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Fig. 1 · Structure
Cat. No.
A10412
CAS No.
54-31-9
Formula
C12H11ClN2O5S
Mol. Weight
330.74

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Furosemide

Cat. No. A10412 CAS 54-31-9
Furosemide is a non-competitive antagonist at GABAA receptors with ~ 100-fold greater selectivity for α6-containing receptors than α1-containing receptors.

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Biological Activity

Bioactivity / Mechanism

Furosemide inhibits human NKCC1-mediated ion transport. A cryo-EM study of phospho-activated NKCC1 bearing affinity-enhancing K289N/A492E mutations placed furosemide in the extracellular ion-entry vestibule, with its carboxyl group coordinating a K+ ion. This mutant structure should not be treated as a wild-type or NKCC2 potency measurement. [1,2]

Literature-reported observations and conditions; not Adooq-tested protocols or general treatment recommendations.

Target & Activity

Target Activity Value
NKCC2

Cellular Effect

Cell Line Activity Value Description Reference
CHO EC50 11.2 μM Agonist activity at C-terminal beta-galactosidase tagged human recombinant GPR35 expressed in CHO cells after 90 mins by beta-arrestin recruitment assay [PMID: 23888932]
HEK293 IC50 > 500 μM Inhibition of human MATE1-mediated ASP+ uptake expressed in HEK293 cells after 1.5 mins by fluorescence assay [PMID: 23241029]
HEK293 IC50 3.76 μM Inhibition of human 11beta-HSD1 expressed in HEK293 cells assessed as conversion of [3H]cortisone to [3H]cortisol by scintillation proximity assay in presence of NADPH [PMID: 18653260]
HEK293 IC50 3.76 μM Inhibition of mouse 11beta-HSD1 expressed in HEK293 cells assessed as conversion of [3H]cortisone to [3H]cortisol by scintillation proximity assay in presence of NADPH [PMID: 18653260]

In Vitro Studies

In human NKCC1 splice variants expressed in Xenopus oocytes, furosemide gave median IC50 values of 5.15 µM for NKCC1A and 5.82 µM for NKCC1B, without significant splice preference. Potency also depended on assay conditions: a separate HEK293 NKCC1 assay reported approximately threefold higher potency after K+-containing than K+-free activation; transport itself was measured with K+ present. [1,2]

Furosemide-bound phospho-activated human NKCC1 structure

Biochemical assay · Purified phospho-activated human NKCC1 K289N/A492E, expressed with WNK1–SPAK pathway components in HEK293 cells [1]

The structure places furosemide in the extracellular ion-entry vestibule of outward-open NKCC1, with its carboxyl group coordinating a co-occluded K+ ion.

Measured endpoints — not treatment concentrations
EndpointReported resultReference
Furosemide binding location and K+ coordinationStructural observation Extracellular vestibule; outward-open state; carboxyl group coordinates co-occluded K+[1]

Literature-Reported Treatment Conditions

Treatment concentration
2 mM furosemide for cryo-EM complex formation
Method
Cryo-electron microscopy of purified transporter–drug complex
Reported limits
Drug incubation duration and drug solvent are not specified in the read purification subsection.
Methods and source locations

Compound reported in the paper: Furosemide; Compound label in the paper: Furosemide

A binding pose in an affinity-enhanced, phospho-activated NKCC1 mutant; not an NKCC2 co-structure or a wild-type potency measurement.

Results: Two modes of NKCC1 antagonism; Fig. 2A–B and EV3A; Methods: Expression and purification of phospho-activated human NKCC1; reagent table

Furosemide binding location and K+ coordination: Cryo-EM density and fitted structure; Fig. 2A–B and EV3A viewed — Results: Two modes of NKCC1 antagonism; Fig. 2A–B and EV3A; Methods: Expression and purification of phospho-activated human NKCC1; reagent table

Potassium dependence of furosemide inhibition in an NKCC1 cell assay

Cell-based study · NKCC1-expressing HEK293 cells with membrane-targeted chloride-sensitive YFP; reporter-only cells included as negative controls in all experiments [1]

Furosemide was approximately threefold more potent after K+-containing activation than after K+-free activation. Both groups subsequently received K+-containing assay buffer.

