Randi B. Silver

Cardiac mast cell–derived renin promotes local angiotensin formation, norepinephrine release, and arrhythmias in ischemia/reperfusion

Having identified renin in cardiac mast cells, we assessed whether its release leads to cardiac dysfunction. In Langendorff-perfused guinea pig hearts, mast cell degranulation with compound 48/80 released Ang I-forming activity. This activity was blocked by the selective renin inhibitor BILA2157, indicating that renin was responsible for Ang I formation. Local generation of cardiac Ang II from mast cell-derived renin also elicited norepinephrine release from isolated sympathetic nerve terminals. This action was mediated by Ang II-type 1 (AT1) receptors. In 2 models of ischemia/reperfusion using Langendorff-perfused guinea pig and mouse hearts, a significant coronary spillover of renin and norepinephrine was observed. In both models, this was accompanied by ventricular fibrillation. Mast cell stabilization with cromolyn or lodoxamide markedly reduced active renin overflow and attenuated both norepinephrine release and arrhythmias. Similar cardioprotection was observed in guinea pig hearts treated with BILA2157 or the AT1 receptor antagonist EXP3174. Renin overflow and arrhythmias in ischemia/reperfusion were much less prominent in hearts of mast cell-deficient mice than in control hearts. Thus, mast cell-derived renin is pivotal for activating a cardiac renin-angiotensin system leading to excessive norepinephrine release in ischemia/reperfusion. Mast cell-derived renin may be a useful therapeutic target for hyperadrenergic dysfunctions, such as arrhythmias, sudden cardiac death, myocardial ischemia, and congestive heart failure.

H+-K+-ATPases: regulation and role in pathophysiological states

Molecular cloning experiments have identified the existence of two H+-K+-ATPases (HKAs), colonic and gastric. Recent functional and molecular studies indicate the presence of both transporters in the kidney, which are presumed to mediate the exchange of intracellular H+ for extracellular K+. On the basis of these studies, a picture is evolving that indicates differential regulation of HKAs at the molecular level in acid-base and electrolyte disorders. Of the two transporters, gastric HKA is expressed constitutively along the length of the collecting duct and is responsible for H+ secretion and K+ reabsorption under normal conditions and may be stimulated with acid-base perturbations and/or K+ depletion. This regulation may be species specific. To date there are no data to indicate that the colonic HKA (HKAc) plays a role in H+ secretion or K+ reabsorption under normal conditions. However, HKAc shows adaptive regulation in pathophysiological conditions such as K+ depletion, NaCl deficiency, and proximal renal tubular acidosis, suggesting an important role for this exchanger in potassium, HCO-3, and sodium (or chloride) reabsorption in disease states. The purpose of this review is to summarize recent functional and molecular studies on the regulation of HKAs in physiological and pathophysiological states. Possible signals responsible for regulation of HKAs in these conditions will be discussed. Furthermore, the role of these transporters in acid-base and electrolyte homeostasis will be evaluated in the context of genetically altered animals deficient in HKAc.Molecular cloning experiments have identified the existence of two H+-K+-ATPases (HKAs), colonic and gastric. Recent functional and molecular studies indicate the presence of both transporters in the kidney, which are presumed to mediate the exchange of intracellular H+ for extracellular K+. On the basis of these studies, a picture is evolving that indicates differential regulation of HKAs at the molecular level in acid-base and electrolyte disorders. Of the two transporters, gastric HKA is expressed constitutively along the length of the collecting duct and is responsible for H+ secretion and K+ reabsorption under normal conditions and may be stimulated with acid-base perturbations and/or K+ depletion. This regulation may be species specific. To date there are no data to indicate that the colonic HKA (HKAc) plays a role in H+ secretion or K+ reabsorption under normal conditions. However, HKAc shows adaptive regulation in pathophysiological conditions such as K+ depletion, NaCl deficiency, and proximal renal tubular acidosis, suggesting an important role for this exchanger in potassium, [Formula: see text], and sodium (or chloride) reabsorption in disease states. The purpose of this review is to summarize recent functional and molecular studies on the regulation of HKAs in physiological and pathophysiological states. Possible signals responsible for regulation of HKAs in these conditions will be discussed. Furthermore, the role of these transporters in acid-base and electrolyte homeostasis will be evaluated in the context of genetically altered animals deficient in HKAc.

