The biosynthesis of hepatic cholesterol esters and triglycerides is impaired in mice with a disruption of the gene for stearoyl-CoA desaturase 1.

Stearoyl-CoA desaturase (SCD) is a microsomal enzyme required for the biosynthesis of oleate and palmitoleate, which are the major monounsaturated fatty acids of membrane phospholipids, triglycerides, and cholesterol esters. Two well characterized isoforms of SCD, SCD1 and SCD2, exist in the mouse. Most mouse tissues express SCD1 and 2 with the exception of the liver, which expresses mainly the SCD1 isoform. We found that asebia mice homozygous for a natural mutation of the gene for SCD1 (SCD-/-) are deficient in hepatic cholesterol esters and triglycerides despite the presence of normal activities of acyl-CoA:cholesterol acyltransferase and glycerol phosphate acyltransferase, the enzymes responsible for cholesterol ester and triglyceride synthesis, respectively, in the liver of these mice. Feeding diets supplemented with triolein or tripalmitolein to the SCD-/- mice resulted in an increase in the levels of 16:1 and 18:1 in the liver but failed to restore the 18:1 and 16:1 levels of the cholesterol ester and triglycerides to the levels found in normal mice. The SCD-/- mouse had very low levels of triglycerides in the VLDL and LDL lipoprotein fractions compared with the normal animal. Transient transfection of an SCD1 expression vector into Chinese hamster ovary cells resulted in increased SCD activity and esterification of cholesterol to cholesterol esters. Taken together, our observations demonstrate that the oleoyl-CoA and palmitoleyl-CoA produced by SCD1 are necessary to synthesize enough cholesterol esters and triglycerides in the liver and suggest that regulation of SCD1 activity plays an important role in mechanisms of cellular cholesterol homeostasis.

Tonotopic variations of calcium signalling in turtle auditory hair cells.

Turtle cochlear hair cells are electrically tuned by a voltage-dependent Ca2+ current and a Ca2+-dependent K+ current (IBK(Ca)). The effects of intracellular calcium buffering on electrical tuning were studied in hair cells at apical and basal cochlear locations tuned to 100 and 300 Hz, respectively. Increasing the intracellular BAPTA concentration changed the hair cell's resonant frequency little, but optimized tuning at more depolarized membrane potentials due to a positive shift in the half-activation voltage (V ) of the IBK(Ca). The shift in V depended similarly on BAPTA concentration in basal and apical hair cells despite a 2. 4-fold difference in the size of the Ca2+ current at the two positions. The Ca2+ current amplitude increased exponentially with distance along the cochlea. Comparison of V values and tuning properties using different BAPTA concentrations with values measured in perforated-patch recordings gave the endogenous calcium buffer as equivalent to 0.21 mM BAPTA in low-frequency cells, and 0.46 mM BAPTA in high-frequency cells. High conductance Ca2+-activated K+ (BKCa) channels recorded in inside-out membrane patches were 2-fold less Ca2+ sensitive in high-frequency than in low-frequency cells. Confocal Ca2+ imaging using the fluorescent indicator Calcium Green-1 revealed about twice as many hotspots of Ca2+ entry during depolarization in high-frequency compared to low-frequency hair cells. We suggest that each BKCa channel is gated by Ca2+ entry through a few nearby Ca2+ channels, and that Ca2+ and BKCa channels occupy, at constant channel density, a greater fraction of the membrane area in high-frequency cells than in low-frequency cells.

Longitudinal spread of second messenger signals in isolated rod outer segments of lizards.

