Library

Notes: Three enzymes (acid phosphatase, peroxidase, and tyrosinase) were localized by electron microscopy within the retina of crayfish Orconectes limosus. Peroxidase activity was observed only in lamellar bodies, which are secondary lysosomes and degrade photosensory membrane. After H2O2 was omitted from the reaction medium, peroxidase activity in lamellar bodies was partly inhibited but was not missing completely. After addition of sodium pyruvate, which inhibits endogenous generation of H2O2, staining of lamellar bodies was absent. Tyrosinase activity was found in lamellar bodies and in small vesicles within the rhabdoms similar to those found positive for acid phosphatase. Granules (500–700 nm in diameter) with an electron opaque matrix and mature screening pigment granules showed tyrosinase activity. Moreover, lamellar structures within membrane-bound organelles that additionally contained screening pigment-like granules were electron dense because of tyrosinase activity. After addition of phenylthiourea (PTU) to the incubation medium, lamellar bodies did not generally contain electron dense deposits, although weak staining of single membranes still was sometimes observed. After addition of sodium pyruvate in combination with PTU, no staining was detected. The possible role of tyrosinase in ommochrome synthesis within secondary lysosomes that degrade photosensory membrane is discussed.

Notes: Zusammenfassung 1. Eine Methode zur elektrophysiologischen Untersuchung der aus dem Krebs-Auge isolierten Retina wird beschrieben. 2. FürEupagurus und andere marine Krebse wird eine physiologische Salzlösung angegeben. 3. Das Belichtungspotential der isolierten Retina vonEupagurus bernhardus — ein einfaches negatives Sehzellenpotential — wird in seiner Abhängigkeit von Dauer und Stärke des Reizlichts sowie vom Adaptationszustand beschrieben. 4. Mit der gleichen Methode wird aus Gründen des Vergleichs das Belichtungspotential des gesamten vom Tier abgetrennten Augenstiels untersucht. 5. Das Bestandpotential wird am Gesamtauge ca. 0,5 mV corneapositiv gemessen. Das Bestandpotential der isolierten Retina ist kleiner und hat in den Versuchen verschiedenes Vorzeichen. 6. Der Widerstand der Retina nimmt bei Belichtung ab. 7. Anodische Polarisation (Distalseite der Retina positiv gegenüber der proximalen) vergrößert und verlängert das Belichtungspotential. Hohe anodische Polarisation verkleinert es. 8. Geringe kathodische Polarisation verkleinert und verkürzt das Belichtungspotential. Bei hoher kathodischer Polarisation wird das Belichtungspotential invers gemessen. 9. Bei positiver sowie bei negativer Polarisation im Dunkeln werden in einigen Fällen mehr oder weniger regelmäßige Oszillationen beobachtet (Abb. 22), deren Amplituden in ähnlicher Weise wie die des Belichtungspotentials von der Polarisationsspannung abhängen. 10. Es wird dargestellt, welchen Einfluß die Orientierung der Retinulae und die Widerstandsänderungen der Retina bei Belichtung auf die Gestalt des von der Retina abgeleiteten Summen-Belichtungspotentials ausüben. 11. Es wird angenommen, daß das Belichtungspotential desEupagurus-Auges ein Membranpotential der Sehzellen ist. 12. Die Widerstandsabnahme der Retina wird als Widerstandsabnahme der Membranen der Sehzellen interpretiert. 13. Die Gestalt des Belichtungspotentials kann gedeutet werden als Folge der Kinetik des Membrangeschehens und der Kinetik der fotochemischen Reaktionen des Sehfarbstoffs. 14. Am Belichtungspotential lassen sich zwei Komponenten (K I und K II) unterscheiden, die vermutlich den Komponenten R und S desDytiscus-Retinogramms (Bernhard, 1942) entsprechen. K II hat bei Ableitung vom Gesamtauge umgekehrtes Vorzeichen wie an der isolierten Retina. 15. Die durch Polarisation bedingten Veränderungen des registrierten Belichtungspotentials sind weitgehend eine Folge der Widerstandsänderung der Retina bei Belichtung. Hohe anodische (wahrscheinlich auch kathodische) Polarisation beeinflußt aber außerdem die Eigenschaften der erregbaren Membran selbst. 16. Es wird angenommen, daß neben der Abnahme der Konzentration des Sehfarbstoffs eine elektrotonische Wirkung von K II die adaptativen Veränderungen des Belichtungspotentials verursacht.

