David R. Giovannucci, Ph.D.
Professor
Director, Raymond & Beverly Sackler Laboratory for Neuroendocrine Tumor Research
Office: 105 Block Health Sciences Building
Tel: 419-383-5004
Lab: 419-383-4023
Email: david.giovannucci@utoledo.edu
Education:
1993: Ph.D., Wayne State University, Detroit, MI.
1998: Postdoctoral Fellow, University of Michigan Medical School, Ann Arbor, MI
2002: Research Assistant Professor, University of Rochester Medical Center, Rochester,
NY.
Research Interests:
Mechanisms of neurotransmitter, neuropeptide and protein secretion; calcium signaling
mechanisms; Neural control of peripheral organs. Work in our lab is focused on defining
the cellular and molecular mechanisms that underlie the cytosolic calcium signals
that control peptide and protein secretion. The exocytotic secretion of neuropeptide
has profound consequences for neuronal function, cardiovascular homeostasis and GI
tract regulation in health and disease. Our lab is also advancing the use of biomarkers
in saliva as indicators of human performance or pathophysiology. This work has significance
for human health concerns such as GI cancer, hypertension and dry mouth. Work in our
lab has been supported by government, military and private funding.
Research Techniques:
Electrophysiology; Confocal and multiphoton microscopy; Molecular biology and protein
biochemistry.
Research Summary:
1. Calcium-dependence, activity-dependence and phosphoregulation of vasoactive peptide
exocytosis. My contributions in this area began with several studies (over 200 total
citations in the literature) that I published as a postdoc in Edward Stuenkel’s laboratory
at the University of Michigan. By exploiting the unique architecture of the rat hypothalamo-neurohypophyseal
axis we were able to make pure preparations of neuroendocrine nerve terminals without
contamination by post-synaptic membrane. Many of these terminals were large enough
to patch clamp thus allowing electrophysiological assessment of secretory activity
at the single nerve terminal level using time-resolved membrane capacitance (Cm) measurements
to probe the kinetic fine structure of calcium-dependent secretory activity and functionally
define the roles of secretory SNARE proteins in this process. In addition to our work
on nerve endings, we were also one of two groups to directly demonstrate patterns
of action potentials could induce somatodendritic secretory activity in cell bodies
of supraoptic nucleus neurons. In another study using rat adrenal chromaffin cells,
I made the novel observation that mitochondria can control exocytotic activity by
both responding to and shaping intracellular calcium signals. We have continued these
studies to probe the relationship to patterns of electrical activity and clarified
how patterns of action potential bursting optimally tuned secretory output. For example,
in collaboration with Dr. Arun Anantharam at University of Michigan we are probing
the calcium-dependent regulation of catecholamine and neuropeptide exocytosis mediated
by synaptotagmin-1 and synaptotagmin-7. These two isoforms appear to differentially
regulate the release of catecholamine or neuropeptide from the adrenal gland. During
this time it became apparent through the work of many others that the molecular players
(eg. SNARE and SNARE-associated proteins) that control the release of neurotransmitter
were not necessarily unique to neurons. Thus, we broadened our studies, taking advantage
of our expertise in the study of calcium dependent release mechanisms and began to
investigate the exocytotic release of atrial natriuretic peptide (ANP) from a subset
of cardiomyocytes that comprise the endocrine heart. ANP is an important vasoactive
peptide that acts as a counter-balance to the renin-angiotensin system and vasopressin
signaling. These observations were published in 2006 in the Journal of Cellular and
Molecular Cardiology. Using single cell physiological approaches, immunoassays and
protein biochemistry, we defined the mechanistic basis for ouabain-induced exocytosis.
We demonstrated that ouabain treatment induced Src-dependent tyrosine phosphorylation
of the exocytotic calcium-sensor protein synaptotagmin-1. The consequence of this
signaling was to increase the Ca2+ affinity of this normally low affinity sensor such
that the exocytotic process could respond to beat-to-beat cardiomyocyte calcium dynamics.
