Calcium compartments in vascular smooth muscle cells as detected by aequorin signal

Abstract

2 A noradrenaline (10 pM)-induced sustained contraction was associated with a sustained increase in the fura-PE3 signal, or a transient increase followed by small sustained increase in the aequorin signal. A high K+-induced contraction was associated with a sustained increase in both the fura-PE3 and aequorin signals. 3 A second application of noradrenaline or high K+ induced reproducible contractions and fura-PE3 signals. In contrast, the aequorin signal resulting from a second application of noradrenaline or high K+ was much smaller than the first signal. 4 Following a 13 h but not a 3 h resting period, the aequorin signal stimulated by noradrenaline or high K+ recovered, without any change in the contractile response.5 In Ca2"-free solution, high K+ was ineffective, whereas noradrenaline induced only a small aequorin signal and contraction compared to those obtained in the presence of external Ca2". After the addition of Ca2", the first application of noradrenaline induced a large aequorin signal and a large contraction, although a second application induced a much smaller aequorin signal accompanied by a large contraction.6 These results suggest that high K+ and noradrenaline increase Ca2" in at least two cytosolic compartments; a compartment that is coupled to the contractile mechanism ('contractile' Ca2+ compartment; major portion of cytoplasm containing contractile elements) and a compartment that is not coupled to contractile mechanisms ('non-contractile' Ca2+ compartment; small sub-membrane area that does not contain contractile elements). On stimulation, the Ca2+ level in the 'contractile' compartment may increase to a level high enough to stimulate myosin light chain kinase but not so high as to consume aequorin rapidly. In contrast, the Ca2+ level in the 'non-contractile' compartment may increase so greatly that aequorin in this compartment is rapidly consumed. These two compartments may be separated by a diffusion barrier and, during a resting period, aequorin may slowly diffuse from the 'contractile' compartment to the 'non-contractile' compartment and thus restore the full aequorin signal. An increase in Ca2+ in the 'non-contractile' compartment seems to be dependent mainly on Ca2+ influx and partly on Ca2+ release.

Pertussis toxin‐sensitive Gi‐proteins and intracellular calcium sensitivity of vasoconstriction in the intact rat tail artery

“…Although the parameters generally taken as criteria for a satisfactory use of fura-2 were not modi®ed by PTX (see above) this does not exclude the fact that there may be di erences between the fura-2 signal and the [Ca 2+ ] i associated with the contractile apparatus of the cell. Van Breemen (1977) Other authors have suggested that the latter domain does not contain contractile elements (Abe et al, 1995;. As (1) sarcoplasmic reticulum function regulates both [Ca 2+ ] pm and [Ca 2+ ] c and (2) agonists such as histamine inhibit whereas high KCl enhances sarcoplasmic ®lling with Ca 2+ (Rembold & Chen, 1998) (Rembold & Chen, 1998), determination of the aequorin/fura-2 ratio (as an indicator of focal increases in [Ca 2+ ] i (Van Riper et al, 1996)), could be used to evaluate potentially di erent e ects of NA and KCl on calcium microdomains.…”

Effects of cyclopiazonic acid and ryanodine on cytosolic calcium and contraction in vascular smooth muscle

“…In brief, muscle strips were treated with Ca2+-free physiological salt solution with 10 mM EGTA at 2°C for 15 min followed by an incubation with the medium containing 1 mM EGTA at 2°C for 20 min. The latter solution was similar to that developed for organ transplantation (Beltzer & Southard, 1988;Maurer et al, 1990) (Abe et al, 1995b). In the present study, experiments were started when reproducible steady responses had been reached after several applications of NA or high K+.…”

“…Aequorin was loaded as reported previously (Abe et al, 1995b). In brief, muscle strips were treated with Ca2+-free physiological salt solution with 10 mM EGTA at 2°C for 15 min followed by an incubation with the medium containing 1 mM EGTA at 2°C for 20 min.…”

“…changed (Abe et al, 1995b). From these results, we postulated that high K+ and NA may increase Ca2" in at least two cytosolic compartments: a compartment that is responsible for the contractile activation ('contractile' Ca2+ compartment; the major portion of cytoplasm containing contractile elements) and a compartment that is unrelated contractile activation ('non-contractile' Ca21 compartment; a small sub-membrane area that does not contain contractile elements).…”

“…Such a superficial buffer barrier may generate a high Ca2" compartment in restricted diffusion spaces between the plasma membrane and SR (for review see van Breemen et al, 1995). This Ca2" space may correspond to the non-contractile Ca2+ compartment that has been postulated by Abe et al (1995b). To examine whether the non-contractile Ca2" compartment is located adjacent to SR and whether the Ca2+ concentration in this compartment is regulated by SR, we studied the effects of an inhibitor of the SR Ca21 pump, cyclopiazonic acid (Kurebayashi & Ogawa, 1991;Uyama et al, 1992;Darby et al, 1993), an inhibitor of the Ca2"-induced Ca2 release from SR, ryanodine (Ito et al, 1986;Hwang & van Breemen, 1987), and an activator of Ca2+-induced Ca2+ release, caffeine (Endo, 1977), on [Ca2+], measured with aequorin and muscle tension in the ferret isolated portal vein.…”