Synthesis, accumulation, and release of d-aspartate in the Aplysia californica CNS.

d-Aspartate (d-Asp) is an endogenous molecule that is often detected in CNS and endocrine tissues. Using capillary electrophoresis and a variety of radionuclide detection techniques, we examine the synthesis, release, and uptake/accumulation of d-Asp in the CNS of the marine mollusk Aplysia californica. We observe the preferential synthesis and accumulation of d-Asp over l-aspartate (l-Asp) in neuron-containing ganglia compared to surrounding sheath tissues. Little conversion of d-Asp to l-Asp is detected. The Ca(2+) ionophore ionomycin and elevated extracellular potassium stimulates release of d-Asp from the cerebral ganglia. Lastly, radioactive d-Asp in the extracellular media is efficiently taken up and accumulated by individual F-cluster neurons. These observations point to a role for d-Asp in cell-to-cell signaling with many characteristics similar to classical transmitters.

D-Aspartate as a putative cell-cell signaling molecule in the Aplysia californica central nervous system.

The content, synthesis and transport of D-aspartate (D-Asp) in the CNS of Aplysia californica is investigated using capillary electrophoresis (CE) with both laser-induced fluorescence and radionuclide detection. Millimolar concentrations of D-Asp are found in various regions of the CNS. In the cerebral ganglion, three adjacent neuronal clusters have reproducibly different D-Asp levels; for example, in the F- and C-clusters, up to 85% of the free Asp is present in the D-form. Heterogeneous distribution of D-Asp is also found in the individual identified neurons tested, including the optical ganglion top-layer neurons, metacerebral cells, R2 neurons, and F-, C- and G-cluster neurons. The F-cluster neurons have the highest percentage of D-Asp (approximately 58% of the total Asp), whereas the lowest value of approximately 8% is found in R2 neurons. In pulse-chase experiments with radiolabeled D-Asp, followed by CE with radionuclide detection, the synthesis of D-Asp from L-aspartate (L-Asp) is confirmed. Is D-Asp in the soma, or is it transported to distantly located release sites? D-Asp is clearly detected in the major nerves of A. californica, including the pleuroabdominal and cerebrobuccal connectives and the anterior tentacular nerves, suggesting it is transported long distances. In addition, both D-Asp and L-Asp are transported in the pleuroabdominal connectives in a colchicine-dependent manner, whereas several other amino acids are not. Finally, d-Asp produces electrophysiological effects similar to those induced by L-Asp. These data are consistent with an active role for D-Asp in cell-to-cell communication.

Structural and functional analysis of Aplysia attractins, a family of water-borne protein pheromones with interspecific attractiveness.

Mate attraction in Aplysia involves a long-distance water-borne signal (the protein pheromone attractin), which is released during egg laying. Aplysia californica attractin attracts species that produce closely related attractins, such as Aplysia brasiliana, whose geographic distribution does not overlap that of A. californica. This finding suggests that other mollusks release attractin-related pheromones to form and maintain breeding aggregations. We describe four additional members of the attractin family: A. brasiliana, Aplysia fasciata, Aplysia depilans (which aggregates with A. fasciata aggregations), and Aplysia vaccaria (which aggregates with A. californica aggregations). On the basis of their sequence similarity with A. californica attractin, the attractin proteins fall into two groups: A. californica, A. brasiliana, and A. fasciata (91-95% identity), and A. depilans and A. vaccaria (41-43% identity). The sequence similarity within the attractin family, the conserved six cysteines, and the compact fold of the NMR solution structure of A. californica attractin suggest a common fold for this pheromone family containing two antiparallel helices. The second helix contains the IEECKTS sequence conserved in Aplysia attractins. Mutating surface-exposed charged residues within this heptapeptide sequence abolishes attractin activity, suggesting that the second helix is an essential part of the receptor-binding interface.