Characterization of potassium cyanide-induced IP(3) formation in PC12 cells
Abstract
Hypoxia/ischemia increases cellular inositol trisphosphate (IP$\sb3)$ levels and thereby mobilizes intracellular Ca$\sp{2+}$. Cyanide which produces histotoxic hypoxia also mobilizes intracellular Ca$\sp{2+}$. It was hypothesized that cyanide's action is similar to that of hypoxia/ischemia and that the IP$\sb3$ system has an important role in cyanide neurotoxicity. KCN was found to increase IP$\sb3$ levels in rat pheochromocytoma (PC12) cells. This observation led to mechanistic studies which revealed that KCN activates phospholipase C (PLC) to generate IP$\sb3$. PLC activation by KCN is a complex process partly dependent on Ca$\sp{2+}$ and partly Ca$\sp{2+}$ independent. Ca$\sp{2+}$-dependent IP$\sb3$ formation was inhibited by pretreatment with the non-specific Ca$\sp{2+}$ channel blocker, LaCl$\sb3$ or nifedipine, showing involvement of voltage-sensitive Ca$\sp{2+}$ channels. Since KCN-induced IP$\sb3$ formation was not additive with that elicited by glutamate, KCN and glutamate may share a common step in the transduction process that increases formation of IP$\sb3$. Also extracellular Ca$\sp{2+}$ removal and a selective metabotropic glutamate receptor (mGluR) antagonist (scL-AP3 or (+)-MCPG) caused greater inhibition of IP$\sb3$ formation compared to either condition alone. These results indicate that KCN induces glutamate release and subsequently mGluR activation may contribute to Ca$\sp{2+}$-independent IP$\sb3$ formation. Glutamate release by KCN was shown to be dependent on extracellular Na$\sp+$ and blocked by Na$\sp+$-glutamate transporter inhibitors (THA or t-PDC) indicating KCN releases glutamate via reversal of the Na$\sp+$ -glutamate transporter. BW 1003C87, a Na$\sp+$ channel-mediated glutamate release inhibitor blocked KCN-induced glutamate release. However, neither amiloride, a Na$\sp+$/H$\sp+$ and Na$\sp+$/Ca$\sp{2+}$ antiporter inhibitor, nor tetrodotoxin, a voltage-sensitive Na$\sp+$ channel inhibitor blocked glutamate release in response to KCN. These results indicate that the Na$\sp+$ channel may be involve in KCN-induced glutamate release. Since cellular energy status does not change during the course of cellular toxicity, ATP depletion does not play a role in KCN-induced glutamate release and IP$\sb3$ formation. In summary, KCN stimulates PLC-coupled polyphosphoinositide hydrolysis in PC12 cells via two distinct pathways. First, KCN increases cytosolic Ca$\sp{2+}$ which activates PLC directly. Secondly, KCN induces glutamate release which interacts with mGluR resulting in PLC activation.
Degree
Ph.D.
Advisors
Isom, Purdue University.
Subject Area
Pharmacology|Cellular biology|Anatomy & physiology|Animals
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