Presynaptic and postsynaptic pathways underlying the sympathetic stress response
The adrenal medulla is a key effector arm of the sympathetic nervous system in the periphery. It responds to sympathetic stimulation by discharging a cocktail of powerful agents – including epinephrine, norepinephrine, opioid hormones, and vasoactive peptides – directly into the circulation. Secretion from the adrenal medulla is dependent on input from sympathetic, “splanchnic” fibers which terminate on adrenomedullary chromaffin cells. Despite the central role of this synapse in regulating the acute stress response in the periphery, remarkably little is known about its molecular operation. Our current goals are to define the mechanisms of calcium-sensing in splanchnic neurons, and to learn how synaptic modulation and plasticity are regulated using stimulation paradigms that model stress. This work is complemented by multi-photon imaging of synaptic architecture and computer simulations, all performed with the goal of understanding how communication between splanchnic neurons and chromaffin cells is shaped by variations in sympathetic tone.
3D rendering of an adrenal chromaffin cell. Green and Red spots indicate positions of different populations of hormone-laden dense core granules.
In addition to the studies described above, we have a long-standing interest in the mechanisms of stimulus-secretion coupling in chromaffin cells, including those that couple activation of ionotropic and metabotropic (GPCRs) receptors to calcium influx and exocytosis. To interrogate these mechanisms, we utilize pharmacological and genetic approaches, in conjunction with high resolution imaging methods (e.g., conventional, polarization-based, and super-resolution TIRF microscopy). We expect these studies will provide a coherent molecular and physiological framework for understanding how presynaptic activity, receptor-coupled signaling pathways, and chromaffin cell exocytosis are mechanistically linked to regulate the stress response.
The molecular mechanisms underlying the secretory pathology of Type-2 Diabetes
Exocytosis of insulin secretory granules from pancreatic beta cells in the islets of Langerhans is required for control of blood glucose — both in basal conditions to maintain normoglycemia, and even more importantly, post-prandially when insulin exocytosis helps to limit excessive excursion of blood glucose into the diabetic range. Initial investigations into the mechanisms important for proper sorting, trafficking, and secretion of the insulin hormone have revealed that the formation and function of insulin storage granules depend critically on the regulation of cholesterol dynamics. Two proteins of particular interest to us are the ATP binding cassette proteins ABCG1 and ABCA1, which work to maintain appropriate levels of cholesterol in intracellular membranes. We have shown that genetic suppression of ABC protein expression severely disrupts both insulin sorting and granule fusion. Our work in this area has direct implications for not only type-2 diabetes, but a wider range of disorders associated with dysregulation of cholesterol dynamics.
Pancreatic beta cells expressing INSULIN C-peptide GFP undergoing glucose-stimulated insulin secretion. Movie captured on a tirf microscope.
A second line of investigation in beta cells concerns the role of the ubiquitous calcium sensors — the synaptotagmins — in regulating insulin hormone secretion. Some 17 different isoforms of synaptotagmin have been identified. Although roles for many of these proteins in synaptic terminals and neuroendocrine cells are established, their function in beta cells remains poorly described. We are investigating the role of the synaptotagmins in insulin secretion using a combination of genetically-modified cell lines as well as a novel transgenic animal in which endogenous insulin is fluorescently-tagged with GFP in the pancreatic islet.