Notes: Resümee Zusammenfassend kann gesagt werden, daß Rhodopsin in den Sehzellen von Verbetraten und Evertebraten nach Photonenabsorption unter Vermittlung eines G-Proteins Enzymkaskaden startet. Diese führen zu einer gewaltig verstärkten Abnahme (Vertebraten) bzw. Zunahme (Evertebraten) der intrazellulären Transmitter-Moleküle, die als Liganden von innen an Proteine der Plasmamembran reversibel gebunden werden können. Die Transmitter-Bindung führt in den Wirbeltier-Photorezeptoren zur Öffnung von Ionenkanälen, durch die bevorzugt Natrium-Ionen passieren können. Cyclisches GMP ist der interne Transmitter, der als Ligand die Öffnung der Kanäle verursacht. Seine intrazelluläre Konzentration wird bei Belichtung durch die Wirkung eines cGMP-spaltenden Enzyms verringert. Bei den Sehzellen von Wirbellosen wird bei Belichtung die Konzentration an IP3 erhöht. Dies führt zu einem Anstieg der intrazellulären Calcium-Ionen-Konzentration. Möglicherweise sind Calcium-Ionen die kanalöffnenden Liganden. Es kann aber auch sein, daß parallel dazu die Rhodopsin-Aktivierung die Produktion eines anderen terminalen Transmitters verursacht — möglicherweise ist dieser cGMP. Eine so komplizierte, aus vielen Schritten bestehende Transduktionskette kann an vielen Stellen steuernd beeinflußt werden. Die Vorgänge, die den Verstärkungsgrad und damit die Empfindlichkeit regeln, spielen bei der Anpassung der Empfindlichkeit der Sehzelle an die mittlere vorhandene Helligkeit, d.h. bei der Hell/Dunkel-Adaptation, eine Schlüsselrolle.

Notes: Abstract The effect of low external Na+ concentrations on the light-induced K+ release from crayfish photoreceptor cells was tested by labelling intracellular K+ with the isotope86Rb. The amount of isotope released per light stimulus is roughly proportional to the external Na+ concentration if the osmolarity is kept constant by replacing Na+ with Tris, choline or sucrose. When sucrose is used to replace the depleted Na+ the light-induced K+ release is a linear function of the external Na+ concentration and is reduced by approx. 95% at an external Na+ concentration of 5 mmol/l. For choline and Tris substitutions the relationships are less clear but at Na+ concentrations ≤56 mmol/l it seems that in comparison with sucrose the light-induced K+ release is smaller in a Tris solution and larger in a choline solution. It is suggested that the light-induced K+ release is due mainly to an activation of voltage sensitive K+ channels.

Notes: Abstract It is assumed that “dark channels” determine a permanent dark conductance of the arthropod visual cell membrane. The light stimulus causes a transient opening of “light channels”. The ion selectivity of dark channels and light channels is roughly described. Factors influencing the activation of light channels, as membrane energy metabolism, membrane potential and adjusted calcium ion concentration are specified. The mechanism of the action of calcium ions on the conductance of the visual cell membrane is discussed.

Notes: Abstract Transient elementary currents, bumps, stimulated by short dim light flashes were measured in ventral nerve photoreceptors of Limulus. It is demonstrated that light activates two types of bumps, which form two distinct components of the receptor current at higher light intensities. The two bump types, which are both assumed to be activated by single absorbed photons, differ in current amplitude and kinetic parameters. The current amplitude of one bump type is smaller than 0.3 nA and that of the other type is in the usual current range of up to several nanoamperes. The average latency of small bumps measured from the short stimulus flash is shorter than that of the large bumps. The small bumps have slower activation kinetics than the large bumps. It is demonstrated that with increasing flash intensity the small bumps overlap first and form a macroscopic current, on top of which the large bumps are superimposed. Results indicate that a single absorbed photon selectively activates only one kind of the enzyme cascades evoking one bump type. We conclude that the active meta conformation of a rhodopsin molecule selectively binds a specific type of G-protein, which is involved in the stimulation of one of the transduction cascades. The two bump types, which are the elements of two macroscopic current components support the previous assumption that light activates different transduction mechanisms in Limulus photoreceptors.

