A Simulation Study on the Pacing and Driving of the Biological Pacemaker.

The research on the biological pacemaker has been very active in recent years. And turning nonautomatic ventricular cells into pacemaking cells is believed to hold the key to making a biological pacemaker. In the study, the inward-rectifier K+ current (I K1) is depressed to induce the automaticity of the ventricular myocyte, and then, the effects of the other membrane ion currents on the automaticity are analyzed. It is discovered that the L-type calcium current (I CaL) plays a major part in the rapid depolarization of the action potential (AP). A small enough I CaL would lead to the failure of the automaticity of the ventricular myocyte. Meanwhile, the background sodium current (I bNa), the background calcium current (I bCa), and the Na+/Ca2+ exchanger current (I NaCa) contribute significantly to the slow depolarization, indicating that these currents are the main supplementary power of the pacing induced by depressing I K1, while in the 2D simulation, we find that the weak electrical coupling plays a more important role in the driving of a biological pacemaker.

Physiology of Electrolyte Transport in the Gut: Implications for Disease.

We now have an increased understanding of the genetics, cell biology, and physiology of electrolyte transport processes in the mammalian intestine, due to the availability of sophisticated methodologies ranging from genome wide association studies to CRISPR-CAS technology, stem cell-derived organoids, 3D microscopy, electron cryomicroscopy, single cell RNA sequencing, transgenic methodologies, and tools to manipulate cellular processes at a molecular level. This knowledge has simultaneously underscored the complexity of biological systems and the interdependence of multiple regulatory systems. In addition to the plethora of mammalian neurohumoral factors and their cross talk, advances in pyrosequencing and metagenomic analyses have highlighted the relevance of the microbiome to intestinal regulation. This article provides an overview of our current understanding of electrolyte transport processes in the small and large intestine, their regulation in health and how dysregulation at multiple levels can result in disease. Intestinal electrolyte transport is a balance of ion secretory and ion absorptive processes, all exquisitely dependent on the basolateral Na+ /K+ ATPase; when this balance goes awry, it can result in diarrhea or in constipation. The key transporters involved in secretion are the apical membrane Cl- channels and the basolateral Na+ -K+ -2Cl- cotransporter, NKCC1 and K+ channels. Absorption chiefly involves apical membrane Na+ /H+ exchangers and Cl- /HCO3 - exchangers in the small intestine and proximal colon and Na+ channels in the distal colon. Key examples of our current understanding of infectious, inflammatory, and genetic diarrheal diseases and of constipation are provided. © 2019 American Physiological Society. Compr Physiol 9:947-1023, 2019.

Sex-Dependent Signaling Pathways Underlying Seizure Susceptibility and the Role of Chloride Cotransporters.

Seizure incidence, severity, and antiseizure medication (ASM) efficacy varies between males and females. Differences in sex-dependent signaling pathways that determine network excitability may be responsible. The identification and validation of sex-dependent molecular mechanisms that influence seizure susceptibility is an emerging focus of neuroscience research. The electroneutral cation-chloride cotransporters (CCCs) of the SLC12A gene family utilize Na+-K+-ATPase generated electrochemical gradients to transport chloride into or out of neurons. CCCs regulate neuronal chloride gradients, cell volume, and have a strong influence over the electrical response to the inhibitory neurotransmitter GABA. Acquired or genetic causes of CCCs dysfunction have been linked to seizures during early postnatal development, epileptogenesis, and refractoriness to ASMs. A growing number of studies suggest that the developmental expression of CCCs, such as KCC2, is sex-dependent. This review will summarize the reports of sexual dimorphism in epileptology while focusing on the role of chloride cotransporters and their associated modulators that can influence seizure susceptibility.

Na+ -K+ -2Cl- Cotransporter (NKCC) Physiological Function in Nonpolarized Cells and Transporting Epithelia.