Measured endpoints — not treatment concentrations
EndpointReported resultReference
Relative furosemide inhibitory potency with versus without K+ during activationRelative potency Threefold higher potency in a K+ buffer than in a K+-free buffer (reported text)[1]

Literature-Reported Treatment Conditions

Treatment concentration
Furosemide concentration series; exact tested concentrations not enumerated in the methods text
Exposure duration
Activation 2–3 h; transport rate from initial 20 s
Method
Activation in 70 mM K2SO4 or 70 mM (NMDG)2SO4; common assay buffer contains 90 mM NaCl and 50 mM KCl, without furosemide
Reported limits
Drug solvent and numerical IC50 estimates are not given in the read text/caption; none inferred from curves.
Methods and source locations

Compound reported in the paper: Furosemide; Compound label in the paper: Furosemide

An assay-condition dependence of apparent inhibitory potency; K+-free activation is not K+-free transport.

Fig. 2F and caption; Results: Two modes of NKCC1 antagonism; Methods: Cl− influx assay in HEK293 cells

Relative furosemide inhibitory potency with versus without K+ during activation: YFP chloride influx dose–response; n = 4 biological repeats, mean ± SD in caption; three-parameter log-inhibitor curve fit — Fig. 2F and caption; Results: Two modes of NKCC1 antagonism; Methods: Cl− influx assay in HEK293 cells

Furosemide inhibition of human NKCC1 splice variants

Cell-based study · Xenopus laevis oocytes expressing hNKCC1A or hNKCC1B, with batch-matched uninjected oocytes [2]

Furosemide inhibited both splice variants in the micromolar range without a significant preference for either variant.

Measured endpoints — not treatment concentrations
EndpointReported resultReference
Inhibition of hNKCC1A-mediated 86Rb+ uptakeIC50 5.15 µM (range 4.34–13.7 µM)[2]
Inhibition of hNKCC1B-mediated 86Rb+ uptakeIC50 5.82 µM (range 4.73–7.27 µM)[2]

Literature-Reported Treatment Conditions

Treatment concentration
Furosemide concentration series; reported IC50 values are assay endpoints, not treatment concentrations
Exposure duration
30 min K+-free preactivation; 5 min uptake at room temperature
Method
86Rb+ uptake as a K+ transport tracer; test solution contains 5 mM KCl; endogenous uptake subtracted using drug-matched uninjected oocytes
Vehicle
DMSO: Drugs subsection reports <1% final; assay subsection specifies ≤1% with matched solvent exposure
Reported limits
Exact furosemide concentration series and furosemide-specific experiment count are not enumerated in Table 1; paper reports 3–7 independent experiments per drug.
Methods and source locations

Compound reported in the paper: Furosemide; Compound label in the paper: Furosemide

Cell-based human NKCC1A/B transport inhibition, not isolated NKCC2 potency or an in vivo dose.

Table 1 furosemide row and footnote; Methods: Drugs, NKCC1A and NKCC1B activity assay, Data analysis; Results: Inhibitory potency of other loop diuretics

Inhibition of hNKCC1A-mediated 86Rb+ uptake: Median of experiment-level fitted IC50 values; ranges across independent experiments (3–7 per drug); Table 1 — Table 1 furosemide row and footnote; Methods: Drugs, NKCC1A and NKCC1B activity assay, Data analysis; Results: Inhibitory potency of other loop diuretics

Inhibition of hNKCC1B-mediated 86Rb+ uptake: Median of experiment-level fitted IC50 values; ranges across independent experiments (3–7 per drug); Table 1 — Table 1 furosemide row and footnote; Methods: Drugs, NKCC1A and NKCC1B activity assay, Data analysis; Results: Inhibitory potency of other loop diuretics

In Vivo Studies

A single 25 mg/kg intraperitoneal furosemide challenge increased urine volume and sodium excretion in control and smooth-muscle AT1A receptor knockout mice, with smaller responses in the knockout group. In a separate mouse study using 15 mg/kg, the potassium-clearance response depended on diet: it increased on standard chow but decreased on a low-sodium, high-potassium diet. These results support model-dependent renal effects rather than a universal electrolyte response. [3,4]

Genotype-dependent acute diuretic response to furosemide

Animal study · Male C57BL/6 control and smooth-muscle AT1A receptor knockout (SMKO) mice, age 10–20 weeks; n = 9 per genotype [3]

Vehicle urine volume and sodium excretion were similar between genotypes. Furosemide increased both endpoints in each genotype, with smaller responses in SMKO mice.

