An enzymatically enhanced recording technique for Limulus ventral photoreceptors: physiology, biochemistry, and morphology.

Enzymatic treatments that facilitated whole-cell electrophysiological recordings were used on Limulus ventral photoreceptor cells. Ventral optic nerves were treated with either collagenase or collagenase, papain, and trypsin. Either treatment greatly increased the ease of making whole-cell recordings of transmembrane potentials. Light responses obtained from enzyme-treated photoreceptor cells were nearly identical to results obtained without enzyme treatment and compared favorably to in vivo recordings of light responses from the compound lateral eye. Enzyme-treated cells also responded to applied octopamine, as do untreated cells, with an increased phosphorylation of a 122-kD protein. This suggests that the external receptors and internal biochemical machinery required for at least one second-messenger cascade are present after enzyme treatment. The morphological integrity of enzyme-treated photoreceptor cells was examined with light microscopy as well as with scanning and transmission electron microscopy. In general, we found that each enzyme treatment greatly reduced the integrity of the layers of glial cells that surround the photoreceptor cells thereby making these cells easily accessible for whole-cell recordings of transmembrane potentials. The morphology of the rhabdomere was normal after enzymatic degradation of the adjacent glial covering.

Light-regulated proteins in Limulus ventral photoreceptor cells.

The protein intermediates of the photoresponse and the modulation of this response in invertebrate photoreceptors are largely unknown. As a first step toward identifying these proteins, we have examined light-stimulated changes in protein phosphorylation in preparations of Limulus photoreceptors. Here we show that light modulates the level of phosphorylation of three proteins associated with Limulus ventral photoreceptors: the upper band of a 46-kD protein doublet (46A) and a 122-kD protein, which become more heavily phosphorylated in response to light, and the lower component of the 46-kD doublet (46B), which is phosphorylated in dark-adapted cells, but not in cells maintained in the light. In dark-adapted preparations, 46A is phosphorylated within 30 s after a flash of light and dephosphorylates over a period of many minutes. It is also a major substrate for calcium/calmodulin-dependent protein kinase (Wiebe et al., 1989); therefore, we speculate that 46A is involved in some aspect of dark adaptation. Interestingly, the level of phosphorylation of 46A is the same when measured from preparations maintained in complete darkness or ambient light for at least 1.5 h. The 122-kD phosphoprotein is the same protein which becomes phosphorylated in response to efferent innervation to Limulus eyes (Edwards et al., 1988) and the efferent neurotransmitter, octopamine (Edwards and Battelle, 1987). It may be involved in the increase in retinal sensitivity and the enhanced response of photoreceptors to light that is initiated by efferent innervation. Its role in light-stimulated processes is not clear. The level of phosphorylation of 46B may be most relevant to the long-term state of adaptation of the photoreceptor cell to light and dark.

Calcium/calmodulin-stimulated phosphorylation of photoreceptor proteins in Limulus.

Calcium (Ca2+) is thought to play a major role in the photoresponse of both vertebrates and invertebrates, but the mechanisms through which Ca2+ exerts its effects are unclear. In many systems, some effects of Ca2+ on cellular processes are thought to be mediated via activation of calcium/calmodulin protein kinase (CaCAM-PK) and the phosphorylation of specific proteins. Thus, protein substrates for CaCAM-PK in photoreceptor cells may be important in mediating the effects of Ca2+ on the photoresponse. In this study, we identify eight substrates for CaCAM-PK found in both the ventral and lateral eyes of Limulus. We focus on a characterization of one of these, a 46-kD substrate. We show that its subcellular distribution in ventral photoreceptors and its isoelectric forms are identical to the 46-kD light-stimulated phosphoprotein (46A) described by Edwards et al. (1989). Furthermore, we present evidence that 46A is unique to photoreceptor cells, and that it is present throughout the cell. Based on the results of this study, and the previous study by Edwards et al. (1989), we propose that 46A is involved in mediating the effects of Ca2+ on Limulus photoreceptor cell function, and that it may be involved in dark adaptation.

Identification and function of octopamine and tyramine conjugates in the Limulus visual system.

Major metabolites of octopamine and tyramine in the Limulus nervous system are identified here as gamma-glutamyl octopamine and gamma-glutamyl tyramine. We show that these conjugates are normal products of amine metabolism in Limulus, and that they are normally present in octopamine-rich Limulus tissues. The synthesis of these conjugates is not restricted to nervous tissue, but the highest activity of gamma-glutamyl amine synthetase was measured in the CNS. Our interest in these molecules stems from our previous observations which showed that they were synthesized and stored in, and released from, the efferent fibers to Limulus eyes which modulate the sensitivity of the eyes to light. Here we provide direct evidence for the release of the conjugates from Limulus eyes in response to depolarization, and that gamma-glutamyl octopamine can increase the sensitivity of the lateral eye to light. Our observations lend support to the hypothesis that gamma-glutamyl octopamine may serve as an intercellular messenger in the Limulus visual system.