Two genes encode the Na+ -K+ -2Cl- cotransporters, NKCC1 and NKCC2, that mediate the tightly coupled movement of 1Na+ , 1K+ , and 2Cl- across the plasma membrane of cells. Na+ -K+ -2Cl- cotransport is driven by the chemical gradient of the three ionic species across the membrane, two of them maintained by the action of the Na+ /K+ pump. In many cells, NKCC1 accumulates Cl- above its electrochemical potential equilibrium, thereby facilitating Cl- channel-mediated membrane depolarization. In smooth muscle cells, this depolarization facilitates the opening of voltage-sensitive Ca2+ channels, leading to Ca2+ influx, and cell contraction. In immature neurons, the depolarization due to a GABA-mediated Cl- conductance produces an excitatory rather than inhibitory response. In many cell types that have lost water, NKCC is activated to help the cells recover their volume. This is specially the case if the cells have also lost Cl- . In combination with the Na+ /K+ pump, the NKCC's move ions across various specialized epithelia. NKCC1 is involved in Cl- -driven fluid secretion in many exocrine glands, such as sweat, lacrimal, salivary, stomach, pancreas, and intestine. NKCC1 is also involved in K+ -driven fluid secretion in inner ear, and possibly in Na+ -driven fluid secretion in choroid plexus. In the thick ascending limb of Henle, NKCC2 activity in combination with the Na+ /K+ pump participates in reabsorbing 30% of the glomerular-filtered Na+ . Overall, many critical physiological functions are maintained by the activity of the two Na+ -K+ -2Cl- cotransporters. In this overview article, we focus on the functional roles of the cotransporters in nonpolarized cells and in epithelia. © 2018 American Physiological Society. Compr Physiol 8:871-901, 2018.

Molecular mechanisms underlying active desalination and low water permeability in the esophagus of eels acclimated to seawater.

Marine teleosts can absorb imbibed seawater (SW) to maintain water balance, with esophageal desalination playing an essential role. NaCl absorption from luminal SW was enhanced 10-fold in the esophagus of SW-acclimated eels, and removal of Na+ or Cl- from luminal SW abolished the facilitated absorption, indicating coupled transport. Mucosal/serosal application of various blockers for Na+/Cl- transporters profoundly decreased the absorption. Among the transporter genes expressed in eel esophagus detected by RNA-seq, dimethyl amiloride-sensitive Na+/H+ exchanger (NHE3) and 4,4'-diisothiocyano-2,2'-disulfonic acid-sensitive Cl-/[Formula: see text] exchanger (AE) coupled by the scaffolding protein on the apical membrane of epithelial cells, and ouabain-sensitive Na+-K+-ATPases (NKA1α1c and NKA3α) and diphenylamine-2-carboxylic acid-sensitive Cl- channel (CLCN2) on the basolateral membrane, may be responsible for enhanced transcellular NaCl transport because of their profound upregulation after SW acclimation. Upregulated carbonic anhydrase 2a (CA2a) supplies H+ and [Formula: see text] for activation of the coupled NHE and AE. Apical hydrochlorothiazide-sensitive Na+-Cl- cotransporters and basolateral Na+-[Formula: see text] cotransporter (NBCe1) and AE1 are other possible candidates. Concerning the low water permeability that is typically seen in marine teleost esophagus, downregulated aquaporin genes (aqp1a and aqp3) and upregulated claudin gene (cldn15a) are candidates for transcellular/paracellular route. In situ hybridization showed that these upregulated transporters and tight-junction protein genes were expressed in the absorptive columnar epithelial cells of eel esophagus. These results allow us to provide a full picture of the molecular mechanism of active desalination and low water permeability that are characteristic to marine teleost esophagus and gain deeper insights into the role of gastrointestinal tracts in SW acclimation.

The roles of the Na+/K+-ATPase, NKCC, and K+ channels in regulating local sweating and cutaneous blood flow during exercise in humans in vivo.

Na+/K+-ATPase has been shown to regulate the sweating and cutaneous vascular responses during exercise; however, similar studies have not been conducted to assess the roles of the Na-K-2Cl co-transporter (NKCC) and K+ channels. Additionally, it remains to be determined if these mechanisms underpinning the heat loss responses differ with exercise intensity. Eleven young (24 ± 4 years) males performed three 30-min semirecumbent cycling bouts at low (30% VO2peak), moderate (50% VO2peak), and high (70% VO2peak) intensity, respectively, each separated by 20-min recovery periods. Using intradermal microdialysis, four forearm skin sites were continuously perfused with either: (1) lactated Ringer solution (Control); (2) 6 mmol·L-1 ouabain (Na+/K+-ATPase inhibitor); (3) 10 mmol·L-1 bumetanide (NKCC inhibitor); or (4) 50 mmol·L-1 BaCl2 (nonspecific K+ channel inhibitor); sites at which we assessed local sweat rate (LSR) and cutaneous vascular conductance (CVC). Inhibition of Na+/K+-ATPase attenuated LSR compared to Control during the moderate and high-intensity exercise bouts (both P Ë‚ 0.01), whereas attenuations with NKCC and K+ channel inhibition were only apparent during the high-intensity exercise bout (both P ≤ 0.05). Na+/K+-ATPase inhibition augmented CVC during all exercise intensities (all P Ë‚ 0.01), whereas CVC was greater with NKCC inhibition during the low-intensity exercise only (P Ë‚ 0.01) and attenuated with K+ channel inhibition during the moderate and high-intensity exercise conditions (both P Ë‚ 0.01). We show that Na+/K+-ATPase, NKCC and K+ channels all contribute to the regulation of sweating and cutaneous blood flow but their influence is dependent on the intensity of dynamic exercise.

