Cyclic AMP modulation of adrenoreceptor-mediated arterial smooth muscle contraction

We examined the effects of cyclic AMP (cAMP) on the intracellular Ca2+ release in both the intact and skinned arterial smooth muscle. The amount of Ca2+ in the sarcoplasmic reticulum (SR) was estimated indirectly by caffeine-induced contraction of the skinned preparation and directly by caffeine-stimulated 45Ca efflux from the previously labeled skinned preparation. The norepinephrine-induced release contraction was markedly enhanced by dibutyryl cAMP (dbcAMP) and reduced by propranolol. The stimulatory effect of dbcAMP was best observed when the muscle was exposed to 10(-5) M dbcAMP and 2 X 10(-6) M norepinephrine was used to induce the release contraction. 10(-5) M cAMP had no effect on the Ca2+-induced contraction or on the pCa-tension relationship in the skinned preparation. This concentration of cAMP increased Ca2+ uptake into the SR of the skinned preparation when the Ca2+ in the SR was first depleted. 10(-5) M cAMP stimulated Ca2+-induced Ca2+ release from the SR after optimal Ca2+ accumulation by the SR. The results indicate that the stimulatory effect of cAMP on the norepinephrine-induced release contraction could be due to enhancement of the Ca2+-induced Ca2+ release from the SR in arterial smooth muscle.     

Ca2+ movements in sarcoplasmic reticulum of rat soleus fibers after hindlimb suspension.

The functional capacity of skeletal muscle sarcoplasmic reticulum (SR) was examined in the slow soleus of rats submitted to 15 days of disuse produced by hindlimb suspension (HS). By using caffeine-induced contractions of single skinned fibers, Ca2+ uptake, Ca2+ release, and passive Ca2+ leakage through the SR membrane were investigated. In the SR of atrophied muscles, the amounts of Ca2+ uptake and Ca2+ release were significantly higher than in the control muscles and were close to those found for a fast muscle, the plantaris. Moreover, the study of the Ca2+ leakage showed that the time required to empty the SR previously loaded with Ca2+ was reduced by a factor of two after HS. Such disturbances of the Ca2+ movements in the SR suggested that alterations of the SR membrane occurred after HS. The results supported the idea that after hindlimb unweighting the slow soleus muscle acquired SR properties that were very much like those of a faster muscle.     

GTP requirement for inositol-1,4,5-trisphosphate-induced Ca2+ release from sarcoplasmic reticulum in smooth muscle

We have examined inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release from the sarcoplasmic reticulum (SR) in the skinned vascular smooth muscle. The amount of Ca2+ in the SR was estimated indirectly by caffeine-induced contraction of the skinned preparation. The Ca2+ release from the SR by IP3 required GTP. A non-hydrolyzable analogue of GTP, guanosine 5'-(beta gamma-imido) triphosphate (GppNHp) could substitute for GTP in the IP3-induced Ca2+ release. These results suggest an involvement of GTP-binding protein in the mechanism of Ca2+ release from the SR by IP3 in smooth muscle.     

Ca2+ regulation in the near-membrane microenvironment in smooth muscle cells

The microenvironment between the plasma membrane and the near-membrane sarcoplasmic reticulum (SR) may play an important role in Ca(2+) regulation in smooth muscle cells. We used a three-dimensional mathematical model of Ca(2+) diffusion and regulation and experimental measurements of SR Ca(2+) uptake and the distribution of the SR in isolated smooth muscle cells to predict the extent that the near-membrane SR could load Ca(2+) after the opening of single plasma membrane Ca(2+) channels. We also modeled the effect of SR uptake on 1), single-channel Ca(2+) transients in the near-membrane space; 2), the association of Ca(2+) with Ca(2+) buffers in this space; and 3), the amount of Ca(2+) reaching the central cytoplasm of the cell. Our results indicate that, although single-channel Ca(2+) transients could increase SR Ca(2+) to a certain extent, SR Ca(2+) uptake is not rapid enough to greatly affect the magnitude of these transients or their spread to the central cytoplasm unless the Ca(2+) uptake rate of the peripheral SR is an order-of-magnitude higher than the mean rate derived from our experiments. Immunofluorescence imaging, however, did not reveal obvious differences in the density of SR Ca(2+) pumps or phospholamban between the peripheral and central SR in smooth muscle cells.     

Drug action of thapsigargin on the Ca2+ pump protein of sarcoplasmic reticulum.

