In human coronary vascular smooth muscle cells under AT1/AT2 receptor blockade, 10 μM aliskiren did not prevent renin- or prorenin-induced ERK1/2 phosphorylation. This acute signaling result should not be generalized to every prorenin-receptor response. [2]
Inhibition of recombinant human renin
Biochemical assay · Purified recombinant, glycosylated human renin generated in HEK-293 cells; synthetic fluorogenic angiotensinogen-sequence substrate. [1]
Aliskiren inhibited substrate cleavage with a reported IC50 of 0.81 ± 0.17 nM. SR 42128 and pepstatin A provided comparator inhibitors. This is an enzyme-assay endpoint, not a cellular treatment concentration or a binding constant.
Literature-Reported Treatment Conditions
- Treatment concentration
- Aliskiren concentration-response series; exact series not enumerated in the text. IC50 is the fitted endpoint.
- Exposure duration
- Fluorescence followed over 1–2 h; reaction velocities used for inhibition analysis.
- Method
- Functional assay: 0.3 nM renin, 1 μM FRET substrate, 50 mM BES pH 7.0, 0.5 mg/ml BSA and 150 mM NaCl; room temperature.
- Vehicle
- 2% DMSO in the functional assay mixture.
- Reported limits
- Aliskiren supplier, salt form, analytical identity, replicate n and meaning of the reported ± are not specified in the acquired article. Figure 2b uses 1 nM renin for a separate progress-curve demonstration; that condition is not assigned to the inhibitor assay.
Methods and source locations
Compound reported in the paper: Aliskiren
Evidence concerns the named aliskiren compound in the reported experimental material. Salt equivalence, analytical stereochemical confirmation and Adooq lot performance are not established.
Results and discussion, inhibitor characterization; Figure 2c and caption; Methods, Functional assay
Inhibition of recombinant human renin catalytic activity: FRET reaction velocities; four-parameter fit of percent inhibition versus inhibitor concentration. Uninhibited renin was the activity control; no enzyme or renin inactivated with 2 μM SR 42128 supplied the negative control. — Results and discussion, inhibitor characterization; Figure 2c and caption; Methods, Functional assay
Aliskiren did not prevent renin/prorenin-induced ERK phosphorylation
Cell-based study · Primary human coronary vascular smooth muscle cells under AT1 and AT2 receptor blockade; AT1A-deficient mouse VSMCs served as an auxiliary signaling control. [2]
At 10 μM, aliskiren did not prevent ERK1/2 phosphorylation induced by renin or prorenin. The MEK1/2 inhibitor PD98059 reduced the response. The result limits receptor-signaling claims to this acute, angiotensin-receptor-blocked model.
Literature-Reported Treatment Conditions
- Treatment concentration
- Aliskiren 10 μM; renin 10 nM or prorenin 2 nM.
- Exposure duration
- 24 h serum-free conditions; 30 min blocker preincubation; 10 min renin/prorenin stimulation for Figure 4D-F.
- Method
- Western blot for phosphorylated and unphosphorylated ERK1/2.
- Co-treatment
- Losartan 10 μM plus PD123319 10 μM to block AT1/AT2 signaling. HRP 1 μM and PD98059 100 nM were separate inhibitor comparators, not aliskiren combination treatments.
- Reported limits
- Aliskiren solvent and salt form not specified; no quantified aliskiren inhibition estimate or exact replicate count beyond at least three different experiments.
Methods and source locations
Compound reported in the paper: Aliskiren; Compound label in the paper: Novartis, Basel, Switzerland
Evidence concerns the named aliskiren compound in the reported experimental material. Salt equivalence, analytical stereochemical confirmation and Adooq lot performance are not established.
Results, Neither Aliskiren nor HRP Affects Renin and Prorenin-Induced ERK1/2 Phosphorylation in Human Coronary VSMC; Figure 4A-F; Concise Methods, Cell Culture and Western Blot
Renin- or prorenin-induced ERK1/2 phosphorylation: Phosphorylated ERK1/2 immunoblot with unphosphorylated ERK loading control; at least three different stimulation experiments per protocol. No numerical equivalence margin reported. — Results, Neither Aliskiren nor HRP Affects Renin and Prorenin-Induced ERK1/2 Phosphorylation in Human Coronary VSMC; Figure 4A-F; Concise Methods, Cell Culture and Western Blot
