Graduation Year

2012

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Medical Sciences

Major Professor

Javier Cuevas

Keywords

arachidonic acid, intracardiac ganglia, mu-opioid receptor, Orai channels

Abstract

Proper autonomic regulation of mammalian cardiac function is dependent upon very complex and precise communication among the intracardiac ganglia and individual neurons within the ganglia. An array of neuromodulators is found within the ganglia that direct neuronal activity by modulating the movement of calcium. The current study determines that opioidergic agonists, which have been found to contribute to severe cardiac disease states and intracellular calcium mobilization, are also responsible for changes in the function of the intracardiac neuron via their effects on store-operated calcium channels (SOCs).

Previous studies suggest that phosphorylation plays a role in SOC regulation. Using Fura-2 calcium fluorometry, we determined that protein kinase A (PKA), protein kinase C (PKC), and cyclic adenosine monophosphate (cAMP) had no effect on store-operated calcium entry in the presence of antagonists, phorbol 12, 13 dibutyrate (PDBu), forskolin, and 8-Br cAMP, respectively. We also found pharmacologically that using both electrophysiology and calcium imaging that μ-opioid agonists, met-enkephalin (ME) and endomorphin (EM) depress SOC activity in intracardiac neurons. Arachidonic acid (AA), which has been found to depress SOC function in rat liver cells and μ-opioid receptor activation (MOR), blocked both store-operated calcium entry (SOCE) and the calcium release-activated current (ICRAC) significantly. Contrastingly, AA metabolites, prostaglandin E2)(PGE2) and prostaglandin D2 (PGD2), do not significantly influence SOCE which suggests that the effects of AA may be direct. The block elicited by EM was partially reversed by pertussis toxin (PTX), indicative of activation of a PTX-sensitive G-protein following MOR activation. Similarly, PLA2 inhibitors, OBAA and AACOCF3, decreased the percent block of SOCE due to opioid agonist-induced inhibition.

Using the perforated-patch method of I-clamp electrophysiology, we demonstrated that gadolinium, at low micromolar concentrations, reversibly reduced action potential firing. Importantly, these results suggest that SOCs may influence action potential firing in mammalian intracardiac neurons. Similarly, AA and EM depressed action potential firing. Taken together, these experiments suggest that a pathway involving EM and AA influences repetitive firing through SOC inhibition.

The importance of SOCs in the maintenance of action potential firing and more specifically, the expression and biophysical functionality of the individual pore-forming subunits (Orai1, 2, and 3) in any neuronal cell type has previously not been explored. Quantitative RT-PCR along with I-clamp electrophysiology revealed that Orai3 was exclusive to repetitively firing neurons. As a result, we hypothesize that robust Ca2+-dependent fast inactivation, also associated Orai3, is a factor in the maintenance of repetitive action potential firing.

Using Fura-2 calcium fluorometry and patch-clamp electrophysiology, we determined pharmacologically that μ-opioid receptor activation precedes an intracellular cascade that is dependent on a PTX-sensitive G-protein and AA but independent of prostaglandin and protein kinase activity. Finally, we used RT-PCR to determine the Orai subunits expressed in the intracardiac neurons and their influence on neuronal firing patterns. This study is the first to determine the role expressed subunits has in the maintenance of the electrical activity of the neuron.

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