1. In vertebrate rods activation of the phototransduction cascade by light triggers changes in the concentrations of at least two diffusible intracellular second messengers (cGMP and Ca2+) whose actions depend on how far they spread from their site of production or entry. To address questions about their spatial spread, cell-attached patch current recording and fluorescence imaging of Calcium Green-dextran were used to measure the longitudinal spread of cGMP and Ca2+, respectively, in functionally intact isolated Gecko gecko lizard rod outer segments under whole-cell voltage clamp. 2. The light-evoked changes in cGMP and Ca2+ concentrations decayed with distance from a site of steady focal activation by two-photon absorption of 1064 nm light with similar decay lengths of approximately 3.5 microm. 3. These results can be understood on the basis of a quantitative model of coupled diffusible intracellular messengers, which is likely to have broad relevance for second messenger signalling pathways in general. 4. The decay length for the spread of adaptation from a site of steady local illumination was about 8 microm, i.e. substantially longer than the decay lengths measured for the spread of cGMP and Ca2+. There are a number of factors, however, that could broaden the apparent relationship between functional changes in the light response and the concentration of a diffusible messenger. For these reasons the measured decay length is an upper limit estimate of the spread of adaptation and does not rule out the possibility that Ca2+ and/or cGMP carry the adaptation signal.

The role of Ca2+-activated K+ channel spliced variants in the tonotopic organization of the turtle cochlea.

1. Turtle auditory hair cells contain multiple isoforms of the pore-forming alpha-subunit of the large-conductance Ca2+-activated K+ (KCa) channel due to alternative splicing at two sites. Six splice variants were studied by expression in Xenopus oocytes. 2. The isoforms possessed differences in apparent Ca2+ sensitivity and kinetics. The lowest Ca2+ sensitivity was observed in a novel variant resulting from a 26 amino acid deletion around one of the splice sites. 3. Co-expression of a bovine beta-subunit slowed the current relaxation 10-fold compared with channels formed from alpha-subunits alone but preserved the original order of kinetic differences. The beta-subunit also increased the Ca2+ sensitivity of isoforms to bring them nearer the range of sensitivity of the native KCa channels of the hair cell. 4. With channels formed from alpha-subunits or alpha + beta-subunits, the half-activation voltage in a fixed Ca2+ concentration, and the time constant of the current relaxation, varied linearly with the combined size of the insertions/deletions at the splice sites. 5. Experiments in which the beta/alpha concentration ratio was varied indicated that the beta-subunit exerts an all-or-none effect on the Ca2+ sensitivity and kinetics of the channel. 6. Co-expression of an avian beta2-subunit had effects on kinetics and Ca2+ sensitivity of several alpha-isoforms which were qualitatively similar to those produced by the bovine beta-subunit. 7. We conclude that differential expression of alternatively spliced alpha-subunit variants and a non-uniform distribution of a beta-subunit can produce a range of KCa channel properties needed to explain the tonotopic organization of the turtle cochlea.

The functional role of alternative splicing of Ca(2+)-activated K+ channels in auditory hair cells.

Turtle auditory hair cells are frequency tuned by the activity of large-conductance calcium-activated potassium (KCa) channels, the frequency range being dictated primarily by the channel kinetics. Seven alternatively spliced isoforms of the KCa channel alpha-subunit, resulting from exon insertion at two splice sites, were isolated from turtle hair cells. These, when expressed in Xenopus oocytes, produced KCa channels with a range of apparent calcium sensitivities and channel kinetics. However, most expressed channels were less calcium sensitive than the hair cells' native KCa channels. Coexpression of alpha-subunit with a bovine beta-subunit substantially increased the channel's calcium sensitivity while markedly slowing its kinetics, but kinetic differences between isoforms were preserved. These data suggest a molecular mechanism for hair cell frequency tuning involving differential expression of different KCa channel alpha-subunits in conjunction with an expression gradient of a regulatory beta-subunit.

Rescue of excitation by inositol following Li(+)-induced block in Limulus ventral photoreceptors.