Intestinal Na+, K+, 2Cl- cotransporter 2 plays a crucial role in hyperosmotic transitions of a euryhaline teleost.

Euryhaline fishes, such as the red drum (Sciaenops ocellatus), must quickly transition between hyperosmotic and hypoosmotic physiological strategies. When freshwater individuals transition to seawater they are exposed to increased diffusive water loss and ion gain. To maintain osmoregulatory balance these animals must drink and absorb seawater through the intestine, followed by ion excretion at the gills. The ability of fishes to transition between strategies can limit the magnitude of osmotic shock that can be tolerated. Here, we demonstrate that red drum can tolerate direct transfer from freshwater to full-strength seawater with marginal impacts on osmotic balance, as indicated by plasma and muscle ion concentration, as well as muscle water. Seawater transition is concurrent with a significant increase in intestinal fluid volume. Typical patterns of osmoregulatory plasticity were observed in the gill with increased expression of nkcc1 and cftr Expression changes in the anterior intestine were observed after 24 h for nkcc2 with smaller and later responses observed for slc26a3, slc26a6, and nbc Immunofluorescence staining demonstrated similar patterns of NKCC localization in freshwater and seawater intestines; however, reduced basolateral staining of V-type ATPase was observed in seawater. Electrophysiological preparations demonstrated that seawater fish had increased absorptive current in the anterior intestine, which was significantly reduced in the presence of 10 µmol/L bumetanide. Overall, these results suggest that nkcc2 plays a crucial role during hyperosmotic transitions, and may be a more important complement to the well-known bicarbonate secretion pathway than generally considered.

Characterization of Rat Meibomian Gland Ion and Fluid Transport.

PURPOSE: We establish novel primary rat meibomian gland (MG) cell culture systems and explore the ion transport activities of the rat MG. METHODS: Freshly excised rat MG tissues were characterized as follows: (1) mRNA expression of selected epithelial ion channels/transporters were measured by RT-PCR, (2) localization of epithelial sodium channel (ENaC) mRNAs was performed by in situ hybridization, and (3) protein expression and localization of ßENaC, the Na+/K+/Cl- cotransporter (NKCC), and the Na+/K+ ATPase were evaluated by immunofluorescence. Primary isolated rat MG cells were cocultured with 3T3 feeder cells and a Rho-associated kinase (ROCK) inhibitor (Y-27632) for expansion. Passaged rat MG cells were cultured as planar sheets under air-liquid interface (ALI) conditions for gene expression and electrophysiologic studies. Passaged rat MG cells also were cultured in matrigel matrices to form spheroids, which were examined ultrastructurally by transmission electron microscopy (TEM) and functionally using swelling assays. RESULTS: Expression of multiple ion channel/transporter genes was detected in rat MG tissues. ß-ENaC mRNA and protein were localized more to MG peripheral acinar cells than central acinar cells or ductular epithelial cells. Electrophysiologic studies of rat MG cell planar cultures demonstrated functional sodium, chloride, and potassium channels, and cotransporters activities. Transmission electron microscopic analyses of rat MG spheroids revealed highly differentiated MG cells with abundant lysosomal lamellar bodies. Rat MG spheroids culture-based measurements demonstrated active volume regulation by ion channels. CONCLUSIONS: This study demonstrates the presence and function of ion channels and volume transport by rat MG. Two novel primary MG cell culture models that may be useful for MG research were established.

Calcineurin and Sorting-Related Receptor with A-Type Repeats Interact to Regulate the Renal Na⁺-K⁺-2Cl⁻ Cotransporter.

