Vanadate, tungstate and molybdate activate rod outer segment phosphodiesterase in the dark

Biochimica et Biophysica Acta 845 (1985) 81-85

81

Elsevier BBA 11441

Vanadate, tungstate and molybdate activate rod outer segment phosphodiesterase in the dark Lucian V. Del Priore * and A a r o n Lewis ** School of Applied and Engineering Physics, Cornell University, Ithaca, N Y 14853 (U.S.A.)

(ReceivedNovember7th, 1984)

Key words: Vanadate;Molybdate;Tungstate; Phosphodiesteraseactivation;(Rod outer segment)

Anionic activation of rod outer segment phosphodiesterase by vanadate, molybdate and tungstate is demonstrated. Comparisons are made to adenylate cyclase, which is known to be activated by vanadate and molybdate but not by tungstate. In view of the differences in anionic activation between these two important enzymatic regulators of intracellular cyclic nucleotide metabolism, it is possible that tungstate can be used as a selective probe for the effects of phosphodiesterase activity in photoreceptors and other cells. The known electrophysiological stimulation of Limulus photoreceptors by these anions is also interpreted in light of our results. If anionic production of quantum bumps in Limulus photoreceptors is mediated by changes in cyclic nucleotides, then the electrophysiological response of Limulus photoreceptors to tungstate may indicate a role for phosphodiesterase rather than adenylate cyclase in mediating light-induced cyclic nucleotide alterations in this cell.

Introduction Vertebrate retinal rod outer segments contain a light-activated phosphodiesterase that is highly specific for cyclic GMP [1-6]. Photoexcited rhodopsin does not bind to phosphodiesterase directly, but activates this enzyme via an intermediary protein called the G-protein or transducin [7]. The absorption of light induces a conformational change in rhodopsin that results in the binding of the G-protein to rhodopsin [8] and the catalytic exchange of GTP for GDP on the G~ subunit of the G-protein [7]. G~-GTP dissociates from rhodopsin and the Gay complex, binds to phosphodiesterase, and relieves the inhibitory con* Current address: B-23, The Wilmer OphthalmologicalInstitute, The Johns HopkinsHospital,Baltimore,MD 21205, U.S.A. ** To whom reprint requests should be addressed. Abbreviation: Hepes, 4-(2-hydroxyethyi)-l-piperazineethanesulfonic acid.

straint imposed by the y-subunit of phosphodiesterase [9]. Activated phosphodiesterase then catalyzes the hydrolysis of cyclic GMP to G M P [1-6]. In vitro, the response of this system to light is highly amplified, since the absorption of a single photon results in the catalytic exchange of GTP for GDP on approx. 500 G-proteins [7], and these G-proteins each activate approx. 800 phosphodiesterase molecules leading to the hydrolysis of 4- 10 5 cyclic GMP within 1 s of photon absorption [2]. The activation of phosphodiesterase by light has many similarities to the activation of adenylate cyclase by hormone receptors located on the surface of many types of eukaryotic cells [10]. The absorption of light by rhodopsin and the binding of catecholamine hormones to cell surface receptors both catalyze the exchange of GTP for GDP bound to the respective G-protein of each system [10]. In the adenylate cyclase system, the G-GTP complex activates the catalytic subunit of this en-

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82 zyme, leading to the synthesis of cyclic AMP from ATP [11]. Recently, there has been a report of a functional exchange of the components of the adenylate cyclase and rod outer segment phosphodiesterase systems [12]. Fluoride ions are known to activate both adenylate cyclase [13-17] and rod outer segment phosphodiesterase [12,18]. Vanadate [19] and molybdate [20,21] activate membrane adenylate cyclase purified from a number of different tissues, but tungstate does not activate this enzyme [20]. In this paper, we demonstrate that vanadate, molybdate and tungstate activate rod outer segment phosphodiesterase in the absence of light. Since tungstate stimulates rod outer segment phosphodiesterase at low concentrations but does not activate adenylate cyclase, this anion may be employed as a selective stimulator of phosphodiesterase-mediated cyclic nucleotide hydrolysis in photoreceptors and other cells. Materials and Methods