Thapsigargin is found to be a potent inhibitor of the intracellular Ca2+ pump proteins from skeletal muscle sarcoplasmic reticulum (SR), cardiac SR, and brain microsomes. For skeletal muscle SR, the molar ratio of thapsigargin to Ca2+ pump protein for complete inhibition (MRc) of the Ca2+ loading rate, Ca(2+)-dependent ATPase activity, and formation of phosphorylated intermediate (EP) was approximately 1. When the Ca2+ pump protein of low affinity to Ca2+ (E2 state) was pretreated with thapsigargin, ATP and Ca2+ binding to the Ca2+ pump protein was completely inhibited. In the presence of Ca2+ (E1 state), Ca2+ pump protein was protected from inactivation by thapsigargin with respect to Ca2+ binding and EP formation. The MRc for brain microsomes, which mediate Ca2+ uptake into intracellular (inositol 1,4,5-trisphosphate-releasable) Ca2+ pools, is likewise stoichiometric. Approximately 30% of Ca2+ loading activity of brain microsomes was insensitive to thapsigargin, indicating the presence of other Ca2+ pumping system(s). The MRc for heart is 3.8, indicating that the Ca2+ pump of cardiac SR is less sensitive to thapsigargin. Phosphorylation of cardiac SR with protein kinase A increased the sensitivity to thapsigargin to MRc of 2.8. In summary, we find that: 1) thapsigargin is the most effective inhibitor of the Ca2+ pump protein of intracellular membranes (SR and endoplasmic reticulum); 2) its primary inhibitory action appears to inactivate the E2 form of the enzyme preferentially; 3) cardiac SR shows lesser sensitivity to thapsigargin than skeletal muscle SR and brain microsomes; protein kinase A treatment of cardiac SR enhances the sensitivity to the drug.     

Chloride channel blockers inhibit Ca2+ uptake by the smooth muscle sarcoplasmic reticulum.

Despite the fact that Ca2+ transport into the sarcoplasmic reticulum (SR) of muscle cells is electrogenic, a potential difference is not maintained across the SR membrane. To achieve electroneutrality, compensatory charge movement must occur during Ca2+ uptake. To examine the role of Cl- in this charge movement in smooth muscle cells, Ca2+ transport into the SR of saponin-permeabilized smooth muscle cells was measured in the presence of various Cl- channel blockers or when I-, Br-, or SO42- was substituted for Cl-. Calcium uptake was inhibited in a dose-dependent manner by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and by indanyloxyacetic acid 94 (R(+)-IAA-94), but not by niflumic acid or 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Smooth muscle SR Ca2+ uptake was also partially inhibited by the substitution of SO42- for Cl-, but not when Cl- was replaced by I- or Br-. Neither NPPB nor R(+)-IAA-94 inhibited Ca2+ uptake into cardiac muscle SR vesicles at concentrations that maximally inhibited uptake in smooth muscle cells. These results indicate that Cl- movement is important for charge compensation in smooth muscle cells and that the Cl- channel or channels involved are different in smooth and cardiac muscle cells.     

Ca2+-induced Ca2+ release in skinned single smooth muscle cells isolated from guinea-pig taenia caeci

The Ca2+ release from intracellular Ca2+ storage sites of skinned single smooth muscle cells isolated from guinea-pig taenia caeci was studied. The Ca2+ release from intracellular Ca2+ storage sites of the skinned single cells was enhanced by the presence of submicromolar concentrations of Ca2+ in the solution. The Ca2+ release was enhanced by caffeine and adenine, and suppressed by Mg2+ and procaine. These results suggest that the Ca2+-induced Ca2+ release mechanism may play an important role in the release of Ca2+ from intracellular storage sites of guinea-pig taenia caeci smooth muscle cells.     

Characterization of sarcoplasmic reticulum in skinned heart muscle cultures

The plasma membranes of rat heart muscle, grown in cell culture, were made permeable with saponin in a Ca-free solution. The cells were then supplied with a medium resembling the cytosol, and the adenosine triphosphate (ATP)-dependent Ca2+ sequestration was measured in the presence of oxalate. The nonmitochondrial component accounts for about 50% of the total Ca2+ uptake. The nonmitochondrial accumulation of Ca2+ within myocardial cells was found to be reversible by addition of the Ca2+ ionophore A23187. On the other hand, the Ca2+ antagonist D-600 (50 microM) had almost no effect on Ca2+ accumulation. Caffeine reduced Ca2+ accumulation in the skinned cardiomyocytes in a concentration-dependent manner. In addition, the anticalmodulin drug trifluoperazine (TFP) reduced Ca2+ accumulation in the skinned cells. Because of the analogy between nonmitochondrial ATP-dependent Ca2+ accumulation and the sarcoplasmic reticulum (SR) function with regard to the influence of various agents, it is assumed that we actually measure Ca2+ accumulation in the SR. The rate of Ca2+ accumulation into the SR measured during the development of the cardiomyocytes in culture shows an almost linear increase as a function of culture age. Amiodarone, a potent antiarrhythmic agent, and its metabolite, desethylamiodarone, inhibited Ca2+ accumulation into SR, which may explain their therapeutic effect.     

