A, a representative tension trace showing that 10 μm nifedipine completely abolished an 80 mm [K⁺]_o-mediated tonic contraction in rings of rabbit IVC pretreated with 10 μm phentolamine. B, L-type VGCC blockade with 10 μm nifedipine partially inhibited a PE-mediated tonic contraction (P < 0.05, Wilcoxon signed-rank test), while additional ROC/SOC blockade with 50 μm SKF96365 completely abolished the remaining tonic contraction.

Blockade of Ca²⁺ entry through NCX with 100 μm 2′,4′-dichlorobenzamil (2,4-DCB) led to a large inhibition of PE-induced tonic contraction (A) and a complete inhibition of PE-induced [Ca²⁺]_i oscillations (B) in rings pretreated with 10 μm nifedipine. The remaining contraction was abolished by application of 50 μm SKF96365 (SKF) while the non-oscillating [Ca²⁺]_i was correspondingly reduced. (* Control refers to the amplitude of PE-mediated tonic contraction in rings pretreated with 10 μm nifedipine.)

IP₃ generated as a consequence of α-adrenergic activation releases Ca²⁺ towards the myoplasm to activate the myofilaments. Subsequent Ca²⁺ removal occurs partially through the PMCA, while the remainder is taken up into the SR by the SERCA. ROCs/SOCs, activated by receptor activation and SR depletion, allow entry of mainly Na⁺ and some Ca²⁺. This Na⁺ entry raises [Na⁺]_i in a restricted space between the plasma membrane and the SR membrane and drives NCX in the reverse mode to supply Ca²⁺ to SERCA to refill the SR lumen, thereby completing the cycle. PE, phenylephrine; NCX, sodium/calcium exchanger; ROC/SOC, receptor-operated channel/store-operated channel; SERCA, sarcoplasmic- endoplasmic reticulum Ca²⁺-ATPase; L-type VGCC, L-type voltage-gated Ca²⁺ channel; PMCA, plasma membrane Ca²⁺-ATPase; IP₃R, IP₃-sensitive SR Ca²⁺ release channel; SR, sarcoplasmic reticulum.

A, sets of time-series images (1-3) are displayed to identify the spatiotemporal patterns of [Ca²⁺]_i rise elicited by different stimuli. Still images of a field of smooth muscle cells are shown to illustrate the rectangular regions (outlined in white in the leftmost images) from which each set of time-series images was derived. The rectangular regions contain a segment of one representative ribbon-shaped SMC and are enlarged and contrast-enhanced to facilitate the visualization of the spatiotemporal patterns of [Ca²⁺]_i rise in these time-series. Variations in fluorescence intensity (F₅₂₅) directly reflect changes in [Ca²⁺]_i. In addition, the changes in F₅₂₅ over time from selected areas (area 1, 2 and 3 outlined in white) spaced out across the longitudinal axis of each depicted SMC are illustrated on the right in the F₅₂₅-time traces (a.u., arbitrary units). SR Ca²⁺ release with 25 mm caffeine caused an initial [Ca²⁺]_i elevation that was initiated at a distinctive intracellular locus (time 1.07 s) and subsequently propagated along the longitudinal axis of the SMC in a regenerative, wave-like fashion. The F₅₂₅-time traces (set 1) from the three intracellular areas indicate a sequential rise in [Ca²⁺]_i over time as the Ca²⁺ signal was initiated at area 1 and subsequently propagated in a wave-like fashion through area 2 and finally to area 3. Stimulation by 5 μm PE also elicited a wave-like Ca²⁺ signal as the F₅₂₅-time traces (set 2) demonstrated a sequential rise of [Ca²⁺]_i over time in the three intracellular areas. In contrast, Ca²⁺ influx across the plasma membrane (PM) stimulated with 80 mm extracellular K⁺ PSS (80 mm [K⁺]_o), caused an initial Ca²⁺ elevation that appears to be non-wave-like in nature. Such a non-wave-like pattern is clearly demonstrated by the F₅₂₅-time traces (set 3), which indicate a synchronized rise in [Ca²⁺]_i from the three intracellular areas. Thus, the wave-like pattern of the [Ca²⁺]_i rise within a series of PE-induced [Ca²⁺]_i oscillations resembles that seen in response to caffeine. This suggests that individual Ca²⁺ spikes elicited by PE are the result of regenerative SR Ca²⁺ release. The scale bars shown represent 5 μm. B, a representative Ca²⁺ trace is shown that displays the temporal changes in [Ca²⁺]_i determined in a 1.36 μm² cytoplasmic region of a single SMC from the rabbit IVC. SERCA blockade with 10 μm CPA resulted in both the abolishment of PE-induced [Ca²⁺]_i oscillations and a sustained elevation in [Ca²⁺]_i. C, PE-elicited transient [Ca²⁺]_i oscillations in tissues pretreated with 10 μm nifedipine (L-type VGCC blocker) and 50 μm SKF96365 (ROC/SOC blocker); additional pretreatment with 75 μm 2-APB (IP₃R channel blocker) abolished this transient Ca²⁺ signal. Pretreatment of the tissues with 75 μm 2-APB alone also abolished PE-mediated Ca²⁺ signals completely.

As shown in the representative traces, which indicate temporal changes in [Ca²⁺]_i from a 1.36 μm² sub-cellular region, L-type VGCC blockade with 10 μm nifedipine (Nif) did not abolish PE-induced [Ca²⁺]_i oscillations. It reduced the frequency of PE-induced [Ca²⁺]_i oscillations, but did not affect the amplitude or the apparent velocity of the oscillatory Ca²⁺ waves. ROC/SOC blockade with 50 μm SKF96365 (SKF) in addition to L-type VGCC blockade abolished PE-induced [Ca²⁺]_i oscillations completely. The frequency of the [Ca²⁺]_i oscillations was derived by counting the number of Ca²⁺ waves generated in a single SMC over a time period of 30-60 s. For estimation of the apparent velocity of the Ca²⁺ wave, two subcellular 1.36 μm² regions separated by distance x (Δx) were selected and the time lag (Δt) in the onset of Ca²⁺ rise between the two regions was determined. The fraction of Δx over Δt yields the apparent velocity of the Ca²⁺ wave. (* Statistical significance with Mann-Whitney U test.)

Blockade of the NCX operating in the reverse with 10 μm KB-R7943 led to a large inhibition of PE-induced tonic contraction (A) in rings pretreated with 10 μm nifedipine and complete inhibition of PE-induced [Ca²⁺]_i oscillations (B). The remaining contraction was abolished with 50 μm SKF96365 (SKF). (* Control refers to the amplitude of PE-mediated tonic contraction in rings pretreated with 10 μm nifedipine.)