Limulus Photoreceptor Cells Research Articles

Bump amplitude, but not latency and time-course, can depend on position of illumination in Limulus photoreceptor cells

Bumps, the responses evoked by single photons in the ventral photoreceptor of Limulus polyphemus, were measured under voltage clamp conditions. The bumps were evoked by illuminating the photoreceptor either with a global flash or a small light spot (diameter about 5 μm) which covers only 0.25% of the light-sensitive part of the cell membrane. The light energy of both flash types was adjusted so that each flash on average evoked one bump. Parameters of bumps evoked by local light spots in various membrane areas were compared with those evoked by light flashes which illuminated the whole photoreceptor. The results show that the bump amplitude depends on the location of the illumination. Membrane areas were found where the average value of the bump amplitude was either smaller or larger for a spot illumination than for a whole cell illumination. The latency and the shape (e.g. width) of the bumps does not depend on the location of the illumination.

Inositol 1,2-cyclic 4,5-trisphosphate concentration relative to inositol 1,4,5-trisphosphate in pancreatic minilobules on stimulation with carbamylcholine in the absence of lithium. Possible role as a second messenger in long- but not short-term responses.

A method for the extraction of cyclic inositol phosphates in the absence of acid after short incubation times is described. A modified high pressure liquid chromatography method is also described which separates inositol 1,2-cyclic 4,5-trisphosphate (IcP3), inositol 1,4,5-trisphosphate (I(1,4,5)P3), and inositol 1,3,4-trisphosphate (I(1,3,4)P3). Mouse pancreatic minilobules were preincubated with [3H]inositol for 1 h in the absence of lithium, washed, and incubated without and with carbamylcholine without lithium for various times. On adding carbamylcholine, I(1,4,5)P3 peaked at 10 s, followed by a fall to a steady-state level which was two-thirds the peak value. This level was maintained for 20 min. IcP3, on the other hand, rose very slowly; at 10 s, it was only 5% of I(1,4,5)P3. It continued to rise until it equaled the steady-state level of I(1,4,5)P3 at 20 min. I(1,3,4)P3 rose gradually but at a faster rate than IcP3, peaking at 40 s at the same level as that achieved by I(1,4,5)P3 at that time and then falling in parallel with I(1,4,5)P3. Assuming equal potencies of IcP3 and I(1,4,5)P3 in mobilizing intracellular stores of Ca2+ in pancreatic minilobules, as seen in Limulus photoreceptor cells, platelets, and 3T3 cells, IcP3 would appear to play no messenger role at very early times. Thus, I(1,4,5)P3 should be responsible for almost all of Ca2+ release at very early times (10 s), while at later times (20 min) IcP3 and I(1,4,5)P3 should contribute equally to Ca2+ release. The significance of these results is discussed.

Isolation and characterization of the inositol cyclic phosphate products of polyphosphoinositide cleavage by phospholipase C. Physiological effects in permeabilized platelets and Limulus photoreceptor cells.

Cleavage of the polyphosphoinositides, catalyzed by phospholipase C purified from ram seminal vesicles, produces phosphorylated inositols containing cyclic phosphate esters (Wilson, D. B., Bross, T. E., Sherman, W. R., Berger, R. A., and Majerus, P. W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4013-4017). In the present study we describe the isolation and characterization of inositol 1:2-cyclic 4-bisphosphate and inositol 1:2-cyclic 4,5-trisphosphate, the two cyclic phosphate products of phospholipase C catalyzed cleavage of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, respectively. We established the structures of these two cyclic compounds through 18O labeling of phosphate moieties, phosphomonoesterase digestion, and fast atom bombardment-mass spectrometry. We examined the physiological effects of these compounds in two systems: saponin-permeabilized platelets loaded with 45Ca2+ and intact Limulus photoreceptors. Both inositol 1:2-cyclic 4,5-trisphosphate and the noncyclic inositol 1,4,5-trisphosphate, but not inositol 1:2-cyclic 4-bisphosphate, release 45Ca2+ from permeabilized platelets in a concentration-dependent manner. Injection of inositol 1:2-cyclic 4,5-trisphosphate into Limulus ventral photoreceptor cells induces both a change in membrane conductance and a transient increase in intracellular calcium ion concentration similar to those induced by light. We injected inositol 1,4,5-trisphosphate and inositol 1:2-cyclic 4,5-trisphosphate into the same photoreceptor cell and found that the cyclic compound is approximately five times more potent than the noncyclic compound in stimulating a conductance change. We speculate that inositol 1:2-cyclic 4,5-trisphosphate may function as a second messenger in stimulated cells.

