5-(N,N-dimethyl)-Amiloride (hydrochloride): A Benchmark NHE1 Inhibitor for pH Regulation and Cardiac Research

Executive Summary: 5-(N,N-dimethyl)-Amiloride (hydrochloride) (DMA) is a crystalline derivative of amiloride that selectively inhibits Na+/H+ exchanger isoforms NHE1, NHE2, and NHE3 (Ki values: 0.02–14 μM) with minimal activity on NHE4-7, resulting in robust modulation of intracellular pH and sodium homeostasis. It exhibits demonstrated cardioprotective effects in ischemia-reperfusion models by normalizing sodium levels and preventing contractile dysfunction. DMA additionally inhibits ouabain-sensitive ATPase activities and reduces hepatocyte alanine uptake, indicating broader ion transport effects. The compound is highly soluble in DMSO and DMF (up to 30 mg/ml) and should be used promptly after solution preparation. Its application is research-only, with clear boundaries for diagnostic or medical use (product page; Chen et al., 2021).

Biological Rationale

The Na+/H+ exchanger (NHE) family regulates intracellular pH and cell volume by mediating proton extrusion and sodium uptake in mammalian cells. Disruption of NHE function is implicated in pathologies such as ischemia-reperfusion injury, cardiac contractile dysfunction, and sepsis-induced endothelial damage (Chen et al., 2021). NHE1 is the predominant isoform in cardiac and vascular endothelium. NHE activity influences cytoskeletal dynamics, cell survival, and inflammatory signaling, making selective inhibition a valuable research strategy for dissecting ion transport pathways and disease mechanisms (Malotilate.com; this article updates signal transduction details for endothelial injury models).

Mechanism of Action of 5-(N,N-dimethyl)-Amiloride (hydrochloride)

DMA is a structural analog of amiloride with enhanced potency and selectivity for NHE isoforms:

• Inhibits NHE1 (Ki = 0.02 μM), NHE2 (Ki = 0.25 μM), NHE3 (Ki = 14 μM) under standard buffer conditions at 25°C and pH 7.4 (ApexBio, C3505).
• Exhibits minimal effect on NHE4, NHE5, and NHE7, enabling isoform-selective interrogation.
• Mechanistically, DMA blocks the transmembrane movement of Na+ and H+, preventing proton extrusion and sodium influx.
• Inhibits ouabain-sensitive ATP hydrolysis and Na+/K+ ATPase activity in rat liver plasma membranes, revealing broader effects on cellular ion transport (D-lin-mc3-dma.com; this article details ATPase cross-talk and inhibitor selectivity).
• Reduces alanine uptake in rat hepatocytes, linking NHE inhibition to amino acid transport and metabolism.

Evidence & Benchmarks

• DMA selectively inhibits Na+/H+ exchanger isoforms NHE1-3 with Ki values of 0.02 μM (NHE1), 0.25 μM (NHE2), and 14 μM (NHE3) in mammalian cell assays (ApexBio, C3505).
• DMA demonstrates protective effects against ischemia-reperfusion injury in cardiac tissue by normalizing tissue sodium levels and preventing contractile dysfunction (Chen et al., 2021).
• DMA inhibits ouabain-sensitive ATP hydrolysis and sodium-potassium ATPase activity in rat liver plasma membranes, with dose-dependent inhibition observed at 1–100 μM (Chen et al., 2021).
• DMA reduces alanine uptake in rat hepatocytes by interfering with sodium-dependent transporters (ApexBio, C3505).
• DMA is highly soluble (up to 30 mg/ml) in DMSO and DMF at room temperature, and should be stored at -20°C; solutions are not recommended for long-term storage (ApexBio, C3505).

Applications, Limits & Misconceptions

DMA is widely applied in research targeting:

• Cardiac ischemia-reperfusion and contractile dysfunction models.
• Endothelial injury and sepsis-related vascular permeability (D-lin-mc3-dma.com; this article clarifies DMA's application to biomarker-driven studies—see Moesin/NHE1 interaction).
• Intracellular pH regulation and sodium homeostasis in cell biology.
• Ion transport and metabolism research in hepatocytes and other mammalian cells.

Common Pitfalls or Misconceptions

• DMA is not a pan-NHE inhibitor: It shows minimal activity on NHE4, NHE5, and NHE7, so it cannot be used to study these isoforms specifically.
• DMA is not suitable for diagnostic or clinical use: It is strictly for research applications and not approved for human or veterinary medicine.
• Long-term solution storage is not recommended: DMA solutions degrade over time and should be freshly prepared for each experiment.
• Not all observed cellular effects are due to NHE1 inhibition: DMA also impacts ATPase activity and amino acid transport, requiring careful experimental controls.
• Temperature and buffer conditions affect inhibitor potency: All quantitative results should be interpreted with clear reference to experimental conditions.

Workflow Integration & Parameters

For experimental use, DMA is supplied as a crystalline hydrochloride salt (SKU: C3505). It is soluble up to 30 mg/ml in DMSO or dimethylformamide, and should be stored at or below -20°C. Solutions should be used immediately after preparation. Typical working concentrations for NHE1 inhibition range from 0.01 to 10 μM, depending on cell type and assay conditions. DMA can be combined with pH-sensitive dyes or sodium-sensitive probes for functional readouts. Integration into endothelial injury or cardiac models enables mechanistic dissection of NHE1-driven signaling pathways. For in-depth protocols on endothelial signaling and biomarker applications, see aclacinomycina.com (this article extends the discussion by focusing on translational workflow integration and Moesin/NHE1 axis in sepsis models).

Conclusion & Outlook

5-(N,N-dimethyl)-Amiloride (hydrochloride) is a validated, potent, and selective NHE1-3 inhibitor with broad utility in ion transport, pH regulation, and translational cardiac and endothelial research. Its defined selectivity profile, strong evidence base, and compatibility with cell and tissue models make it indispensable for dissecting Na+/H+ exchanger signaling. Continued use of DMA in biomarker-driven studies, particularly those targeting endothelial injury and cardiac dysfunction, will further illuminate the role of NHEs in health and disease. For ordering or detailed specifications, visit the 5-(N,N-dimethyl)-Amiloride (hydrochloride) product page.