PFI-3 induces vasorelaxation with potency to reduce extracellular calcium influx in rat mesenteric artery

Agents

PFI-3 (E)-1-(2-hydroxyphenyl)-3-((1R,4R)-2-pyridin-2-yl-2,5-diazabicyclo[2.2.1] heptan-5-yl) prop-2-en-1-one, ODQ 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, thapsigargin (TG) and phenylephrine (PE) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Acetylcholine chloride (Ach) was purchased from Harvest Pharmaceutical Co. Ltd. (Shanghai, China). L-NAME, N(ω)-nitro-L-arginine methyl ester, glibenclamide (Gli), and tetraethylammonium (TEA) were purchased from MedChemExpress LLC (Shanghai, China). Fluo-3/AM was purchased from Life Technologies (Invitrogen, Waltham, MA, USA). Ach, PE, physiological salt solution (PSS), and high-K⁺ salt solution (KPSS) were dissolved in double distilled water, and PFI-3, ODQ, L-NAME, TG, Gli and TEA were dissolved in DMSO (Tianjin Fuyu Fine Chemical Co., Ltd, Wuqing District, China). Arterial smooth muscle cells (A10) were purchased from the American Type Culture Collection (ATCC). KPSS, was composed of (in mM): 74.4 NaCl, 60 KCl, 1.17 MgSO₄ ⋅ 7H₂O, 1.18 KH₂PO₄, 14.9 NaHCO₃, 1.6 CaCl₂, 5.5 D-glucose, and 0.026 EDTA. The PSS solution was composed of (in mM): 130 NaCl, 4.7 KCl, 1.17 MgSO₄ ⋅7H₂O, 1.18 KH₂PO₄, 14.9 NaHCO₃, 1.6 CaCl₂, and 5.5 D-glucose.

Animals and mesenteric artery tension measurement

Adult male Sprague-Dawley (SD) rats (12 weeks old, male, body weight 320–350 g) were purchased from the Experimental Animal Center of Harbin Medical University (Grade II). The adult male SD rats were maintained on a standard light/dark cycle (12 h/12 h) at 30–70% humidity and a constant temperature (23 ± 3 °C). Standard chow and water were freely provided to the SD rats. All animal experiments were performed in accordance with NIH guidelines (Guide for the Care and Use of Laboratory Animals) and approved by the Institutional Animal Care and Use Committee of Harbin Medical University (IRB3102619). The adult male SD rats were anesthetized with sodium pentobarbital (40 mg/kg, ip). The animals were euthanized via cervical dislocation following anesthesia, and then the intact mesenteric artery was then quickly isolated and transferred to a cold PSS (at 4 °C) preoxygenated with 95% O₂ and 5% CO₂. The adipose tissue and residual blood on the mesenteric artery were promptly removed, and a 2 mm long section of the vessel ring was excised. To construct an endothelium-denuded vascular ring model, the mechanical method was employed; specifically, rat whiskers with a minimum diameter of 80 µm were slowly rubbed on the inner surface of the vascular ring to remove the endothelial cells. The absence of vasorelaxation in response to 1 µM Ach indicated successful endothelial cell disruption. The mesenteric artery ring was fixed with two metal wires threaded through it in a bath containing a precooled PSS solution continuously supplied with 95% O₂ and 5% CO₂ using a microvascular tension measurement device (DMT620, Danish Myo Technology, Aarhus, Denmark). The mesenteric artery rings were allowed to equilibrate for 60 min before undergoing a wake-up procedure involving reactivation of the mechanical, functional, and signaling functions of the vessel via stimuli with PE and KPSS. The isometric systolic tension of the mesenteric artery was recorded using a DMT620 microvascular tension measurement device.

MTT assays

After the cells adhered to 96-well plates, they were treated with varying concentrations of PFI-3. Subsequently, 200 µL of MTT solution was added to each well and the cells were incubated for 20 min at 37 °C. After incubation, the supernatant was removed, and 150 µL of DMSO was added to dissolve the formazan crystals formed by the viable cells. The optical density was measured at 490 nm using an absorbance spectrophotometer.

Measurement of the cytosolic [Ca²⁺]_i in VSMCs

A10 cells were cultured and washed three times with serum-free high glucose DMEM. Then, 5 µM fluo-3/AM was added and the cells were incubated at 37 °C in darkness for 30 min. The culture medium was discarded, and the cells were washed three times with warm Ca²⁺-free KPSS (at 37 °C). Subsequently, 1 ml of warm calcium-free KPSS (at 37 °C) was added, and the fluorescence intensity of fluo-3 in the cells was measured.

Whole-cell patch clamp experiments

All whole-cell patch-clamp experiments were performed using an Axopatch 700B amplifier. Whole-cell currents were sampled at 1 kHz and filtered at 2 kHz. The I_Ca,L in A10 cells was recorded in extracellular solution containing (in mM) 120 TEA, 10 HEPES, 1 MgCl₂•6H₂O, 10 CsCl, 10 glucose, and 1.8 CaCl₂ (pH adjusted to 7.30 with CsOH). The pipette solution contained (in mM) 120 CsCl, 1 MgCl₂•6H₂O, 40 CsOH, 5 Mg-ATP, 11 EGTA, and 10 HEPES (pH adjusted to 7.30 with CsOH). The cells were maintained at a potential of −50 mV.

Statistical analysis

All data are presented as mean ± standard deviation (SD). Data were analyzed using the GraphPad Prism (v8.0; GraphPad Software, USA) software.

Two-tailed Student’s paired and unpaired t tests were used to analyze the differences between two variables. Differences among groups were evaluated using one-way ANOVA followed by Tukey’s test or Dunnett’s test for post-hoc comparison when appropriate. P-values <0.05 were considered statistically significant.