3',5'-cyclic adenosine monophosphate and adenylate cyclase in phototransduction by limulus ventral photoreceptors.

Biochemical and electrophysiological measurements were made on photoreceptor cells from Limulus ventral eyes to investigate the possible role of cyclic AMP and adenylate cyclase in the visual transduction mechanism. Cyclic AMP content in a photoreceptor-enriched fraction (the end organs) of Limulus ventral eyes was approximately 15 pmol/mg protein. The cyclic AMP content was increased by bathing eyes in 1-methyl-3-isobutyl xanthine or forskolin and was increased almost 100-fold when bathed in both. Illumination did not change cyclic AMP content significantly in any of these conditions. Discrete events that can be recorded electrophysiologically occur spontaneously in darkness. An increase in the frequency of discrete events is evoked by dim illumination. The discrete events are a sign of excitation of Limulus photoreceptor cells. Drug-induced changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness were not correlated with changes in cyclic AMP content. Adenylate cyclase activity measured from a small number of pooled photoreceptor clusters was stimulated by fluoride and vanadate ions, hydrolysis-resistant analogues of GTP, cholera toxin and forskolin. The Limulus enzyme is similar pharmacologically to mammalian and avian adenylate cyclases. Activation of adenylate cyclase by drugs was not correlated with changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness. A heat-treated Lubrol extract of membranes from Limulus ventral eyes reconstituted the adenylate cyclase activity of membranes from S49 mouse lymphoma cyc- mutant cells which lack a functional regulatory protein. These findings suggest that Limulus ventral eye photoreceptors contain a regulatory protein that mediates the activation of adenylate cyclase by guanine nucleotides, fluoride or cholera toxin. This regulatory protein is homologous with that found in mammalian and avian adenylate cyclases. Our findings suggest that neither cyclic AMP nor adenylate cyclase activation is a necessary or obligatory component of the excitation mechanism in Limulus ventral photoreceptors.

Quantitative pressure injection of picoliter volumes into Limulus ventral photoreceptors

Many pharmacological probes must be applied to the interior of cells to produce their effects. Ideally, a method for injecting such materials should be simple, rapid, and independent of the chemical properties of the material to be injected. In addition, one might desire to confirm immediately that an injection occurred and to estimate the volume injected shortly thereafter. We report that these conditions are fulfilled when the injection of materials from micropipettes by pressure pulses is confirmed by visualization of injection-induced disturbances in cells viewed on a video monitor. Volumes of aqueous droplets subsequently injected into a nearby oil pool may be used to estimate the volumes injected into cells. We have obtained a calibration curve for these quantitative estimates of injected volumes by injecting radioactively labeled sulfate into Limulus photoreceptor cells. We find that the estimates are accurate within a range covering one order of magnitude. We assess the sources of systematic and random errors in making these estimates.

Energy, quanta and limulus vision

The threshold for evoking nerve impulses from single ommatidia was measured in the darkadapted, lateral eye of Limulus in situ. Our results are consistent with the notion that under dim illumination the eccentric cell fires a nerve impulse for every absorbed photon. However, the threshold is probably not constant and may vary as a result of the spontaneous activity in the eye. This variability could produce deceptively low estimates for the threshold. Measurements of the incident light intensity and corneal attenuation, together with an estimate of the photopigment absorption, suggest that the quantum efficiency for eliciting a nerve impulse is between 0.05 and 0.2. Perhaps the large, regenerativelike potential fluctuations we have recorded from the Limulus photoreceptor cells in situ amplify the effect of absorbed photons, and enable the eye to respond to single quanta.

All-or-none responses of Limulus photoreceptor cells in presence of barium ions.

All-or-none responses of Limulus photoreceptor cells in presence of barium ions.