Light-induced decreases in cGMP concentration precede changes in membrane permeability in frog rod photoreceptors.

We used an apparatus in which pieces of dark-adapted amphibian retinas (Rana pipiem, Bufo marinus) obtained under infrared illumination were exposed to precise intervals of 500-nm illuminations, and then frozen by contact of their outer segment surface with a liquid helium-cooled copper mirror. Sections of the frozen outer segment layer were obtained in a cryostat and then assayed for total extractable cyclic 3',5'-guanosine monophosphate (cGMP). Significant losses of cGMP with respect to the dark level were evident as early as 60 ms after light onset. With dim subsecond illuminations these losses were surprisingly large, which suggests a previously underestimated magnification in the cGMP cascade, or a transient substantial inhibition of guanylate cyclase activity in combination with increased cyclic GMP phosphodiesterase activity. Within the subsecond period, significant losses that were proportional to light intensity (2-log-unit range) and duration (60-550 ms) were generally not evident. However, losses significandy proportional to these factors became evident with durations of 1 s or longer. When pieces of retina were first illuminated (10 or 60 ms), then held in darkness for increasing periods before freezing, we observed a continuous loss of cGMP during the early postillumination dark period, followed by a recovery of the total cGMP level. The times for recovery to the preillumination level appear to be significantly longer than times reported for the recovery of the photoreceptor membrane potential after similar light exposures.

Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 98%

See 1 more Smart Citation

Light-induced losses and dark recovery rates of guanosine 3',5'-cyclic monophosphate in rod outer segments of intact amphibian photoreceptors.

Cohen

Blazynski

1988

“…THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 89 " 1987 Recent observations on the effects of Ca 2+ and cyclic GMP (cGMP) on the lightsensitive conductance in vertebrate rod photoreceptors have caused a dramatic shift in the thinking about the role of Ca 2+ in visual transduction in vertebrate rod photoreceptors, from that of an excitatory messenger to a more indirect regulatory role (Fesenko et al, 1985 ;Matthews et al, 1985 ;Cobbs and Pugh, 1985 ;Yau and Nakatani, 1985b;Nicol et al, 1987). Changes in the extracellular Ca 21 concentration have strong effects on a number of important aspects of rod physiology and rod biochemistry, such as the magnitude of the dark current flowing into the outer segment (Hodgkin et al ., 1984), the light sensitivity of rods (Bastian and Fain, 1982), and the rod cGMP content (Cohen et al, 1978;Woodruff and Fain, 1982 ;Cote et al ., 1984). Indirect evidence suggests that changes in intracellular Ca 2+ may influence the waveshape of the photoresponse, particularly its duration and falling phase (Hodgkin et al, 1985;Matthews et al, 1985).…”

Section: Introductionmentioning

confidence: 99%

Na+- and cGMP-induced Ca2+ fluxes in frog rod photoreceptors.

Schnetkamp¹,

Bownds²

1987

Self Cite

We have examined the Cal ' content and pathways of Ca l ' transport in frog rod outer segments using the Ca"-indicating dye arsenazo III . The experiments employed suspensions of outer segments of truncated, but physiologically functional, frog rods (OS-IS), intact isolated outer segments (intact OS), and leaky outer segments (leaky OS with a plasma membrane leaky to small solutes, but with sealed disk membranes). We observed the following . (a) Intact OS or OS-IS isolated and purified in Percoll-Ringer's solution contained an average of 2.2 mM total Ca 21, while leaky OS contained 2 .0 mM total Ca 21 . This suggests that most of the Cat' in OS-IS is contained inside OS disks . (b) Phosphodiesterase inhibitors increased the Ca t' content to^-4 .2 mM in intact OS or OS-IS, whereas the Cat ' content of leaky OS was not altered. (c) Na-Ca exchange was the dominant pathway for Ca t' efflux in both intact and leaky OS/OS-IS . The rate of Na-Ca exchange in intact OS/OS-IS was halfmaximal between 30 and 50 mM Na' ; at 50 mM Na', this amounted to 5.8 X 10' Cat'/OS . s or 0.05 mM total Ca t '/s. This is much larger than the Call component of the dark current . (d) Other alkali cations could not replace Na' in Na-Ca exchange in either OS-IS or leaky OS . They inhibited the rate of NaCa exchange (K >_ Rb > Cs ? Li > TMA) and, as the inhibition became greater, a delay developed in the onset of Na-Ca exchange. The inhibition of Na-Ca exchange by alkali cations correlates with the prolonged duration of the photoresponse induced by these cations (Hodgkin, A . L., P. A . McNaughton, and B. J. Nunn. 1985. journal of Physiology. 358 :447-468) . (e) In addition to Na-Ca exchange, disk membranes in leaky OS showed a second pathway of Ca t' transport activated by cyclic GMP (cGMP) . The cGMP-activated pathway required the presence of alkali cations and had a maximal rate of 9 .7 X 10 6 Cat'/OS-s. cGMP caused the release of only 30% of the total Ca l' from leaky OS . The rate of Na-Ca exchange in leaky OS amounted to 1 .9 X 10' Ca 21/ OS-s . (f) Even though the ratio of plasma to disk membrane surface area in frog OS is -1/100, the measured Cal ' flux across the plasma membrane would be expected to change the cytoplasmic Cat' levels three times faster than Call fluxes across the disk membrane .Address reprint requests to Dr . Paul P. M . Schnetkamp,

“…Schobert, and M . D. Bownds, manuscript submitted for publication) has correspondingly fast kinetics and a high stoichiometry (Cote et al ., 1984).…”

Section: Introductionmentioning

confidence: 99%

A monoclonal antibody to guanine nucleotide binding protein inhibits the light-activated cyclic GMP pathway in frog rod outer segments.

Hamm¹,

Bownds²

1984

Self Cite

A monoclonal antibody that blocks the light-activated cyclic GMP (cGMP) pathway in frog photoreceptor outer segments (ROS) has been obtained . The antibody (4A) inhibits guanine nucleotide binding to G-protein, the intermediate that links rhodopsin excitation to cGMP phosphodiesterase (PDE), inhibiting light-induced PDE activity as a consequence . Antibody inhibition of the light-activated cGMP pathway is complete at a stoichiometry of approximately one antibody per G-protein in the mixture, which indicates high specificity of the inhibition . Inhibition is more pronounced than that caused by PDE inhibitors such as isobutylmethylxanthine (IBMX) or Ro 20-1724 . Antibody 4A has the further effect of inhibiting the phosphorylation of two low molecular weight proteins, components I and 11, whose phosphorylation normally can be stimulated by raising cGMP levels . The inhibition is not overridden by adding cGMP, which suggests that the G-protein influences these phosphorylations by a pathway distinct from its action on cGMP concentration . Antibody 4A may prove useful as a probe of the relevance of the cGMP pathway to visual transduction in living photoreceptors . Six other monoclonal antibodies to Gprotein, as well as six monoclonal antibodies to rhodopsin and one to PDE, do not block light-activated guanine nucleotide binding, PDE activity, or ROS protein phosphorylations .