Calcium ions play essential roles in most cellular activities, including fertilization, cell division, motility and contraction, excitability and secretion. Moreover, altered Ca2+ regulation and signaling play central roles in many pathological conditions. My current research concerns the regulation of the intracellular Ca2+ concentration and its role in normal and pathological cell signaling in vascular smooth muscle, with a focus on the pathogenesis of salt-dependent hypertension.
The sarcoplasmic/endoplasmic reticulum (S/ER) accumulates and stores Ca2+ for subsequent release as "signal Ca2+". We identified a "signaling complex" region, termed the "PlasmERosome", that regulates Ca2+ storage and signaling. The PLasmERosome consists of three main elements: certain plasma membrane (PM) microdomains, the adjacent "junctional" S/ER (jS/ER), and the tiny pocket of cytosol between the PM and jS/ER. Ca2+ is regulated within this cytosolic region by specific ion channels, transporter isoforms and receptors contained in the PM microdomains. This, in turn, regulates Ca2+ storage and Ca2+ release (i.e., the Ca2+ signals) from the S/ER. We are identifying the component transporters within these complexes and determining how the complexes are organized and how they influence local and global Ca2+ concentrations and signaling. We employ a variety of molecular and cellular biological methods, including digital imaging. Several transgenic mouse lines are used to study cardiovascular parameters in intact animals, and the properties of isolated small arteries, individual myocytes, and cultured myocytes. Our findings are unraveling the molecular links between salt and hypertension.
Specific questions currently being addressed include:
How do mutations in the human alpha-2 and alpha-3 Na+ pumps explain, respectively, the manifestations of familial hemiplegic migraine and rapid onset dystonia with parkinsonism? The native alpha-2 Na+ pumps in primary cultured human and rodent arterial myocytes are being replaced (via transfection) by pumps bearing the human mutations. Digital imaging will be used to determine how cytosolic Na+ and Ca2+ concentrations and Ca2+ storage are altered in the transfected cells.
How does Na+ pump inhibition influence intracellular Ca2+ storage and cell signaling? Low dose ouabain (Na+ pump inhibitor), or knock-out of specific Na+ pump catalytic subunit isoforms, enhance Ca2+ signaling in most cells [Ref. 4, 7], and augment arterial vasoconstriction [Ref. 4]. Novel near-membrane ion-sensitive dyes such as the Na+ pump alpha subunit-conjugated Ca2+-sensitive protein, "G-CaMP" [Ref. 7] and Total Internal Reflective Fluorescence (TIRF) methods are being used to determine the Na+ and Ca2+ concentrations in the sub-PM cytoplasmic compartment between the PM and theS/ER. We are testing the hypothesis that these local ion concentration changes play a critical role in regulating Ca2+ signaling in vascular smooth muscle cells.
How does Ca2+ signaling regulate myogenic tone in small arteries?And, how does this control long-term blood pressure? Our goal is to understand how the mechanisms mentioned above operate in intact preparations. Ca2+ signaling within intact small arteries is being investigated with "real time" confocal microscopy and simultaneous diameter measurement [Refs. 1, 3, 7]. Arteries from normal rats and mice, and from transgenic mice with reduced Na+ pump activity, or reduced or increased Na/Ca exchanger activity, are being studied. Blood pressure and cardiac output are measured in intact, free-moving mice. Our findings are providing novel insight into the molecular mechanisms that link salt retention to high blood pressure [Ref. 1, 4, 5].
Lab Techniques and Equipment:
The biological preparations we use include: intact rats and mice, isolated small arteries,freshly isolated or cultured arterial smooth muscle cells, primary cultured neurons and glial cells, and neuronal slice cultures. Transgenic animals (e.g., those with knock-out or overexpression of specific Na+ and Ca2+ transporters) as well as normal animals are used.
The techniques we employ include: application of molecular and cell biological methods (e.g., immunoblotting, immunoprecipitation, PCR, and construction of DNAs, adenoviral vectors and anti-sense probes or silencer RNA, patch clamping, and high-resolution digital (fluorescence) microscopy. The latter is used for immunocytochemical detection of ion transporters, and for ion concentration monitoring with ion-sensitive dyes (including novel Ca2+ sensors introduced by transfection). Microscopy methods include standard wide-field and confocal fluorescence, 3-D image reconstruction, and analysis of near-membrane phenomena with lipophilic cation-sensitive indicators, targeted Ca2+-sensitive fluorescent proteins and TIRF. Hemodynamics is studied with telemetry in intact, conscious animals and with echocardiography in lightly anesthetized mice.
Training and Lab Personnel:
Exciting opportunities are available for pre- and post-doctoral fellows to characterize, at the molecular, cellular, tissue and whole organism levels, the fundamental mechanisms that link salt to hypertension. Trainees will work with the group that first identified this novel pathway. These studies entail the use of state-of-the-art digital imaging, patch clamp electrophysiology, molecular biology and whole animal (mouse) hemodynamic methods.
Zhang, J., J.M. Hamlyn, E. Karashima, H. Raina, J.R.H. Mauban, M. Izuka, R. Berra-Romani, A. Zulian, W.G. Wier, and M.P. Blaustein. Low Dose Ouabain Constricts Small Arteries from Ouabain-Hypertensive Rats: Implications for Sustained Elevation of Vascular Resistance. Am. J. Physiol., Heart Circ. Physiol. 297:H1140-H1150, (2009). PMCID: PMC2755988.