Measured endpoints — not treatment concentrations
EndpointReported resultReference
Urine volume and urinary sodium excretion after vehicle or furosemideDirection of response Increased in both genotypes; attenuated in SMKO versus controls[3]

Conditions shared by the study groups

Route
Intraperitoneal injection
Frequency
Single administration
Treatment course
4 h urine collection
Vehicle
Methanol followed by saline, adjusted with NaOH to approximately pH 7.7
Study groups and reported outcomes
Group / doseComparatorObserved resultReference
Control mice
25 mg/kg		Vehicle condition in the same genotype; n = 9 mice per genotypeFurosemide increased urine volume and Na+ excretion relative to vehicle.[3]
SMKO mice
25 mg/kg		Vehicle condition in the same genotype; n = 9 mice per genotypeUrine volume and Na+ excretion increased, but post-furosemide responses were smaller than in controls.[3]
Control mice
Model: Male C57BL/6 age-matched control littermates, 10–20 weeks
Reported limits: Final methanol fraction not specified in the read methods.
SMKO mice
Model: Male C57BL/6 mice with smooth-muscle-specific AT1A receptor deletion, 10–20 weeks
Reported limits: Final methanol fraction not specified in the read methods.
Methods and source locations

Compound reported in the paper: Furosemide; Compound label in the paper: Furosemide

Acute renal responses in these mouse genotypes; neither clinical efficacy nor direct NKCC2 binding potency.

Methods: Mouse model details, Natriuretic Response to Furosemide, Statistical Analysis; Results: Impaired Acute Response to Furosemide; Fig. 5A–B and caption

Urine volume and urinary sodium excretion after vehicle or furosemide: n = 9 per genotype; blinded experiments; repeated-measures two-way ANOVA with Sidak post-tests; group summaries mean ± SE — Methods: Mouse model details, Natriuretic Response to Furosemide, Statistical Analysis; Results: Impaired Acute Response to Furosemide; Fig. 5A–B and caption

Control mice · Methods: Mouse model details, Natriuretic Response to Furosemide, Statistical Analysis; Results: Impaired Acute Response to Furosemide; Fig. 5A–B and caption

SMKO mice · Methods: Mouse model details, Natriuretic Response to Furosemide, Statistical Analysis; Results: Impaired Acute Response to Furosemide; Fig. 5A–B and caption

Diet modifies the potassium-clearance response to acute furosemide

Animal study · Wild-type C57BL/6 mice, age 10–12 weeks, on standard or low-Na/high-K diet for 4–7 days [4]

Acute furosemide increased sodium clearance on both diets. Potassium clearance increased on standard chow but decreased on the low-Na/high-K diet.

Measured endpoints — not treatment concentrations
EndpointReported resultReference
12 h urinary sodium and potassium clearancesDiet-dependent direction Na+ clearance increased on both diets; K+ clearance increased on CTRL but decreased on LNaHK[4]

Conditions shared by the study groups

Route
Intraperitoneal injection
Frequency
Single administration
Treatment course
Diet for 4–7 days; 12 h urine collection after injection
Vehicle
Polyethylene glycol (PEG)
Study groups and reported outcomes
Group / doseComparatorObserved resultReference
CTRL + vehicle
100 µL polyethylene glycol vehicle		—Diet-matched vehicle control.[4]
CTRL + furosemide
15 mg/kg furosemide		Diet-matched vehicleFurosemide increased Na+ and K+ clearance versus vehicle.[4]
LNaHK + vehicle
100 µL polyethylene glycol vehicle		—Diet-matched vehicle control.[4]
LNaHK + furosemide
15 mg/kg furosemide		Diet-matched vehicleFurosemide increased Na+ clearance but decreased K+ clearance versus vehicle.[4]
CTRL + vehicle
Model: C57BL/6 wild-type mice, 10–12 weeks; standard chow (0.3% Na, 0.6% K)
Reported limits: Exact salt, supplier, purity, sex, and drug-solution preparation not specified in the read main methods.
CTRL + furosemide
Model: C57BL/6 wild-type mice, 10–12 weeks; standard chow (0.3% Na, 0.6% K)
Reported limits: Exact salt, supplier, purity, sex, and drug-solution preparation not specified in the read main methods.
LNaHK + vehicle
Model: C57BL/6 wild-type mice, 10–12 weeks; low-Na/high-K diet (0.01% Na, 5% K)
Reported limits: Exact salt, supplier, purity, sex, and drug-solution preparation not specified in the read main methods.
LNaHK + furosemide
Model: C57BL/6 wild-type mice, 10–12 weeks; low-Na/high-K diet (0.01% Na, 5% K)
Reported limits: Exact salt, supplier, purity, sex, and drug-solution preparation not specified in the read main methods.
Methods and source locations

