Kooichi Saida

Mechanism of Ca++ antagonist-induced vasodilation. Intracellular actions.

We studied the effects of Ca++ antagonists on intact and skinned muscles of rabbit mesenteric artery. Intact muscle contractions were inhibited by 10−6M diltiazem, whereas greater levels were required to abolish contractions in skinned muscle fibers. In contrast, nisoldipine had no effect on skinned muscle contractions, although it inhibited, almost completely, the contraction of intact muscle at concentrations below 10−6M. In the presence of EGTA, norepinephrine-induced contractions result from a release of Ca++ from an intracellular store. Diltiazem inhibited these contractions at concentrations between 10−6 and 10−4M. Higher doses were required in studies with skinned muscle preparations. Unlike diltiazem, nisoldipine only partially inhibited the Ca++-free norepinephrine-induced contractions in the range of 10−7 to 10−5 M. From these results, we assumed that at low concentrations (below 10−6 M), diltiazem induced relaxation by blocking Ca++ influx, whereas at relatively high concentrations (above 10−6 M), an inhibition of Ca++ release from an intracellular store also occurred. A similar conclusion was reached regarding the mechanism whereby nisoldipine inhibits force developments.

Extracellular Ca2+ dependence and diltiazem inhibition of contraction in rabbit conduit arteries and mesenteric resistance vessels.

The dependence of norepinephrine-(NE-) and high potassium (80 mM K) depolarization-induced contractions on extracellular versus intracellular calcium (Ca2+) pools was studied in strips of rabbit superior mesenteric artery and its branches, and in perfused mesenteric resistance vessels. 80 mM K contractions were abolished in the presence of 0 Ca2+ 2 mM EGTA in all arteries studied, whereas 10(-5) M NE-induced contractions and stimulated 45Ca efflux in 0 Ca2+ 2 mM EGTA decreased in a graded fashion from proximal to distal arteries. These data indicate a decreasing release of intracellular Ca2+ and an increasing dependence on extracellular Ca2+ for NE-induced contractions as one proceeds from proximal to distal arteries. This pattern was paralleled by increasing sensitivity of NE-induced contractions to inhibition by diltiazem (10(-9)-10(-4) M) from proximal to distal arteries, while inhibition of 80 mM K contractions was similar in all vessels. 45Ca influx induced by 10(-5) M NE in the resistance vessels, wherein the NE does not release intracellular Ca2+, is approximately 10,000-fold more sensitive to the action of diltiazem than that in the aorta. However, when the aorta is activated by 10(-8) M NE, it becomes more sensitive to inhibition by diltiazem than when it is activated by 10(-5) M NE. The former NE concentration does not release intracellular Ca2+ in the aorta, whereas the latter does. Thus, it appears that NE-induced 45Ca influx is most susceptible to inhibition by diltiazem when the NE has not also released intracellular Ca2+. We suggest that the release of intracellular Ca2+ by NE may make its stimulated Ca2+ influx less susceptible to inhibition by diltiazem.

Effects of Ca antagonists on Ca fluxes in resistance vessels

We have examined contractions and 45Ca fluxes induced by norepinephrine (NE) and 80 mM potassium (high K) depolarization and their inhibition by diltiazem in rabbit mesenteric resistance vessels. Contraction induced by both NE and high K depended almost completely on extracellular Ca. Dose-response curves for diltiazem inhibition of NE (10(-5) M) and high K contractions showed ED50 values of 1 X 10(-8) and 6 X 10(-7) M, respectively, indicating that the receptor-operated channel (ROC) was more sensitive than the potential-operated channel (POC) to the action of diltiazem. Diltiazem (10(-6) M) was shown to inhibit NE- and 80 mM K-stimulated 45Ca influx effectively by 87 +/- 15 and 85 +/- 10%, respectively. Comparison of these data to those obtained from aorta suggest that although the sensitivity of the POC is approximately the same in aorta and mesenteric resistance vessels, the sensitivity of the ROC is much greater in the latter. This increased sensitivity is paralleled by a greatly decreased role of intracellular Ca release in NE contraction in mesenteric resistance vessels.

