoic acid (DPA, 22: 5n-6), an n-6 series FA, in a near stoichiometric manner (3Galli C. Trzeciak H.I. Paoletti R. Biochim. Biophys. Acta. 1971; 248: 449-454Google Scholar). n-3 deficiency is associated with visual and cognitive deficits, as observed in animal and human infant studies (2Salem N. Spiller G.A. Scala J. Current Topics in Nutrition and Disease: New Protective Roles for Selected Nutrients. Alan R. Liss Inc., New York1989: 109-228Google Scholar). Studies in reconstituted systems demonstrate that visual signaling, the best characterized GPCR system, is sensitive to membrane phospholipid acyl chain composition and appears to be optimal in DHA membranes (4Litman B.J. Niu S.L. Polozova A. Mitchell D.C. J. Mol. Neurosci. 2001; 16: 237-242Google Scholar). An important question in determining the underlying mechanism responsible for the deficits observed in n-3 FA deficiency is whether the loss of DHA in vivo results in suboptimal GPCR signaling. In visual signaling, absorption of a photon by rhodopsin triggers the formation of the active form of the receptor, metarhodopsin II (MII) (5Matthews R.G. Hubbard R. Brown P.K. Wald G. J. Gen. Physiol. 1963; 47: 215-240Google Scholar). MII binds and activates several hundred molecules of transducin (Gt), the visual G protein (6Kibelbek J. Mitchell D.C. Beach J.M. Litman B.J. Biochemistry. 1991; 30: 6761-6768Google Scholar), which subsequently activates the effector enzyme, a cGMP phosphodiesterase (PDE). PDE catalyzes cGMP hydrolysis, triggering closure of cGMP-gated Na+/Ca2+ channels in the rod outer segment (ROS) plasma membrane. This hyperpolarizes the rod cell and initiates the visual response, as observed in the a-wave of the electroretinogram (ERG). Thus, the ERG a-wave time course and amplitude are determined by the kinetics and coupling parameters of the individual steps in the visual signaling pathway. Early studies of the FA dependence of the ERG showed that decreased levels of n-3 FAs in the diet led to decreased a-wave and b-wave amplitudes in the rat ERG (7Wheeler T.G. Benolken R.M. Anderson R.E. Science. 1975; 188: 1312-1314Google Scholar). Similar changes were observed in the ERGs of rodents (8Weisinger H.S. Vingrys A.J. Sinclair A.J. Ann. Nutr. Metab. 1996; 40: 91-98Google Scholar, 9Weisinger H.S. Armitage J.A. Jeffrey B.G. Mitchell D.C. Moriguchi T. Sinclair A.J. Weisinger R.S. Salem Jr., N. Lipids. 2002; 37: 759-765Google Scholar) and cats (10Pawlosky R.J. Denkins Y. Ward G. Salem Jr., N. Am. J. Clin. Nutr. 1997; 65: 465-472Google Scholar). n-3 FA deficiency was also associated with increased implicit times in the ERG and decreased visual acuity in nonhuman primates (11Connor W.E. Neuringer M. Karnosky M.L. Lent A. Bolls L.C. Biological Membranes: Abbreviations in Membrane and Function Structure. Alan R. Liss Inc., New York1988: 275-276Google Scholar, 12Neuringer M. Connor W.E. Lin D.S. Barstad L. Luck S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4021-4025Google Scholar, 13Jeffrey B.G. Mitchell D.C. Hibbeln J.R. Gibson R.A. Chedester A.L. Salem Jr., N. Lipids. 2002; 37: 839-848Google Scholar). The most dramatic change observed in the composition of the retinal tissue is the replacement of DHA by DPA, suggesting that depletion of retinal DHA is responsible for the observed suboptimal responses, likely through changes in membrane physical properties that lead to down-regulation of GPCR signaling pathways. In this study, we provide explicit experimental evidence linking the changes in phospholipid acyl chain composition associated with n-3 FA deficiency to the down-regulation of individual steps in a GPCR signaling pathway using the visual signaling pathway as a model system. The replacement of DHA by DPA in n-3 FA-deficient rat ROS resulted in slower kinetics and reduced levels of receptor activation, receptor-G protein coupling, and an attenuation of the integrated signaling pathway, as evidenced by a 3-fold reduction in PDE activity at physiologically relevant bleach levels. In addition, ROS membranes from n-3-deficient rats had a higher degree of phospholipid acyl chain order than those obtained from n-3-adequate rats. These studies provide a mechanistic basis for the reduced amplitude and delayed response (increased latency) of the