Alan Fein

Feedback inhibition by calcium limits the release of calcium by inositol trisphosphate in Limulus ventral photoreceptors

Injection of inositol 1,4,5 trisphosphate (InsP3) into Limulus ventral photoreceptors elevates the concentration of intracellular calcium ions and as a consequence depolarizes the photoreceptor. This InsP3-induced elevation can be inhibited by a prior injection of calcium or InsP3 delivered 1 s earlier. Recovery from this inhibition has a half-time of between 1.5 and 5 s at 20 degrees C. Calcium released by InsP3 therefore inhibits further release of calcium from InsP3-sensitive calcium stores. This feedback inhibition may protect the calcium stores from depletion during prolonged bright illumination. Feedback inhibition, rather than periodic depletion of calcium stores, may also underlie the oscillatory bursts of InsP3-induced calcium release that have been observed in many cell types.

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.

cGMP inhibits IP3‐induced Ca2+ release in intact rat megakaryocytes via cGMP‐ and cAMP‐dependent protein kinases

1 Inhibition of inositol 1,4,5‐trisphosphate (IP3) receptor‐mediated Ca2+ release by cGMP was examined in intact rat megakaryocytes, by using a combination of single cell fluorescence microscopy to monitor intracellular free calcium ([Ca2+]i) and flash photolysis of caged second messengers. 2 Sodium nitroprusside (SNP), a nitric oxide (NO) donor, and the hydrolysis‐resistant cGMP analogue 8‐(4‐chlorophenylthio)guanosine 3′,5′‐cyclic monophosphate (pCPT‐cGMP) inhibited Ca2+ release induced by photolysis of caged IP3. Neither of them affected the rate of Ca2+ removal from the cytoplasm following photolysis of caged Ca2+. 3 Photolysis of the caged NO donor 3‐morpholinosydnonimine (SIN‐1) during agonist‐induced [Ca2+]i oscillations inhibited Ca2+ release without affecting the rate of Ca2+ uptake and/or extrusion. 4 We conclude that the inhibition of IP3‐induced Ca2+ release is the principal mechanism of NO‐cGMP‐dependent inhibition of [Ca2+]i mobilization. 5 IPG, a specific peptide inhibitor of cGMP‐dependent protein kinase (cGMP‐PK), blocked the inhibitory effect of pCPT‐cGMP, indicating that the inhibition of IP3‐induced Ca2+ release by pCPT‐cGMP is mediated by cGMP‐PK. However, the simultaneous application of both IPG and IP20, a specific peptide inhibitor of cAMP‐dependent protein kinase (cAMP‐PK), was required to block the inhibitory effect of SNP. These data strongly suggest that NO‐cGMP‐dependent inhibition of [Ca2+]i mobilization is mediated via the activation of both cGMP‐PK and cAMP‐PK.

Absorptance and spectral sensitivity measurements of rod photoreceptors of the tiger salamander, Ambystoma tigrinum.

The spectral sensitivity of the extracellularly-recorded photoresponse of isolated rods of the tiger salamander, Ambystoma tigrinum, was compared to the absorptance spectrum. Both measurements were made with the same optical system on the same portion of each cell to avoid errors that could occur when the two kinds of measurement were made under different conditions. The relative spectral sensitivity and absorptance spectrum were found to be in excellent agreement between 450 and 700 nm.

[Ca2+]i oscillations and [Ca2+]i waves in rat megakaryocytes

ATP activated [Ca2+]i oscillations were measured in single rat megakaryocytes using fluorescence ratio microscopy. With increasing ATP concentration the duration of the [Ca2+]i oscillations increased, however, there was considerable variation from cell to cell in the absolute value of the peak [Ca2+]i and the frequency and duration of the oscillations. This variation depended, in part, on the level of Fura-2 loading suggesting that megakaryocytes are sensitive to buffering of [Ca2+]i by Fura-2. Agents, that increase the level of intracellular cGMP (sodium nitroprusside and 8-pCPT-cGMP) or cAMP (prostacyclin, IBMX, forskolin and 8-bromo-cAMP) inhibited [Ca2+]i oscillations. Despite the large cell to cell variation in the patterns of [Ca2+]i oscillations, reapplication of the agents that elevated cAMP or cGMP inhibited the oscillations similarly. Using video rate fluorescence ratio imaging we found that the agonist-induced [Ca2+]i oscillations were the result of a well-defined [Ca2+]i wave, which spread across the cell with an average speed of about 35 microns/s, during the rising phase of each oscillatory spike. After reaching a peak, [Ca2+]i decreased uniformly across the whole cell during the falling phase of the spike. Analysis of the temperature dependence of [Ca2+]i waves showed that the rate of [Ca2+]i decay exhibited a strong temperature dependence (Q10 approximately 4), whereas, the rate of rise exhibited a weak temperature dependence (Q10 approximately 1.3), suggesting, that the rate limiting process for [Ca2+]i wave propagation in rat megakaryocytes is the rate of [Ca2+]i diffusion.

