Diacylglycerol content of Chaetopterus oocytes during maturation and fertilization.

The resumption of meiotic maturation in Chaetopterus oocytes is believed to be initiated by activation of protein kinase C (pkC). Since the physiological activator of pkC is diacylglycerol (DG), we examined the DG content of the oocytes before and during germinal vesicle breakdown (GVBD). Unstimulated oocytes contained 366 +/- 34 fmol DG per cell. This value increased to approximately 430-500 fmol 3-5 min after normal induction of GVBD and declined thereafter to 147 +/- 7 fmol per cell. Fertilization increased the DG levels slightly, 3-5 min after insemination. These results strongly support the hypothesis that pkC is a physiological activator of GVBD in this species.

Concentrations of phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate within the distal segment of squid photoreceptors.

Although inositol trisphosphate (InsP3) is a key substance in phototransduction of invertebrate photoreceptors, its intracellular concentration remains unknown. The purpose of this study was to assay its concentration and the concentration of its precursor, phosphatidylinositol bisphosphate (PtdInsP2), within squid photoreceptors. Rhabdomeric membranes were purified and their PtdInsP2 content measured with a phosphate assay after the extracted phospholipids were deacylated and separated by ion-exchange chromatography. At least 75% of the total PtdInsP2 found in the retinal homogenate was associated with the plasma membranes of the rhabdomeric microvilli, where PtdInsP2 was 3.1 +/- 0.7% of the total phospholipids, a level comparable to values published for rat brain. In terms of rhodopsin, microvillar membranes contained 3.7 +/- 0.9 mol PtdInsP2/mol rho. The InsP3 content of living retinas was measured with a radioreceptor assay. The basal content of dark-adapted retinas was 0.15 +/- 0.05 InsP3/rho, equivalent to 30 +/- 9 nmol/g tissue that is about twice that of rat brains. Flash illumination (approximately 1 ms in duration) that photoactivated 1% of rhodopsin increased the level about fivefold to 0.68 +/- 0.22 InsP3/rho. Corresponding decrease in PtdInsP2 was undetectable as it was within measurement errors. For PtdInsP2, the measured content corresponds to 5.6 +/- 1.4 mM within the volume of rhabdomere. Maximal light-induced concentration of InsP3 is calculated to be 1.2 +/- 0.4 mM within the cytoplasm of the distal segment. Each photoactivated rhodopsin leads to the formation of < or = 500 InsP3 molecules when measured 15 s after the flash.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-independent deactivation of squid rhodopsin.

Deactivation of light-activated squid rhodopsin was studied in vitro using GTP gamma S binding by G-protein as a direct measure of rhodopsin activity. Deactivation was inhibited by dilution of the retinal suspension or by removal of soluble components. Deactivation could be restored by addition of soluble material to washed membranes. These results indicate that the deactivation is not due entirely to a conformational transition within rhodopsin itself, but depends on the interaction with other molecules. The possibility that phosphorylation is involved in the deactivation was studied. Deactivation occurred in the presence and absence of added ATP. Deactivation also occurred in the presence of kinase inhibitors and after addition of apyrase, which reduced residual ATP levels to below 1 microM. These results indicate that light-induced phosphorylation is not required for deactivation of squid rhodopsin. In this regard deactivation of squid rhodopsin is different from that of vertebrate rhodopsin, which requires phosphorylation.

Submicrovillar tubules in distal segments of squid photoreceptors detected by rapid freezing.

