Quantitative pressure injection of picoliter volumes into Limulus ventral photoreceptors

Corson, D. Wesley; Fein, Alan M.

doi:10.1016/s0006-3495(83)84303-3

Cited by 66 publications

(49 citation statements)

References 13 publications

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“…Injections were monitored using the infrared video camera (Corson and Fein, 1983a). InsP 3 was obtained from Research Biochemicals (Natick, M A), dissolved into a 10 mM stock solution, and kept at Ϫ20°C.…”

Section: Methodsmentioning

confidence: 99%

Protein Kinase C Activators Inhibit the Visual Cascade inLimulusVentral Photoreceptors at an Early Stage

Dabdoub

Payne

1999

J. Neurosci.

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The phosphoinositide cascade mediates visual transduction in invertebrate photoreceptors. Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol bisphosphate, producing inositol trisphosphate (InsP 3 ) and diacylglycerol (DAG). Protein kinase C (PKC) is a major target of DAG in many cell types. We have used PKC activators to investigate the function of the kinase in the phototransduction cascade in Limulus polyphemus ventral photoreceptors. Extracellular application of (Ϫ)-indolactam V (0.03-30 M) or phorbol-12,13-dibutyrate (10 M) reversibly reduced the sensitivity of the electrical response of the photoreceptors to light by up to 1000-fold. The inert stereoisomer (ϩ)-indolactam V and 4␣-phorbol had no effect. The effect of (Ϫ)-indolactam V was antagonized by the PKC inhibitors bisindolylmaleimide I and Gö 6976. Coapplication of bisindolylmaleimide V, used as a negative control compound for PKC inhibition, did not reduce the effectiveness of (Ϫ)-indolactam V. These findings are consistent with (Ϫ)-indolactam V activating PKC and desensitizing the light response. Furthermore, our pharmacological results indicate that PKC activation does not appear to play a role in light adaptation. We localized the position of the target of PKC in the visual cascade. We chemically excited the cascade at various stages to determine the kinase's target. PKC activation by (Ϫ)-indolactam V decreased the light-induced elevation of intracellular calcium but had no effect on the photoreceptor's excitatory response to intracellular injection of InsP 3 . However, the PKC activator greatly reduced the excitation caused by GTP-␥-S injection. We propose that PKC inhibits the visual transduction cascade at the G-protein and/or PLC stage.

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Section: Methodsmentioning

confidence: 99%

Protein Kinase C Activators Inhibit the Visual Cascade inLimulusVentral Photoreceptors at an Early Stage

Dabdoub

Payne

1999

J. Neurosci.

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“…For the injection pressures and durations used in this study, ~ 1 pl of solution was typically ejected. This was used to estimate a volume of 1-10 pl injected into the cell, according to the method of Corson and Fein (1983). This represents a small percentage of the cell's volume of ~400 pl (Caiman and Chamberlain, 1982) and a 40-400-fold dilution once the injected material has dispersed within the cytoplasm.…”

Section: Methodsmentioning

confidence: 99%

A lingering elevation of Cai accompanies inhibition of inositol 1,4,5 trisphosphate-induced Ca release in Limulus ventral photoreceptors.