Fluorescence and differential light absorption recordings with calcium probes and potential-sensitive dyes in skinned cardiac cells

This report describes an optical system for microspectrophotometry in a single cardiac cell from which the sarcolemma has been removed by microdissection (skinned cardiac cell). This system is attached to the high power inverted microscope used for the microdissection and includes (a) a single variable wavelength microspectrophotometer used to define the spectrum of a given dye or Ca2+ probe; and (b) a dual wavelength, differential microspectrophotometer used to record differentially between the optimum wavelength and a wavelength separated by 25--30 nm. Results are presented using the following optical methods: (a) fluorescence measurements with chlorotetracycline to monitor the amount of Ca2+ bound to the inner face of the sarcoplasmic reticulum (SR) membrane; (b) differential absorption measurements with arsenazo III to measure changes of myoplasmic [Ca2+]free resulting from Ca2+ release from the SR; (c)fluorescence and (or) differential absorption measurements with the potential-sensitive dyes merocyanine 540, NK 2367, and di-S-C3(5) to monitor changes of charge distribution on the SR membrane during Ca2+ accumulation in the SR, as well as before and during Ca2+-induced release of Ca2+ from the SR. A small and rapid signal is observed which precedes the Ca2+-induced release of Ca2+ from the SR. It is detected as an increase of CA2+ binding inside the SR with chlorotetracycline and as a "hyperpolarization" with potential-sensitive dyes, while no transient change of myoplasmic [Ca2+]free is detected with arsenazo III. This small and rapid signal preceding the Ca2+ release may be a first hint to an understanding of the mechanism whereby a small increase of [Ca2+]free outside the SR triggers Ca2+ release from the SR.     

Effects of ryanodine in skinned cardiac cells

Ryanodine (1 X 10(-5) M) did not affect the Ca2+ sensitivity of the myofilaments of skinned (sarcolemma removed by microdissection) cardiac cells from the rat ventricle. Ryanodine (1 X 10(-5) M) inhibited three types of Ca2+ release from the sarcoplasmic reticulum (SR), which have different mechanisms: 1) Ca2+-induced release of Ca2+ triggered by a rapid and transient increase of [free Ca2+] at the outer surface of the SR; 2) caffeine-induced release of Ca2+; 3) spontaneous cyclic release of Ca2+ occurring in the continuous presence of a [free Ca2+] sufficient to overload the SR. These results suggest that the three types of Ca2+ release are through the same channel across the SR membrane, although the gating mechanisms are different for the three types. Ryanodine also diminished the rate of Ca2+ accumulation into the SR. Even in the presence of 1 X 10(-5) M ryanodine the SR accumulated Ca2+ that could be released when the SR was sufficiently overloaded with Ca2+. Thus, ryanodine pretreatment did not permit the direct activation of the myofilaments by externally applied Ca2+. The approximately 1000-fold difference in the effective concentrations of ryanodine in intact vs. skinned cardiac cells suggests that low concentrations of ryanodine act in the intact cardiac tissues through processes or on structures that are destroyed by the skinning procedure. No significant differences were observed in the effects of ryanodine in skinned cardiac cells from different adult mammalian species.     