Invertebrate phototransduction is believed to involve an inositol trisphosphate (InsP3)-mediated release of calcium from intracellular storage compartments. Although light-induced production of InsP3 has been demonstrated for squid retinas, morphological evidence for the presence of internal calcium stores has been lacking. Because squid retinas are about 1 mm thick and composed of densely packed receptor cells, conventional aldehyde fixatives may not penetrate rapidly enough to preserve subcellular organelles. To reduce the time for fixative penetration, receptor cells were isolated from intact retinas before fixation, but these techniques provided little improvement in the preservation of membrane-bound compartments. Alternatively, the distal ends of the receptors were ultra-rapidly frozen by dropping 1 mm2 pieces of intact retina against a liquid helium-cooled copper block. Electron micrographs of thick sections from rapidly frozen and freeze-substituted retinas showed elongated saccules oriented parallel to the long axis of the receptor cell and located about 40 nm from the microvillar openings. Freeze-fracture and etch views of rapidly frozen cells showed that the saccules are 130 nm diameter tubules and extend for at least several micrometers along the length of the receptor cell. We call these organelles submicrovillar tubules (SMT). The gap between the SMT and the plasma membrane contains a network of filaments that appear to be actin. Freeze-fracture and etch views of the rhabdomeres also indicate that adjacent microvilli are separated by a 6-8-nm-wide extracellular space along most of their length. This space is spanned by extracellular connections linking adjacent microvilli. The position and orientation of the SMT suggest that these organelles may serve the same function as the more voluminous and highly convoluted submicrovillar cisternae found in other invertebrates. The SMT is likely to be the intracellular compartment that stores and releases calcium as part of the InsP3-mediated light response.

Solubility of retinoids in water.

Spectrophotometric and radioactive techniques were used to measure the water solubility of retinaldehyde, retinol (vitamin A), and retinoic acid under physiological conditions. Hydration decreases the molar extinction coefficient of these substances and shifts their absorption peak bathochromically (10 nm for retinal and approximately 1 nm for the rest). We find their solubility to be about 0.1 microM at room temperature, pH 7.3 (with experimental values being 0.11 microM for retinaldehyde, 0.06 microM for retinol, and 0.21 microM for retinoic acid). To prevent oxidative degradation of retinol, which is the most labile retinoid, our argon-saturated buffer solutions contained physiological levels of ascorbate or alpha-tocopherol. To the best of our knowledge, water solubility of these compounds has not yet been previously reported. Although the measured solubilities are relatively low, they are significant and may account for the movement of retinoids through the aqueous phase as observed by others during exchange with binding proteins and during intervesicular transfer in the absence of binding proteins. Diffusion of uncomplexed retinoids through the aqueous phase can be a major pathway for transport over subcellular distances.

Light-dependent GTP-binding proteins in squid photoreceptors.

Previous biochemical and electrophysiological evidence suggests that in invertebrate photoreceptors, a GTP-binding protein (G-protein) mediates the actions of photoactivated rhodopsin in the initial stages of transduction. We find that squid photoreceptors contain more than one protein (molecular masses 38, 42 and 46 kDa) whose ADP-ribosylation by bacterial exotoxins is light-sensitive. Several lines of evidence suggest that these proteins represent distinct alpha subunits of G-proteins. (1) Pertussis toxin and cholera toxin react with distinct subsets of these polypeptides. (2) Only the 42 kDa protein immunoreacts with the monoclonal antibody 4A, raised against the alpha subunit of the G-protein of vertebrate rods [Hamm & Bownds (1984) J. Gen. Physiol. 84. 265-280]. (3) In terms of ADP-ribosylation, the 42 kDa protein is the least labile to freezing. (4) Of the 38 kDa and 42 kDa proteins, the former is preferentially extracted with hypo-osmotic solutions, as demonstrated by the solubility of its ADP-ribosylated state and by the solubility of the light-dependent binding of guanosine 5'-[gamma-thio]triphosphate. The specific target enzymes for the observed G-proteins have not been established.

Metabolism of inositol 1,4,5-trisphosphate in squid photoreceptors.

Inositol 1,4,5-trisphosphate (InsP3) is rapidly formed in squid photoreceptors in response to light, where it is converted sequentially into inositol bisphosphate (InsP2) and inositol monophosphate (InsP1). All of the InsP3 appears to be degraded to inositol 1,4-bisphosphate via an InsP3-phosphatase, which is characterized in this study. The enzyme is water-soluble and present in the light-transducing distal segments of squid photoreceptors. It has a Km of 50 microM for InsP3, requires Mg++ for its activity, is maximally active at neutral pH, specifically hydrolyses the 5-phosphate and is inhibited by 2,3-diphosphoglycerate. In these respects, InsP3-phosphatase of squid is very similar to the enzymes of other cells. Since no InsP4 or more highly phosphorylated inositols are found in squid photoreceptors, the InsP3-phosphatase may be important in the regulation of InsP3 concentration within these cells.

