I-force Relationship Research Articles

Cytosolic calcium concentration-force relation during contractions in the rabbit femoral artery: time-dependency and stimulus specificity.

1. By use of front-surface fluorometry with fura-2-loaded rabbit femoral arterial strips, both the cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored. By utilizing the [Ca2+]i-force curves, we were thus able to examine the temporal changes in the relationships between [Ca2+]i and force ([Ca2+]i-force relationship) during contractions induced by a high external K+ solution, noradrenaline (NA) and 5-hydroxytryptamine (5-HT). 2. The 'basic' [Ca2+]i-force relationship of the Ca(2+)-induced contractions was obtained by the cumulative applications of extracellular Ca2+ (0-10 mM) during 118 mM K(+)-depolarization (Ca(2+)-contractions). 3. When each vascular strip was exposed to high external K+ (30 mM K(+)-118 mM K+) solutions, the [Ca2+]i abruptly increased until it reached a peak, and then slightly decreased and eventually reached a steady-state level. The force also rapidly rose to reach a maximum plateau level. The changes in [Ca2+]i were more rapid than those in the force. Thus, the [Ca2+]i-force curves observed during the contractions induced by high+ (30 mM-118 mM) solutions showed a counter-clockwise rotation, over time. The entire curve shifted to the right, in a concentration-dependent manner, as compared with the line of the 'basic' [Ca2+]i-force relationship of the Ca(2+)-contraction. However, the [Ca2+]i-force relationship of the steady-state of contractions induced by the single dose applications of high K+ (30 mM-118 mM) overlapped with the line of the 'basic' [Ca2+]i-force relationship of Ca(2+)-contractions. 4. As references, the levels of [Ca2+]i and the force at rest (without stimulation) and at the steady-state of the contractions induced by a single dose application of 118 mM K+ solution were designated as 0% and 100%, respectively. When the vascular strips were exposed to NA (10(-5) M) and to 5-HT (10(-4) M), the [Ca2+]i abruptly rose, and reached a peak (107.1 +/- 5.8%) and 101.3 +/- 2.8%, respectively) after 1 min and 2 min, respectively (the [Ca2+]i-rising phase), and thereafter declined with a similar time course (the [Ca2+]i-declining phase) until reaching a low steady level (the steady-state phase). The force induced by 10(-5) M NA and 10(-4) M 5-HT reached a peak at 4 min (129%) and at 2 min (115%), respectively, and thereafter gradually declined. In contrast to the similarity in the [Ca2+]i transient between NA and 5-HT, the force induced by NA declined more slowly and reached higher steady levels than that seen with 5-HT. The level of force 20 min after the application of NA and 5-HT was 112% and 72%, respectively.5. In the entire time course of the 5-HT-induced contraction, i.e., in [Ca2+]i-rising, [Ca2+]i-declining and the steady-state phases, the [Ca2+]i-force relation was almost the same as that of the Ca2+-contractions.In the [Ca2+]i-rising phase of NA-induced contraction, the [Ca2+]i-force relation was similar to that of the Ca2+-contractions. However, in the [Ca2+] -declining and the steady-state phases, NA produced a greater force than that expected from a given change in the [Ca2+]i of the Ca2+-contractions, which resulted in a leftward shift of the [Ca2+]i-force relation. The extent of the leftward shift depended on the concentration of NA.6. These results suggest that (1) changes in [Ca2+]i precede changes in the force during the high K+-induced contraction, (2) in the initial [Ca2+]i-rising phase of the contractions induced by NA or by 5-HT, the [Ca2+]i-force relation is similar to that of Ca2+-contractions, and (3) in the subsequent[Ca2+]i-declining and the steady-state phases of the contractions, 5-HT demonstrated little enhancement in force for the given levels of [Ca2+]i, while NA induced a greater force for the given levels of [Ca2+],, in the rabbit femoral artery. Based on the above findings we suggest the presence of a time-dependent and stimulus-specific modulation of the Ca2+ sensitivity in the contractile apparatus of arterial smooth muscles.

P2-52 - The effect of terfenadine(TF), a histamine Hlantagonist, on [Ca2+]i-force relationship in porcine coronary artery

P2-52 - The effect of terfenadine(TF), a histamine Hlantagonist, on [Ca2+]i-force relationship in porcine coronary artery

Role of protein kinase C in relationship between Ca2+ and contractile elements in rat alpha-toxin-permeabilized mesenteric artery.