Complex effects of ryanodine on the sarcoplasmic reticulum Ca2+ levels in smooth muscle cells

We have studied the effects of ryanodine and inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) with thapsigargin, on both [Ca(2+)](i) and the sarcoplasmic reticulum (SR) Ca(2+) level during caffeine-induced Ca(2+) release in single smooth muscle cells. Incubation with 10 microM ryanodine did not inhibit the first caffeine-induced [Ca(2+)](i) response, although it abolished the [Ca(2+)](i) response to a second application of caffeine. To assess whether ryanodine was inducing a permanent depletion of the internal Ca(2+) stores, we measured the SR Ca(2+) level with Mag-Fura-2. The magnitude of the caffeine-induced reduction in the SR Ca(2+) level was not augmented by incubating cells with 1 microM ryanodine. Moreover, on removal of caffeine, the SR Ca(2+) levels partially recovered in 61% of the cells due to the activity of thapsigargin-sensitive SERCA pumps. Unexpectedly, 10 microM ryanodine instead of inducing complete depletion of SR Ca(2+) stores markedly reduced the caffeine-induced SR Ca(2+) response. It was necessary to previously inhibit SERCA pumps with thapsigargin for ryanodine to be able to induce caffeine-triggered permanent depletion of SR Ca(2+) stores. These data suggest that the effect of ryanodine on smooth muscle SR Ca(2+) stores was markedly affected by the activity of SERCA pumps. Our data highlight the importance of directly measuring SR Ca(2+) levels to determine the effect of ryanodine on the internal Ca(2+) stores.     

Ca2+ compartments in saponin-skinned cultured vascular smooth muscle cells

A method for saponin skinning of primary cultured rat aortic smooth muscle cells was established. The saponin-treated cells could be stained with trypan blue and incorporated more 45Ca2+ than the nontreated cells under the same conditions. At low free Ca2+ concentration, greater than 85% of 45Ca2+ uptake into the skinned cells was dependent on the extracellularly supplied MgATP. In the intact cells, both caffeine and norepinephrine increased 45Ca2+ efflux. In the skinned cells, caffeine increased 45Ca2+ efflux, whereas norepinephrine did not. The caffeine-releasable 45Ca2+ uptake fraction in the skinned cells appeared at 3 X 10(-7) M Ca2+, increased gradually with the increase in free Ca2+ concentration, and reached a plateau at 1 X 10(-5) M Ca2+. The 45Ca2+ uptake fraction, which was significantly suppressed by sodium azide, appeared at 1 X 10(-5) M Ca2+ and increased monotonically with increasing free Ca2+ concentration. The results suggest that the caffeine-sensitive Ca2+ store, presumably the sarcoplasmic reticulum, plays a physiological role by releasing Ca2+ in response to norepinephrine or caffeine and by buffering excessive Ca2+. The 45Ca2+ uptake by mitochondria appears too insensitive to be important under physiological conditions.     

Caffeine inhibition of calcium accumulation by the sarcoplasmic reticulum in mammalian skinned fibers

Summary Oxalate-supported Ca accumulation by the sarcoplasmic reticulum (SR) of chemically skinned mammalian skeletal muscle fibers is activated by MgATP and Ca²⁺ and partially inhibited by caffeine. Inhibition by caffeine is greatest when Ca²⁺ exceeds 0.3 to 0.4 m, when free ATP exceeds 0.8 to 1mm, and when the inhibitor is present from the beginning of the loading period rather than when it is added after Ca oxalate has already begun to precipitate within the SR. Under the most favorable combination of these conditions, this effect of caffeine is maximal at 2.5 to 5mm and is half-maximal at approximately 0.5mm. For a given concentration of caffeine, inhibition decreases to one-half of its maximum value when free ATP is reduced to 0.2 to 0.3mm. Varying free Mg²⁺ (0.1 to 2mm) or MgATP (0.03 to 10mm) has no effect on inhibition. Average residual uptake rates in the presence of 5mm caffeine atpCa 6.4 range from 32 to 70% of the control rates in fibers from different animals. The extent of inhibition in whole-muscle homogenates is similar to that observed in skinned fibers, but further purification of SR membranes by differential centrifugation reduces their ability to respond to caffeine. In skinned fibers, caffeine does not alter the Ca²⁺ concentration dependence of Ca uptake (K _0.5, 0.5 to 0.8 m; Hilln, 1.5 to 2.1). Reductions in rate due to caffeine are accompanied by proportional reductions in maximum capacity of the fibers, and this configuration can be mimicked by treating fibers with the ionophore A23187. Caffeine induces a sustained release of Ca from fibers loaded with Ca oxalate. However, caffeine-induced Ca release is transient when fibers are loaded without oxalate. The effects of caffeine on rate and capacity of Ca uptake as well as the sustained and transient effects on uptake and release observed under different conditions can be accounted for by a single mode of action of caffeine: it increases Ca permeability in a limited population of SR membranes, and these membranes coexist with a population of caffeine-insensitive membranes within the same fiber.     