retinal ERG a-wave observed in n-3 FA-deficient animals. Animal Procedures—The protocol and all animal procedures used in this experiment were approved by the Animal Care and Use Committee of the National Institute on Alcohol Abuse and Alcoholism. Female Long-Evans rats were obtained from Charles River (Portage, MI) at weaning (3 weeks of age). Weaning females were semirandomly divided into two dietary groups with the constraint that both groups had the same mean body weight. One group of females was fed with the n-3 FA-adequate diet, and the second group was fed with the n-3 FA-deficient diet (14Moriguchi T. Greiner R.S. Salem Jr., N. J. Neurochem. 2000; 75: 2563-2573Google Scholar). The females in both dietary groups were mated with 12-week-old males when they were 11 weeks of age. Offspring were culled to a maximum of 12/dam, and the dams were maintained on their respective diets during lactation. At the age of 21 days, the pups were dark-adapted overnight and sacrificed by decapitation under dim red light. Diet Composition—Diets were patterned after those of the American Institute of Nutrition (AIN93) with the fat source modified to provide either a low or an adequate level of n-3 FAs. Both diets had the same basal macronutrients, vitamins, minerals, and basal fats (hydrogenated coconut and safflower oils) (14Moriguchi T. Greiner R.S. Salem Jr., N. J. Neurochem. 2000; 75: 2563-2573Google Scholar). However, the n-3 FA-adequate diet also contained flaxseed oil and DHASCO® (Martek Biosciences, Columbia, MD), fats that supply α-linolenic acid and DHA, respectively, as their principal component. FA composition of the diets was balanced for saturated FAs, monounsaturated FAs, and linoleic acid; the key difference between diets was a substitution of a small amount of flaxseed and DHASCO® oils for a portion of the hydrogenated coconut oil in the n-3 FA-adequate diet. ROS Membrane Sample Preparation—Retinas were dissected immediately after sacrifice under dim red light and placed in 10 ml of TBS (10 mm Tris, 60 mm KCl, 30 mm NaCl, 2 mm MgCl2, 50 μm DTPA, 2 mm dithiothreitol, and 15 μg of aprotinin, pH 8.0) at 4 °C. ROS were prepared using a sucrose gradient method as described previously (15Organisciak D.T. Wang H. Kou A.L. Exp. Eye Res. 1982; 34: 401-412Google Scholar). The endogenous peripheral membrane proteins, Gt and PDE, in ROS were removed by two washes in hypotonic buffer (5 mm Tris and 50 μm DTPA, pH 8.0). The purified ROS membranes were assayed for rhodopsin contents and the concentration of total phospholipids. Aliquots of Gt and PDE isolated from bovine retinas were added back to the hypotonically stripped ROS membranes at the physiological ratio of 100:10:1 of rhodopsin:Gt:PDE to restore the signaling cascade (16Miller J.L. Litman B.J. Dratz E.A. Biochim. Biophys. Acta. 1987; 898: 81-89Google Scholar). Phospholipid Analysis—Phospholipid molecular species were analyzed using reversed phase HPLC-electrospray mass spectrometry (17Kim H.Y. Wang T.C. Ma Y.C. Anal. Chem. 1994; 66: 3977-3982Google Scholar). Lipids were extracted from ROS membranes using the Bligh and Dyer procedure in the presence of deuterium-labeled phospholipid internal standards. Samples were injected into a C18 column and separated using a mobile phase containing 0.5% NH4OH in water, methanol, and hexane and a gradient from 12:88:0 to 0:88:12 in 17 min at a flow rate of 0.4 ml/min after holding at the initial composition for 3 min. The separated phospholipid molecular species were detected using an Agilent HPLC-MS 1100 Series mass selective detector instrument. For electrospray ionization the capillary voltage and the exit voltage were set at 4000 and 200 V, respectively. The drying gas temperature was set at 350 °C, the flow rate of the drying gas was 13 liters/min, and the gas pressure of the nebulizer was 32 p.s.i. Quantification was based on the area ratios calculated against the internal standard of the same phospholipid class. Assays—Light-activated PDE activity was assayed using a real time pH method (18Yee R. Liebman P.A. J. Biol. Chem. 1978; 253: 8902-8909Google Scholar) with the following modifications. A high sensitivity pH meter with built-in temperature compensation (Model 370 from Thermo Orion, Beverly, MA) coupled