Suppression of miRNA-708 by Polycomb Group Promotes Metastases by Calcium-Induced Cell Migration

The progression of cancer to metastatic disease is a major cause of death. We identified miR-708 being transcriptionally repressed by polycomb repressor complex 2-induced H3K27 trimethylation in metastatic breast cancer. miR-708 targets the endoplasmic reticulum protein neuronatin to decrease intracellular calcium level, resulting in reduction of activation of ERK and FAK, decreased cell migration, and impaired metastases. Ectopic expression of neuronatin refractory to suppression by miR-708 rescued cell migration and metastasis defects. In patients with breast cancer, miR-708 expression was decreased in lymph node and distal metastases, suggesting a metastasis-suppressive role. Our findings uncover a mechanistic role for miR-708 in metastasis and provide a rationale for developing miR-708 as a therapeutic agent against metastatic breast cancer.

Decreased intracellular calcium mediates the histamine H3-receptor-induced attenuation of norepinephrine exocytosis from cardiac sympathetic nerve endings

Activation of presynatic histamine H3 receptors (H3R) down-regulates norepinephrine exocytosis from cardiac sympathetic nerve terminals, in both normal and ischemic conditions. Analogous to the effects of α2-adrenoceptors, which also act prejunctionally to inhibit norepinephrine release, H3R-mediated antiexocytotic effects could result from a decreased Ca2+ influx into nerve endings. We tested this hypothesis in sympathetic nerve terminals isolated from guinea pig heart (cardiac synaptosomes) and in a model human neuronal cell line (SH-SY5Y), which we stably transfected with human H3R cDNA (SH-SY5Y-H3). We found that reducing Ca2+ influx in response to membrane depolarization by inhibiting N-type Ca2+ channels with ω-conotoxin (ω-CTX) greatly attenuated the exocytosis of [3H]norepinephrine from both SH-SY5Y and SH-SY5Y-H3 cells, as well as the exocytosis of endogenous norepinephrine from cardiac synaptosomes. Similar to ω-CTX, activation of H3R with the selective H3R-agonist imetit also reduced both the rise in intracellular Ca2+ concentration (Cai) and norepinephrine exocytosis in response to membrane depolarization. The selective H3R antagonist thioperamide prevented this effect of imetit. In the parent SH-SY5Y cells lacking H3R, imetit affected neither the rise in Cai nor [3H]norepinephrine exocytosis, demonstrating that the presence of H3R is a prerequisite for a decrease in Cai in response to imetit and that H3R activation modulates norepinephrine exocytosis by limiting the magnitude of the increase in Cai. Inasmuch as excessive norepinephrine exocytosis is a leading cause of cardiac dysfunction and arrhythmias during acute myocardial ischemia, attenuation of norepinephrine release by H3R agonists may offer a novel therapeutic approach to this condition.

Aldehyde Dehydrogenase Activation Prevents Reperfusion Arrhythmias by Inhibiting Local Renin Release From Cardiac Mast Cells

BACKGROUND Renin released by ischemia/reperfusion from cardiac mast cells activates a local renin-angiotensin system (RAS). This exacerbates norepinephrine release and reperfusion arrhythmias (ventricular tachycardia and fibrillation), making RAS a new therapeutic target in myocardial ischemia. METHODS AND RESULTS We investigated whether ischemic preconditioning (IPC) prevents cardiac RAS activation in guinea pig hearts ex vivo. When ischemia/reperfusion (20 minutes of ischemia/30 minutes of reperfusion) was preceded by IPC (two 5-minute ischemia/reperfusion cycles), renin and norepinephrine release and ventricular tachycardia and fibrillation duration were markedly decreased, a cardioprotective anti-RAS effect. Activation and blockade of adenosine A(2b)/A(3) receptors and activation and inhibition of protein kinase Cepsilon (PKCepsilon) mimicked and prevented, respectively, the anti-RAS effects of IPC. Moreover, activation of A(2b)/A(3) receptors or activation of PKCepsilon prevented degranulation and renin release elicited by peroxide in cultured mast cells (HMC-1). Activation and inhibition of mitochondrial aldehyde dehydrogenase type-2 (ALDH2) also mimicked and prevented, respectively, the cardioprotective anti-RAS effects of IPC. Furthermore, ALDH2 activation inhibited degranulation and renin release by reactive aldehydes in HMC-1. Notably, PKCepsilon and ALDH2 were both activated by A(2b)/A(3) receptor stimulation in HMC-1, and PKCepsilon inhibition prevented ALDH2 activation. CONCLUSIONS The results uncover a signaling cascade initiated by A(2b)/A(3) receptors, which triggers PKCepsilon-mediated ALDH2 activation in cardiac mast cells, contributing to IPC-induced cardioprotection by preventing mast cell renin release and the dysfunctional consequences of local RAS activation. Thus, unlike classic IPC in which cardiac myocytes are the main target, cardiac mast cells are the critical site at which the cardioprotective anti-RAS effects of IPC develop.