Inositol trisphosphate production in squid photoreceptors. Activation by light, aluminum fluoride, and guanine nucleotides.

The light-stimulated production of inositol triphosphate (IP3), via hydrolysis of phosphatidylinositol bisphosphate (PIP2), can be demonstrated in an in vitro preparation of isolated distal segments of squid photoreceptors. The retina is labeled with [3H]inositol (Szuts, E. Z., Wood, S. F., Reid, M. S., and Fein, A. (1986) Biochem. J. 240, 929-932), and the rhodopsin-containing distal segments are isolated in artificial cytosol. Within 2 s after a flash, IP3 levels increase 200% (corresponding to an intracellular increase of approximately 5 microM), and the lipid precursor PIP2 decreases by 50%. Inositol bisphosphate (IP2) levels increase later, as a breakdown product of IP3. IP3 response is light-dependent, saturating when 0.5% of the rhodopsin is photoactivated. Guanosine-5'-O-(3-thiotriphosphate (GTP gamma S) binding demonstrates that the plasma membrane of most of the photoreceptor distal segments is intact or only transiently permeable. Membrane permeabilization enhances light-activated GTP gamma S binding but abolishes the light-activated IP3 production. Receptor-mediated production of IP3 is believed to be the result of a receptor-G-protein-phospholipase C cascade (i.e. Cockcroft, S., and Gomperts, B. D. (1985) Nature 314, 534-536). To test for G-proteins, we incubated the photoreceptors in AlF4- (an activator of G-proteins) in the dark. IP3 and IP2 were produced with a corresponding decrease in PIP2. Incubation with GTP or GTP gamma S, in hypotonic buffer, which causes transient leakiness, increased dark levels by IP3 by 50%. Addition of GTP in isotonic buffer enhanced the light-induced increase of IP3. These results localize the light-stimulated phospholipase C activity to the distal segments and suggest that a G-protein couples rhodopsin to phospholipase C.

A novel eye in 'eyeless' shrimp from hydrothermal vents of the Mid-Atlantic Ridge.

Rimicaris exoculata is a shrimp that swarms over high-temperature (350 degrees C) sulphide chimneys at Mid-Atlantic Ridge hydrothermal fields (3,600 m). This shrimp lacks an externally differentiated eye, having instead a pair of large organs within the cephalothorax immediately beneath the dorsal surface of the transparent carapace, connected by large nerve tracts to the supraesophageal ganglion. These organs contain a visual pigment with an absorption spectrum characteristic of rhodopsin. Ultrastructural evidence for degraded rhabdomeral material suggests the presence of photoreceptors. No image-forming optics are associated with the organs. We interpret these organs as being eyes adapted for detection of low-level illumination and suggest that they evolved in response to a source of radiation associated with the environment of hydrothermal vents.

Inositol trisphosphate, inositol phospholipid metabolism, and germinal vesicle breakdown in surf clam oocytes.

Others have reported that microinjection of inositol 1,4,5-trisphosphate (InsP3) releases stored intracellular Ca2+ and causes fertilization envelope elevation, part of the activation process normally initiated by fertilization in deuterostome eggs. In the protostome, Spisula solidissima, germinal vesicle breakdown (GVBD) is the first visible response of the egg to fertilization. To test the effects of InsP3 on egg activation in this organism, we microinjected the compound into oocytes. Microinjection of 0.4-7.0 x 10(-21) moles of InsP3 (equivalent to 5-80 pM if distributed throughout the cell) elicited GVBD in a dose-dependent manner, demonstrating that increased oocyte InsP3 can mimic part of the activation process in this protostome. Synthesis of InsP3 occurs in vivo when phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is hydrolyzed by phospholipase C. To determine whether stimulus-induced synthesis of InsP3 occurs after fertilization of Spisula oocytes, we labeled oocyte lipids with [32P]orthophosphate and measured the radioactivity in phospholipids after insemination. Fertilization resulted in a rapid, transient loss of radioactivity from PtdInsP2. Because the radioactivity in phosphatidylinositol 4-phosphate and other phospholipids did not change, the loss of radioactivity from PtdInsP2 is most likely due to its hydrolysis, yielding InsP3 and diacylglycerol. The latter compound activates protein kinase C which has also been shown to be involved in regulating Spisula oocyte GVBD. Since both of these compounds appear to be early products of fertilization, they could coordinately activate Ca2+- and protein kinase C-dependent processes involved in Spisula oocyte GVBD. These data indicate that egg activation in this protostome includes pathways similar to those found in deuterostome eggs and in other eukaryotic cells.