to a microelectrode (MI-710 from Microelectrodes, Inc., Bedford, NH) was used to monitor pH. The signal output from the pH meter was acquired by a computer through a 12-bit A/D board (Lab-PC-1200/AI, National Instruments, Austin, TX) operated at a 1-kHz rate. Samples that contained 5 μm rhodopsin from either n-3 FA-adequate or -deficient ROS membranes with replenished bovine Gt and PDE, 50 μm GTP, and 1 mm cGMP in TBS buffer (pH 8.0) were preincubated at 37 °C in a thermo-regulated microcuvette in the dark for 10 min. A set of 20 data points was collected as the base-line activity prior to sample activation by a flash lamp (FX1131 from EG&G; pulse width = 1 μs) synchronized by the computer. The light intensity was attenuated using neutral density filters to vary the level of rhodopsin activation in samples, which was determined by rhodopsin concentrations before and after light exposure. The PDE activity is obtained from the shift in pH and is expressed as cGMP hydrolyzed (mm)/s. Rhodopsin activation was characterized by the MI-MII equilibrium constant, Keq, using a spectroscopic method (19Straume M. Mitchell D.C. Miller J.L. Litman B.J. Biochemistry. 1990; 29: 9135-9142Google Scholar, 20Niu S.L. Mitchell D.C. Litman B.J. J. Biol. Chem. 2001; 276: 42807-42811Google Scholar). Time-resolved Fluorescence Measurements—Fluorescence lifetime and differential polarization measurements were performed with a K2 multifrequency cross-correlation phase fluorometer (ISS, Urbana, IL) as described previously (21Mitchell D.C. Litman B.J. Biophys. J. 1998; 74: 879-891Google Scholar). For lifetime measurements 12 modulation frequencies were used, logarithmically spaced from 5 to 200 MHz, and differential polarization measurements were made at 15 modulation frequencies logarithmically spaced from 5 to 200 MHz. Both total intensity decay and differential polarization measurements were repeated a minimum of three times. Measured polarization-dependent differential phases and modulation ratios for each sample were combined with the measured total intensity decay to yield the anisotropy decay, r(t). Anisotropy decay data of diphenylhexatriene (DPH) were analyzed using the Brownian rotational diffusion model (21Mitchell D.C. Litman B.J. Biophys. J. 1998; 74: 879-891Google Scholar). This model characterizes the anisotropy decay of DPH in terms of the orientational distribution function, f(θ), and the diffusion coefficient for rotation about the long axis of DPH. Flash Photolysis Measurements—Samples for flash photolysis measurements were prepared by diluting concentrated ROS suspensions with pH 7.5 TBS buffer, dividing the solution in half, and adding concentrated Gt to one half and an identical amount of Gt buffer to the other half. Samples were then incubated for 4 h on ice to ensure binding of Gt to the bilayer. Final concentrations were 5.0 μm rhodopsin and 1.0 μm Gt. Kinetics of MII and MII-Gt formation were assessed by measuring the transient absorption at 380 nm using a flash photolysis system as described previously (22Mitchell D.C. Niu S.L. Litman B.J. J. Biol. Chem. 2001; 276: 42801-42806Google Scholar). Excitation was provided by a high pressure flash lamp (EG&G; pulse width = 1 μs) filtered with a broad (±25 nm) bandpass filter centered at 500 nm. The current from a thermoelectrically cooled photomultiplier tube (R928, Hamamatsu) was passed to a low noise current amplifier (Stanford Research). The amplifier output voltage was acquired at 2–10 μs/point by a 1.25-MHz, 12-bit analog-to-digital converter (National Instruments) installed in a personal computer. The detailed kinetics of MII formation were extracted from the changes in absorbance observed at 380 nm in the absence of Gt via analysis in terms of the microscopic rate constants of a branched photoreaction model (19Straume M. Mitchell D.C. Miller J.L. Litman B.J. Biochemistry. 