Phorbol ester, which activates protein kinase C (PKC), modulates vasoconstrictor-induced tension in vascular smooth muscle. Recently, Staphylococcal aureus alpha-toxin, which produces too small pores in the plasma membrane to allow passage of proteins, such as PKC, is used to investigate the signal transduction system in vascular smooth muscle cells. In order to elucidate the role of PKC on vascular smooth muscle contraction, we examined whether PKC activation influences the relationship between intracellular Ca2+ ([Ca2+]i) and tension in Wistar rat superior mesenteric artery (SMA) using vascular smooth muscle permeabilized with Staphylococcal alpha-toxin. [Ca2+]i was clamped at specified values (10(-8.5)-10(-4) mol/L) using EGTA-Ca2+ buffer. In alpha-toxin non-treated rings of SMA, isometric tension was evoked by 10 mmol/L caffeine and 10-30 mmol/L external potassium (high K+) in the absence or presence of phorbol 12, 13-dibutyrate (PDBu), a PKC activator, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and staurosporine (PKC inhibitors). PDBu significantly augmented caffeine- and high K(+)-evoked contractions. H-7 and staurosporine significantly attenuated caffeine- and high K(+)-evoked contractions augmented by PDBu. Moreover, H-7 significantly suppressed high K(+)-induced contraction in the absence of PDBu. In alpha-toxin permeabilized artery, PDBu shifted the [Ca2+]i-force relationship curve to the left. These results suggest that PKC activates vascular smooth muscle contraction by increasing the sensitivity of the contractile apparatus to Ca2+.

Ca2+]i signaling in pregnant human myometrium

The purpose of the present study was to determine the changes in intracellular ionized calcium concentration ([Ca2+]i) or [Ca2+]i sensitivity accompanying spontaneous and agonist-induced contraction of human myometrium at term pregnancy, as well as to quantify the response to three prototypical agonists: 1) oxytocin, 2) vasopressin, and 3) phenylephrine. Uterine biopsies were obtained at the time of cesarean section from patients who delivered at or near full-term gestation. These preparations were used to measure isometric force development and [Ca2+]i levels with the luminescent calcium indicator aequorin. Concentration-response relationships were determined with respect to isometric force development in the presence of the agonist. [Ca2+]i-force relationships were determined with respect to spontaneous phasic contractions, as well as agonist-induced phasic and tonic contractions. The results provide evidence that the phasic nature of term human myometrium is due to 1) the resting [Ca2+]i level being less than the calcium threshold for contractions and 2) the inability of the tissue to maintain high [Ca2+]i levels for prolonged periods of time. In addition, calcium-independent mechanisms of regulation were suggested by the relatively minor calcium sensitizing action of oxytocin and the observation that relaxation of tonic contractions preceded the fall in [Ca2+]i levels.

Mechanisms of vasoconstriction induced by endothelin-1 in smooth muscle of rabbit mesenteric artery.

1. The mechanism underlying the vasoconstriction induced by endothelin-1 (ET-1) was investigated by measuring the intracellular concentration of Ca2+ ([Ca2+]i), isometric force and phosphorylation of the myosin light chain (MLC) in endothelium-denuded unskinned and beta-escin-treated skinned smooth muscle from resistance vessels of the rabbit mesentery. The role of protein kinase C (PKC) in the action of ET-1 was studied in skinned smooth muscle using a synthetic peptide, PKC19-36, which corresponds to the autoinhibitory domain of PKC. 2. ET-1 (> 0.1 nM) induced slowly developing, maintained increases in [Ca2+]i and force. Nicardipine completely blocked the ET-1-induced increase in [Ca2+]i. BQ-123 (an inhibitor of the ETA receptor) blocked the ET-1-induced contraction but IRL 1620 (Suc-[Glu9,Ala11,15]-ET-1(8-21), an agonist of the ETB receptor) failed to induce contraction. 3. In ionomycin- and 70 mM K(+)-treated strips, ET-1 shifted the [Ca2+]i-force relationship to the left and enhanced the maximum amplitude of contraction induced by 2.6 mM Ca2+. In skinned smooth muscle treated with ionomycin, Ca2+ (0.1-3 microM) increased both force and MLC phosphorylation, in a concentration-dependent manner. ET-1 with GTP shifted both the Ca(2+)-force and Ca(2+)-MLC phosphorylation relationships to the left without significant changes in the maximum responses. ET-1 with GTP did not change the relationship between force and MLC phosphorylation. Similar effects were observed with phorbol 12,13-dibutyrate (PDBu, an activator of PKC). These results indicate that the sensitivity of MLC phosphorylation to Ca2+ is enhanced both by ET-1 with GTP and by PDBu. 4. PKC19-36 (an inhibitor of PKC) modified neither the contraction nor MLC phosphorylation induced by 0.3 microM Ca2+ but blocked the PDBu-induced enhancement of both these Ca(2+)-induced responses. However, PKC19-36 only partly inhibited the enhancement produced by ET-1 with GTP on the Ca(2+)-induced responses. PKC19-36 did not modify the relationship between force and MLC phosphorylation in the presence either of ET-1 with GTP or of PDBu. By contrast, BQ-123, neomycin and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) each abolished the ET-1-induced enhancement of the contraction induced by 0.3 microM Ca2+. 5. These results suggest that ET-1 acts on the ETA receptor and increases Ca2+ influxes through an activation of the dihydropyridine-sensitive Ca2+ channel, causing a long-lasting and maintained contraction in resistance vessels of the rabbit mesentery.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of extracellular and intracellular sources of Ca2+ in sarafotoxin S6b-induced contraction of strips of the rat aorta.

