Renewal of visual pigment in photoreceptors of the blowfly

Summary Spectrophotometric measurements of photoreceptors 1–6 in the blowfly demonstrate that rhodopsin undergoes a continuous renewal. This involves, in the dark, the slow degradation of rhodopsin whereas metarhodopsin is degraded at a much faster rate. The effect of light is to reduce the rate at which metarhodopsin is degraded, i.e. the rate is inversely related to the intensity of the light. Rhodopsin synthesis is dependent on the presence of 11-cis retinal which is formed via a photoreaction from all-trans retinal resulting from the breakdown of rhodopsin and/or metarhodopsin: the biosynthesis of rhodopsin is therefore a light dependent process. Light of the blue/violet spectral range was found to mediate the isomerization of all-trans retinal into the 11-cis form. It is proposed that this stereospecificity is the result of all-trans retinal being bound to a protein. On the basis of the results a visual pigment cycle is proposed.     

Linear and non-linear contrast coding in light-adapted blowfly photoreceptors

The response properties of R1–6 type photoreceptors of the blowfly (Calliphora vicina) were investigated using 1- to 600-ms light steps with contrasts from -1.00 to +1.12 at different adapting backgrounds. To prevent activation of voltage-dependent K⁺-channels, some photoreceptors were treated with ionophoretically injected tetraethylammonium. The linearity and time-course of the photoresponses depended on the adapting background and the duration of the contrast stimulus. At low backgrounds photoresponses were approximately linear regardless of the contrast step duration. However, at higher backgrounds photoreceptors produced, with long contrast steps of opposite polarities, larger and slower hyperpolarizing responses than depolarizing ones, but linear responses with equal time-courses to transient (2ms) contrast changes. The early rising phases of photoresponses were independent of the contrast signal duration, deviated to the same extent from the steady-state potential and steepened as the value of contrast was increased. The photoreceptor contrast gain increased with the background, but its value depended on the duration, the magnitude, and the polarity of the contrast step. Tetraethylammonium linearised the photoreceptor membrane, increased and delayed photoresponses probably by blocking the shunting of the outwardly rectifying potassium channels. These effects did not strongly influence on the photoreceptors' characteristic non-linear contrast dependence on light adaptation.Abbreviations LED light emitting diode - TEA tetraethylammonium ion     

Some properties of pyruvate and 2-oxoglutarate oxidation by blowfly flight-muscle mitochondria

1. High rates of state 3 pyruvate oxidation are dependent on high concentrations of inorganic phosphate and a predominance of ADP in the intramitochondrial pool of adenine nucleotides. The latter requirement is most marked at alkaline pH values, where ATP is profoundly inhibitory. 2. Addition of CaCl(2) during state 4, state 3 (Chance & Williams, 1955) or uncoupled pyruvate oxidation causes a marked inhibition in the rate of oxygen uptake when low concentrations of mitochondria are employed, but may lead to an enhancement of state 4 oxygen uptake when very high concentrations of mitochondria are used. 3. These properties are consistent with the kinetics of the NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) from this tissue, which is activated by isocitrate, citrate, ADP, phosphate and H(+) ions, and inhibited by ATP, NADH and Ca(2+). 4. Studies of the redox state of NAD and cytochrome c show that addition of ADP during pyruvate oxidation causes a slight reduction, whereas addition during glycerol phosphate oxidation causes a ;classical' oxidation. Nevertheless, it is concluded that pyruvate oxidation is probably limited by the respiratory chain in state 4 and by the NAD-linked isocitrate dehydrogenase in state 3. 5. The oxidation of 2-oxoglutarate by swollen mitochondria is also stimulated by high concentrations of ADP and phosphate, and is not uncoupled by arsenate.     

Model of red blood cell membrane skeleton: electrical and mechanical properties

A theoretical membrane skeleton model of erythrocyte has been developed and successfully applied to interpret electrical and mechanical properties of the red blood cell spectrin-actin network. The model is based on the structure of the membrane skeleton that is comprised of unit cells each containing an actin protofilament and shooting forth a few spectrin heterodimers. The loose ends of the heterodimers of adjacent cells can form bonds with each other giving rise to an integrated network. The number of bonds depends on the temperature. The bond length being excessive (2.6 times the distance between the centers of adjacent cells), the bonds are flexible, and can thus be regarded as entropy springs. The advanced model has been employed to calculate the shear modulus of the membrane skeleton as well as to establish its temperature dependence. In a wide range of temperatures mu(T) is a decreasing function well fitting the experimental data. The relationship between the membrane bilayer-free size of the skeleton and the ionic strength of the solution has been derived to appear in good agreement with the results obtained previously. Experimental data combined with the advanced theory yield the average number of heterodimers per unit cell, m0, as equal to ca. 5; the spectrin heterodimer charge has been estimated.     