The furosemide-sensitive Na(+)-K(+)-2Cl(-)-cotransporter (NKCC2) is crucial for NaCl reabsorption in kidney thick ascending limb (TAL) and drives the urine concentrating mechanism. NKCC2 activity is modulated by N-terminal phosphorylation and dephosphorylation. Serine-threonine kinases that activate NKCC2 have been identified, but less is known about phosphatases that deactivate NKCC2. Inhibition of calcineurin phosphatase has been shown to stimulate transport in the TAL and the distal convoluted tubule. Here, we identified NKCC2 as a target of the calcineurin Aß isoform. Short-term cyclosporine administration in mice augmented the abundance of phospho-NKCC2, and treatment of isolated TAL with cyclosporine increased the chloride affinity and transport activity of NKCC2. Because sorting-related receptor with A-type repeats (SORLA) may affect NKCC2 phosphoregulation, we used SORLA-knockout mice to test whether SORLA is involved in calcineurin-dependent modulation of NKCC2. SORLA-deficient mice showed more calcineurin Aß in the apical region of TAL cells and less NKCC2 phosphorylation and activity compared with littermate controls. In contrast, overexpression of SORLA in cultured cells reduced the abundance of endogenous calcineurin Aß. Cyclosporine administration rapidly normalized the abundance of phospho-NKCC2 in SORLA-deficient mice, and a functional interaction between calcineurin Aß and SORLA was further corroborated by binding assays in rat kidney extracts. In summary, we have shown that calcineurin Aß and SORLA are key components in the phosphoregulation of NKCC2. These results may have clinical implications for immunosuppressive therapy using calcineurin inhibitors.

Integrated compensatory network is activated in the absence of NCC phosphorylation.

Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase-deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type H⁺-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG-activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy.

[Pathophysiological aspects of K+: Cl- cotransporters]. / Aspectos fisiopatologicos de los cotransportaores de K+ :CI- .

The K+:Cl- cotransporters or KCCs are membrane proteins that move K+ and Cl- ions across the membrane without changing the transmembrane potential. KCCs belong to the SLC12 (Solute Carrier Family 12) family of electroneutral cation-chloride cotransporters (CCC), and they are secondary active ion transporters because use the established gradients from the primary active transporter through the Na+/K+- ATPase. Although there are nine members identify in this family, up today only seven genes had been characterized. Among them are two loop diuretics-sensitive Na+:K+:2Clcotransporters (NKCC1/NKCC2), the thiazide-sensitive Na+:Cl- cotransporter (NCC), and finally the K+:Cl- cotransporters (KCC), encoded for at least four homologous genes (KCC1-KCC4), and from which there are many isoforms due to alternative splicing. KCC1 is a ubiquitous isoform, KCC3 and KCC4 isoforms are widely expressed, particularly in epithelial cells, while KCC2 is restricted to the central nervous system (CNS). All these cotransporters play an essential role in many physiological processes such as cell volume regulation, transepithelial salt transport and regulation of the intraneuronal chloride concentration. This review has the purpose to show briefly the molecular characteristics as well as the physiological importance and roles of the KCCs in several pathologies.

Gitelman syndrome with hiponatremia, a rare presentation

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Cation-chloride cotransporters: regulation, physiological significance, and role in pathogenesis of arterial hypertension.

This review summarizes the data on the functioning of carriers providing electroneutral symport of sodium, potassium, and chloride (Na(+),K(+),2Cl(-) cotransport), potassium and chloride (K(+),Cl(-) cotransport), and sodium and chloride (K(+),Cl(-) cotransport) as well as molecular mechanisms of the regulation of these carriers and their physiological significance. We emphasized the involvement of chloride-coupled carriers in the regulation of cell volume and intracellular chloride concentration and novel data on the role of ubiquitous isoform of Na(+),K(+),2Cl(-) cotransporter NKCC1 in regulation of vascular smooth muscle contraction and activity of GABA(A) receptors. Finally, we analyzed the data on activation of NKCC1 in patients with essential hypertension and its role in the long-term maintenance of elevated systemic blood pressure and myogenic response in microcirculatory beds.

Heterozygous disruption of renal outer medullary potassium channel in rats is associated with reduced blood pressure.

The renal outer medullary potassium channel (ROMK, KCNJ1) mediates potassium recycling and facilitates sodium reabsorption through the Na(+)/K(+)/2Cl(-) cotransporter in the loop of Henle and potassium secretion at the cortical collecting duct. Human genetic studies indicate that ROMK homozygous loss-of-function mutations cause type II Bartter syndrome, featuring polyuria, renal salt wasting, and hypotension; humans heterozygous for ROMK mutations identified in the Framingham Heart Study have reduced blood pressure. ROMK null mice recapitulate many of the features of type II Bartter syndrome. We have generated an ROMK knockout rat model in Dahl salt-sensitive background by using zinc finger nuclease technology and investigated the effects of knocking out ROMK on systemic and renal hemodynamics and kidney histology in the Dahl salt-sensitive rats. The ROMK(-/-) pups recapitulated features identified in the ROMK null mice. The ROMK(+/-) rats, when challenged with a 4% salt diet, exhibited a reduced blood pressure compared with their ROMK(+/+) littermates. More importantly, when challenged with an 8% salt diet, the Dahl salt-sensitive rats with 50% less ROMK expression showed increased protection from salt-induced blood pressure elevation and signs of protection from renal injury. Our findings in ROMK knockout Dahl salt-sensitive rats, together with the previous reports in humans and mice, underscore a critical role of ROMK in blood pressure regulation.

