Inorganic phosphate regulates the contraction‐relaxation cycle in skinned muscles of the rabbit mesenteric artery.

Abstract

SUMMARY1. The effects were investigated of inorganic phosphate (Pi) on the Ca2+-dependent and Ca2+-independent contractions evoked in chemically skinned smooth muscles of the rabbit mesenteric artery.2. The relation between the concentration of Ca2+ and tension showed a sigmoidal curve in the range ofpCa 7-5 5. Pi (over 1 mM) inhibited the Ca2+-induced contraction, shifted the pCa-tension curve to the right and increased the Hill number from 2 to 3. Calmodulin did not change the Hill number and attenuated the inhibitory action of Pi as estimated from the shift of the curve, but this agent did not modify the increased Hill number in the presence of Pi.3. P1 consistently inhibited the Ca2+-independent contractions provoked by application of trypsin-treated myosin light chain kinase, of MgATP following adenosine-5'-o-(3-thiotriphosphate) (ATP S) and Ca2+, and of a solution containing high Mg2+. These inhibitory actions of Pi were inversely proportional to the amplitude of the contraction. When Pi was applied simultaneously with ATP S and Ca2+, there was no change in the amplitude of Ca2+-independent contractions provoked by the application of MgATP.4. The amplitude of the rigor contraction evoked by ATP-free solution was less than 70 ofthat of the 10 /uM-Ca2+-induced contraction. When ATP was removed from the solution during the Ca2+ contraction, the rigor contraction was also generated.Pi did not inhibit either type of contraction.5. With a decrease in the concentration of Ca2+ from 10/M to below 1 nm, the tissue relaxed at a slower rate than the rate of rise of the Ca2+-induced contraction. The slow relaxation was not modified by a change in the concentration of EGTA or addition of 1 /tM-calmodulin.6. Pi reduced, and high Mg2+ prolonged the time required for the relaxation. This action of Pi was not prevented in the presence of calmodulin or of high Mg2+.

Effects of pinacidil on contractile proteins in high K+‐treated intact, and in β‐escin‐treated skinned smooth muscle of the rabbit mesenteric artery

“…Following application of 10pM Ca2 + to the strips, the solution was rinsed off with Ca2 +-free solution containing 4mM EGTA and no ATP and thus a rigor contraction was produced, as described previously (Itoh et al, 1986a). Pinacidil (10pM) had no effect on this rigor contraction (not shown) suggesting that pinacidil did not inhibit actin-myosin interactions directly.…”

“…The methods used to prepare skinned muscle and the composition of the solutions have been described elsewhere (Itoh et al, 1986a;Kobayashi et al, 1989). lonomycin (1,IuM) was present throughout the experiment to deplete the intracellularly stored Ca2 .…”

“…The concentration of K+ was modified as required by replacing NaCl with KCl, For experiments on skinned muscle, the composition of the relaxing solution was (mM): potassium methanesulphonate (KMs) 87, piperazine-N-N'-bis-(2-ethanesulphonic acid) (PIPES) 20, Mg(Ms)2 5.1, ATP 5.2, phosphocreatine 10, EGTA 4 and ionomycin 1 M. Various Ca2 + concentrations were prepared by adding appropriate amounts of Ca(Ms)2 to 4mM EGTA, based on the calculation given previously (Itoh et al, 1986a). The pH of the solution was adjusted to 7.1 at 25°C with KOH and the ionic strength was standardized at 0.2 by changing the amount of KMs added.…”

Consequences of metabolic inhibition in smooth muscle isolated from guinea‐pig stomach

“…This was presumably caused by a decrease in [Pé], which attenuates Ca¥-induced tension by shifting the Ca¥-tension relationship (e.g. Itoh, Kanmura & Kuriyama, 1986), and by an increase in the free-energy change for ATP hydrolysis. Similar potentiation of the tension has been reported in sheep aorta under exposure to 2-deoxyglucose-containing solutions (Hardin et al 1992b).…”

Effect of KC399, a newly synthesized K+ channel opener, on acetylcholine‐induced electrical and mechanical activities in rabbit tracheal smooth muscle

“…The methods used to skin the muscle strips and the compositions of the solutions used have been described elsewhere (Itoh et al, 1986;1992;1994b). Briefly, skinned muscle preparations were obtained by treatment with 25 gM P-escin in Ca2"-free solution containing EGTA and ATP ('relaxing solution') for 25 min, after the constant amplitude of contraction induced by 128 mM K+ had been recorded.…”