Protein kinase C activity, protein phosphorylation and germinal vesicle breakdown in Spisula oocytes.

To test the possible role of protein kinase C (C-kinase) in regulating germinal vesicle breakdown (GVBD) in Spisula oocytes, we studied the effects of phorbol esters and antagonists of C-kinase on GVBD and protein phosphorylation. Responses to these agents were compared to those elicited by fertilization or increased extracellular K+. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent agonist of C-kinase, elicited GVBD with half-maximal stimulation at 20 nM. By contrast, 4 alpha-phorbol-12,13-didecanoate, a phorbol ester which does not stimulate C-kinase, did not trigger GVBD. TPA accelerated GVBD when induced by excess K+, but it did not affect the time course of the process when initiated by fertilization. Three structurally different antagonists of C-kinase (W-7, H-7, and retinol) all blocked GVBD when induced by fertilization or TPA. When oocytes were preincubated with [32P]orthophosphate and then stimulated to undergo GVBD by fertilization, TPA, or 45 mM K+, protein phosphorylation was greatly increased, especially for a polypeptide(s) of about 45 kDa. Phosphorylation increased prior to GVBD. Retinol inhibited phosphorylation in activated eggs. C-kinase activity was demonstrated in oocyte extracts. These results strongly suggest that protein phosphorylation by C-kinase is involved in the pathway that regulates GVBD in Spisula oocytes.

Light stimulates the rapid formation of inositol trisphosphate in squid retinas.

To test the hypothesis that inositol trisphosphate (InsP3) mediates adaptation and excitation in invertebrate photoreceptors, we measured its formation on a rapid time scale in squid retinas. For squid, excitation and adaption occurs within 0.1 and 1-2 s respectively. We could detect an elevation in InsP3 within 200 ms of a bright flash. This increase is about 240% over dark basal levels and is maintained for at least 2 min after a flash. The increase probably occurs in the photoreceptors, which are the only neurons in squid retinas. Analysis by h.p.l.c. indicates that the light-regulated isomer is Ins(1,4,5)P3, which is formed by the hydrolysis of phosphatidylinositol bisphosphate (PtdInsP2).

Light stimulates phosphorylation of two large membrane proteins in frog photoreceptors.

By photoactivating rhodopsin, light indirectly initiates a series of biochemical reactions within photoreceptors as part of the visual process. I herein report that one of the light-stimulated reactions in bullfrog photoreceptors is the phosphorylation of two previously unreported proteins (220 and 240 kDa). Their phosphorylation by endogenous kinase(s) is readily observed in freshly isolated, fragmented rods. On subcellular fractionation, the labeled proteins copurify with the membranes of the outer segments, from which they cannot be extracted with low ionic strength. They appear to be integral membrane proteins of the disk or plasma membranes. Their light-induced phosphorylation is also observed in intact receptors when excised frog retinas are incubated under in vivo conditions with 32PO4. Thus, appropriate kinase(s) is (are) present within outer segments and presumably is (are) the one(s) responsible for phosphorylation in fragmented cells. In the presence of adenosine 5'-(gamma-[35S]thiotriphosphate) [( 35S] ATP-gamma-S), light can also stimulate thiophosphorylation, leading to preferential labeling of the 220-kDa protein. On the basis of four criteria (electroporetic mobility, membrane location, binding of concanavalin A, and mobility shifts with SH oxidation), the 220-kDa protein appears to correspond to the membrane protein previously identified at the rims of rod disks [Papermaster, D.S., Schneider, B.G., Zorn, M.A. & Kraehenbuhl, J.P. (1978) J. Cell Biol. 78, 415-425]. Identity of the other substrate protein is unknown. When fragmented cells are illuminated with a flash of 1-ms duration, the half-time for phosphorylation is about 1 min with ATP at 0.1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium flux across disk membranes. Studies with intact rod photoreceptors and purified disks.