1990; 29: 9135-9142Google Scholar). The kinetics of MII-Gt formation were determined by analyzing absorbance changes at 380 nm obtained in the presence of Gt in terms of the branched model plus a reaction between MII and Gt to form the MII-Gt complex. In both cases the observed absorbance increase at 380 nm was directly analyzed in terms of the appropriate microscopic rate constants using NONLIN with subroutines specifying each model written by the authors of this paper. Flash photolysis measurements in the presence of Gt produced an equilibrium mixture of MI, free MII, and MII-Gt complex. MI and free MII were assumed to be present in the ratio corresponding to their equilibrium mixture measured in the absence of Gt, and the concentrations of free MII and MII-Gt complex were determined from the kinetic analysis. The association constant, Ka, between MII and Gt was determined using the measured concentrations of free MII and MII-Gt complex and assuming that [free Gt] = [total Gt] – [MII-Gt complex]. Lipid and Protein Analysis—To characterize the effect of the dietary regimes on ROS membrane lipid composition, phospholipid molecular species and total acyl chain composition for each phospholipid class were determined for both n-3 FA-adequate and -deficient dietary groups. The n-3 FA-adequate group contained the highest levels of DHA in phosphatidylserine (PS) and phosphatidylethanolamine (PE) at 44.7 (±0.9)% and 49.8 (±0.4)% of total acyl chains, respectively. DHA levels in phosphatidylcholine (PC) and plasmalogen PE (PLE) were lower at 29.4 (±0.9)% and 24.5 (±0.4)%, respectively (Table I). Only minor levels of DPA were detected in all classes analyzed. Relative to the n-3 FA-adequate diet, the n-3 FA-deficient diet resulted in a major loss of DHA in PC, PS, PE, and PLE of 79, 79, 73, and 82%, respectively. The reduction in DHA was accompanied by a compensatory replacement by the n-6 FA, DPA, a close structural analog of DHA with one less double bond at the n-3 position. This is consistent with the essentially reciprocal replacement of DHA by DPA associated with n-3 FA deficiency that was observed by others (3Galli C. Trzeciak H.I. Paoletti R. Biochim. Biophys. Acta. 1971; 248: 449-454Google Scholar). Differences for key phospholipid molecular species are illustrated in Fig. 1. DHA is the major phospholipid component in the n-3 FA-adequate ROS. A significant amount of PC, PS, and PE contains DHA in both the sn-1 and sn-2 acyl chain positions. In contrast, ROS from n-3 FA-deficient rats show a major loss of DHA, which is accompanied by the appearance of DPA-containing species as the primary constituents. One of the interesting findings is that the di-22:6n-3 species of PE and PC are not well replaced by di-22:5n-6 species; instead, there is an increased level of 18:0,22:5n-6 PC in the ROS from n-3 FA-deficient rats. There is also a failure in the replacement of 16:0,22:6n-3 PLE species by the corresponding DPA species. The ratio of membrane phospholipid to rhodopsin was determined to be the same for both dietary groups, suggesting that rhodopsin expression was not altered by n-3 FA deficiency.Table IPhospholipid compositions of ROS membranesLipid classn-3 adequaten-3 deficient22:6n-3aPercentages of 22:6n-3 or 22:5n-6 are expressed as the mole percentage of total fatty acid in each phospholipid class. Values in parentheses are S.D.22:5n-6aPercentages of 22:6n-3 or 22:5n-6 are expressed as the mole percentage of total fatty acid in each phospholipid class. Values in parentheses are S.D.22:6n-3aPercentages of 22:6n-3 or 22:5n-6 are expressed as the mole percentage of total fatty acid in each phospholipid class. Values in parentheses are S.D.22:5n-6aPercentages of 22:6n-3 or 22:5n-6 are expressed as the mole percentage of total fatty acid in each phospholipid class. Values in parentheses are S.D.%%PC29.4 (± 0.9)0.8 (± 0.0)6.2 (± 0.1)22.2 (± 0.2)PS44.7 (± 0.9)2.4 (± 0.2)9.4 (± 0.8)36.2 (± 0.2)PE49.8 (± 0.4)1.8 (± 0.1)13.5 (± 0.1)31.3 (± 0.4)PLE24.5 (± 0.4)1.8 (± 0.1)4.5 (± 0.2)15.0 (± 0.4)a Percentages of 22:6n-3 or 22:5n-6 are expressed as the mole percentage of total fatty acid in each phospholipid class. Values in parentheses are S.D. Open table in a new tab Light-stimulated PDE Activity—The PDE activity, which is a measure of the integrated activity of the visual signaling pathway encompassing rhodopsin activation, MII-Gt coupling, Gtα-PDE coupling, and PDE-catalyzed cGMP hydrolysis, was assayed for ROS membranes isolated from n-3 FA-adequate and -deficient rat retinas. ROS membranes were hypotonically stripped of endogenous Gt and PDE and subsequently reconstituted with equivalent aliquots of