These observations have significance for dysregulation of blood volume homeostasis
and heart failure. In addition, our data point to a broader significance and novel
aspect of phosphoregulation of exocytosis.
a. Giovannucci DR & Stuenkel EL. (1997) Regulation of secretory granule recruitment and exocytosis at rat neurohypophysial nerve endings. Journal of Physiology 498.3, 735-751.
b. Giovannucci DR, Hlubek MD, & Stuenkel EL. (1999) Mitochondria Regulate the Ca2+-exocytosis Relationship of Bovine Adrenal Chromaffin Cells. Journal of Neuroscience 19(21): 9261-9270.
c. Soldo BL*, Giovannucci DR*, Stuenkel EL, Moises HC. (2004) Ca2+ and frequency dependence of exocytosis in isolated somata of magnocellular supraoptic neurones of the rat hypothalamus. Journal of Physiology. 16;555(Pt 3):699-711.
d. Peters CG, Miller DF, Giovannucci DR. (2006) Identification, localization and interaction of SNARE proteins in atrial cardiac myocytes, Journal of Molecular and Cellular Cardiology 40(3): 361-374.
e. Rao TC, Santana Rodriguez Z, Bradberry MM, Ranski AH, Dahl PJ, Schmidtke MW, Jenkins PM, Axelrod D, Chapman ER, Giovannucci DR, Anantharam A. Synaptotagmin isoforms confer distinct activation kinetics and dynamics to chromaffin cell granules. J Gen Physiol. (2017), published ahead of print.
2. Calcium signaling and exocrine gland secretion. Over the years I have had a long-standing and productive collaboration with Dr. David Yule at the University of Rochester Medical Center. We published a series of papers on the compartmentalization of calcium signaling and phosphoregulation controlling fluid secretion in pancreatic and parotid acinar cells. My laboratory was one of the first to apply novel optical techniques like time-differentiated imaging and multi-photon microscopy to assess exocytotic activity in and to develop an organotypic slice preparation of the salivary gland. This preparation represented a significant step forward in the repertoire of tools available to study salivary gland because primary cultures of enzymatically-dispersed acini rapidly lose their morphological and functional characteristics over several hours. Subsequently, I extended our studies to assess the roles of calcium entry pathways and of non-canonical intracellular calcium release pathways in the control of salivation in health and in salivary hypofunction diseases. This approach has focused on alternative purinergic and pyrimidinergic signaling pathways for generating calcium signals in salivary gland cells to identify new potential targets for augmenting or restoring function for patients suffering reduced saliva formation. Our lab demonstrated that activation of ATP-gated ionotropic P2X receptors can evoke protein exocytosis and salt secretion in parotid glands via influx of extracellular calcium or through calcium induced calcium release from the endoplasmic reticulum. In our 2012 Journal of Physiology paper and a 2015 follow-up paper exploring signaling crosstalk, we functionally characterized P2X4 and P2X7 receptors and our findings suggested that, although both channel types could contribute to acinar cell calcium increases and exocytosis, the P2X4 receptor was a more attractive, drugable target for augmentation therapy. My lab is also clarifying the role of a novel intracellular calcium release pathway in salivation. In collaboration with Drs. James Slama and Katherine Wall in the Department of Medicinal Chemistry in the School of Pharmacy I have begun to define the role of nicotinic acid adenine dinucleotide 2’-phosphate (NAADP), a centrally important but here-to-fore enigmatic intracellular messenger and highly potent factor that mobilizes calcium from acidic stores to regulate complex calcium signals and diverse physiological endpoints. Using pharmacological approaches and photorelease of caged NAADP analogs, we showed that this pathway is important in mouse salivary gland following activation of sympathetic input and demonstrated that the dominant acidic store for NAADP-mediated Ca2+ release are the secretory granules. These findings indicated that NAADP mediated signals played an important role in coupling autonomic input to generate calcium signals critical for driving salt and fluid secretion. Although current therapeutic sialogogues can override the reduced sensitivity to salivation in patients, these drugs are not without systemic effects. In contrast, restorative therapy at the level of the NAADP pathway has the advantage of improving sensitivity to endogenous neural input to augment function. It is envisioned that small molecule/genetic therapies that leverage NAADP signaling could be used to improve function where parasympathetic input is diminished. In addition, we have addressed this pathway in murine T cells and human cancer cell lines using a variety of biochemical and functional techniques.