1. The effect of sarafotoxin S6b (sarafotoxin), a vasoconstrictor peptide, on cytosolic Ca2+ concentration ([Ca2+]i) and force in rat aortic strips loaded with fura-2 was determined by front-surface fluorometry. The objective was to elucidate the role of extracellular and intracellular Ca2+ in the mechanism of action of this peptide. 2. In the presence of extracellular 1.25 mM Ca2+, sarafotoxin induced a biphasic response consisting of an initial rapid increase in [Ca2+]i followed by a secondary sustained increase. Tension developed slowly but was sustained during the application of sarafotoxin. Diltiazem (10 nM-0.1 mM) partially inhibited both the increases in [Ca2+]i and tension. 3. In the presence of extracellular Ca2+, the force developed in relation to the increase in [Ca2+]i ([Ca2+]i-force relationship) observed with sarafotoxin was much greater than that observed upon K+ depolarization. In the presence of diltiazem the sarafotoxin-induced [Ca2+]i-force relationship was shifted even further to the left. 4. In the absence of extracellular Ca2+, sarafotoxin induced a transient increase in [Ca2+]i and a sustained contraction. Extending the incubation time in Ca(2+)-free physiological solution, resulted in smaller responses. However, after 60 min in Ca(2+)-free solution, sarafotoxin induced a sustained contraction but no change in [Ca2+]i. This residual contraction was inhibited by H-7, which is known to inhibit protein kinase C. 5. After treatment with caffeine to reduce intracellular stored Ca2+, sarafotoxin could still elicit increases in [Ca2+]i and in tension, showing that the caffeine-sensitive intracellular Ca2+ store partially overlaps with the sarafotoxin-sensitive store. 6. We conclude that, in addition to those components of contraction dependent on extracellular- and on intracellularly stored Ca2 , sarafotoxin can also induce contraction without increasing [Ca2+],. This component may be partially linked to the activation of protein kinase C and may contribute, in part, to the leftward shift of the [Ca2+]i-force relationship in the presence of sarafotoxin.

E4080 has a dual action, as a K + channel opener and a Ca 2+ channel blocker, in canine coronary artery smooth muscle

To clarify the vasodilating mechanism of action of D4080, which possesses vasodilating and bradycardic effects, we investigated its effects on intracellular Ca 2+ concentrations ([Ca 2+] i), as measured with fura-2, and force of contraction in canine coronary artery. E4080 reduced the increase in [Ca 2+] i and force of contraction induced by 30 and 90 mM KCl physiological salt solution (PSS) in a concentration-dependent manner. The effects of E4080 in 30 mM KCl-PSS were inhibited by 10 −5 M glibenclamide. In 30 mM KCl-PSS, the slope of the [Ca 2+] i-force relationship in the presence of E4080 was steeper than that of control, suggesting that E4080 decreased the sensitivity of contractile elements to Ca 2+, as an effect which was also inhibited by glibenclamide. However, the [Ca 2+] i-force curve was not changed by E4080 in 90 mM KCl-PSS. These results suggest that E4080 is a vasodilator in canine coronary artery, having K + channel opening and Ca 2+ channel blocking actions. The membrane hyperpolarization induced by E4080 may reduce the sensitivity of contractile elements to Ca 2+.

Mechanisms of vasodilation induced by NKH477, a water-soluble forskolin derivative, in smooth muscle of the porcine coronary artery.