Notes: Summary 1.The action potential of the isolated retina of the hermit crab Eupagurus bernhardus L. resulting from exposure to light has been measured with external electrodes under constant stimulus conditions. Three measurements of the retinal action potentials (RAP's) were taken to observe the changes of the RAP's quantitatively: a) The amplitude h max of the maximum; b) the amplitude h e of the plateau, measured at the end of the stimulus, for taking the shape quotient h max /h e ; c) the peak-amplitude-time t max. 2.The RAP's of the retina in a standard physiological saline are compared with those of the retina in salines of different ionic composition while osmotic pressure and p h were kept constant. 3.Increasing K+-concentration reduces the amplitude h max of the RAP's gradually, which is zero at 500 mM K+/l. The peak-time t max decreases with increasing K+-concentration up to 50 mM K+/l, whereas at higher concentrations it increases. In contrast to this fact h max/h e increases up to 50 mM K+/l and decreases above this concentration (Fig. 3). 4.Increasing Ca++-concentration reduces h max (zero above 350 mM Ca++/l) and t max. h max/h e rises up to a Ca++-concentration of about 30 mM Ca++/l; whereas at a higher Ca++-concentration it decreases again (Fig. 6). When the Ca++-concentration is very low the fall of the RAP is much slowed down and the plateau h e extremly rises. In a Ca++-free saline which contained 1 mM/l Ethylendiamintetraacetic acid (EDTA) the retina lost its irritability reversibly (Fig. 8). 5.The amplitude of the RAP's is augmented with increasing Mg++-concentration up to 10–30 mM Mg++/l and decreases above this concentration (Fig. 11). When the saline contains virtually no other cations but Mg++ (367 mM/l) the amplitude of the RAP is small (20%) but not zero. 6.In a buffered isotonic NaCl-solution as well as in a saline in which all the Cl--ions are substituted by SO4 ---ions the amplitude of the RAP's is higher but the shape of the RAP's is changed in the same way as in other solutions with very low concentrations of Ca++. 7.All the changes of the RAP's described so far are reversible. 8.Even when the retina is kept in a salt solution containing sodium in a very low concentration (ca. 3–5 mM/l, the sodium substituted by choline+-ions) for 5 hours the amplitude h max of the RAP does not change significantly but the shape: the peaktime t max is longer, h max/ h e is much greater. Afterwards when the retina is brought into standard saline again, the amplitude h max increases, t max remains almost unchanged and h max/h e , decreases strongly (Fig. 17). 9.Substitution of all the Na+-, Ca++- and Mg++-ions by choline+-ions results in a decrease of the amplitude h max, a lengthening of t max and a small increase of h max/h e . 10.Substitution of all the NaCl by glucose decreases the amplitude h max, lengthens t max very much and decreases the value of h max/h e but little. Afterwards, when the retina is brought into standard saline again the effect of the glucose solution on the amplitude h max is only little reversible: h max increases very little, t max decreases strongly and h max/h e increases (Fig. 21). 11.With increasing external K+-concentration the resting potential decreases. The changes of the resting potential cause the changes in the shape of the RAP's. 12.The presence of a small concentration of Ca++-ions outside of the cell membrane is obviously necessary for the ability of the cell membrane of the photoreceptor to increase its ionic permeability consequent to stimulation by light. Above a Ca++-concentration of about 1 mM/l the raise of permeability of the cell membrane during illumination is smaller with increasing Ca++-concentration. The velocity of the changes in permeability is augmented, especially of those changes concerned with the fall of the RAP. The effect of Mg++-ions is somewhat similar to that of Ca++-ions, but much weaker. 13.The changes of the RAP are mainly determined by the low Ca++-content when the retina stays in the NaCl- or sulfate saline. 14.Choline+-ions probably greatly increase the raise of permeability of the cell membrane for the divalent cations Ca++ and Mg++ during excitation. 15.It is suggested that under normal conditions in Eupagurus the amplitude of the RAP is determined primarily by the Na+-concentration gradient over the receptor cell membrane. But also the divalent cations Ca++ and Mg++ contribute to the amplitude of the RAP, especially after the retina has been treated with choline chloride.

Notes: Summary 1. The action potential of the retina of the hermit crab Eupagurus bernhardus L. resulting from exposure to light has been measured with external electrodes. 2. Three measurements of the retinal action potentials (RAP's) were taken to observe the changes of the RAP's quantitatively: a) the amplitude of the maximum h max, to characterize the height of the RAP's; b) the amplitude of the plateau h e,measured at the end of the stimulus for taking the shape quotient h max/h e; c) the peak amplitude time t max. 3. The changes of the RAP's under the influence of metabolic inhibitors (2,4-Dinitrophenol 0,2 mM/1; Potassiumcyanide 1,0 mM/1; Sodiumazide 1,0 mM/1) have been studied under constant stimulus conditions. 4. Under the influence of the metabolic inhibitors the RAP's soon become smaller and finally under these conditions the excitability of the retina is lost. 5. Some minutes after washing out the inhibitor RAP's again can be evoked, these increasing in size with time. 6. The three inhibitors manifest similar effects on the RAP's; however, they differ in reaction time. 7. The excitability of the retina, which has been lost by poisoning, was not restorable by use of positive and negative polarizing currents. 8. The changes of the RAP's in the course of dark adaptation under constant stimulus conditions and the changes by different intensities of the stimulating light are measured and compared with the alterations of the RAP's induced by metabolic inhibitors. 9. When the intensity of the light stimuli increases, the amplitude h max of the RAP's and the shape quotient h max/h egrow in size and the peak amplitude time t max decreases. 10. In the first phase of the influence of the metabolic inhibitors the changes of the height of the RAP's are similar to those produced by decreasing intensities of the stimuli, whereas the changes of the shape measured by the shape quotient h max/h eand t max are similar to those produced by increasing intensities of the light stimuli. In the second phase of the inhibitors' action, both, the changes of height and shape of the RAP's are similar to those produced by decreasing intensity of the stimuli. 11. The changes of the RAP's under the influence of the inhibitors are very similar to the changes of the RAP's in the course of dark adaptation, when compared in the reverse direction of time. They are also rather similar to the changes of the RAP's when the resting potential of the photoreceptor cells is decreased under the influence of increasing potassium concentration in the extracellular solution. 12. It is suggested that under the influence of metabolic inhibitors the resting potential of the photoreceptor cells will decrease and the concentration of rhodopsin probably also will decrease.