The phosphoinositide (PI) intracellular signaling pathway, which triggers Ca2+ release from intracellular stores, appears to be a central feature of phototransduction in most invertebrate species studied. Procedures designed to inhibit PI-pathway reactions cause suppression of excitation to dim lights. However, in Limulus photoreceptors, responses to bright stimuli are in fact enhanced by some of these procedures, suggesting that PI metabolism is not obligatory for light-induced excitation. Other studies, however, suggest that Ca2+ release is obligatory for excitation. We studied this issue by examining the effects of PI-pathway inhibitor, Li+, on electrophysiological responses to light in Limulus photoreceptors. Li+ is reported to cause depletion of intracellular PI-pathway intermediate, inositol; and it offers the pharmacological advantage that its block can be bypassed by introducing exogenous inositol. Introduction of Li+ caused a very slowly developing but complete suppression of responses to dim stimuli. In contrast, Li+ caused a rapidly developing but partial suppression of responses to bright stimuli. Li(+)-induced suppression was reversed by exogenous introduction of inositol. In addition, inositol prevented Li(+)-induced suppression of excitation. Li+ enhanced light adaptation (light-induced desensitization) but slowed response deactivation, indicating a difference in the processes underlying these phenomena. Li+ slowed dark adaptation, the recovery of sensitivity following light adaptation. All of these effects were prevented or rescued by extracellularly applied inositol, suggesting the presence of a transmembrane inositol transport system. The overall results suggest that PI-dependent signaling is central and obligatory for excitation in Limulus, at least for responses to dim to moderate illumination. The failure of Li+ to suppress bright light-induced excitation completely may be due to a failure of Li+ to block PI metabolism completely, as in other systems; however, it may point to a parallel, PI-independent excitation pathway possessing very low light sensitivity when PI metabolism is inhibited.

Arrestin with a single amino acid substitution quenches light-activated rhodopsin in a phosphorylation-independent fashion.

Arrestins are members of a superfamily of regulatory proteins that participate in the termination of G protein-mediated signal transduction. In the phototransduction cascade of vertebrate rods, which serves as a prototypical G protein-mediated signaling pathway, the binding of visual arrestin is stimulated by phosphorylation of the C-terminus of photoactivated rhodopsin (Rh*). Arrestin is very selective toward light-activated phosphorhodopsin (P-Rh*). Previously we reported that a single amino acid substitution in arrestin, Arg175Gln, results in a dramatic increase in arrestin binding to Rh* [Gurevich, V. V., & Benovic, J. L. (1995) J. Biol. Chem. 270, 6010-6016]. Here we demonstrate that a similar mutant, arrestin(R175E), binds to light-activated rhodopsin independent of phosphorylation. Arrestin(R175E) binds with high affinity not only to P-Rh* and Rh* but also to light-activated truncated rhodopsin in which the C-terminus phosphorylation sites have been proteolytically removed. In an in vitro assay that monitored rhodopsin-dependent activation of cGMP phosphodiesterase (PDE), wild type arrestin quenched PDE response only when ATP was present to support rhodopsin phosphorylation. In contrast, as little as 30 nM arrestin(R175E) effectively quenched PDE activation in the absence of ATP. Arrestin(R175E) had no effect when the lifetime of Rh* no longer contributed to the time course of PDE activity, suggesting that it disrupts signal transduction at the level of rhodopsin-transducin interaction.

The mechanisms of vertebrate light adaptation: speeded recovery versus slowed activation.

Light adaptation in vertebrate photoreceptors is commonly attributed to a feedback mechanism that reduces the amplitude of the receptor potential by speeding the inactivation of the transduction cascade and hastening the recovery process. Recent studies have challenged this model and suggest instead that desensitization originates mainly from changes in the activation phase rather than the recovery phase of the response. This has important implications for understanding the molecular mechanisms that underlie the control of sensitivity in this G-protein-coupled, signal-transduction pathway.

Ca2+ dependence of dark- and light-adapted flash responses in rod photoreceptors.