Characteristics of the Norepinephrine-Sensitive Ca2+ Store in Vascular Smooth Muscle

A comparison was made between the properties of the norepinephrine- and caffeine-sensitive Ca2+ store in both intact and skinned smooth muscle of the rabbit mesenteric artery. After a first application of 10(-5) M norepinephrine, reapplication of norepinephrine did not induce a second contraction in Ca2+-free medium. However, following this sequence 25 mM caffeine still induced a large contraction. The rates of Ca2+ leakage and Ca2+ filling of the norepinephrine-sensitive store were much faster than those of the caffeine-sensitive one. The amplitude of the norepinephrine-induced contraction in Ca2+-free medium also depended on the amount of Ca2+ present in the caffeine-sensitive store. In the saponin-treated skinned muscle caffeine induced a Ca2+ release only after loading with Ca2+, whereas norepinephrine was unable to induce Ca2+ release in the skinned preparation even after loading with Ca2+. The release of Ca2+ from the caffeine-sensitive store could be activated by Ca2+ itself when the skinned muscle was loaded with Ca2+ above 10(-6) M. These results suggest that the norepinephrine-sensitive Ca2+ store is distinct from a large fraction of the caffeine-sensitive one, and that the norepinephrine-sensitive store is close to the cell membrane. In vascular smooth muscle, under physiological conditions, Ca2+ released from the norepinephrine-sensitive store by norepinephrine may induce Ca2+ release from the caffeine-sensitive Ca2+ store which may be comprised of the sarcoplasmic reticulum.

A possible Ca2+-induced Ca2+ release mechanism mediated by norepinephrine in vascular smooth muscle

In vascular smooth muscle, the norepinephrine-sensitive Ca2+ store is distinct from the sarcoplasmic reticulum. Under physiological conditions, Ca2+ released from the norepinephrine-sensitive store may induce Ca2+ release from the sarcoplasmic reticulum, i.e. norepinephrine mediated activation may involve a Ca2+-induced Ca2+ release mechanism.

The specific GTP requirement for inositol 1,4,5-trisphosphate-induced Ca2+ release from skinned vascular smooth muscle.

Exogenous GTP was required for the induction of Ca2+ release from smooth muscle SR by IP3 if endogenous GTP was depleted. NaN3 could function as a partial substitute for GTP as a cofactor for the IP3-induced Ca2+ release from the SR. In contrast to the IP3-induced Ca2+ release, caffeine-induced Ca2+ release from the SR did not require GTP. Pertussis toxin inhibited the IP3- induced Ca2+ release from the SR, whereas it had no effect on caffeine-induced Ca2+ release. These results indicate that in smooth muscle two different Ca2+ release-channels exist in the SR: (a) activated by IP3, and (b) activated by caffeine or Ca2+.

Modulation of ryanodine-induced Ca2+ release in amphibian skeletal muscle

We examined effects of ryanodine on tension in intact and skinned amphibian skeletal muscle. 100 microM ryanodine (RY) alone in the frog Ringers solution (FR) produced tension in the intact muscle reaching its peak by 1 h; 10 min treatment with RY augmented depolarization-induced tension and prevented a subsequent caffeine-induced contraction. In contrast, RY in Ca2+-free FR was unable to produce tension, after which caffeine produced irreversible tension. In skinned fibers, RY at pCa 6.5 produced tension and abolished a subsequent caffeine-induced contraction; while Ry in 2 mM EGTA did not produce tension. These data indicate that RY, in the presence of CA2+, releases CA2+ from the SR resulting in subsequent depletion of CA in the SR.

Sarcoplasmic Reticulum of Mesenteric Arteries Buffers Stimulated Ca 2+ Entry

The effect of sarcoplasmic reticulum (SR) Ca2+ depletion on the efficacy of Ca2+ entry to cause contraction was investigated by sequentially exposing small mesenteric arteries to Ca2+-free experimental solutions containing EGTA with or without agonist. The agonist-mediated Ca2+ depletion of the SR markedly reduced the rate and magnitude of the contraction due to Ca2+ influx through voltage-gated channels. This buffering of Ca2+ influx by the norepinephrine-sensitive SR fits in with a two compartment model for the SR in mesenteric arteries in which norepinephrine, through the intermediate action of inositol-1, 4, 5-trisphosphate, releases Ca2+ from the superficial SR, which can be replenished from the deeper SR. This functional model was deduced from earlier observations in small mesenteric arteries that norepinephrine in Ca2+-free experimental solutions activated a biphasic contraction with an amplitude of half the magnitude of the rapid monophasic contraction induced by caffeine. It is concluded that norephinephrine potentiates the contractile effect of Ca2+ entry in mesenteric arteries by shunting out the superficial buffer barrier.