Characterization of constitutive and acid-induced outwardly rectifying chloride currents in immortalized mouse distal tubular cells

Thiazides block Na+ reabsorption while enhancing Ca2 + reabsorption in the kidney. As previously demonstrated in immortalized mouse DCT (MDCT) cells, chlorothiazide application induced a robust plasma membrane hyperpolarization, which increased Ca2 + uptake. This essential thiazide-induced hyperpolarization was prevented by the Cl− channel inhibitor 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), implicating NPPB-sensitive Cl− channels, however the nature of these Cl− channels has been rarely described in the literature. Here we show that MDCT cells express a dominant, outwardly rectifying Cl− current at extracellular pH 7.4. This constitutive Cl− current was more permeable to larger anions (Eisenman sequence I; I− > Br− ≥ Cl−) and was substantially inhibited by > 100 mM [Ca2 +]o, which distinguished it from ClC-K2/Barttin. Moreover, the constitutive Cl− current was blocked by NPPB, along with other Cl− channel inhibitors (DIDS, FFA). Subjecting the MDCT cells to an acidic extracellular solution (pH < 5.5) induced a substantially larger outwardly rectifying NPPB-sensitive Cl− current. This acid-induced Cl− current was also anion permeable (I− > Br− > Cl−), but was distinguished from the constitutive Cl− current by its rectification characteristics, ion sensitivities, and response to FFA. In addition, we have identified similar outwardly rectifying and acid-sensitive currents in immortalized cells from the inner medullary collecting duct (mIMCD-3 cells). Expression of an acid-induced Cl− current would be particularly relevant in the acidic IMCD (pH < 5.5). To our knowledge, the properties of these Cl− currents are unique and provide the mechanisms to account for the Cl− efflux previously speculated to be present in MDCT cells

New Methods for the Analysis of Heartbeat Behavior in Risk Stratification

Developing better methods for risk stratification for tachyarrhythmic sudden cardiac remains a major challenge for physicians and scientists. Since the transition from sinus rhythm to ventricular tachycardia/fibrillation happens by different mechanisms in different people, it is unrealistic to think that a single measure will be adequate to provide a good index for risk stratification. We analyze the dynamical properties of ventricular premature complexes over 24 h in an effort to understand the underlying mechanisms of ventricular arrhythmias and to better understand the arrhythmias that occur in individual patients. Two dimensional density plots, called heartprints, correlate characteristic features of the dynamics of premature ventricular complexes and the sinus rate. Heartprints show distinctive characteristics in individual patients. Based on a better understanding of the natures of transitions from sinus rhythm to sudden cardiac and the mechanisms of arrhythmia prior to cardiac arrest, it should be possible to develop better methods for risk stratification

Aldosterone upregulates transient receptor potential melastatin 7 (TRPM7)

Transient receptor potential melastatin 7 (TRPM7) is a ubiquitously expressed Mg2+-permeable ion channel fused to a C-terminal α-kinase domain. Recently, aldosterone was shown to increase intracellular Mg2+ levels and alter inflammatory signaling in TRPM7-expressing HEK293 cells. This study was undertaken to assess whether these effects were related to an aldosterone-mediated increase of TRPM7 current and/or plasma membrane localization. Using HEK293 cells stably expressing WT-TRPM7, we found that 18-h application of aldosterone significantly increased TRPM7 current and TRPM7 plasma membrane protein expression by 48% and 34%, respectively. The aldosterone-mediated increase of TRPM7 current was inhibited by eplerenone, a mineralocorticoid receptor (MR) blocker, and GSK-650394, an inhibitor of the serum- and glucocorticoid-regulated kinase 1 (SGK1). SGK1 blockade also prevented the aldosterone-induced increase of TRPM7 plasma membrane protein. It was further determined that K1648R-TRPM7, the phosphotransferase-inactive TRPM7 mutant, was unresponsive to aldosterone. Therefore, chronic aldosterone treatment increases the plasma membrane expression of TRPM7, which is associated with an increase of TRPM7 current. This process occurs via an MR-dependent, genomic signaling cascade involving SGK1 and a functioning TRPM7 α-kinase domain. We suggest that this mechanism may be of general relevance when interpreting the effects of aldosterone because the MR receptor is found in multiple tissues, and TRPM7 and SGK1 are ubiquitously expressed

Subcellular localization of the Na ϩ /H ϩ exchanger NHE1 in rat myocardium

localization of the Na ϩ /H ϩ exchanger NHE1 in rat myocardium. Am. J. Physiol. 276 (Heart Circ. Physiol. 45): H709-H717, 1999.-The Na ϩ /H ϩ exchanger NHE1 isoform is an integral component of cardiac intracellular pH homeostasis that is critically important for myocardial contractility. To gain further insight into its physiological significance, we determined its cellular distribution in adult rat heart by using immunohistochemistry and confocal microscopy. NHE1 was localized predominantly at the intercalated disk regions in close proximity to the gap junction protein connexin 43 of atrial and ventricular muscle cells. Significant labeling of NHE1 was also observed along the transverse tubular systems, but not the lateral sarcolemmal membranes, of both cell types. In contrast, the Na ϩ -K ϩ -ATPase ␣ 1 -subunit was readily labeled by a specific mouse monoclonal antibody (McK1) along the entire ventricular sarcolemma and intercalated disks and, to a lesser extent, in the transverse tubules. These results indicate that NHE1 has a distinct distribution in heart and may fulfill specialized roles by selectively regulating the pH microenvironment of pH-sensitive proteins at the intercalated disks (e.g., connexin 43) and near the cytosolic surface of sarcoplasmic reticulum cisternae (

