Natural and Selected Synthetic Toxins - American Chemical Society

Chapter 3

Cellular Signaling in PC12 Affected by Pardaxin 1

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S. Abu-Raya , E. Bloch-Shilderman , H. Jiang , K. Adermann , E. M . Schaefer , E. Goldin , E. Yavin , and P. Lazarovici Downloaded by NORTH CAROLINA STATE UNIV on September 23, 2012 | http://pubs.acs.org Publication Date: December 20, 1999 | doi: 10.1021/bk-2000-0745.ch003

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Department of Pharmacology, School of Pharmacy, Faculty of Medicine of the Hebrew University, Jerusalem, Israel Section of Growth Factors, NICHD and NINDS, National Institutes of Health, Bethesda, MD 20892 Niedersaechsisches Institut fuer Peptide-Forschung, Feodor-Lynen-Strasse 31, D-30625, Hannover, Germany Promega Company, 2800 Words Hollow Road, Madison, WI 53711-5399 Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel 2

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Pardaxins, a family of polypeptide, excitatory neurotoxins, isolated from the gland's secretion of Pardachirus fish are used as pharmacological tools to investigate calcium-dependent and calcium-independent signaling leading to neurotransmitter release. In PC12 cells, a neural model to study exocytosis, pardaxin forms voltage-dependent pores which are involved in pardaxin-induced increase in intracellular calcium and calcium-dependent dopamine release. Pardaxin-induced calcium-independent, dopamine release from PC12 cells is attributed to the stimulation of the arachidonic acid cascade and the increased release of the arachidonic acid metabolites produced by the lipoxygenase pathways. Pardaxin rapidly stimulates MAPK phosphorylation activity, which is proposed to be involved in pardaxin-induced arachidonic acid and dopamine release. It seems likely that pardaxin delayed stimulation of stress-kinases JNK and p38 will play a prominent role in triggering cell death. Collectively, these results demonstrate that pardaxin selectively modulates neuronal signaling to achieve massive exocytosis and neurotoxicity. The ordered growth, and functioning of multicellular organisms requires the transfer of informationfromone cell to another and from one part of the cell to another. This information comes from a variety of sources both within and outside the cell. Hormones, growth factors and neurotransmitters interact with specific receptors on the plasma membrane or in the cytoplasm. Inside the cell, information is relayed and transduced by intracellular messengers including calcium, cyclic nucleotides, diacylglycerol, inositol polyphosphates, etc. (/). Finally, balanced interactions between these different signaling pathways ensure that Corresponding author. 22

©2000 American Chemical Society

In Natural and Selected Synthetic Toxins; Tu, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.

Downloaded by NORTH CAROLINA STATE UNIV on September 23, 2012 | http://pubs.acs.org Publication Date: December 20, 1999 | doi: 10.1021/bk-2000-0745.ch003

23 the required biological response occurs. It is not surprising, therefore, that many toxins interfere with the chemical communication in the body. A simple way to achieve intercellular communication in the nervous system involves release of neurotransmitter which signals through receptor molecules that exist on the same or neighbouring cell. This principle lies at the heart of the signaling process utilized by the synapse, the basic unit on which an integral nervous system is built (2). At synapses, the elementary signaling event involves a depolarization dependent calcium influx into the presynaptic terminal that triggers the release of the neurotransmitter to be sensed by receptors in the postsynaptic cells (3). Neurotoxins are commonly defined as chemicals which interfere with synaptic activity. They can be classified into ionic channel toxins which modify ionic conductance, presynaptic toxins which affect neurotransmitter release, and post-synaptic toxins which block the neurotransmitter receptors (4). For the last two decades we investigate the mechanism of action of pardaxin, a presynaptic neurotoxin isolated from the Red Sea flatfish Pardachilus marmoratus (5). Elucidation of pardaxin's mode of action is essential to its effective use as a pharmacological tool and is also expected to provide new understanding into the molecular steps involved in the mechanism of neurotransmitter release. Isolation, Characterization and Synthesis of a Novel Pardaxin Isoform Sole fish of the genus Pardachirus produce an exocrine secretion from epithelial glands located at the base of the dorsal and anal fins (Figure la). This secretion contains toxic components that play an important role in the defense of predatory fish. Beside aminoglycosteroids (6), peptides, know as pardaxins (5), represent the major components of the secretion (7-9). Pardaxins have been shown to form voltage-dependent ion channels (10-12) at low concentrations in a variety of artificial membranes and cytolysis at high concentrations (5, 13). The membranal activity of pardaxins such as binding, insertion and rearrangement for pore formation, is dependent on the a-helix content as well as the intramolecular interaction between the amino- and carboxy-terminal domains (12,14,15). Thus, this peptide family is structurally and functionally related to other membranally active peptides, e.g. cecropins (16), magainins (17) and melittin (18). To date, pardaxins have been isolated and characterized from the western Pacific Peacock sole, P. pavoninus (PI, P2 and P3) (7), and the Red Sea Moses sole, P. marmoratus (P4) (5,9) (Figure lb). All know pardaxins contain one single peptide chain composed of 33 amino acids and show a high tendency of aggregation in aqueous solution (5,9). Their sequences are very homologous, differing only at positions 5, 14, or 31 (Figure lb). The membranal effects of pardaxins are rather sensitive to structural variations such as the net charge, attached side chains, the stereochemistry of a-carbons and chain truncation (19,13,20). Recently we reported the isolation of a novel pardaxin isoform (P5)fromP. marmoratus (49). The primary structure of the native peptide has been determined by amino acid sequencing, endoproteinase cleavage and mass spectrometric techniques. The exchange of Gly31 of Pardaxin P4 by Asp (Figure lb) is the major difference between P4 and P5. The sequence of the novel pardaxin P5 differs by one In Natural and Selected Synthetic Toxins; Tu, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.