Light adaptation is thought to be orchestrated by a Ca2+ feedback signal that desensitizes the response by speeding recovery. To evaluate the role of Ca2+ in adaptation, we compared the effect of lowered Ca2+ on response properties in darkness and during adaptation. Internal Ca2+ was reduced from its normal resting dark level (535 nM) by either background illumination or exposure to Ringer's solution containing low Ca2+ and/or cyclic GMP-gated channel blockers in darkness. Ca2+ reductions in light decreased the activation gain of the transduction process and speeded recovery kinetics, while equivalent Ca2+ reductions in darkness caused similar gain reduction without accelerating recovery. This indicates that adaptational changes in the response are not due purely to feedback effects on recovery.

The calcium feedback signal in the phototransduction cascade of vertebrate rods.

Intracellular free Ca (Cai) was measured in functionally intact rod outer segments in darkness and during light responses using the fluorescent Ca indicator Indo-dextran. In darkness, Cai was 554 +/- 25 nM (n = 28) for -85 +/- 2 pA of circulating dark current (Id) and declined in saturating light to a minimum value of approximately 50 nM with a time course that paralleled the fall in Na:Ca,K exchange current. During a subsaturating flash response that reduced Id by 70%, Cai fell to a minimum of approximately 325 nM and recovered incompletely to a plateau of approximately 450 nM that lasted approximately 15 s after full recovery of Id. During a 60 s step that caused approximately 7-fold reduction in sensitivity of superimposed flash responses, Cai reached a steady-state level of approximately 252 nM.

Purification and physiological evaluation of a guanylate cyclase activating protein from retinal rods.

In retinal rods light triggers a cascade of enzymatic reactions that increases cGMP hydrolysis and generates an electrical signal by causing closure of cGMP-gated ion channels in the photoreceptor outer segment. This leads to a decrease in internal Ca, which activates guanylate cyclase and promotes photoresponse recovery by stimulating the resynthesis of cGMP. We report here that the activation of guanylate cyclase by low Ca is mediated by an approximately 20-kDa protein purified from bovine rod outer segments by using DEAE-Sepharose, hydroxylapatite, and reverse-phase chromatographies. In a reconstituted system, this protein restores the Ca-sensitive regulation of guanylate cyclase and when dialyzed into functionally intact lizard rod outer segment decreases the sensitivity, time to peak, and recovery time of the flash response.

The effect of recoverin-like calcium-binding proteins on the photoresponse of retinal rods.

The rod photoresponse is triggered by an enzyme cascade that stimulates cGMP hydrolysis. The resulting fall in cGMP leads to a decrease in Ca2+, which promotes photoresponse recovery by activating guanylate cyclase, causing cGMP resynthesis. In vitro biochemical studies suggest that Ca2+ activation of guanylate cyclase is medicated by recoverin, a 26 kd Ca(2+)-binding protein. To evaluate this, exogenous bovine recoverin and two other homologous Ca(2+)-binding proteins from chicken and Gecko retina were dialyzed into functionally intact Gecko rods using whole-cell recording. All three proteins prolonged the rising phase of the photoresponse without affecting the kinetics of response recovery. These results suggest that recoverin-like proteins affect termination of the transduction cascade, rather than mediate Ca(2+)-sensitive activation of guanylate cyclase.

Some unresolved issues in the physiology and biochemistry of phototransduction.

A number of recent review articles have discussed what is known about the events responsible for generating the electrical light response in vertebrate photoreceptors. The similarity of the material covered and the unanimity of the conclusions drawn have given rise to the popular, but false, impression that visual transduction is understood fully. The purpose of the present review is to dispell this notion by focusing on some of the unresolved issues.

cGMP suppresses GTPase activity of a portion of transducin equimolar to phosphodiesterase in frog rod outer segments. Light-induced cGMP decreases as a putative feedback mechanism of the photoresponse.