Transient receptor potential melastatin 7 cation channel kinase new player in angiotensin II–induced hypertension

Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg2+)/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase–deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg2+ was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (P<0.05). Acetylcholine-induced vasorelaxation was reduced in Ang II–infused TRPM7Δkinase mice, an effect associated with Akt and endothelial nitric oxide synthase downregulation. Vascular cell adhesion molecule–1 expression was increased in Ang II–infused TRPM7 kinase–deficient mice. TRPM7 kinase targets, calpain, and annexin-1, were activated by Ang II in WT but not in TRPM7Δkinase mice. Echocardiographic and histopathologic analysis demonstrated cardiac hypertrophy and left ventricular dysfunction in Ang II–treated groups. In TRPM7 kinase–deficient mice, Ang II–induced cardiac functional and structural effects were amplified compared with WT counterparts. Our data demonstrate that in TRPM7Δkinase mice, Ang II–induced hypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II–induced hypertension and associated vascular and target organ damage

Aldosterone signaling through transient receptor potential melastatin 7 cation channel (TRPM7) and its α-kinase domain

We demonstrated a role for the Mg2 + transporter TRPM7, a bifunctional protein with channel and α-kinase domains, in aldosterone signaling. Molecular mechanisms underlying this are elusive. Here we investigated the function of TRPM7 and its α-kinase domain on Mg2 + and pro-inflammatory signaling by aldosterone. Kidney cells (HEK-293) expressing wild-type human TRPM7 (WThTRPM7) or constructs in which the α-kinase domain was deleted (ΔKinase) or rendered inactive with a point mutation in the ATP binding site of the α-kinase domain (K1648R) were studied. Aldosterone rapidly increased [Mg2 +]i and stimulated NADPH oxidase-derived generation of reactive oxygen species (ROS) in WT hTRPM7 and TRPM7 kinase dead mutant cells. Translocation of annexin-1 and calpain-II and spectrin cleavage (calpain target) were increased by aldosterone in WT hTRPM7 cells but not in α-kinase-deficient cells. Aldosterone stimulated phosphorylation of MAP kinases and increased expression of pro-inflammatory mediators ICAM-1, Cox-2 and PAI-1 in Δkinase and K1648R cells, effects that were inhibited by eplerenone (mineralocorticoid receptor (MR) blocker). 2-APB, a TRPM7 channel inhibitor, abrogated aldosterone-induced Mg2 + responses in WT hTRPM7 and mutant cells. In 2-APB-treated ΔKinase and K1648R cells, aldosterone-stimulated inflammatory responses were unchanged. These data indicate that aldosterone stimulates Mg2 + influx and ROS production in a TRPM7-sensitive, kinase-insensitive manner, whereas activation of annexin-1 requires the TRPM7 kinase domain. Moreover TRPM7 α-kinase modulates inflammatory signaling by aldosterone in a TRPM7 channel/Mg2 +-independent manner. Our findings identify novel mechanisms for non-genomic actions of aldosterone involving differential signaling through MR-activated TRPM7 channel and α-kinase

Spatial distribution of nerve processes and β-adrenoreceptors in the rat atrioventricular node

Atrioventricular (AV) nodal conduction time is known to be modulated by the autonomic nervous system. The presence of numerous parasympathetic and sympathetic nerve fibres in association with conduction tissue in the heart is well authenticated. In this study, confocal microscopy was used to image the distribution of antibodies directed against the general neuronal marker PGP 9.5, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and β(1) and β(2)-adrenoreceptors. Serial 12 μm sections of fresh frozen tissue taken from the frontal plane of the rat atrioventricular node, His bundle and bundle branches were processed for histology, acetylcholinesterase (AChE) activity and immunohistochemistry. It was found that the AV and ventricular conduction systems were more densely innervated than the atrial and ventricular myocardium as revealed by PGP 9.5 immunoreactivity. Furthermore, the transitional cell region was more densely innervated than the midnodal cell region, while spatial distribution of total innervation was uniform throughout all AV nodal regions. AChE-reactive nerve processes were found throughout the AV and ventricular conduction systems, the spatial distribution of which was nonuniform exhibiting a paucity of AChE-reactive nerve processes in the central midnodal cell region and a preponderance in the circumferential transitional cell region. TH-immunoreactivity was uniformly distributed throughout the AV and ventricular conduction systems including the central midnodal and circumferential transitional cell regions. β(1)-adrenoreceptors were found throughout the AV and ventricular conduction systems with a preponderance in the circumferential transitional cell region. β(2)-adrenoreceptors were localised predominantly in AV and ventricular conduction systems with a paucity of expression in the circumferential transitional cell region. These results demonstrate that the overall uniform distribution of total nerve processes is comprised of nonuniformly distributed subpopulations of parasympathetic and sympathetic nerve processes. The observation that the midnodal cell region exhibits a differential spatial pattern of parasympathetic and sympathetic innervation suggests multiple sites for modulation of impulse conduction within this region. Moreover, the localisation of β(2)-ARs in the AV conduction system, with an absence of expression in the circumferential transitional cell layer, suggests that subtype-specific pharmacological agents may have distinct effects upon AV nodal conduction