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Item Isolation and Cannulation of Cerebral Parenchymal Arterioles(2016) Pires, Paulo W.; Dabertrand, Fabrice; Earley, ScottIntracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves - innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.Item Novel Biomarkers and Treatments of Cardiac Diseases(2016) Zhu, Hua; Han, Renzhi; Duan, Dayue D.Cardiac diseases are mainly caused by malfunction of or injuries to the hearts. Although significant advances have been made during the past decades to improve the successful rate of treatments of these diseases, they still remain the top leading cause of morbidity and mortality in the world. Currently, cardiac diseases are defined according to the traditional system- or organ-based classification and the identification of diagnostic and therapeutic biomarkers has been focused on the heart. Therefore, the “golden standard” biomarkers are mainly cardiac muscle-related. For example, the conventional troponin (cTn) has been widely utilized for the diagnosis of acute myocardial infarction in the clinics. However, multiple limitations exist with clinical applications of these types of biomarkers. The ELISA based cTn detection assay is time consuming, and the dynamics of the biomarkers are not sensitive enough to represent the development of the diseases. Clearly, there is an urgent need for identifying sensitive, specific biomarkers of different types of cardiac diseases and development of new therapies for this unmet medical need. In this special issue, we have assembled a series of articles of reviews, perspectives, and original contributions from experts in current research of novel biomarkers and treatments of cardiac diseases in both basic research and clinical practice.Item Mechanisms underlying prorenin actions on hypothalamic neurons implicated in cardiometabolic control(2016) Pitra, Soledad; Feng, Yumei; Stern, Javier E.Hypertension and obesity are highly interrelated diseases, being critical components of the metabolic syndrome. Despite the growing prevalence of this syndrome in the world population, efficient therapies are still missing. Thus, identification of novel targets and therapies are warranted. An enhanced activity of the hypothalamic renin-angiotensin system (RAS), including the recently discovered prorenin (PR) and its receptor (PRR), has been implicated as a common mechanism underlying aberrant sympatho-humoral activation that contributes to both metabolic and cardiovascular dysregulation in the metabolic syndrome. Still, the identification of precise neuronal targets, cellular mechanisms and signaling pathways underlying PR/PRR actions in cardiovascular-and metabolic related hypothalamic nuclei remain unknown. Methods and results: Using a multidisciplinary approach including patch-clamp electrophysiology, live calcium imaging and immunohistochemistry, we aimed to elucidate cellular mechanisms underlying PR/PRR actions within the hypothalamic supraoptic (SON) and paraventricular nucleus (PVN), key brain areas previously involved in cardiometabolic regulation. We show for the first time that PRR is expressed in magno-cellular neurosecretory cells (MNCs), and to a lesser extent, in presympathetic PVN neurons (PVNPS). Moreover, we show that while PRR activation efficiently stimulates the firing activity of both MNCs and PVNPS neurons, these effects involved AngII-independent and AngII-dependent mechanisms, respectively. In both cases however, PR excitatory effects involved an increase in intracellular Ca2+ levels and a Ca2+-dependent inhibition of a voltage-gated K+ current. Conclusions: We identified novel neuronal targets and cellular mechanisms underlying PR/PRR actions in critical hypothalamic neurons involved in cardiometabolic regulation. This fundamental mechanistic information regarding central PR/PRR actions is essential for the development of novel RAS-based therapeutic targets for the treatment of cardiometabolic disorders in obesity and hypertension. (C) 2016 The Author(s). Published by Elsevier GmbH.Item Mechanisms Restricting Diffusion of Intracellular cAMP(2016) Agarwal, Shailesh R.; Clancy, Colleen E.; Harvey, Robert D.Although numerous receptors stimulate cAMP production in a wide array of cells, many elicit distinct, highly localized responses, implying that the subcellular distribution of cAMP is not uniform. One often used explanation is that phosphodiesterases, which breakdown cAMP, act as functional barriers limiting diffusion. However, several studies refute the notion that this is sufficient, suggesting that phosphodiesterase-independent movement of cAMP must occur at rates slower than free diffusion. But, until now this has never been demonstrated. Using Raster Image Correlation Spectroscopy (RICS), we measured the diffusion coefficient of a fluorescently-labeled cAMP derivative (phi 450-cAMP) as well as other fluorescent molecules in order to investigate the role that molecular size, cell morphology, and buffering by protein kinase A (PKA) play in restricting cAMP mobility in different cell types. Our results demonstrate that cytosolic movement of cAMP is indeed much slower than the rate of free diffusion and that interactions with PKA, especially type II PKA associated with mitochondria, play a significant role. These findings have important implications with respect to cAMP signaling in all cells.Item The critical role of the central nervous system (pro)renin receptor in regulating systemic blood pressure(2016) Xu, Quanbin; Jensen, Dane D.; Peng, Hua; Feng, YumeiThe systemic renin-angiotensin system (RAS) has long been recognized as a critically important system in blood pressure (BP) regulation. However, extensive evidence has shown that a majority of RAS components are also present in many tissues and play indispensable roles in BP regulation. Here, we review evidence that RAS components, notably including the newly identified (pro)renin receptor (PRR), are present in the brain and are essential for the central regulation of BP. Binding of the PRR to its ligand, prorenin or renin, increases BP and promotes progression of cardiovascular diseases in an angiotensin II-dependent and -independent manner, establishing the PRR a promising antihypertensive drug target. We also review the existing PRR blockers, including handle region peptide and PRO20, and propose a rationale for blocking prorenin/PRR activation as a therapeutic approach that does not affect the actions of the PRR in vacuolar H+-ATPase and development. Finally, we summarize categories of currently available antihypertensive drugs and consider future perspectives. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license.