To study the mechanism of vasodilation induced by 6-(3-dimethylaminopropionyl) forskolin (NKH477), a water-soluble forskolin derivative, its effects on the acetylcholine (ACh)-induced contraction of muscle strips of porcine coronary artery were examined. [Ca2+]i, isometric force, and cellular concentrations of cAMP and inositol 1,4,5-trisphosphate were measured. NKH477 (0.1-1.0 microM), isoproterenol (0.01-0.1 microM), or forskolin (0.1-1.0 microM) increased cAMP and attenuated the contraction induced by 128 mM K+ or 10 microM ACh in a concentration-dependent manner. These agents, at concentrations up to 0.3 microM, did not change the amount of cGMP. NKH477 (0.1 microM) attenuated the contraction induced by 128 mM K+ without corresponding changes in the evoked [Ca2+]i responses. ACh (10 microM) produced a large phasic increase followed by a small tonic increase in [Ca2+]i and produced a sustained contraction. The ACh-induced phasic increase in [Ca2+]i, but not the tonic increase, disappeared after application of 0.1 microM ionomycin. NKH477 (0.1 microM) attenuated both the increase in [Ca2+]i and the force induced by 10 microM ACh in muscle strips that were not treated with ionomycin and inhibited the ACh-induced contraction without corresponding changes in [Ca2+]i in ionomycin-treated muscle strips. These results suggest that NKH477 inhibits ACh-induced Ca2+ mobilization through its action on ionomycin-sensitive storage sites. In ionomycin-treated and 128 mM K(+)-treated muscle strips, 0.1 microM NKH477 shifted the [Ca2+]i-force relation to the right in the presence or absence of 10 microM ACh. In beta-escin-skinned smooth muscle strips, 0.1 microM NKH477 shifted the pCa-force relation to the right but had no effects on Ca(2+)-independent contraction. We conclude that in smooth muscle of porcine coronary artery, NKH477 inhibits ACh-induced contraction by both attenuating ACh-induced Ca2+ mobilization and reducing the sensitivity of the contractile machinery to Ca2+, possibly by activating cAMP-dependent mechanisms.

KCl-Depolarization Potentiates the Ca2+Sensitization by Endothelin-1 in Canine Coronary Artery

In Ca2+-free solution containing endothelin-1 (ET-1), depolarization by high KC1 increased the force of contraction without any changes in intracellular calcium concentration ((Ca2 +]i) in canine coronary artery. The [Ca2+]i-force relationships were examined in the absence or presence of ET-1 in 5 or 90 mM KC1. The relation curve in the presence of ET-1 in 90 mM KC1-PSS was shifted to the left and upward compared with that in 5 mM KC1. These results suggest that KCl-depolarization potentiates the Ca2+sensitization by ET-1.

Calcium-sensitizing inotropic agents in the treatment of heart failure: A critical view

Interventions that augment the contractile state of the heart are associated with, or caused by, alterations in Ca2+ exchange in heart muscles. New inotropic agents have been developed that increase the sensitivity of the myofilaments to Ca2+. To examine the effect of calcium-sensitizing agents on force development, we measured systolic and diastolic intracellular Ca2+ concentration [( Ca2+]i) and constructed [Ca2+]i-force relationships in normal (n = 6) and myopathic human hearts (n = 10). Using the bioluminescent calcium indicator aequorin, we found that the diastolic [Ca2+]i was 225 +/- 52 nM in normal muscles, whereas in myopathic muscles diastolic [Ca2+]i was significantly higher at 361 +/- 68 nM. Calcium-sensitizing agents that shift the [Ca2+]i-force relationship toward lower [Ca2+]i increase the diastolic force of myopathic hearts significantly more than in normal human hearts. This leads us to the conclusion that inotropic agents that increase the sensitivity of the myofilaments to Ca2+ further impair relaxation in myopathic hearts, resulting in a reduced contractile reserve and diminished active force production.

An analysis of the nitrate‐like and K channel opening actions of KRN2391 in canine coronary arterial smooth muscle