Renin: at the heart of the mast cell

Summary:  Cardiac mast cells proliferate in cardiovascular diseases. In myocardial ischemia, mast cell mediators contribute to coronary vasoconstriction, arrhythmias, leukocyte recruitment, and tissue injury and repair. Arrhythmic dysfunction, coronary vasoconstriction, and contractile failure are also characteristic of cardiac anaphylaxis. In coronary atherosclerosis, mast cell mediators facilitate cholesterol accumulation and plaque destabilization. In cardiac failure, mast cell chymase causes myocyte apoptosis and fibroblast proliferation, leading to ventricular dysfunction. Chymase and tryptase also contribute to fibrosis in cardiomyopathies and myocarditis. In addition, mast cell tumor necrosis factor‐α promotes myocardial remodeling. Cardiac remodeling and hypertrophy in end‐stage hypertension are also induced by mast cell mediators and proteases. We recently discovered that cardiac mast cells contain and release renin, which initiates local angiotensin formation. Angiotensin causes coronary vasoconstriction, arrhythmias, fibrosis, apoptosis, and endothelin release, all demonstrated mechanisms of mast‐cell‐associated cardiac disease. The effects of angiotensin are further amplified by the release of norepinephrine from cardiac sympathetic nerves. Our discovery of renin in cardiac mast cells and its release in pathophysiological conditions uncovers an important new pathway in the development of mast‐cell‐associated heart diseases. Several steps in this novel pathway may constitute future therapeutic targets.

IL-10 Suppresses Calcium-Mediated Costimulation of Receptor Activator NF-κB Signaling during Human Osteoclast Differentiation by Inhibiting TREM-2 Expression

Induction of effective osteoclastogenesis by RANK (receptor activator of NF-κB) requires costimulation by ITAM-coupled receptors. In humans, the TREM-2 (triggering receptor expressed on myeloid cells 2) ITAM-coupled receptor plays a key role in bone remodeling, as patients with TREM-2 mutations exhibit defective osteoclastogenesis and bone lesions. We have identified a new rapidly induced costimulatory pathway for RANK signaling that is dependent on TREM-2 and mediated by calcium signaling. TREM-2-dependent calcium signals are required for RANK-mediated activation of calcium/calmodulin-dependent protein kinase (CaMK)II and downstream MEK and ERK MAPKs that are important for osteoclastogenesis. IL-10 inhibited RANK-induced osteoclastogenesis and selectively inhibited calcium signaling downstream of RANK by inhibiting transcription of TREM-2. Down-regulation of TREM-2 expression resulted in diminished RANKL-induced activation of the CaMK-MEK-ERK pathway and decreased expression of the master regulator of osteoclastogenesis NFATc1. These findings provide a new mechanism of inhibition of human osteoclast differentiation. The results also yield insights into crosstalk between ITAM-coupled receptors and heterologous receptors such as RANK, and they identify a mechanism by which IL-10 can suppress cellular responses to TNFR family members.