Rod outer segments were isolated from frozen bovine retinas by sucrose flotation techniques that are described elsewhere [6]. The rod outer segment suspension was diluted to a rhodopsin concentration of 4-15 /~M in 250 /~1 of reaction media (4 mM cyclic G M P / 1 mM CAC12/60 mM KC1/30 mM NaC1/2 mM MgC12/1 mM dithiothreitol/10 mM Hepes at pH 7.8) and rapidly stirred with a magnetic spin bar. The phosphodiesterase activity was determined by measuring the rate of evolution of protons accompanying cyclic GMP hydrolysis with a pH meter and a chart recorder [6,22]. For dark trials, 2.5-/~1 aliquots of a concentrated stock solution containing the anion of interest were added to the rod outer segment suspension at approx. 1-min intervals, with up to eight serial additions of anion per sample. In this manner, each rod outer segment solution could be used to determine the phosphodiesterase activity at several different anion concentrations. The number of serial additions was limited by two factors: (1) all measurements were completed before the pH changed by 0.2 units, and (2) the total added volume was limited to less than 8% of the initial volume. The effect of diluting the rod outer segment sample on the phosphodiesterase activity was

corrected for in all of the results presented here [22]. Light trials were conducted by either fully bleaching the sample in room lights or exposing it to a calibrated commercial photoflash unit (Vivitar) placed 20 cm from the sample prior to the addition of anions. Either 40/~M p[NH]ppG or 1 mM GTP was added to the rod outer segment suspension prior to illumination, since light activation of phosphodiesterase requires a guanyl nucleotide cofactor [10]. When bright flash illumination and a GTP cofactor were used, the initial measurement of light-activated phosphodiesterase activity was made 30 s after the flash, since we observed that the activity of phosphodiesterase remains constant for several minutes after this time. Although the absolute light a n d / o r dark phosphodiesterase activity (mol cyclic GMP hydrolyzed/mol rhodopsin per s) varied with the lighting conditions employed, the GTP cofactor used, and the rod outer segment preparation, the activation and inhibition of phosphodiesterase by various ions discussed below is independent of these variables. Results

Vanadate (K m = 100 /~M), tungstate (K m = 1 mM) and molybdate ( K m = 3 mM) each activate rod outer segment phosphodiesterase in the dark (Figs. 1-3). 2 mM vanadate or 10 mM tungstate activate dark phosphodiesterase to nearly the same extent that this enzyme is activated by light, but 10-20 mM molybdate induces only 30-35% of the maximum activity of light-activated phosphodiesterase. None of these anions can further stimulate phosphodiesterase maximally activated by light (Figs. 1-3). Concentrations of vanadate greater than 2 mM markedly inhibit the phosphodiesterase activity elicited by either light or lower concentrations of vanadate in the dark, but high concentrations of tungstate or molybdate either do not affect or slightly inhibit the activity of phosphodiesterase. We cannot be certain of the slight inhibitory effect of tungstate or molybdate, because the addition of large amounts of these anions limited the accuracy of our measurements by changing the buffering capacity of the medium. Vanadate inhibition of phosphodiesterase does not appear to be due to a nonspecific ionic perturba-

83

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Fig. 1. Vanadate activates rod outer segment phosphodiesterase. In the dark, low concentrations of vanadate stimulate the activity of rod outer segment phosphodiesterase, with 2 mM vanadate stimulating this enzyme to greater than 90% of the light-induced activity. Concentrations of vanadate greater than 2 mM inhibit the activity of phosphodiesterase elicited by light or lower vanadate concentrations in the dark. The light curve was determined by bleaching 6% of the rhodopsin present in rod outer segment suspensions containing 1 mM GTP as a cofactor. The effect of vanadate on the activity of phosphodiesterase in rod outer segment samples bleached in room lights was also investigated, and the results obtained were similar to the results obtained using flash illumination (results not shown). Assay mixtures contained 1 mM CaC12 and 4 mM cyclic G M P in buffer. All suspensions were 10.2 # M in rhodopsin. Experiments were performed at room temperature at an initial pH of 7.8, and all measurements were completed before the p H had changed by 0.2 units. A relative phosphodiesterase activity = 1.0 corresponds to 14.3 laM of cyclic G M P hydrolyzed/s. PDE, phosphodiesterase.