State-dependent contribution of the hyperpolarization-activated Na+/K+ and persistent Na+ currents to respiratory rhythmogenesis in vivo.

How rhythms are generated by neuronal networks is fundamental to understand rhythmic behaviors such as respiration, locomotion, and mastication. Respiratory rhythm is generated by the preBötzinger complex (preBötC), an anatomically and functionally discrete population of brainstem neurons, central and necessary for respiratory rhythm. In specific in vitro conditions, preBötC neurons depend on voltage-dependent inward currents to generate respiratory rhythm. In the mature and intact organism, where preBötC neurons are deeply embedded in the respiratory network, the contribution of ionic currents to respiratory rhythm is unclear. We propose that a set of ionic currents plays a key role in generating respiratory rhythm in the mature organism in vivo. By microperfusing ionic current blockers into the preBötC of adult rats, we identify the hyperpolarization-activated cation current as a critical component of the mechanism promoting respiratory rhythm, and that this current, in combination with the persistent sodium current, is essential to respiratory rhythm in vivo. Importantly, both currents contribute to rhythmic activity in states of anesthesia, quiet wakefulness, and sleep, but not when the organism is engaged in active behaviors. These data show that a set of ionic currents at the preBötC imparts the network with rhythmicity in reduced states of arousal, although the network can override their contribution to adjust its activity for nonrhythmic behaviors in active wakefulness.

The SLC12 family of electroneutral cation-coupled chloride cotransporters.

The SLC12 family encodes electroneutral cation-coupled chloride cotransporters that are critical for several physiological processes including cell volume regulation, modulation of intraneuronal chloride concentration, transepithelial ion movement, and blood pressure regulation. Members of this family are the targets of the most commonly used diuretic drugs, have been shown to be the causative genes for inherited disease such as Gitelman, Bartter and Andermann syndromes, and potentially play a role in polygenic complex diseases like arterial hypertension, epilepsy, osteoporosis, and cancer.

Loss of WNK3 is compensated for by the WNK1/SPAK axis in the kidney of the mouse.

WNK3 kinase is expressed throughout the nephron and acts as a positive regulator of NKCC2 and NCC in vitro. Here we addressed the in vivo relevance of WNK3 using WNK3-deficient mice. WNK3-/- mice were viable and showed no gross abnormalities. The net tubular function was similar in wild-type (WT) and WNK3-/- mice as assessed by determination of 24-h urine output (1.63 ± .06 in WT and 1.55 ± .1 ml in WNK3-/-, n=16; P=0.42) and ambient urine osmolarity (1,804 ± 62 in WT vs. 1,819 ± 61 mosmol/kg in WNK3-/-, n=40; P=0.86). Water restriction (48 h) increased urine osmolarity similarly in both genotypes to 3,440 ± 220 and 3,200 ± 180 mosmol/kg in WT and WNK3-/- mice, respectively (n=11; P=0.41). The glomerular filtration rate (343 ± 22 vs. 315 ± 13 ml/min), renal blood flow (1.35 ± 0.1 vs. 1.42 ± 0.04 ml), and plasma renin concentration (94 ± 18 vs. 80 ± 13 ng ANG I·ml(-1)·h(-1)) were similar between WT and WNK3-/- mice (n=13; P=0.54). WNK1 was markedly upregulated in WNK3-deficient mice, whereas the expression of WNK4 was similar in both genotypes. When the mice were fed a salt-restricted diet [0.02% NaCl (wt/wt)] the levels of pSPAK/OSR1, pNKCC2, and pNCC were enhanced in both genotypes compared with the baseline conditions, with the levels in WNK3-/- exceeding those in WT mice. The upregulation of pSPAK/OSR1, pNKCC2, and pNCC in WNK3-/- mice relative to the levels in WT mice when fed a low-salt diet was paralleled by an increased diuresis in response to hydrochlorothiazide. In summary, the overall relevance of WNK3 for the renal reabsorption of NaCl appears to be limited and can be largely compensated for by the activation of WNK3-independent pathways. Consequently, our data suggest that WNK3 may serve as a member of a kinase network that facilitates the fine-tuning of renal transepithelial NaCl transport.