Coupling of histamine H3 receptors to neuronal Na+/H+ exchange: A novel protective mechanism in myocardial ischemia

In myocardial ischemia, adrenergic nerves release excessive amounts of norepinephrine (NE), causing dysfunction and arrhythmias. With anoxia and the concomitant ATP depletion, vesicular storage of NE is impaired, resulting in accumulation of free NE in the axoplasm of sympathetic nerves. Intraneuronal acidosis activates the Na+/H+ exchanger (NHE), leading to increased Na+ entry in the nerve terminals. These conditions favor availability of the NE transporter to the axoplasmic side of the membrane, causing massive carrier-mediated efflux of free NE. Neuronal NHE activation is pivotal in this process; NHE inhibitors attenuate carrier-mediated NE release. We previously reported that activation of histamine H3 receptors (H3R) on cardiac sympathetic nerves also reduces carrier-mediated NE release and alleviates arrhythmias. Thus, H3R activation may be negatively coupled to NHE. We tested this hypothesis in individual human SKNMC neuroblastoma cells stably transfected with H3R cDNA, loaded with the intracellular pH (pHi) indicator BCECF. These cells possess amiloride-sensitive NHE. NHE activity was measured as the rate of Na+-dependent pHi recovery in response to an acute acid pulse (NH4Cl). We found that the selective H3R-agonist imetit markedly diminished NHE activity, and so did the amiloride derivative EIPA. The selective H3R antagonist thioperamide abolished the imetit-induced NHE attenuation. Thus, our results provide a link between H3R and NHE, which may limit the excessive release of NE during protracted myocardial ischemia. Our previous and present findings uncover a novel mechanism of cardioprotection: NHE inhibition in cardiac adrenergic neurons as a means to prevent ischemic arrhythmias associated with carrier-mediated NE release.

RATIO IMAGING : PRACTICAL CONSIDERATIONS FOR MEASURING INTRACELLULAR CALCIUM AND PH IN LIVING TISSUE

Publisher Summary For the maintenance of steady state there must be mechanisms in place in the cell to ensure the appropriate balance between the intracellular and extracellular milieu. The study of intracellular ion concentrations in the steady-state and the role of various ions in regulating cellular processes was until recently limited to systems that could be impaled with a microelectrode. This chapter focuses on the measurement of two intracellular ions: (1) intracellular H+ (pH i ) and (2) Ca 2+ (Ca i ), at the single-cell level with a relatively noninvasive technique, quantitative ratio imaging. The development and availability of Ca i - and pH i -specific fluorescent probes coupled to major advances in the technology and design of low-light level charge-coupled devices geared toward biological applications and improved microscope optics has made it possible to visualize a two-dimensional (2-D) fluorescence signal—that is related to the free concentration of Ca and pH inside a cell. This chapter describes the basis for using the dual excitation ratio-imaging technique. The uncertainties associated with quantitating single-wavelength fluorescence imaging are apparent from the standard fluorescence equation as presented by Bright et al .

Mast cell renin and a local renin–angiotensin system in the airway: Role in bronchoconstriction

We previously reported that mast cells express renin, the rate-limiting enzyme in the renin–angiotensin cascade. We have now assessed whether mast cell renin release triggers angiotensin formation in the airway. In isolated rat bronchial rings, mast cell degranulation released enzyme with angiotensin I-forming activity blocked by the selective renin inhibitor BILA2157. Local generation of angiotensin (ANG II) from mast cell renin elicited bronchial smooth muscle contraction mediated by ANG II type 1 receptors (AT1R). In a guinea pig model of immediate type hypersensitivity, anaphylactic mast cell degranulation in bronchial rings resulted in ANG II-mediated constriction. As in rat bronchial rings, bronchoconstriction (BC) was inhibited by a renin inhibitor, an AT1R blocker, and a mast cell stabilizer. Anaphylactic release of renin, histamine, and β-hexosaminidase from mast cells was confirmed in the effluent from isolated, perfused guinea pig lung. To relate the significance of this finding to humans, mast cells were isolated from macroscopically normal human lung waste tissue specimens. Sequence analysis of human lung mast cell RNA showed 100% homology between human lung mast cell renin and kidney renin between exons 1 and 10. Furthermore, the renin protein expressed in lung mast cells was enzymatically active. Our results demonstrate the existence of an airway renin–angiotensin system triggered by release of mast-cell renin. The data show that locally produced ANG II is a critical factor governing BC, opening the possibility for novel therapeutic targets in the management of airway disease.