Contrast gain, signal-to-noise ratio, and linearity in light-adapted blowfly photoreceptors

Response properties of short-type (R1-6) photoreceptors of the blowfly (Calliphora vicina) were investigated with intracellular recordings using repeated sequences of pseudorandomly modulated light contrast stimuli at adapting backgrounds covering 5 log intensity units. The resulting voltage responses were used to determine the effects of adaptational regulation on signal-to-noise ratios (SNR), signal induced noise, contrast gain, linearity and the dead time in phototransduction. In light adaptation the SNR of the photoreceptors improved more than 100-fold due to (a) increased photoreceptor voltage responses to a contrast stimulus and (b) reduction of voltage noise at high intensity backgrounds. In the frequency domain the SNR was attenuated in low frequencies with an increase in the middle and high frequency ranges. A pseudorandom contrast stimulus by itself did not produce any additional noise. The contrast gain of the photoreceptor frequency responses increased with mean illumination and the gain was best fitted with a model consisting of two second order and one double pole of first order. The coherence function (a normalized measure of linearity and SNR) of the frequency responses demonstrated that the photoreceptors responded linearly (from 1 to 150 Hz) to the contrast stimuli even under fairly dim conditions. The theoretically derived and the recorded phase functions were used to calculate phototransduction dead time, which decreased in light adaptation from approximately 5-2.5 ms. This analysis suggests that the ability of fly photoreceptors to maintain linear performance under dynamic stimulation conditions results from the high early gain followed by delayed compressive feed-back mechanisms.     

Na+/ K+-pump activity in photoreceptors of the blowfly Calliphora : a model analysis based on membrane potential measurements

Na⁺/K⁺-pump activity and intracellular Na⁺ and K⁺ concentration changes in blowfly photoreceptors are derived from intracellular potential measurements in␣vivo with a model based on the Goldman-Hodgkin-Katz theory for membrane currents. The relation between the intracellular Na⁺ concentration and the pump activity appears to follow a Hill function with a Hill coefficient of 1 and a maximal possible Na⁺ current due to pump activity of about −4 nA. The developed photoreceptor model incorporates the slow and fast voltage-dependent K⁺ channels of the blowfly photoreceptor of which the properties were taken from the literature. Accepted: 5 August 1996     

The contribution of tonoplast and plasma membrane to the electrical properties of a higher-plant cell

Translocation of ¹⁴C-labelled assimilates down the petioles was studied in intact plants of Pelargonium zonale (L.) L'Hérit ex Ait. The central bundle of the petiole was dissected out and treated with solutions of various inhibitors. Whereas cytochalasin B had no effect on ¹⁴C-translocation, a distinct and localized inhibition was caused by CCCP (10^-7 M), antimycin (5×10^-5 M), atractylate (5×10^-5 M), and valinomycin (10^-5 M) without any significant change in the proportion of [¹⁴C]sucrose in the translocate. The inhibition of translocation is inferred both by accumulation of ¹⁴C distal to and a decrease in ¹⁴C concentration basal to the treated petiolar region. If valinomycin was fed into the transpiration stream by flapping the peripheral bundles of the petioles an increased labeling of sugar phosphates occurred in the ¹⁴C fed leaf. Plasmolysis tests indicated that whereas CCCP interfered with the semipermeability of phloem cell membranes, valinomycin had no such effect. The results with valinomycin suggest a compartmentation of potassium ions for the translocation process but are ambiguous as to whether or not a potassium pump is involved.Dedicated to Wilhelm Halbsguth, Kiel to his 65th birthday     

Immunocytochemical localization of opsin in the cell membrane of developing rat retinal photoreceptors

Mature retinal rod photoreceptors sequester opsin in the disk and plasma membranes of the rod outer segment (ROS). Opsin is synthesized in the inner segment and is transferred to the outer segment along the connecting cilium that joins the two compartments. We have investigated early stages of retinal development during which the polarized distribution of opsin is established in the rod photoreceptor cell. Retinas were isolated from newborn rats, 3-21 d old, and incubated with affinity purified biotinyl-sheep anti-bovine opsin followed by avidin- ferritin. At early postnatal ages prior to the development of the ROS, opsin is labeled by antiopsin on the inner segment plasma membrane. At the fifth postnatal day, as ROS formation begins opsin was detected on the connecting cilium plasma membrane. However, the labeling density of the ciliary plasma membrane was not uniform: the proximal cilium was relatively unlabeled in comparison with the distal cilium and the ROS plasma membrane. In nearly mature rat retinas, opsin was no longer detected on the inner segment plasma membrane. A similar polarized distribution of opsin was also observed in adult human rod photoreceptor cells labeled with the same antibodies. These results suggest that some component(s) of the connecting cilium and its plasma membrane may participate in establishing and maintaining the polarized distribution of opsin.     