In rod photoreceptor cells, the light response is triggered by an enzymatic cascade that causes cGMP levels to fall: excited rhodopsin (Rho*)----rod G-protein (transducin, Gt)----cGMP-phosphodiesterase (PDE). This results in the closure of plasma membrane channels that are gated by cGMP. PDE activation by Gt occurs when GDP bound to the alpha-subunit of Gt (Gt alpha) is exchanged with free GTP. The interaction of Gt alpha-GTP with the gamma-subunits of PDE releases their inhibitory action and causes cGMP hydrolysis. Inactivation is thought to be caused by subsequent hydrolysis of Gt alpha-GTP by an intrinsic Gt-GTPase activity. Here we report that there are two portions of Gt in frog rod outer segments (ROS) expressing different rates of GTP hydrolysis: 19.5 +/- 3 mmol of Gt/mol of Rho, equivalent to that amount which participates in PDE activation, hydrolyzing GTP at a rate of approximately 0.6 turnover/s ("fast") and the remaining Gt (80.5 +/- 3 mmol/mol Rho) hydrolyzing GTP at a rate of 0.058 +/- 0.009 turnover/s. Fast GTPase activity is abolished in the presence of cGMP. This effect occurs over the physiological range of cGMP concentration changes in ROS, half-saturating at approximately 2 microM and saturating at 5 microM cGMP. cGMP-dependent suppression of GTPase is specific for cGMP; cAMP in millimolar concentration does not affect GTPase, while the poorly hydrolyzable cGMP analogue, 8-bromo-cGMP, mimics the effect. GTPase regulation by cGMP is not affected by Ca2+ over the concentration range 5-500 nM, which spans the physiological changes in cytoplasmic Ca2+ in rod cells. We suggest that the fast cGMP-sensitive GTPase activity is a property of the Gt that activates PDE. In this model, cGMP serves not only as a messenger of excitation but also modulates GTPase activity, thereby mediating negative feedback regulation of the pathway via PDE turnoff: a light-dependent decrease in cGMP accelerates the hydrolysis of GTP bound to Gt, resulting in the rapid inactivation of PDE.

Physiological roles of Na+/Ca2+ exchange in Limulus ventral photoreceptors.

In previous work we have presented evidence for electrogenic Na+/Ca2+ exchange in Limulus ventral photoreceptors (1989. J. Gen. Physiol. 93:473-492). This article assesses the contributions to photoreceptor physiology from Na+/Ca2+ exchange. Four separate physiological processes were considered: maintenance of resting sensitivity, light-induced excitation, light adaptation, and dark adaptation. (a) Resting sensitivity: reduction of [Na+]o caused a [Ca2+]o-dependent reduction in light sensitivity and a speeding of the time courses of the responses to individual test flashes; this effect was dependent on the final value to which [Na+]o was reduced. The desensitization caused by Na+ reduction was dependent on the initial sensitivity of the photoreceptor; in fully dark-adapted conditions no desensitization was observed; in light-adapted conditions, extensive desensitization was observed. (b) Excitation: Na+ reduction in fully dark-adapted conditions caused a Ca2+o-dependent depolarizing phase in the receptor potential that persisted beyond the stimulus duration and was evoked by a bright adapting flash. (c) Light adaptation: the degree of desensitization induced by a bright adapting flash was Na+o dependent, being larger with lower [Na+]o. Na+ reduction enhanced light adaptation only at intensities brighter than 4 x 10(-6) W/cm2. In addition to being Na+o dependent, light adaptation was Ca2+o dependent, being greater at higher [Ca2+]o. (d) Dark adaptation: the recovery of light sensitivity after adapting illumination was Na+o dependent. Dark adaptation after bright illumination in voltage-clamped and in unclamped conditions was faster in normal-Na+ saline than in reduced Na+ saline. The final sensitivity to which photoreceptors recovered was lower in reduced-Na+ saline when bright adapting illumination was used. The results suggest the involvement of Na+/Ca2+ exchange in each of these physiological processes. Na+/Ca2+ exchange may contribute to these processes by counteracting normal elevations in [Ca2+]i.