,,

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Fig. 2. Tungstate activates rod outer segment phosphodiesterase. In the dark, 10 mM tungstate stimulates the activity of rod outer segment phosphodiesterase to a level greater than 90% of the light-induced activity. Concentrations of tungstate greater than 10 m M may exert a slight inhibitory effect on phosphodiesterase activity, but this is uncertain because of the change in buffering capacity introduced by this anion. The effect of tungstate on the light-induced activity of phosphodiesterase was determined with 40/~M p[NH]ppG as a cofactor, and with the sample activated by a flash bleaching 0.2% of the rhodopsin present. Assay mixtures contained 1 mM CaC1 z and 4 mM cyclic GMP. Experiments were performed at an initial pH of 7.8. Suspensions were 4 / t M in rhodopsin, and a relative phosphodiesterase activity = 1.0 corresponds to 17.7 /~M cyclic GMP hydrolyzed/s. PDE, phosphodiesterase. 1.2

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tion of the medium, since high concentrations of tungstate or molybdate only produce minimal effects. Chromate, a group VI B tetroxo complex similar to vanadate, molybdate and tungstate, does not significantly activate phosphodiesterase in the dark (data not shown). Flash-activated phosphodiesterase undergoes a first-order deactivation in the presence of ATP and G T P [6]. All of the results reported in this paper were obtained with 1 • 10 - 3 M Ca 2÷ added, but we conducted our assay for anion-induced deactivation of phosphodiesterase in 1 - 1 0 - 9 M Ca 2+ because flash-induced deactivation is more rapid in low calcium [6]. In 1 • 10 - 9 M Ca 2÷ the phosphodiesterase activity remained constant for at least 2 min after addition of 1.2 mM vanadate,

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Fig. 3. Molybdate activates rod outer segment phosphodiesterase. In the dark, 10-20 mM molybdate stimulates phosphodiesterase activity to a level 30-35% of the light-induced activity of this enzyme. The effect of molybdate on the light-induced activity of phosphodiesterase was determined with 1 mM GTP as a cofactor, and with the sample fully activated by bleaching in room lights. Assay mixtures contained 1 mM CaCI 2 and 4 mM cyclic GMP. Suspensions were 10.2 # M in rhodopsin, and experiments were performed at an initial pH of 7.8. A relative phosphodiesterase activity = 1.0 corresponds to 11.4 # M of cyclic G M P hydrolyzed/s. PDE, phosphodiesterase.

84 0.8 m M tungstate and 2 - 4 mM molybdate in the presence of 0.5 m M ATP and 0.5 mM G T P in the dark. This observation is consistent with the hypothesis that ATP-dependent deactivation occurs at the level of opsin phosphorylation or by the phosphorylation of the 48 kDa protein (arrestin) stimulated by photolyzed rhodopsin [30].

Discussion Impfications for the observation of anion-induced quantum bumps in Limulus Fluoride, vanadate, molybdate and tungstate produce an increase in the rate of quantum bumps in Limulus ventral photoreceptors in the dark that resembles the light response [23-25]. Since light alters cyclic nucleotides in visual photoreceptors [1-6,26,27], the role of cyclic nucleotide metabolism in visual excitation and sensory adaptation has been extensively investigated. In view of these observations, it has been suggested that cyclic nucleotides may be involved in the production of quantum bumps in Limulus [23-25]. However, Stern and Lisman [28] have recently performed internal dialysis experiments in Limulus ventral photoreceptors that seriously question the role of cyclic nucleotides in this cell. The concentration of cyclic nucleotides that must be dialyzed into Limulus photoreceptors to affect the electrophysiological response is much larger than the concentration of cyclic nucleotides in other cell types. In addition, Brown et al. [29] have also questioned the role of cyclic nucleotides in mediating the electrophysiological response in Limulus. However, none of these studies definitively exclude a role for cyclic nucleotides in visual transduction in Limulus ventral photoreceptors. It has been suggested that the anionic stimulation of quantum bumps in ventral photoreceptors of Limulus in the dark could be due to the activation of adenylate cyclase by fluoride, vanadate or molybdate [23-25]. However, since these anions also stimulate phosphodiesterase-mediated cyclic nucleotide hydrolysis in vertebrate photoreceptors, the net biochemical effect of these anionic activators on cyclic nucleotide metabolism in the ventral photoreceptor of Limulus is unknown. Significantly, we have demonstrated that tungstate stimulates phosphodiesterase, but this anion does

not stimulate adenylate cyclase [20]. Furthermore, it has been shown that tungstate also stimulates the production of quantum bumps in Limulus in the dark [25]. Therefore, if the production of quantum bumps by tungstate in Limulus is due to its effect on cyclic nucleotide metabolism, this process probably results from the activation of phosphodiesterase rather than adenylate cyclase. Thus, our results indicate that tungstate may be used to selectively stimulate cyclic nucleotide hydrolysis in photoreceptor and other cells, and may therefore be a unique chemical tool for probing the role of cyclic nucleotide hydrolysis in living cells.

Acknowledgements We would like to thank Drs. Jeffrey Walker and George Hess for helpful discussions related to this paper. Supported by U.S. Army contract No. D A M D 17-79-C-9041.

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