Cellular Ca2+ monitored by aequorin in adenosine-mediated smooth muscle relaxation

Abstract

We studied the effect of adenosine on cytoplasmic ionized calcium-force relationships in vascular smooth muscle (VSM) and determined the dose dependence of the observed effects. The bioluminescent protein aequorin was used as an index of cytoplasmic ionized calcium and was chemically loaded into ferret portal vein strips. The VSM strips were contracted with 33 mM potassium (K+), 5 X 10(-6) M phenylephrine (PE), or electrical stimulation. Force and aequorin light, i.e., cytoplasmic ionized calcium, were simultaneously recorded. Adenosine pretreatment (3.7 X 10(-6) M) reduced both force and light responses in contractures with K+, PE, or electrical stimulation. In contrast, the addition of adenosine during PE or K+ contractions decreased force without a change in light. Dose-response curves for the effects of adenosine on K+ contractures indicated that at low doses adenosine decreases force and cytoplasmic ionized calcium but that at high concentrations (greater than 3.7 X 10(-6) M) adenosine increases light and apparently relaxes VSM by desensitizing the myofilaments to cytoplasmic ionized calcium.

The role of calcium in genetic hypertension.

“…These results confirmed but extended the observed association between reduced force generation by adenosine in ferret portal vein strips and the reduced cytoplasmic free calcium concentration, as determined by aqueorin, a bioluminescent protein. 65 They also corroborated the calcium flux data across coronary arteries in adenosine-treated dogs 67 and reinforced the results of calcium uptake in adenosine-treated intact and cultured vascular smooth muscle cells.…”

“…Decreased contractility of vascular smooth muscle in response to a variety of vasodilatory and antihypertensive agents, such as adenosine, 65 Ca fluxes that would predict such a reduction. On the other hand, the increased vascular reactivity observed in SHR, 67 -93~% has been attributed to the membrane leakiness to calcium, similar to that for sodium, potassium, and chloride, 97 • 98 although, as will be discussed, the evidence for defective membrane calcium regulation is largely indirect.…”

Mechanical properties of gastrointestinal smooth muscle

“…In chemically skinned uterus muscle that was completely dephosphorylated by a specific phosphatase, complete relaxation occurred even in the presence of saturation levels of Ca2+ and CaM (130). During normal relaxation it is evident that maintained force can be supported by a cellular Ca2+ concentration significantly lower than that responsible for the initial phosphorylation, because aequorin measurements of Ca2+ activity typically show a large initial peak followed by a decline to near basal values (38,99,216,218,233). The finding that this maintained force is abolished by the relaxation-associated Ca2+ withdrawal mechanisms again points to the presence of additional regulatory mechanism(s) with different Ca2+ sensitivities.…”

MARCKS is a major PKC-dependent regulator of calmodulin targeting in smooth muscle

“…Ferrets were anaesthetized with chloroform and the portal vein from each ferret was quickly removed to an oxygenated (95% O 2 /5% CO 2 ) physiological saline solution (PSS; 120 mM NaCl, 5.9 mM KCl, 25 mM NaHCO 3 , 11.5 mM dextrose, 2.5 mM CaCl 2 , 1.2 mM MgCl 2 , 1.2 mM NaH 2 PO 4 , pH 7.4) for dissection as previously described (Bradley and Morgan, 1985). 51 mM KCl PSS was prepared by adding 45.1 mM KCl instead of an equivalent amount of NaCl.…”