Dissecting out the complex Ca2+-mediated phenylephrine-induced contractions of mouse aortic segments

Abstract

L-type Ca2+ channel (VGCC) mediated Ca2+ influx in vascular smooth muscle cells (VSMC) contributes to the functional properties of large arteries in arterial stiffening and central blood pressure regulation. How this influx relates to steady-state contractions elicited by α1-adrenoreceptor stimulation and how it is modulated by small variations in resting membrane potential (Vm) of VSMC is not clear yet. Here, we show that α1-adrenoreceptor stimulation of aortic segments of C57Bl6 mice with phenylephrine (PE) causes phasic and tonic contractions. By studying the relationship between Ca2+ mobilisation and isometric tension, it was found that the phasic contraction was due to intracellular Ca2+ release and the tonic contraction determined by Ca2+ influx. The latter component involves both Ca2+ influx via VGCC and via non-selective cation channels (NSCC). Influx via VGCC occurs only within the window voltage range of the channel. Modulation of this window Ca2+ influx by small variations of the VSMC Vm causes substantial effects on the contractile performance of aortic segments. The relative contribution of VGCC and NSCC to the contraction by α1-adrenoceptor stimulation could be manipulated by increasing intracellular Ca2+ release from non-contractile sarcoplasmic reticulum Ca2+ stores. Results of this study point to a complex interactions between α1-adrenoceptor-mediated VSMC contractile performance and Ca2+ release form contractile or non-contractile Ca2+ stores with concomitant Ca2+ influx. Given the importance of VGCC and their blockers in arterial stiffening and hypertension, they further point toward an additional role of NSCC (and NSCC blockers) herein.

Fig 1. Kinetic analysis of the isometric contractions of mouse aortic segments by 1 μM PE.

Aortic segments mounted in organ baths produced isometric contractions as shown in (A). Bi-exponential fits of the contractions in A revealed amplitudes (B) and time constants (C) of fast and slow components. Data were acquired at 10 Hz, specific data points (open circles) are shown as mean± sem (n = 23).

Fig 2. Temporal relationship between intracellular Ca²⁺ signal and isometric contraction by PE.

Ca²⁺signal (left, blue) and corresponding isometric tension development (right, red) induced by 1 μM PE (arrow indicates addition) in myograph-mounted endothelium-denuded mouse aortic segments in normal KR solution (A, KR), in Ca²⁺-free KR (B, 0Ca²⁺) and upon re-addition of 3.5 mM Ca²⁺ to the Ca²⁺-free KR solution containing 1 μM PE (C, +Ca²⁺). (D) Sum (B+C) of PE-responses in the absence of Ca²⁺ and upon re-admission of 3.5 mM Ca²⁺ (black) compared with the [Ca²⁺]_i and tension signals by PE in control (blue and red). Data were acquired at 1 Hz, some data points (open circles) are shown as mean± sem (n = 6).

Fig 3. Effects of VGCC blockers on isometric contractions by PE.

Nifedipine, verapamil and diltiazem partly inhibit isometric contractions induced by 1 μM PE in organ bath-mounted aortic segments. A) Contractions induced by 1 μM PE after incubating the segments with 1 to 100 nM nifedipine (n = 4); B) Ca²⁺ channel blocker (CCB) concentration-response curves for the inhibition of contractions induced by 1 μM PE.

Fig 4. VGCC and NSCC contribute to the Ca²⁺ influx-mediated contraction elicited by PE.

The PE-induced contraction following addition of 3.5 mM Ca²⁺ to 0Ca (n = 4) in the absence (black) and presence of 3 μM verapamil (Vera, blue) and B) in the presence of 100 μM 2-APB (green). Verapamil and 2-APB were added before and after the transient contraction by PE respectively. After 600 s, 100 nM levcromakalim (Lev) was added in each condition. C) Mean maximal contractions in control (PE), 3 μM verapamil (+Vera) and 100 μM 2-APB (+2-APB) in the absence (left bars) and presence (+ Lev, hatched bars) of 300 nM levcromakalim. ***: P<0.001, versus PE; ##, ###: P<0.01 and 0.001, + Lev versus control

Fig 5. Modulation of the PE-induced contraction by window VGCC Ca²⁺ influx.

A) The relaxation of the isometric pre-contraction (1 μM PE, red, organ bath) with addition of 300 nM levcromakalim (+ Lev) is reversed by increasing extracellular K⁺ (K_o ⁺) to 11.9 and 17.9 mM (n = 4). B) Representative example of a myograph experiment, showing the increase of intracellular Ca²⁺ (blue) with 1 μM PE (+ PE), decrease with 300 nM levcromakalim (+ Lev) and increase with supplementary addition of 10 mM external K⁺ (+ K_o ⁺) in combination with 30 nM BAY K8644 (+ Bay).

Fig 6. External K⁺- modulation of the PE-induced contraction.