Changes in intramitochondrial adenine nucleotides in blowfly flight-muscle mitochondria

1. With freshly isolated blowfly mitochondria 38% of the intramitochondrial adenine nucleotide was present as AMP. 2. On incubation with oxidizable substrates the AMP and ADP concentrations fell and that of ATP rose; with pyruvate together with proline the ATP concentration reached its maximum value at 6min; with glycerol phosphate the phosphorylation of endogenous nucleotide was more rapid. 3. Addition of the uncoupling agent carbonyl cyanide phenylhydrazone caused a rapid fall of ATP and a parallel rise in ADP, then ADP was converted into AMP. 4. This was in contrast with rat liver mitochondria endogenous AMP concentrations, which were always lower than those of blowfly mitochondria and changed little under different metabolic conditions. 5. Evidence is presented that adenylate kinase (EC 2.7.4.3) has a dual distribution in blowfly mitochondria, a part being located in the matrix space and a part in the space between the outer and inner mitochondrial membranes, as in liver and other mitochondria. 6. The possible regulatory role of changing AMP concentrations in the mitochondrial matrix was investigated. Partially purified pyruvate carboxylase (EC 6.4.1.1) and citrate synthase (EC 4.1.3.7) were inhibited 30% by 2mm-AMP, whereas pyruvate dehydrogenase (EC 1.2.4.1) was unaffected. 7. AMP activated the NAD(+)-linked isocitrate dehydrogenase (EC 1.1.1.41) activity of blowfly mitochondria in the absence of ADP, but in the presence of ADP, AMP caused inhibition. 8. It is suggested that AMP may exert a controlling effect on the oxidative activity of blowfly mitochondria.     

Isolation and properties of the outer membrane of plant mitochondria.

The outer membrane of turnip (Brassica rapa L.) mitochondria was isolated by incubating the mitochondria with a dilute digitonin solution and differential centrifuging. The outer membrane fraction was not contaminated by inner membrane enzymes and lacked an NADPH-cytochrome c reductase. However it possessed very active NADH-cytochrome c, dichloroindophenol and ferricyanide reductases which were insensitive to antimycin A, Amytal and low (less than 10 μm) concentrations of Dicumarol. p-Chloromercuribenzoate (ClHgBzO^?) and high concentrations (greater than 10 μm) of Dicumarol inhibited the reductases, ClHgBzO^? almost completely. Preincubation of the outer membrane with NADH protected it from ClHgBzO^? inhibition. An acid phosphatase and an NADPH-ferricyanide reductase were also detected, but the latter was only loosely bound to the membrane. The NADH dehydrogenase of the outer membrane was insensitive to ethylene glycol-bis(β-aminoethyl ether)N,N′-tetraacetate (1 mm) and was not stimulated by CaCl₂ (0.5 mm), thus differing from the external NADH oxidase of the inner membrane (Coleman, J. O. D., and Palmer, J. M. (1971) FEBS Lett., 17, 203–208). Respiratory-linked oxidation of exogenous NADH by intact mitochondria showed a similar pattern of inhibition by ClHgBzO^? as did the outer membrane, but was inhibited strongly by low concentrations of Dicumarol (5 μm inhibited by 70%).     

Proline synthesis from glutamate in mitochondria from the blowfly Aldrichina grahami

• 1.1. Glutamyl kinase in a homogenate of blowfly abdomens was not inhibited by proline and its analogues, and was found only in the mitochondrial fraction.
• 2.2. Proline was biosynthesized from glutamate only in a cell-free system prepared from blowfly abdomen.