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Figure la. Lateral views of the Pardachirus marmoratus fish; Arrows indicate the location of the toxic glands, at the basis of the fins. Figure lb, Amino acid alignment of pardaxins. P I , P2 and P3 are from P. pavoninus; P4 and P5 are from P. marmoratus. Variable residues are bold-faced.



25 point mutation from the pardaxin PI isolated from P. pavoninus (Figure lb). The change Asp31->Glu31 would require a mutation of the third base of the Asp31 codon. In addition, a mutation C-»U, the first base of the Leu5 codon of PI, would lead to the sequence of P3, another pardaxin found in P. pavonius. In contrast, no single point mutation of either PI, P3 or P5 would lead to P2. The pardaxin P2 is genetically closest to P5, differing in position 31 (Asp-»Gly:C->A exchange of the first base of the Leul4 codon). We suggest that the pardaxin isoform P4 may provide the mutation gap between P5 and P2. It would be one point mutation away from P5 (Asp31-»Gly31) and another point mutation away from P2 (Leul4->Ilel4). However, the investigation of this evolutionary structure-function relationship of paradaxins requires further evaluation using other Pardachirus species. PC12 Pheochromocytoma Cells: A Neuronal Model to Study Secretion Over the past two decades, the clonal PC 12 cell line has become a widely used preparation for various model studies on neurons and adrenal gland chromaffin cells (26). Regulated exocytosis of catecholamines has been largely investigated in bovine adrenal chromaffin cells (27) and in rat PC12 cells (28). The secretory vesicles of these cells, the chromaffin granules, similar to synaptic vesicles store dopamine, norepinephrine, ATP and various proteins. The essential role of calcium in catecholamine secretion from chromaffin cells has been well established (27). Cytosolic calcium ([Ca]j) can be increased due to membrane depolarization, opening of receptor-operated channels or release of calcium from intracellular stores (27). The concentration of ([Ca]0 is strictly regulated, and it is thought that the increase in calcium concentration within the microdomain of the active exocytotic zone allows vesicles to fuse and release their catecholamines content. In contrast to calcium-dependent neurotransmitter release, the signal transduction pathways of calcium-independent neurotransmitter release (29-31) have received scant attention. Characterization of Pardaxin-induced Dopamine Release: The Role of Calcium The ability of Pardaxin versus to other secretagogues to induce dopamine release from PC 12 cells in the presence or absence of extracellular calcium ([Ca]o) is presented in Figure 2a. By subtracting the basal release, Pardaxin (6 uM) stimulated dopamine release by 26 ± 2% and 20 ± 1% of total content in the presence or absence of [Ca]o, respectively. In calcium-containing medium, carbachol (10 ^iM) bradykinin (1 |iM) and KCl (50 mM) stimulated dopamine release by 9.5 ± 2%, 13 ± 3% and 15 ± 2.5% of total content, respectively. In the absence of [Ca]o these compounds did not induce dopamine release. The dose and time course dependence of Pardaxin-induced dopamine release were determined in calcium-containing medium. As shown in Figure 2b, Pardaxin caused dopamine release in a concentration and time dependent manner. The most pronounced effect was obtained at 10 jiiM. At this concentration, Pardaxin stimulated dopamine released by 37 ± 3%, 51 ± 2.5% and 58 ± 2.5% of total content after 15, 30 and 60 min, respectively. These data indicate Pardaxin, in contrast to KCl, carbachol and bradykinin, induced dopamine release in


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Natural and Selected Synthetic Toxins - American Chemical Society

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