the same preparation of purified bovine Gt and PDE so as to restore the native ratio of proteins in the visual pathway. This was done to ensure that n-3 FA-adequate and -deficient ROS membranes contained identical amounts of Gt and PDE so that the only difference in the two samples was the change in ROS membrane phospholipid composition induced by the diet. Thus any changes observed in light-stimulated PDE activity can unequivocally be associated with the observed changes in ROS membrane composition. The ROS membranes from both dietary groups exhibited similar dark PDE activity, as shown from the base line in the absence of light stimulus (Fig. 2a). Upon light stimulation, which activates a fraction of 1.7 × 10–3 rhodopsin molecules, there were considerable differences in the amplitude and the rate of cGMP hydrolysis observed for ROS membranes derived from n-3 FA-adequate and -deficient rat retinas. These differences were dependent on the level of light stimulus, which is shown in the dose-response curve (Fig. 2b), where the PDE activity is expressed as a percentage of the maximal rate of cGMP hydrolysis and the light stimulus level is expressed as the fraction of rhodopsin molecules light-activated. The dose-response curve is shifted toward the right for the n-3 FA-deficient group, indicating a loss of sensitivity of the light-activated signaling pathway (Fig. 2b). High levels of light stimulus saturated the light-activated PDE activity and diminished the difference in PDE activity between the two dietary groups. However, the large difference in PDE activity in the ROS samples at low light stimulus levels is explicitly shown in the plot of the ratio of PDE activity in n-3 FA-adequate ROS to that in n-3 FA-deficient ROS (Fig. 2c). Under physiological conditions, where about 1 in 105 rhodopsin molecules is activated, there was a ∼3-fold difference between light-stimulated PDE activity in n-3 FA-adequate ROS and n-3 FA-deficient ROS. Receptor Activation—To identify the steps involved in the change in visual pathway sensitivity induced by n-3 FA deficiency, we measured the level and rate of rhodopsin activation in ROS membranes from both dietary groups. The MI-MII equilibrium constant, Keq = [MII]/[MI], which is a measure of the level of rhodopsin activation, was reduced by 16% at 37 °C in ROS membranes from the n-3 FA-deficient group relative to the n-3-adequate group (Fig. 3a). This observation in the ROS membrane is consistent with the lower Keq values observed previously for rhodopsin reconstituted in membranes containing phospholipids with lower DHA or acyl chain unsaturation levels (20Niu S.L. Mitchell D.C. Litman B.J. J. Biol. Chem. 2001; 276: 42807-42811Google Scholar). The time constant for MII formation at 37 °C, τMII, in the n-3 FA-adequate group was 0.56 ms, compared with 0.67 ms in the deficient group (Fig. 3b). This translates into ∼20% delay in the rate of MII formation induced by n-3 FA deficiency. Previous studies established that DHA-containing membranes have relatively low membrane acyl chain order parameters (23Huster D. Arnold K. Gawrisch K. Biochemistry. 1998; 37: 17299-17308Google Scholar), high compressibility (24Koenig B.W. Strey H.H. Gawrisch K. Biophys. J. 1997; 73: 1954-1966Google Scholar), and high membrane acyl chain packing free volume (21Mitchell D.C. Litman B.J. Biophys. J. 1998; 74: 879-891Google Scholar). Given that a molecular volume expansion is associated with the MI to MII transition, the properties of DHA membranes would likely impose a minimal energy barrier for the formation of MII, whereas a reduction in DHA levels in n-3 FA-deficient ROS membranes would likely raise the energy barrier. This is consistent with the reduced acyl chain packing free volume observed for the ROS membranes from the n-3 FA-deficient group relative to the n-3-adequate group (see “Membrane Structure” below). This variation in membrane properties would affect both the extent and the rate of formation of MII. Receptor-G Protein Coupling—The initial step in signal amplification and information flow from receptor to effector enzyme in GPCR signaling is the formation of an activated receptor-G protein complex, which is the MII-Gt complex in the visual system. The association constant, Ka, for