Prenatal hypoxia inhibited propionate-evoked BK channels of mesenteric artery smooth muscle cells in offspring

Abstract

As a common complication of pregnancy, gestational hypoxia has been shown to predispose offspring to vascular dysfunction. Propionate, one of short-chain fatty acids, exerts cardioprotective effects via reducing blood pressure. This study examined whether prenatal hypoxia impaired propionate-stimulated large-conductance Ca²⁺ -activated K⁺ (BK) channel activities in vascular smooth muscle cells (VSMCs) of offspring. Pregnant rats were exposed to hypoxia (10.5% oxygen) and normoxia (21% oxygen) from gestational day 7-21. At 6 weeks of age, VSMCs in mesenteric arteries of offspring were analysed for BK channel functions and gene expressions. It was shown firstly that propionate could open significantly BK single channel in VSMCs in a concentration-dependent manner. Antagonists of G protein βγ subunits and inositol trisphosphate receptor could completely suppress the activation of BK by propionate, respectively. Gα_i/o and ryanodine receptor were found to participate in the stimulation on BK. Compared to the control, vasodilation and increments of BK NPo (the open probability) evoked by propionate were weakened in the offspring by prenatal hypoxia with down-regulated Gβγ and PLCβ. It was indicated that prenatal hypoxia inhibited propionate-stimulated BK activities in mesenteric VSMCs of offspring via reducing expressions of Gβγ and PLCβ, in which endoplasmic reticulum calcium release might be involved.

Keywords: large-conductance calcium-activated potassium channels; offspring; prenatal hypoxia; propionate.

Figure 1

Vessel tone responding to stimulations in offspring mesenteric arteries. A, KCl‐mediated constriction; n = 15 each group (not consanguineous). B, Cumulative concentration of PE‐induced response curves. The responses of PE are presented as the percentage of maximal contraction induced by 120 mmol/L KCl; n = 15 each group (not consanguineous). *P < .05, ^bP < .001, HY vs CON. C, Dose‐response curves of propionate on 60 mmol/L KCl‐induced contraction; n = 15 each group (not consanguineous). **P < .01, HY vs CON. D, The sensitivity of vasodilation to propionate (pD₂); n = 15 each group (not consanguineous). **P < .01, HY vs CON

Figure 2

Short‐chain fatty acids receptors are present in rat mesenteric arterial VSMCs. A, Representative immumohistochemistry images of Gpr41 and Olr59 in rat mesenteric arteries (Bar: 20 μm); n = 5. B, Representative immunofluorescence staining images of α‐SMA in primary mesenteric VSMCs. Nuclei were counterstained by DAPI. Bar: 50 μm; n = 5. C, Ethidium bromide‐stained agarose gel of RT‐PCR products amplified from SCFAs receptors in rat mesenteric VSMCs with β‐actin as positive control and eNOS as negative control; n = 5

Figure 3

Prenatal hypoxia reduced sensibility of BK to propionate. A, Dose‐response curves for sodium propionate (SP)‐induced single BK channel activities in mesenteric VSMCs. NPo, open probability; n = 10. B, Butyrate failed to trigger the opening of BK; n = 10. C, Representative BK single‐channel current recordings from inside‐out patches and dwell time of open and closed state of BK channels monitored at + 50 mV; n = 15 each group (not consanguineous). *P < .05, **P < .01, SP‐stimulation vs baseline. D, Basal and SP‐stimulated open probability of BK in mesenteric arteries; n = 15 each group (not consanguineous).*P < .05, SP‐stimulation vs baseline. E, NPo of BK stimulated by SP relative to baseline; n = 15 each group (not consanguineous). #, P < .05, HY vs CON

Figure 4

Open probability of BK induced by propionate with pre‐incubation of pertussis toxin (PTX, A), gallein (B), 2‐aminoethoxydiphenyl borate (2‐APB, C) and ryanodine (D); n = 15 each group (not consanguineous). **P < .01, SP‐stimulated BK in the presence of antagonists vs baseline of BK in the presence of antagonists

Figure 5

Expressions of SCFA receptors and BK subunits in mesenteric arteries. A, mRNA expressions of Gpr41 and Olr59 relative to b‐actin. B, Protein expressions of Gpr41 and Olr59 (top blot) and densitometric analysis normalized to presence of b‐actin (down box & whiskers graph). C, mRNA expressions of Kcnma1 and Kcnmb1 relative to b‐actin. D, Protein expressions of Kcnma1 and Kcnmb1 (top blot) and densitometric analysis normalized to presence of b‐actin (down box & whiskers graph); n = 15 each group (not consanguineous). *P < .05, **P < .01, HY vs CON

Figure 6

Expressions of G protein b subunits, phospholipase C, IP3R and ryanodine receptor in mesenteric arteries. Relative mRNA expressions of G protein b subunits (Gnb, A), phospholipase C (Plc, B), IP3R (C), and ryanodine receptor (RyR, C). Protein expressions of Gnb5 (D), Plcb3 (E), IP3R2, RyR3 (F), and densitometric analysis normalized to b‐actin; n = 15 each group (not consanguineous). *P < .05, **P < .01, HY vs CON

Figure 7

Schematic representation of propionate‐dependent BK activation. Binding of propionate to Gpr41 or Olr59 in VSMCs facilitates the exchange of GDP for GTP on the α subunit of G protein complex, releasing G protein βγ dimer from G protein α subunit. Separated Gβγ dimer activates phospholipase C β (PLCβ), catalysing the formations of inositol 1,4,5‐trisphosphate (IP₃) and diacylglycerol (DAG). Consequently, IP3R/ryanodine receptor (RyR)‐operated endoplasmic reticulum calcium release is stimulated. Finally, BK channel is evoked by the increase of intercellular calcium ([Ca²⁺]_i)