Compound reported in the paper: Furosemide; Compound label in the paper: Furosemide

Diet-dependent mouse electrolyte clearance; does not establish a universal potassium effect or clinical treatment guidance.

Accepted manuscript: Results first subsection; Fig. 1A–B and caption; Table 1/footnote; Methods: Animals and Metabolic Cage

12 h urinary sodium and potassium clearances: Fig. 1A–B; caption N = 4–14 per group across plotted conditions; two-way ANOVA with post-hoc Tukey; Table 1 reports mean ± SEM for its separate metabolic endpoints — Accepted manuscript: Results first subsection; Fig. 1A–B and caption; Table 1/footnote; Methods: Animals and Metabolic Cage

CTRL + vehicle · Accepted manuscript: Results first subsection; Fig. 1A–B and caption; Table 1/footnote; Methods: Animals and Metabolic Cage

CTRL + furosemide · Accepted manuscript: Results first subsection; Fig. 1A–B and caption; Table 1/footnote; Methods: Animals and Metabolic Cage

LNaHK + vehicle · Accepted manuscript: Results first subsection; Fig. 1A–B and caption; Table 1/footnote; Methods: Animals and Metabolic Cage

LNaHK + furosemide · Accepted manuscript: Results first subsection; Fig. 1A–B and caption; Table 1/footnote; Methods: Animals and Metabolic Cage

Specifications

Manufacturer Adooq Bioscience
Actions Antagonist
CAS Number 54-31-9
Formula C12H11ClN2O5S
Molecular Weight 330.74
Synonyms Lasix
SMILES
C1=COC(=C1)CNC2=CC(=C(C=C2C(=O)O)S(=O)(=O)N)Cl

Storage & Solubility

In Vitro Solubility

Reported Solubility

SolventReported Solubility
DMSO 63 mg/mL (190.48 mM)
Water Insoluble
Ethanol Insoluble

Preparing Stock Solutions

Stock preparation is unavailable for this product molecular weight.

Calculated stock concentrations do not establish solvent solubility. Refer to the reported solubility data for experimental guidance.

Storage

Store powder at -20°C, keep desiccated.
In powder form, the chemical is stable for 36 months.
In solution, store at -20°C and use within 1 month to prevent loss of potency. Aliquot to avoid multiple freeze/thaw cycles.

Research References

  1. [1] Zhao Y, Vidossich P, Forbush B, et al. Structural basis for human NKCC1 inhibition by loop diuretic drugs The EMBO journal (2025). PMID: 39875725 DOI: 10.1038/s44318-025-00368-6 Source material
  2. [2] Hampel P, Römermann K, MacAulay N, et al. Azosemide is more potent than bumetanide and various other loop diuretics to inhibit the sodium-potassium-chloride-cotransporter human variants hNKCC1A and hNKCC1B Scientific reports (2018). PMID: 29959396 DOI: 10.1038/s41598-018-27995-w Source material
  3. [3] Sparks MA, Dilmen E, Ralph DL, et al. Vascular control of kidney epithelial transporters American journal of physiology. Renal physiology (2021). PMID: 33969697 DOI: 10.1152/ajprenal.00084.2021 Source material
  4. [4] Wang B, Wen D, Li H, et al. Net K<sup>+</sup> secretion in the thick ascending limb of mice on a low-Na, high-K diet Kidney international (2017). PMID: 28688582 DOI: 10.1016/j.kint.2017.04.009 Source material

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