     

Sodium transport and electrical properties of the chick chorioallantoic membrane

Transport and electrical properties of the chick chorioallantoic membrane (CAM) were studied in order to find the osmoregulatory organ which helps to compensate the renal filtration-reabsorption disbalance of chick embryos. It could be shown that CAM resembles Na+ transporting epithelia in that active Na+ absorption is responsible for the potential difference and short circuit current, which could be abolished by ouabain on the ectodermal and amiloride on the endodermal side. The transepithelial conductance rose with increasing sodium concentration in accordance with the Michaelis-Menten kinetics. The allantoic sac thus plays a role similar to the toad urinary bladder despite the low potential difference and resistance which indicate that CAM is a leaky epithelium. CAM is therefore not only a respiratory but also an osmoregulatory organ.     

Extracellular electrical activity from the photoreceptors of midge

The ontogeny of photosensitivity has been studied in a holometabolous insect, the midgeChironomus ramosus. The life cycle of midges shifts from an aquatic environment to a non-aquatic environment. Extracellular electrical activity of photoreceptor organs was recorded at larval and adult stages. We found an increase in photosensitivity as the larva metamorphosed to the adult stage. This is the first report of changes in photosensitivity during the development of any insect described in an ecological context.     

Intracellular ionic compartmentation, electrical membrane properties, and cell membrane permeability before and during first cleavage in the Ambystoma egg.

The intracellular ionic distribution in uncleaved and cleaving Ambystoma eggs was investigated by analysing the influx of ³H₂O, by determining the total content of Na⁺, K⁺ and Cl^? in extracts of eggs at different stages by both flame spectrophotometry and ion-selective microelectrodes, and by the continuous measurement of the Na⁺, K⁺ and Cl^? activities (a_Naⁱ, a_Kⁱ and a_Clⁱ) using intracellular ion-selective microelectrodes. The electrical membrane potential (E_m) and membrane resistance (R_m) were measured continuously in uncleaved and normally cleaving eggs as well as in eggs cleaving after removal of the vitelline membrane. The latter eggs expose their newly formed cleavage membrane to the external medium. Ionic permeability of the cell membrane before and during cleavage was analysed by a statistical comparison of the experimentally determined relationship between E_m and the ionic gradients across the cell membrane with those predicted theoretically from a constant field equation in dependence on the relative permeability, through insertion of the measured intracellular ion activities.³H₂O influx revealed the existence of a single intracellular water compartment (3.06 μl/egg) and a low water permeability (5.35 × 10^?5 cm sec^?1). Na⁺, K⁺ and Cl^? concentrations were constant at 54.1, 72.1 and 73.1 mM respectively, while a_Naⁱ, a_Kⁱ and a_Clⁱ were constant at 5.8, 51.8 and 59.7 mM respectively. It was concluded that all Cl^? ions are in solution, while 12.5% of all K⁺ and 86% of all Na⁺ is bound. The uncleaved egg showed a positive E_m of ca 40 mV and a specific membrane resistance of 39 kOhm cm². E_m could be described by a constant field equation with a permeability ratio $\text{P}{}_{\mathrm{<mtext>K</mtext>}}\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0.073}$. Shortly after the onset of first cleavage, E_m rapidly decreased concomitant with a rise in R_m (68.5 kOhm cm²). This was interpreted as a drop in Na⁺ permeability. During the cleavage process E_m progressively hyperpolarized and R_m decreased due to the insertion of a small fraction (3.3%) of the newly formed intercellular membrane into the cleavage furrow. This new membrane had a low specific resistance (0.69 kOhm cm²). Both in normally cleaving eggs and in eggs cleaving in the absence of the vitelline membrane E_m behaved according to the constant field equation, $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$ being 0.69 and 0.39, respectively. The differences with other amphibian eggs were discussed.     

Cytochemical properties of mitochondria in the gastric parietal cell.

The matrix of some mitochondria in gastric parietal cells of rat and guinea pig evidenced affinity for the high iron diamine method which localizes sulfated complex carbohydrates selectively by light and electron microscopy. Such staining has not been observed elsewhere in the stomach. The high iron diamine reactive mitochondria about equaled in number those which were unreactive, and the two groups were indistinguishable morphologically. The distinction was not apparent either when mitochondria were stained by other cytochemical procedures including dialyzed iron for acidic complex carbohydrates, 3-3' diaminobenzidine-H2O2 at pH 6.0 for cytochrome oxidase, and Kominick's pyroantimonate osmium tetroxide for antimonate precipitable cations. The dialyzed iron method stained acid glycoconjugates in the outer intermembrane space in parietal cell mitochondria. These mitochondria stained more strongly with dialyzed iron than have any others examined heretofore with this method and comprised the only reactive mitochondria in the stomach. Parietal cell mitochondria also stained strongly for cytochrome oxidase but those of other gastric cells failed to evidence this reactivity.