Transducin activation in electropermeabilized frog rod outer segments is highly amplified, and a portion equivalent to phosphodiesterase remains membrane-bound.

An electropermeabilized preparation of frog retinal rod outer segments (ROS) has been developed to examine the light sensitivity and amplification of visual transduction reactions in a minimally disturbed environment. Electropermeabilized ROS are indistinguishable from whole and osmotically intact ROS in the light microscope and retain 3-fold more protein than mechanically disrupted ROS. They differ from mechanically fragmented ROS in several respects. Illumination results in more amplified activation of the GTP-binding protein transducin (Gt) than previously observed: bleaching as little as approximately 1 rhodopsin molecule (Rho*) in every 10 disks within a single ROS activates 37,000 molecules of Gt per Rho*, equivalent to 70% of the light-activatable Gt present on a single disk face. This amplification is maintained over approximately 1 decade of light intensity but drops sharply as disk faces begin to absorb a second photon. Lower amplification is observed in fragmented ROS and derives from the fact that physical disruption of ROS causes Gt to bind GTP and elute from the membrane, thus decreasing the amount remaining and available for light activation. Illumination of electropermeabilized ROS in the presence of GTP or of the nonhydrolyzable substrate guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) causes redistribution of Gt: an amount (approximately 20 mmol/mol Rho) equivalent to the amount of inhibitory gamma subunit of phosphodiesterase (PDE) remains internal and bound to nucleotide, and the remaining activated Gt diffuses out in a manner graded with light intensity. This suggests that PDE activation by Gt alpha may not require dissociation of Gt alpha bound to the gamma subunit of PDE in a form than can elute from ROS. Two further differences between electropermeabilized and mechanically disrupted ROS are noted: the addition of ATP to electropermeabilized ROS does not affect the light sensitivity or kinetics of the GTP binding reaction, and a specificity for light-induced GTP versus GDP binding is observed.

Evidence for electrogenic Na+/Ca2+ exchange in Limulus ventral photoreceptors.

The Ca2+ indicator photoprotein, aequorin, was used to estimate and monitor intracellular Ca2+ levels in Limulus ventral photoreceptors during procedures designed to affect Na+/Ca2+ exchange. Dark levels of [Ca2+]i were estimated at 0.66 +/- 0.09 microM. Removal of extracellular Na+ caused [Ca2+]i to rise transiently from an estimated 0.5-0.6 microM in a typical cell to approximately 21 microM; [Ca2+]i approached a plateau level in 0-Na+ saline of approximately 5.5 microM; restoration of normal [Na+]o lowered [Ca2+]i to baseline with a time course of 1 log10 unit per 9 s. The apparent rate of Nao+-dependent [Ca2+]i decline decreased with decreasing [Ca2+]i. Reintroduction of Ca2+ to 0-Na+, 0-Ca2+ saline in a typical cell caused a transient rise in [Ca2+]i from an estimated 0.36 microM (or lower) to approximately 16.5 microM. This was followed by a decline in [Ca2+]i approaching a plateau of approximately 5 microM; subsequent removal of Cao2+ caused [Ca2+]i to decline slowly (1 log unit in approximately 110 s). Intracellular injection of Na+ in the absence of extracellular Na+ caused a transient rise in [Ca2+]i in the presence of normal [Ca2+]o; in 0-Ca2+ saline, however, no such rise in [Ca2+]i was detected. Under constant voltage clamp (-80 mV) inward currents were measured after the addition of Nao+ to 0-Na+ 0-Ca2+ saline and outward currents were measured after the addition of Cao2+ to 0-Na+ 0-Ca2+ saline. The results suggest the presence of an electrogenic Na+/Ca2+ exchange process in the plasma membrane of Limulus ventral photoreceptors that can operate in forward (Nao+-dependent Ca2+ extrusion) or reverse (Nai+-dependent Ca2+ influx) directions.