the complex is 16.4 μm–1 for the n-3-adequate ROS and 9.9 μm–1 for the n-3-deficient ROS. This demonstrates that lower levels of the MII-Gt complex form in the n-3 FA-deficient ROS. In the n-3 FA-adequate group, the time constant for MII-Gt formation, τMII-Gt, is 0.81 ms, paralleling closely the kinetics of MII formation, which has a time constant of 0.56 ms. The tight coupling of MII appearance and MII-G protein complex formation makes visual transduction one of the most efficiently coupled systems in the GPCR family. However, in the n-3 FA-deficient ROS, the kinetics of MII-Gt formation were slower than those of the adequate group by 38% (Fig. 3c). The ratio of the time constants τMII-Gt:τMII is a measure of the lag time in the coupling of MII to Gt. This ratio is 1.45 for the n-3 FA-adequate ROS and is increased by 16% to 1.67 in the n-3 FA-deficient ROS, indicating a reduced efficiency of MII coupling to Gt. The overall lag time in the initiation of the ERG a-wave is determined by the fact that the time constant for the appearance of the MII-Gt complex is 38% larger in the n-3-deficient ROS than in the n-3-adequate ROS. A previous study, using reconstituted membranes, demonstrated that the rate of MII-Gt formation, which is controlled by lateral diffusion of MII and Gt in the plane of the membrane, is mediated by phospholipid acyl chain composition and optimized in DHA-containing bilayers (22Mitchell D.C. Niu S.L. Litman B.J. J. Biol. Chem. 2001; 276: 42801-42806Google Scholar). A similar dependence would explain the observed delay in the formation of MII-Gt in the n-3 FA-deficient ROS membranes. Membrane Structure—Time-resolved fluorescence anisotropy of the hydrophobic fluorescent probe DPH was shown to be a reliable probe of acyl chain packing properties in both reconstituted rhodopsin-lipid vesicles and ROS disk membranes (22Mitchell D.C. Niu S.L. Litman B.J. J. Biol. Chem. 2001; 276: 42801-42806Google Scholar, 25Niu S.L. Mitchell D.C. Litman B.J. J. Biol. Chem. 2002; 277: 20139-20145Google Scholar). Analyzing the data using a Brownian rotational diffusion model yields a parameter fv, which allows a comparison of relative acyl chain packing free volume in membranes of different lipid composition. This parameter decreases with increasing acyl chain order. Values of fv for ROS membranes from n-3 FA-adequate and -deficient rats are 0.11 and 0.086, respectively. The smaller fv value for the n-3 FA-deficient ROS membranes indicates a more ordered phospholipid acyl chain packing than that of the n-3 FA-adequate ROS membranes. ERGs of n-3 FA-deficient rodents and nonhuman primates are characterized by a reduced amplitude and increased latency in the a-wave, which is determined by the visual transduction pathway in the ROS. The rate and degree of hyperpolarization of the ROS plasma membrane, which results from the closure of cGMP-gated Na+/Ca2+ channels, are determined by the rate and extent of hydrolysis of cGMP by the PDE. Characteristics of the light-stimulated PDE activity in turn depend on the rate and extent of rhodopsin activation to MII, MII activation of Gt, and the resulting activation of the PDE by Gtα. A recently published model relates the generation of the ERG a-wave to the kinetic and equilibrium parameters associated with the various steps in the visual transduction pathway (26Leskov I.B. Klenchin V.A. Handy J.W. Whitlock G.G. Govardovskii V.I. Bownds M.D. Lamb T.D. Pugh Jr., E.N. Arshavsky V.Y. Neuron. 2000; 27: 525-537Scopus (159) Google Scholar). The model explicitly relates the amplitude of the ERG a-wave to the number of activated rhodopsin molecules and the subsequent number of G proteins and PDE catalytic subunits activated, whereas the rate of generation of the a-wave is associated with the kinetics of coupling of these proteins in the signaling pathway. In the current study, we provide i" @default.
• W1993813725 created "2016-06-24" @default.
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• W1993813725 date "2004-07-01" @default.
• W1993813725 modified "2023-09-27" @default.
• W1993813725 title "Reduced G Protein-coupled Signaling Efficiency in Retinal Rod Outer Segments in Response to n-3 Fatty Acid Deficiency" @default.
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