PFI-3 did not exhibit cytotoxic effects on A10 cells

The animal experimental design is presented in Fig. 1. To investigate the cytotoxic effect of PFI-3 on A10 cells, the MTT assay was utilized to examine cell viability (Fig. 2). Consistent with previous studies (Gerstenberger et al., 2016), PFI-3 did not substantially affect cell viability at doses up to 5 µM. The maximum dose of PFI-3 used in this study was 4 µM.

PFI-3 dose-dependently induced vasorelaxation of rat mesenteric artery after PE and high K⁺ -evoked constriction

As depicted in Fig. 3A, the mesenteric arteries were pre-constricted by PE (5 µM), and after the vasoconstriction reached the maximum level and stabilized, the endothelium-dependent vasodilator Ach (1 µM) was administered. The occurrence of Ach-induced relaxation confirmed that the endothelium of the mesenteric arteries was intact. Compared to the DMSO treatment, PFI-3 resulted in concentration-dependent relaxation in rat mesenteric arteries with both intact endothelium (Fig. 3B) and denuded endothelium (Fig. 3D).

Similar to above, high K⁺ could also induce vasoconstriction of rat mesenteric artery, PFI-3 treatment relaxed the vasoconstriction, regardless of the intact or denuded endothelium (Fig. 4).

PFI-3 preincubation inhibited PE and high K⁺-evoked contraction in rat mesenteric artery

The study further investigated the preventive effects of PFI-3 against PE- and high K⁺-induced vasoconstriction. As shown in Figs. 5–6, pretreatment with PFI-3 (4 µM) for 20 min significantly inhibited PE- and KPSS-induced vasoconstriction in endothelium-intact and -denuded rat mesenteric arteries.

PFI-3 induced vasorelaxation without involving the nitric oxide (NO)/ soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway in rat mesenteric arteries

To assess the influences of the nitric oxide synthase inhibitor, L-NAME, and the guanylate cyclase inhibitor ODQ on PFI-3-induced arterial vasodilation, mesenteric arteries were exposed to L-NAME (100 µM) or ODQ (20 µM) for 20 min. As depicted in Figs. 7A and 7B, neither L-NAME nor ODQ showed any effect on PFI-3-induced vasorelaxation.

PFI-3-induced vasorelaxation was irrelevant to K⁺ channel blockers in rat mesenteric arteries

Incubation with K⁺ channel blockers, such as Gli (20 µM), or TEA (1 mM), did not alter PFI-3-induced relaxation of endothelium-intact mesenteric arterial rings pre-contracted by PE (5 µM) (Fig. 8).

PFI-3 affected extracellular Ca²⁺-induced vasoconstriction

High K⁺ induced contractile response of mesenteric arterial rings with denuded endothelium, due to the depolarization of VSMCs and extracellular Ca²⁺ influx through voltage-dependent calcium channels (VDCCs) (Jones, Jones & Channer, 2003). In high-K⁺ PSS solution with Ca ²⁺-free, addition of CaCl₂ (0.3–3 mM) led to a gradual increase of mesenteric arterial ring tension. Pretreatment with PFI-3 reduced the amplitude of contraction induced by extracellular CaCl₂ (Fig. 9A). During PE-induced contraction, intracellular calcium stores are mobilized firstly, and this is followed by Ca²⁺ influx through receptor-operated calcium channels (ROCCs) (Ehrlich, 1988; Arruda-Barbosa et al., 2021). Mesenteric arterial rings with denuded endothelium were treated with PE (5 µM) without Ca²⁺, and the vessel tone gradually increased with the addition of CaCl₂ (0.3–3 mM). However, the increased vessel tone was abolished after PFI-3 pretreatment (Fig. 9B).

PFI-3 induced vasorelaxation without involving intracellular Ca²⁺ release

To investigate the involvement of Ca²⁺ release from sarcoplasmic reticulum (SR) in PFI-3-induced vasorelaxation, the effect of TG on rat mesenteric artery rings was assessed. Whereas, TG did not show a significant effect on PFI-3-induced vasorelaxation (Fig. 10).

PFI-3 affected the cytosolic [Ca²⁺] _i in A10 cells

High K⁺ induced depolarized of the membrane potential of VSMCs, leading to the influx of extracellular Ca²⁺ through VDCCs (Betrie et al., 2021). To further elucidate the effect of PFI-3 on high-K ⁺-induced Ca²⁺ influx, the cytosolic [Ca ²⁺] _i was measured in A10 cells using a Fluo-3/AM fluorescent probe. As illustrated in Fig. 11, in Ca²⁺-free KPSS, PFI-3 did not affect the cytosolic [Ca²⁺] _i, but after treatment of CaCl₂ (2 mM) PFI-3 decreased cytosolic [Ca²⁺] _i.

PFI-3 supressed L-type VDCC currents in A10 cells

The above data showed PFI-3 did not affect intracellular calcium release, but affected extracellular calcium entry. Thus, the effects of PFI-3 on extracellular calcium influx currents were measured by whole-cell patch clamp techniques in A10 cells (Guan et al., 2006). Our research showed that PFI-3 inhibited L-type VDCC current densities in A10 cells (Fig. 12).

Statement of ethics

All animal experiments were performed in accordance with the NIH guidelines (Guide for the Care and Use of Laboratory Animals) and approved by the Institutional Animal Care and Use Committee of Harbin Medical University (IRB3102619). The adult Sprague-Dawley rats used in this study were provided by the Experimental Animal Center of Harbin Medical University (Grade II).