Enhanced Ca2+ uptake and ATPase activity of sarcoplasmic reticulum in the presence of diethyl ether

The presence of diethyl ether enhances the rates of both Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles (SR) isolated from rabbit skeletal muscle. Stopped-flow measurements of Ca2+ transport in SR show that, in the absence of oxalate and other calcium-complexing anions, the initial velocity of the ATP-dependent Ca2+ uptake increases from 60 to 107 nmol of Ca2+/s/mg of protein when 5% (v/v) diethyl ether is present. Similar concentrations of diethyl ether increase steady state levels of Ca2+ accumulation by over 80%. Parallel to the enhancement of the rate of Ca2+ transport, diethyl ether induces an increased rate of Ca2+-dependent ATPase activity. Among four other ether compounds tested, three enhanced the rate of Ca2+ uptake, but none as effectively as diethyl ether, and a fourth reduced the rate of Ca2+ transport by the SR. These results contrast with previous observations concerning the effect of diethyl ether on ATP-dependent Ca2+ transport by SR and are now consistent with a direct pharmacological action of ether as a muscle relaxant at the level of SR Ca2+ transport.     

The role of free calcium ion in calcium release in skinned muscle fibers

Major questions in excitation--contraction coupling of fast skeletal muscle concern the mechanism of signal transmission between sarcolemma and sarcoplasmic reticulum (SR), the mechanism of SR Ca release, and operation of the SR active transport system during excitation. Intracellular Ca movement can be studied in skinned muscle fibers with more direct control, analysis of 45Ca flux, and simultaneous isometric force measurements. Ca release can be stimulated by bath Ca2+ itself, ionic "depolarization," Mg2+ reduction, or caffeine. The effectiveness of bath Ca2+ has suggested a possible role for Ca2+ in physiological release, but this response is difficult to analyze and evaluate. Related evidence emerged from analysis of other responses: with all agents studied, stimulation of 45Ca efflux is highly Ca2+-dependent. The presence of a Ca chelator prevents detectable stimulation by ionic "depolarization" or Mg2+ reduction and inhibits the potent caffeine stimulus; inhibition is graded with chelator concentration and caffeine concentration, and is synergistic with inhibition by increased Mg2+. The results indicate that a Ca2+-dependent pathway mediates most or all of stimulated 45Ca efflux in skinned fibers, and has properties compatible with a function in physiological Ca release.     

Hg2+-induced contracture in mechanically skinned fibers of frog skeletal muscle

In mechanically skinned fibers of the semitendinosus muscle of bullfrogs, we examined the role of membrane sulfhydryl groups on Ca2+ release from the sarcoplasmic reticulum (SR). Hg2+, a sulfhydryl reagent (20-100 microM), induced a repetitive contracture of skinned fibers, and this contracture did not occur in skinned fibers in which the SR had been disrupted by treatment with a detergent (Brij 58). Procaine (10 mM), Mg2+ (5 mM), or dithiothreitol (1 mM) blocked the Hg2+-induced contracture. Ag+ or p-chloromercuribenzenesulfonic acid produced similar contractures to that induced by Hg2+. We conclude that Hg2+ releases Ca2+ from SR of a skinned fiber by modifying sulfhydryl groups on the SR membrane, and suggest that the Ca2+ released by Hg2+ may trigger a greater release of Ca2+ from SR to develop tension.     

Cellular mechanisms of transitory smooth muscle contraction of the coronary arteries during hypoxia: role of intracellular Ca2+

In experiments on isolated porcine and canine coronary artery rings it was shown that vascular smooth muscle (VSM) during hypoxia (decreasing bath PO2 with 147 to 20-15 mm Hg) response to biphasic constriction-dilation reaction. Transient hypoxic contractions (THC) of VSM preserved completely in Ca2+-free solution and partially (up 50-60%) in the presence of Ca2+-channel blockers, but abolished by procaine. THC of VSM skinned by saponin significantly depressed at depletion of Ca2+-store sarcoplasmic reticulum (SR) by caffeine nd abolished after SR destruction. THC is not linked with Na+-K+-ATPase inhibition because it preserved (or increased) at ouabain treatment. THC significantly depressed under selective glycolysis blockade by monoiodoacetic acid and pyruvate and also after inositol-1 monophosphatase inhibition by lithium (the phase of hypoxic relaxation of VSM was augmented in this condition). Our results indicate that transient contraction of coronary arteries under hypoxia may be mediated mainly by release of Ca2+ from SR and linked obviously with production of inositol-1,4,5-trisphosphate. The participation of glycolysis in this process is unknown.     