1. To clarify the mechanism of action of KRN2391, a new vasodilator containing a nitroxy group, its effects on intracellular Ca2+ concentration ([Ca2+]i) and force of contraction in canine coronary artery were compared with those of two derivatives lacking the nitro group. 2. KRN2391, its hydroxy and acetoxy derivative (Compound 2 and Compound 3, respectively) partially reduced [Ca2+]i in 5 or 30 mM KCl physiological salt solution (PSS), effects which were antagonized by glibenclamide. No KRN2391-induced change in [Ca2+]i was observed in 90 mM KCl-PSS. 3. The order of potency in reducing [Ca2+]i and inhibiting the contracture in 30 mM KCl-PSS was: KRN2391 greater than Compound 3 greater than Compound 2. 4. In 30 mM KCl-PSS, KRN2391 shifted the [Ca2+]i-force relationship so that a greater increase in [Ca2+]i was needed to produce force. Compounds 2 and 3 were ineffective. The [Ca2+]i-force curve obtained in 90 mM KCl-PSS was shifted to the right by KRN2391 (10(-4)M). 5. The ability of KRN2391 to reduce the force of contraction in both 30 mM and 90 mM KCl-PSS was inhibited by 10(-5) M methylene blue. 6. KRN2391 inhibited U46619-induced contractions; this effect was associated with a reduction of [Ca2+]i, which decreased below the basal level. 7. Thus, KRN2391 is a potent vasodilator in canine coronary artery. It possesses nitrate-like and potassium channel opening actions and can be designated as a nitrate-potassium channel opener (N-K) hybrid. The denitrate derivatives of KRN2391 are specific K channel openers. The nitroxy moiety in KRN2391 is important for not only its action as a nitrate but also its potency as a K channel opener.

Flosequinan, a vasodilator with a novel mechanism of action

1. The mechanism of action of flosequinan was investigated in ferret aortic smooth muscle by the simultaneous measurement of aequorin luminescence and isometric force. 2. The control calcium-force curve was obtained by plotting the calibrated aequorin luminescence against the force from potassium-depolarized muscles. Flosequinan relaxed potassium-depolarized muscles by causing parallel changes in [Ca2+]i and force with no shift in the control [Ca2+]i-force relationship. 3. The [Ca2+]i-force relationship in the presence of a maximally effective concentration of phenylephrine was significantly shifted to the left of that for the control, potassium-depolarized muscle. Flosequinan relaxed the phenylephrine-contracted muscle by causing a large decrease in force with only a minimal decrease in [Ca2+]i, resulting in an apparent rightward shift of the [Ca2+]i-force relationship, toward the control curve. 4. In comparison, sodium nitroprusside caused relaxation of either the potassium- or phenylephrine-induced contraction solely by a decrease in [Ca2+]i with no shift in either calcium-force relationship. 5. Milrinone caused no significant rightward shift of the calcium-force relationship during phenylephrine- or potassium-induced contractions, but when milrinone was added in the absence of vasoconstrictors, relaxation was obtained with no significant decrease in [Ca2+]i. 6. Flosequinan appears to differ in mechanism of action from both nitroprusside and milrinone. It relaxes depolarization-mediated contractions solely by decreasing [Ca2+]i but also appears to be capable of reversing the apparent calcium sensitizing action of phenylephrine.

Effects of vasoactive intestinal peptide (VIP) on contractile responses of smooth muscle in rat stomach.

1. The effects of vasoactive intestinal peptide (VIP) on contractile responses to carbachol (CCh), KCl and caffeine of the circular smooth muscle in rat stomach were examined by the isometric tension recording method and by measurement of the intracellular Ca level, [Ca]i, with fura 2. 2. Removal of extracellular Ca or nifedipine (0.1 microM) inhibited contractions induced by KCl (40 mM) and a low concentration (1 microM) of CCh but not that induced by caffeine (3 mM). After these treatments, the contraction induced by a high concentration of CCh (100 microM) changed to a phasic response. 3. VIP dose-dependently inhibited the contraction induced by 1 microM CCh, but not those caused by 40 mM KCl or 3 mM caffeine. 4. In Ca-free solution containing 2 mM EGTA, VIP inhibited the phasic contraction induced by 100 microM CCh, but not that induced by 30 mM caffeine. 5. CCh caused dose-dependent tension development concomitant with the increase in [Ca]i. VIP reduced both responses and thus did not affect the [Ca]i-force relation for CCh. In the chemically skinned muscle fibres, VIP had no effect on the pCa-tension relation. 6. It is suggested that the inhibitory effects of VIP on CCh-induced contractions are due to the inhibition of the processes of signal transduction from muscarinic receptors to voltage-dependent Ca channels and to intracellular Ca stores.