Levy

Payne

1993

The Journal of General Physiology

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Injection of inositol 1,4,5 trisphosphate (ImPs) into Limulus ventral photoreceptors causes an elevation of intracellular free Ca concentration (Cai) and depolarizes the photoreceptors. When measured with the photoprotein aequorin, the InsPs-induced Cai increase follows the time course of depolarization and declines within 1-2 s. However, sensitivity to further injections of InsP3 remains suppressed for several tens of seconds. The possibility that the suppression of Ca release (feedback inhibition) is due to a small lingering elevation of Cai, below the existing detection limit of aequorin, was investigated by measuring Cai with Ca-sensitive electrodes. Double-barreled, Ca-selective microelectrodes were used to pressure inject InsPs and measure Cai at the same point. Light or InsPs injections into the light-sensitive compartment depolarized the photoreceptors and induced an elevation of Cai that persisted for tens of seconds. Injections of InsP3 during the decay of Cai showed that sensitivity to InsP3 recovered as resting Cai approached the prestimulus level. The relationship between elevated Cai and feedback inhibition was very steep. An elevation of Cai of 1 o.M or more was associated with inhibitions of 79 +-12.4% (SEM; n = 7) for the InsP~-induced Cai increase and of 76 ± 8% for depolarizations. With a residual Cai elevation of 0.01 o.M or less, the mean inhibition was 10 ± 7.4% for InsPs-induced Cai increase and 6.6 ± 4% for InsPz-induced depolarization. Injections of InsP3 into a light-insensitive compartment within the cell induced elevations of Cai with no associated depolarizations or feedback inhibition. To verify that a sustained elevation of Ca i is necessary for inhibition of InsPz-induced Cai increase and depolarization, we injected ethyleneglycol-bis-(13-aminoethylether)-N,N'-tetraacetic acid (EGTA) between two injections of InsP3. Injection of 1 mM EGTA or the related Ca chelator BAPTA, delivered 750 ms after the first injection of InsPs, restored the peak depolarization caused by the second injection of InsP3 to > 80 ± 3% of control, compared with 13 ± 8% without an intervening injection of EGTA. Measurement of Cai with aequorin showed that an intervening injection of EGTA partially restored the InsP3-induced Ca i increase.

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“…The intensity of the unattenuated beam was 3.5 mW/cm 2, and stimulus intensities throughout this article are given as logt0 I/Io, where Io is the unattenuated light intensity. Cells were illuminated throughout the experiments with infrared light from a substage illuminator, allowing for visualization of the photoreceptors (and substances injected into the cells) with an infrared-sensitive video camera (Corson and Fein, 1983). Cells were impaled with two glass microelectrodes containing the substances to be injected.…”

Section: Recording and Stimulationmentioning

confidence: 99%

“…Cells were impaled with two glass microelectrodes containing the substances to be injected. Injections were achieved by applying brief pressure pulses to the back of the micropipettes according to the method of Corson and Fein (1983).…”

Section: Recording and Stimulationmentioning

confidence: 99%

The role of the inositol phosphate cascade in visual excitation of invertebrate microvillar photoreceptors.

Frank

Fein

1991

The Journal of General Physiology

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The identity of the transmitter(s) involved in visual transduction in invertebrate microvillar photoreceptors remains unresolved. In this study, the role of inositol 1,4,5-trisphosphate (IPs) was examined in Limulus ventral photoreceptors by studying the effects on the light response of heparin and neomycin, agents that inhibit the production or action of IP 3. Both heparin and neomycin reduce responses to brief flashes of light and the transient component of responses to steps of light, and also inhibit IPa-induced calcium release, indicating that IP s plays a direct role in invertebrate visual excitation. The effects of BAPTA, a calcium buffer, were also examined and shown to be consistent with a role for IPa-mediated calcium release in visual excitation. However, all three agents fail to block the plateau component of the response to a step of light, indicating that a single pathway involving IP~ and calcium cannot solely be responsible for visual excitation in invertebrates. We suggest that the inositol phosphate cascade and a second parallel process that is not dependent on IP 3 are involved in the production of the light response.

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Quantitative pressure injection of picoliter volumes into Limulus ventral photoreceptors

Cited by 66 publications

References 13 publications

Protein Kinase C Activators Inhibit the Visual Cascade inLimulusVentral Photoreceptors at an Early Stage

Protein Kinase C Activators Inhibit the Visual Cascade inLimulusVentral Photoreceptors at an Early Stage

A lingering elevation of Cai accompanies inhibition of inositol 1,4,5 trisphosphate-induced Ca release in Limulus ventral photoreceptors.

The role of the inositol phosphate cascade in visual excitation of invertebrate microvillar photoreceptors.

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