Calcium accumulation by rod disks was studied in excised bullfrog retinas with 45Ca tracer-exchange methods. Ca uptake by disks is a necessary requirement if light-induced Ca releases from disks mediate photoreceptor excitation. In an hour-long incubation, disks exchanged less than or equal to 0.01 mole of Ca per mole of rhodopsin, or less than or equal to 10% of their total Ca. This corresponds to a unidirectional flux of less than or equal to 0.01 fmol/cm2 S, or less than or equal to 5 ions/disk-second across the disk membrane. Neither incubation in 10 mM Ca (which increases cytoplasmic activity 10--100-fold) nor photostimulation (which photoactivated up to 50% rhodopsin/h) had measurable effect on exchange rate, though an increase of several orders of magnitude would have been expected according to the hypothesis. The observed exchange could not be explained by: (a) 45Ca losses from disks before measurement (neither the net efflux nor the Ca-Ca exchange property of disks adequately explains such losses), (b) a limited pool of exchangeables Ca from strongly binding intradiskal sites, or (c) rate-limiting flux across the plasma membrane during incubation. For the study of the Ca efflux properties of disks, separate experiments were performed with 45Ca-loaded disks. Intradiskal activity could be estimated from the disks' hyperosmotically sensitive 45Ca pool and from their intradiskal volume (indirectly assayed by density). Ca-Ca exchange was undetectable (less than or equal to 0.1 fmol/cm2 S) in disks whose intradiskal activity was at least 0.3 mM. Net efflux was 0.2 fmol/cm2 S for an intradiskal activity of approximately 1 mM and is comparable to published fluxes for phospholipid vesicles. These results seem to exclude the internal space of disks as the source of Ca for photoreceptor excitation.

Photochemical electron transport oin photosynthetic reaction centers from Rhodopseudomonas spheroides. 3. Effects of orthophenanthroline and other chemicals.

Reaction centers from Rhodopseudomonas spheroides mediate the photochemical oxidation of cytochrome c (cyt c), and show a time-varying fluorescence of P870. Analyses of these effects indicate that the reaction centers contain a primary photochemical electron acceptor capable of holding one electron. Native or added ubiquinone (UQ) can act as a secondary electron acceptor. Orthophenanthroline (o-phen) blocks electron transfer from primary to secondary acceptors, and allows the primary acceptor to be exhibited in the foregoing experiments. Other chelators (with the possible exception of 8-hydroxyquinoline) and dichlorophenyldimethylurea (DCMU) are without apparent effect on reaction centers. o-Phen also inhibits the primary photochemical act in reaction centers; this effect is prevented by the presence of UQ. 2-n-Nonyl-4-hydroxyquinoline-N-oxide (NQNO) inhibits the primary photochemistry in reaction centers but does not affect secondary electron transfer.

Photochemical electron transport in photosynthetic reaction centers from Rhodopseudomonas spheroides. II. Interaction with external electron donors and acceptors and a reevaluation of some spectroscopic data.

Photochemical reaction centers prepared from Rhodopseudomonas spheroides were treated with reduced cytochrome c (cyt c), and in some cases with ubiquinone (UQ), and illuminated. The light-induced oxidation of cy and reduction of UQ were observed, and also the variations in fluorescence of P870. These observations indicated that each reaction center contains a primary photochemical electron acceptor capable of holding just one electron. Depending on the method of preparation, the reaction centers may also contain secondary electron acceptor pools consisting mainly of UQ. The role of native UQ as an electron acceptor could be duplicated by added UQ. The yield of P870 fluorescence increased by a factor of 3-4, at most, during illumination of reaction centers in the presence of an electron donor such as reduced cyt. This suggests that the quantum efficiency for the primary photoact is about 0.7, rather than 0.9-1.0 as concluded in the past from optical absorption measurements. The apparent quantum efficiency for the oxidation of cyt by illuminated reaction centers can be increased by the addition of UQ and is decreased at higher concentrations of the detergent lauryl dimethylamine oxide (LDAO). These treatments do not affect the quantum efficiency of P870 oxidation, measured in the absence of cyt.