Effects of glibenclamide on cytosolic calcium concentrations and on contraction of the rabbit aorta

1. Using fluorometry of fura-2 and rabbit aortic strips, we studied the effects of glibenclamide (GLB), a sulphonylurea anti-diabetic drug and an inhibitor of opening of K+ channels, on cytosolic calcium concentrations ([Ca2+]i) and on force development. 2. Both high K(+)-depolarization and noradrenaline (NA) increased [Ca2+]i and force, in a concentration-dependent manner, in the presence of extracellular Ca2+ (1.25 mM). However, force development in relation to [Ca2+]i ([Ca2+]i-force relationship) observed with NA was much greater than that observed with K(+)-depolarization. 3. Pretreatment with GLB (10(-6)-10(-4) M) for 10 min partially inhibited, in a concentration-dependent manner, both [Ca2+]i elevation and the force development induced by 118 mM K(+)-depolarization or NA 10(-5) M in the presence of extracellular Ca2+. The [Ca2+]i-force relationship induced by both 118 mM K+ physiological salt solutions and by NA 10(-5) M in the GLB-treated strips overlapped that obtained in the non-treated strips, thereby suggesting that GLB has no effect on the Ca2(+)-sensitivity of the intracellular contractile apparatus. Only high concentrations (10(-4) M) of GLB decreased [Ca2+]i and the force, when applied after the force induced by 118 mM K+ PSS or NA 10(-5) M reached the maximum level. 4. In the absence of extracellular Ca2+, NA induced a transient increase in [Ca2+]i and in the force and these increases were inhibited when the vascular strips were pretreated with GLB for 10 min. The [Ca2+]i-force relationship obtained in the GLB-treated strips overlapped that in the non-treated ones. 5. An ATP-sensitive K+ channel opener, cromakalim (10-5M) reduced the increased [Ca2 + ]i and force induced by 25mm K+-depolarization and NA 10-SM. Subsequent application of GLB concentrationdependently reversed this relaxant effect of cromakalim on the NA-induced contraction (IC50 = 2x 10 7 M). Complete reversal of the effect was observed with 10IsM GLB. 6. We suggest that GLB inhibits both high K+-depolarization- and NA-induced contraction of the rabbit aorta, by decreasing [Ca2+]i and with no effect on the [Ca2+]i-force relationship. However, when NA-induced contractions were inhibited by a K+-channel opener, GLB reversed this inhibitory effect by inhibiting K+-channel opening and increasing [Ca2 +].

Cytoplasmic calcium and the relaxation of canine coronary arterial smooth muscle produced by cromakalim, pinacidil and nicorandil

1. In order to investigate the vasodilator mechanisms of the K+ channel openers, cromakalim, pinacidil and nicorandil, we measured changes in cytoplasmic Ca2+ concentration [( Ca2+]i) simultaneously with force by a microfluorimetric method using fura-2, a calcium indicator, in canine coronary arterial smooth muscle cells. 2. The three K+ channel openers all produced a concentration-dependent reduction of [Ca2+]i in 5 and 30 mM KCl physiological salt solution (PSS) but failed to affect [Ca2+]i in 45 and 90 mM KCl-PSS. 3. Cromakalim only partly inhibited (-45%) the 30 mM KCl-induced contractures, whereas pinacidil and nicorandil nearly abolished contractions produced by 45 mM, 90 mM and 30 mM KCl-PSS. 4. Tetrabutylammonium (TBA), a nonselective K+ channel blocker, or glibenclamide, a supposed adenosine 5'-triphosphate (ATP)-sensitive K+ channel blocker, abolished the reduction of [Ca2+]i caused by the three K+ channel openers and the relaxant effect of cromakalim, whereas they only slightly attenuated the relaxant effects of pinacidil and nicorandil. 5. The increase in [Ca2+]i produced by 45 or 90 mM KCl-PSS in the presence of pinacidil or nicorandil was abolished by 10(-5) M verapamil, indicating that the increase in [Ca2+]i was caused by the influx of extracellular Ca2+ and that pinacidil and nicorandil did not affect the voltage-dependent Ca2+ channel directly. 6. The [Ca2+]i-force relationship in the presence of cromakalim was not distinguishable from that of control. 7. The [Ca2+]i-force curve was shifted to the right by pinacidil and nicorandil. 8. These results show that cromakalim is a more specific K+ channel opener than pinacidil and nicorandil, and that vasodilatation produced by cromakalim in this study is predominantly a result of a reduction of [Ca2+]i due to the closure of voltage-dependent Ca21 channels by hyperpolarization. In contrast, additional mechanisms are involved in the vasodilator actions of pinacidil and nicorandil. One of these is related to a reduction in the sensitivity of contractile proteins to Ca2 . The latter mechanism of nicorandil is akin to that of nitroglycerin. K+ channels opened by these K+ channel openers may be ATP-sensitive ones which are blocked by glibenclamide.