Isometric contractions induced by 1 μM PE at different extracellular K⁺ concentrations in the organ bath. Contractions were measured at 2, 5.9, 10 and 15 mM extracellular K⁺ in control (A) and in the presence of 35 μM diltiazem (B), 100 μM 2-APB (C) or 30 nM BAY K8644 (D). Bi-exponential analysis of the force development by PE revealed the amplitudes of fast and slow force components in control (E). The “steady-state” tonic contractions at 650 s in the absence (control) or presence of diltiazem, 2-APB or BAY K8644 are summarized in (F). *, **, ***: P<0.05, 0.01, 0.001 versus 5.9 mM K⁺ (n = 4–5).

Fig 7. CPA and PE release intracellular Ca²⁺, cause Ca²⁺ influx and produce force by different mechanisms.

Intracellular Ca²⁺ (A) and accompanying tension (B) were measured in aortic segments (n = 6) in the absence (0Ca) and after re-addition of 3.5 mM Ca²⁺ (+Ca) in the presence of 1 μM PE alone (black), in the presence of 10 μM CPA alone (blue) or in the presence of the combination (green). C) Force-calcium graph for the data in A and B with the squares referring to the +Ca data (Ca²⁺ influx) and the circles to the 0Ca data (Ca²⁺ release). *, ***: P<0.05, 0.001 CPA or CPA/PE versus PE

Fig 8. Modulation of the PE-induced contraction by NSCC Ca²⁺ influx.

Isometric contractions induced by Ca²⁺ re-addition (+ Ca) to organ bath mounted aortic segments (n = 4) incubated in 0Ca in the presence of 1 μM PE A) with (blue) or without (white) 10 μM CPA (blue) or B) with 10 μM CPA and 35 μM dilitiazem (Dil) at 5.9 (blue), 35 (red), 65 (green) and 124 (purple) mM K⁺. In A) 35 μM diltiazem (+ dil) was added after 10 minutes to measure the relative amount of VGCC Ca²⁺ influx to the contractions. Finally, 50 μM 2-APB (+ 2-APB) was added to inhibit NSCC. ***: P<0.001 CPA versus control. In B) 50 μM 2-APB was added after 16 minutes to inhibit the contraction due to NSCC Ca²⁺ influx. *, **, ***: P<0.05, 0.01, 0.001 K⁺ versus control.

Fig 9. Effects of intracellular Ca²⁺ increase by CPA on contractions by PE in the presence of diltiazem or 2-APB.

A) Representative example of PE-induced contractions after incubation of a segment with 35 μM diltiazem (red) or 50 μM 2-APB (green). 10 μM CPA was added in both conditions as indicated, after which 50 μM 2-APB (arrow, +2-APB)was added to the diltiazem condition and diltiazem (arrow, +dil) to the 2-APB condition. Figure B summarizes the results (n = 5). In C, the segments were incubated with 50 μM 2-APB and 35 μM diltiazem before the challenge with 1 μM PE. Subsequent addition of 10 μM CPA did not increase the PE-induced contraction. Part D summarizes the results with the change in isometric force by the diltiazem/2-APB in baseline conditions or following addition of 1 μM PE alone (PE) or in combination with 10 μM CPA (PE+CPA) (n = 4).*, **: p<0.05, 0.01 2-APB versus diltiazem condition in B or PE/PE+CPA versus baseline in D.

Fig 10. Scheme showing the Ca²⁺ mediated processes involved in α₁-adrenoceptor stimulation of mouse aortic segments with PE.

PE causes phasic Ca²⁺ increase and concomitant contraction by releasing Ca²⁺ from the SR (event 1). This is accompanied by influx of Ca²⁺ via complex interactions between NSCC and VGCC and the steady-state contraction by PE is determined by the relative contribution of window Ca²⁺ influx via VGCC (very voltage-dependent) and Ca²⁺ influx via NSCC (less voltage-dependent) (event 2). Window VGCC Ca² influx and related contraction are inhibited by diltiazem, membrane potential repolarization with K⁺ or levcromakalim and Ca²⁺ release from non-contractile Ca²⁺ stores by CPA and stimulated by high K⁺ and BAY K844 (event 3). NSCC Ca² influx and related contraction are inhibited by 2-APB and very high K⁺ (strong depolarization to −20 mV or less negative) and stimulated by Ca²⁺ release from non-contractile Ca²⁺ stores with CPA (event 4). CPA causes high Ca²⁺ release from a non-contractile compartment of the SR (event 5). Emptying of the non-contractile Ca²⁺ store with CPA causes large Ca²⁺ influx, which is accompanied with minor contraction in the absence of PE, but which turns the PE-induced contraction to one that is mainly mediated by NSCC. This points to a complex interaction between the non-contractile and contractile SR Ca²⁺ stores and their refilling via VGCC and/or different NSCC.