a. Giovannucci DR, Bruce JIE, Straub SV, Arreola J, Shuttleworth TJ, Yule DI. Cytosolic Ca2+ and Ca2+- activated Cl- current dynamics: insights from two functionally distinct exocrine cells. Journal of Physiology (2002) 540.2: 469-484.
b. Chen Y, Warner JD, Yule DI, Giovannucci DR. (2005) Spatiotemporal Analysis of Exocytosis in Mouse Parotid Acinar Cells. American Journal of Physiology (Cell Physiology) 289(5): C1209-19.
c. Warner JD, Peters CG, Saunders R, Wong J-H, Betzenhauser MJ, Gunning III WT, Yule DI & Giovannucci DR. (2008) Visualizing form and function in organotypic slices of the adult mouse parotid gland. American Journal of Physiology (Gastrointestinal and Liver Physiology), 295(3):G629-40.
d. Bhattacharya S, Verrill DS, Carbone KM, Brown S, Yule DI, Giovannucci DR. Distinct contributions by ionotropic purinoceptor subtypes to ATP-evoked calcium signals in mouse parotid acinar cells. J Physiol. (2012) 590(Pt 11):2721-37.
e. Bhattacharya S, Imbery JF, Ampem PT, Giovannucci DR. Crosstalk between purinergic receptors and canonical signaling pathways in the mouse salivary gland. Cell Calcium (2015), 58(6):589-97.
f. Imbery JF, Bhattacharya S, Khuder S, Weiss A, Goswamee P, Iqbal AK, Giovannucci DR. cAMP-dependent recruitment of acidic organelles for Ca2+ signaling in the salivary gland Am J Physiol Cell Physiol. (2016); 311(5):C697-C709.
g. Ali RA, Zhelay T, Trabbic CJ, Walseth TF, Slama JT, Giovannucci DR**, Wall KA**. Activity of nicotinic acid substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs in a human cell line: difference in specificity between human and sea urchin NAADP receptors. Cell Calcium (2014) 55(2):93-103.
h. Ali RA, Camick C, Wiles K, Walseth TF, Slama JT, Bhattacharya S, Giovannucci DR, Wall KA. Nicotinic acid adenine dinucleotide phosphate plays a critical role in naive and effector murine T cells but not natural regulatory T cells. Journal of Biological Chemistry (2016) 291(9):4503-22.
3. Salivary biomarkers of performance. Following the designation of the UT Health Science Campus as a center of excellence for biomarker research and personalized medicine by the State of Ohio and our expertise in salivary gland biology, we embarked on studies to identify salivary biomarkers that correlate with stress, neural trauma and cognitive performance. In humans, three major paired, highly vascularized salivary glands constitutively or actively secrete fluid and protein components of saliva. Specialized cells that take up water, salts and molecules from the blood produce the fluid component of saliva. Although distinct from blood plasma, saliva can mirror the levels of substances in the blood, including nutrients, metabolites, inflammatory cytokines and hormones, as well as non-biological compounds such as metals or drugs. These cells also secrete saliva-specific proteins that combine with the fluid to make a complex “cocktail” that is important for digestion, oral health and speech. Furthermore, the salivary gland is under autonomic neural control that controls fluid and protein secretion. Thus saliva output and composition reflects sympathetic and parasympathetic tone. This places saliva not only as an ideal bio-fluid to assay biomarkers for assessing normal physiological homeostatic mechanisms, but also as a critical diagnostic fluid for a variety of pathologies including brain trauma and cancer. As lead principal investigator on a multi-PI grant, I brought together a collaborative team of experts using proteomic approaches to identify and validate biomarkers that can assess levels of neurotrauma, stress, fatigue and performance degradation and that can serve as targets for the in vitro selection and characterization of high-affinity aptamer ligands for the development of a biomarker-based sensors. This work has the potential to significantly enhance our ability to assess human health and performance in medical and aerospace environments.
Marvin RK, Saepoo MB, Ye S, White DB, Liu R, Hensley K, Rega P, Kazan V, Giovannucci DR, Isailovic D. (2017) Salivary protein changes in response to acute stress in medical residents performing advanced clinical simulations: a pilot proteomics study. Biomarkers: Jun;22(3-4):372-382.
Complete List of Published Work in My Bibliography: