High salt consumption contributes to the development of hypertension and is considered an independent risk factor for vascular remodeling, cardiac hypertrophy, and stroke incidence. In this review, we discuss the molecular origins of primary sensors involved in the phenomenon of salt sensitivity. Based on the analysis of literature data, we conclude that the kidneys and central nervous system (CNS) are two major sites for salt sensing via several distinct mechanisms: 1) [Cl⁻] sensing in renal tubular fluids, primarily by Na⁺-K⁺-Cl⁻cotransporter (NKCC) isoforms NKCC2B and NKCC2A, whose expression is mainly limited to macula densa cells; 2) [Na⁺] sensing in cerebrospinal fluid (CSF) by a novel isoform of Na⁺channels, Na_x, expressed in subfornical organs; 3) sensing of CSF osmolality by mechanosensitive, nonselective cation channels (transient receptor potential vanilloid type 1 channels), expressed in neuronal cells of supraoptic and paraventricular nuclei; and 4) osmolarity sensing by volume-regulated anion channels in glial cells of supraoptic and paraventricular nuclei. Such multiplicity of salt-sensing mechanisms likely explains the differential effects of Na⁺and Cl⁻loading on the long-term maintenance of elevated blood pressure that is documented in experimental models of salt-sensitive hypertension.

Recently, we reported that ouabain kills renal epithelial and vascular endothelial cells independently of elevation of the [Na⁺]_i/[K⁺]_i ratio. These observations raised the possibility of finding cardiotonic steroids (CTS) that inhibit the Na⁺,K⁺ pump without attenuating cell survival and vice versa. To test this hypothesis, we compared CTS action on Na⁺,K⁺ pump, [Na⁺]_i content, and survival of Madin-Darby canine kidney cells. At a concentration of 1 μm, ouabain and other tested cardenolides, as well as bufadienolides such as bufalin, cinobufagin, cinobufotalin, and telobufotoxin, led to ∼10-fold inhibition of the Na⁺,K⁺ pump, a 2-3-fold decrease in staining with dimethylthiazol-diphenyltetrazolium (MTT), and massive death indicated by detachment of ∼80% of cells and caspase-3 activation. In contrast, Na⁺,K⁺ pump inhibition and elevation of [Na⁺]_i seen in the presence of 3 μm marinobufagenin (MBG) and marinobufotoxin did not affect MTT staining and cell survival. Inhibition of the Na⁺,Rb⁺ pump in K⁺-free medium was not accompanied by a decline of MTT staining and cell detachment but increased sensitivity to CTS. In K⁺-free medium, half-maximal inhibition of ⁸⁶Rb influx was observed in the presence of 0.04 μm ouabain and 0.1 μm MBG, whereas half-maximal detachment and decline of MTT staining were detected at 0.03 and 0.004 μm of ouabain versus 10 and 3 μm of MBG, respectively. Both ouabain binding and ouabain-induced [Na⁺]_i,[K⁺]_i-independent signaling were suppressed in the presence of MBG. Thus, our results show that CTS exhibit distinctly different potency in Na⁺,K⁺ pump inhibition and triggering of [Na⁺]_i/[K⁺]_i-independent signaling, including cell death.

Stimulus-dependent elevation of intracellular Ca²⁺ ([Ca²⁺]_i) affects the expression of numerous genes – a phenomenon known as excitation-transcription coupling. Recently, we found that increases in [Na⁺]_i trigger c-Fos expression via a novel Ca²⁺_i-independent pathway. In the present study, we identified ubiquitous and tissue-specific [Na⁺]_i/[K⁺]_i-sensitive transcriptomes by comparative analysis of differentially expressed genes in vascular smooth muscle cells from rat aorta (RVSMC), the human adenocarcinoma cell line HeLa, and human umbilical vein endothelial cells (HUVEC). To augment [Na⁺]_i and reduce [K⁺]_i, cells were treated for 3 hrs with the Na⁺,K⁺-ATPase inhibitor ouabain or placed for the same time in the K⁺-free medium. Employing Affymetrix-based technology, we detected changes in expression levels of 684, 737 and 1839 transcripts in HeLa, HUVEC and RVSMC, respectively, that were highly correlated between two treatments (p0.62). Among these Na⁺_i/K⁺_i-sensitive genes, 80 transcripts were common for all three types of cells. To establish if changes in gene expression are dependent on increases in [Ca²⁺]_i, we performed identical experiments in Ca²⁺-free media supplemented with extracellular and intracellular Ca²⁺ chelators. Surprisingly, this procedure elevated rather than decreased the number of ubiquitous and cell-type specific Na⁺_i/K⁺_i-sensitive genes. Among the ubiquitous Na⁺_i/K⁺_i-sensitive genes whose expression was regulated independently of the presence of Ca²⁺ chelators by more than 3-fold, we discovered several transcription factors (Fos, Jun, Hes1, Nfkbia), interleukin-6, protein phosphatase 1 regulatory subunit, dual specificity phosphatase (Dusp8), prostaglandin-endoperoxide synthase 2, cyclin L1, whereas expression of metallopeptidase Adamts1, adrenomedulin, Dups1, Dusp10 and Dusp16 was detected exclusively in Ca²⁺-depleted cells. Overall, our findings indicate that Ca²⁺_i-independent mechanisms of excitation-transcription coupling are involved in transcriptomic alterations triggered by elevation of the [Na⁺]_i/[K⁺]_i ratio. There results likely have profound implications for normal and pathological regulation of mammalian cells, including sustained excitation of neuronal cells, intensive exercise and ischemia-triggered disorders.

This review summarizes the data on the functional significance of ubiquitous (NKCC1) and renal-specific (NKCC2) isoforms of electroneutral sodium, potassium and chloride cotransporters. These carriers contribute to the pathogenesis of hypertension via regulation of intracellular chloride concentration in vascular smooth muscle and neuronal cells and via sensing chloride concentration in the renal tubular fluid, respectively. Both NKCC1 and NKCC2 are inhibited by furosemide and other high-ceiling diuretics widely used for attenuation of extracellular fluid volume. However, the chronic usage of these compounds for the treatment of hypertension and other volume-expanded disorders may have diverse side-effects due to suppression of myogenic response in microcirculatory beds.

In this study, we examined the effect of Na⁺‐K⁺ pump inhibition on the expression of early response genes in vascular smooth muscle cells (VSMC) as possible intermediates of the massive RNA synthesis and protection against apoptosis seen in ouabain‐treated VSMC in our previous experiments. Incubation of VSMC with ouabain resulted in rapid induction of c‐Fos protein expression with an approximately sixfold elevation after 2 h of incubation. c‐Jun expression was increased by approximately fourfold after 12 h, whereas expression of activating transcription factor 2, cAMP/Ca²⁺ response element binding protein (CREB)‐1 and c‐Myc was not altered. Markedly augmented c‐Fos expression was also observed under Na⁺‐K⁺ pump inhibition in potassium‐depleted medium. Na⁺‐K⁺ pump inhibition triggered c‐Fos expression via elevation of the [Na⁺]_i/[K⁺]_i ratio. This conclusion follows from experiments showing the lack of effect of ouabain on c‐Fos expression in high‐potassium‐low‐sodium medium and from the comparison of dose responses of Na⁺‐K⁺ pump activity, [Na⁺]_i and [K⁺]_i content and c‐Fos expression to ouabain. A fourfold increment of c‐Fos mRNA was revealed 30 min following addition of ouabain to the incubation medium. At this time point, treatment with ouabain resulted in an approximately fourfold elevation of [Na⁺]_i but did not affect [K⁺]_i. Augmented c‐Fos expression was also observed under VSMC depolarization in high‐potassium medium. Increments in both c‐Fos expression and ⁴⁵Ca uptake in depolarized VSMC were abolished under inhibition of L‐type Ca²⁺ channels with 0.1 μM nicardipine. Ouabain did not affect the free [Ca²⁺]_i or the content of exchangeable [Ca²⁺]_i. Ouabain‐induced c‐Fos expression was also insensitive to the presence of nicardipine and [Ca²⁺]_o, as well as chelators of [Ca²⁺]_o (EGTA) and [Ca²⁺]_i (BAPTA). The effect of ouabain and serum on c‐Fos expression was additive. In contrast to serum, however, ouabain failed to activate the Elk‐1, serum response factor, CREB and activator protein‐1 transcription factors identified within the c‐Fos promoter. These results suggest that Na⁺‐K⁺ pump inhibition triggers c‐Fos expression via [Na⁺]_i‐sensitive [Ca²⁺]_i‐independent transcription factor(s) distinct from factors interacting with known response elements of this gene promoter.