Ca2+ regulation of vascular smooth muscle

Regulation of intracellular free Ca2+ concentrations in vascular smooth muscle is accomplished mainly by Ca2+ channels and ATP-dependent Ca2+ pumps in the plasmalemma and sarcoplasmic reticulum (SR). Ca2+ entry through the plasmalemma is apparently mediated by four different pathways: leak; receptor-operated Ca2+ channels; potential sensitive Ca2+ channels; and stretch-activated channels. The agonist releasable intracellular Ca2+ store appears to be identical with the SR. Evidence for the involvement of Ca2+-induced Ca2+ release and inositol-1,4,5-trisphosphate in the release of SR Ca2+ is discussed. Smooth muscle contractions induced by certain agonists may be further enhanced by inhibition of Ca2+ uptake by the SR and of active Ca2+ extrusion across the plasmalemma. At the moment it is not clear from a consideration of the Ca2+ regulatory mechanisms present in vascular smooth muscle how dietary Ca2+ affects vascular tone. The increased Ca2+ permeation through smooth muscle cell membranes of resistance arteries taken from spontaneously hypertensive rats may be relevant to this problem.     

Evidence that a Ca2+ sparks/STOCs coupling mechanism is responsible for the inhibitory effect of caffeine on electro-mechanical coupling in guinea pig ureteric smooth muscle

Recent studies have highlighted the role of the sarcoplasmic reticulum (SR) in controlling excitability, Ca2+ signalling and contractility in smooth muscle. Caffeine, an agonist of ryanodine receptors (RyRs) on the SR has been previously shown to effect Ca2+ signalling but its effects on excitability and contractility are not so clear. We have studied the effects of low concentration of caffeine (1 mM) on Ca2+ signalling, action potential and contractility of guinea pig ureteric smooth muscle. Caffeine produced reversible inhibition of the action potentials, Ca2+ transients and phasic contractions evoked by electrical stimulation. It had no effect on the inward Ca2+ current or Ca2+ transient but increased the amplitude and the frequency of spontaneous transient outward currents (STOCs) in voltage clamped ureteric myocytes, suggesting Ca2+-activated K+ channels (BK) are affected by it. In isolated cells and cells in situ caffeine produced an increase in the frequency and the amplitude of Ca2+ sparks as well the number of spark discharging sites per cell. Inhibition of Ca2+ sparks by ryanodine (50 microM) or SR Ca2+-ATPase (SERCA) cyclopiazonic acid (CPA, 20 microM) or BKCa channels by iberiotoxin (200 nM) or TEA (1 mM), fully reversed the inhibitory effect of caffeine on Ca2+ transients and force evoked by electrical field stimulation (EFS). These data suggest that the inhibitory effect of caffeine on the action potential, Ca2+ transients and force in ureteric smooth muscle is caused by activation of Ca2+ sparks/STOCs coupling mechanism.     

Two novel types of calcium release from skeletal sarcoplasmic reticulum by phosphatidylinositol 4,5-biphosphate.

In both the heavy and light fractions of fragmented sarcoplasmic reticulum (SR) vesicles from the fast skeletal muscle, about 27 min after beginning the active Ca2+ uptake, the extravesicular Ca2+ concentration suddenly increased to reach a steady level (delayed Ca2+ release). Phosphatidylinositol 4,5-bisphosphate (PIP2) not only shortened the time to delayed Ca2+ release but also induced prompt Ca2+ release from the heavy fraction of SR. Delayed Ca2+ release and prompt Ca2+ release stimulated by 100 microM PIP2 were not modified by ruthenium red. PIP2 (>0.1 microM) markedly accelerated the rate of 45Ca2+ efflux from SR vesicles in a concentration-dependent manner. The PIP(2)-induced 45Ca2+ efflux was potentiated by ruthenium red but profoundly inhibited by La3+. The concentration-response curve for Ca2+ or Mg2+ in PIP2-induced 45Ca2+ release was clearly different from that in the Ca(2+)-induced Ca2+ release. PIP2 caused a concentration-dependent increase in Ca2+ release from SR of chemically skinned fibers from skeletal muscle. Furthermore, [3H]ryanodine or [3H]methyl-7-bromoeudistomin D (MBED) binding to SR was increased by PIP2 in a concentration-dependent manner. These observations present the first evidence that PIP2 most likely activates two types of SR Ca2+ release channels whose properties are entirely different from those of Ca(2+)-induced Ca2+ release channels (the ryanodine receptor 1).