Inositol 1,4,5-trisphosphate induces bursts of calcium release inside Limulus ventral photoreceptors

Injection of inositol 1,4,5-trisphosphate (InsP3) into dark-adapted Limulus ventral photoreceptors produces a series of discrete bursts of membrane depolarization. Prior injection of aequorin, a luminescent calcium indicator, reveals that the bursts of depolarization are accompanied by individual bursts of intracellular calcium elevation with a similar time course. Reduction of extracellular calcium increased rather than decreased the InsP3-induced rise in calcium. These results suggest that small numbers of InsP3 molecules can trigger discrete and rapid releases of large amounts of calcium from intracellular stores. In some cells, InsP3 injection induces a delayed and prolonged elevation of intracellular calcium in addition to the brief bursts.

Excitation and adaptation of Limulus photo-receptors by light and Inositol 1,4,5-trisphosphate

Abstract Receptor-mediated signal transduction is a rapidly developing area of research in which the paths of discovery in biochemistry and physiology are converging. A small number of intracellular messengers appear to mediate a variety of responses to stimuli as diverse as light, hormones and neurotransmitters. The main focus of this review will be on intracellular messengers of visual excitation and adaptation in the ventral photoreceptors of Limulus with an emphasis on the possible role that phosphoinositide metabolism may play in these processes. Because of their large size, the ventral photoreceptors have been a favorite preparation for studying the molecular basis of excitation and adaptation. In Limulus the photoreceptive membranes are composed of microvilli, as is the case in the complex eyes of many other invertebrates. The extrapolation of knowledge obtained from Limulus to other microvillar photoreceptors can be justified on the basis of common structure and physiology, but to draw inferences about non-microvillar photoreceptors is less justifiable.

Limulus rhodopsin: rapid return of transient intermediates to the thermally stable state.

Spectral transitions of rhodopsin in single cells of the Limulus ventral eye were observed both with flash photometry and by measuring the early receptor potential. Even with repetitive stimulus flashes the rhodopsin did not bleach; after each flash the spectral intermediates decayed rapidly to the initial thermally stable state. The pigment returns to the stable state in a time comparable with the duration of the late receptor potential.

Divergent mechanisms for phototransduction of invertebrate microvillar photoreceptors.

Recently, it was reported that the polyunsaturated fatty acids (PUFAs), arachidonic acid (AA) and linolenic acid (LNA), activate the light-sensitive channels in Drosophila photoreceptors (Chyb et al. 1999). We have examined whether these PUFAs activate the light-sensitive channels in Limulus ventral photoreceptors. We find that, whether applied from the outside or injected into a Limulus ventral photoreceptor, either AA or LNA fails to activate the light-sensitive channels. Moreover, the synthetic diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol, also fails to activate the light-sensitive channels. We suggest that these findings require us to rethink our view about the generality of the process of phototransduction in invertebrate microvillar photoreceptors. We propose that the photoreceptors of Drosophila and Limulus evolved to utilize different branches of the phosphoinositide pathway for phototransduction: those of Limulus evolved to utilize IP3-mediated calcium release while those of Drosophila evolved to utilize diacylglycerol and its downstream products.

Rapid Increase in Plasma Membrane Chloride Permeability during Wound Resealing in Starfish Oocytes

Plasma membrane wound repair is an important but poorly understood process. We used femtosecond pulses from a Ti-Sapphire laser to make multiphoton excitation–induced disruptions of the plasma membrane while monitoring the membrane potential and resistance. We observed two types of wounds that depolarized the plasma membrane. At threshold light levels, the membrane potential and resistance returned to prewound values within seconds; these wounds were not easily observed by light microscopy and resealed in the absence of extracellular Ca2+. Higher light intensities create wounds that are easily visible by light microscopy and require extracellular Ca2+ to reseal. Within a few seconds the membrane resistance is ∼100-fold lower, while the membrane potential has depolarized from −80 to −30 mV and is now sensitive to the Cl− concentration but not to that of Na+, K+, or H+. We suggest that the chloride sensitivity of the membrane potential, after wound resealing, is due to the fusion of chloride-permeable intracellular membranes with the plasma membrane.