How should ethnicity-related information be included on drug labels? Considerations based on comparison of multiregional clinical trial data on the label between Japan and the United States.

Clinical data on various ethnicities collected in multiregional clinical trials (MRCTs) have increased in regulatory review for drug approval. However, how such data should be included on the drug labels has not been discussed. We compared information related to ethnicities on the labels of drugs that were approved in both Japan and the United States, and discussed the issues to be considered for providing better information to healthcare professionals in this era of globalized drug development.

Representation of older patients in clinical trials for drug approval in Japan.

To examine how target patients seen in clinical practice are represented in clinical trials for approved drugs in Japan, we compared the age distribution of older patients enrolled in confirmatory clinical trials for regulatory approval with that of the estimated actual patient population. Drugs for 6 chronic conditions common among older patients (diabetes mellitus, hypertension, rheumatoid arthritis, non-small cell lung cancer, depression and Alzheimer's disease) launched by 2012 in Japan were selected. The disparity in age distribution between patients in trials and patients seen in clinical practice varied depending on the disease, but older patients, especially those aged 75 or older, were generally underrepresented in clinical trials for regulatory approval in Japan. Under-representation of older patients in hypertension trials was particularly marked compared to other conditions, despite the similarity in age distribution of patients seen in clinical practice. One factor causing this disparity may be an upper age limit in clinical trial protocols. More effort is needed to properly characterize the benefits and risks of drugs for older patients. This should include the active enrollment of older patients in clinical trials, the establishment of better assessment tools such as pharmacometric approaches, and the appropriate planning and conducting of post-marketing surveys and studies.

Significant differences in drug lag in clinical development among various strategies used for regulatory submissions in Japan.

Although the number of global clinical trials (GCTs) conducted in multiple countries including Japan has increased recently, it is not clear how much these GCTs help in reducing the lag in drug development (LDD: difference between the submission dates for new drug applications (NDAs) in the United States and Japan). We examined the effects of various clinical development strategies on LDD because the development period depends on what types of clinical trials were conducted for the Japanese NDA. Although various drug development strategies are available, deciding early on an appropriate strategy is a key to minimizing the LDD in Japan. The inclusion of GCTs in the clinical development strategy is also important; simultaneously, the smaller sample size of the Japanese population should be taken into consideration. Furthermore, reinforcement of Japan's capability to lead drug development may also be important in providing innovative drugs to Japanese patients without any significant LDD.

Regulatory challenges in the review of data from global clinical trials: the PMDA perspective.

Regulatory agencies face challenges in reviewing data from global clinical trials (GCTs) in the era of globalization of drug development. One major challenge is consideration of ethnic factors in evaluating GCT data so as to extrapolate foreign population data to one's own national population. Here, we present the Pharmaceuticals and Medical Devices Agency (PMDA) perspective in reviewing GCT data in new drug applications (NDAs) and discuss future challenges for new drug approval.

Regulatory science as a bridge between science and society.

Development of innovative drugs has recently become more difficult. The case of rosiglitazone shows the extreme difficulty of making the regulatory decision that will best balance the benefits and risks of a drug. There is a high expectation that regulatory science (RS) can improve the situation. However, without user understanding of its basic characteristics, RS will not deliver what is expected.

PMDA's challenge to accelerate clinical development and review of new drugs in Japan.

In recent years, Japan's Pharmaceuticals and Medical Devices Agency (PMDA) has conducted several projects to shorten drug development and review times in Japan to resolve the "drug lag." Key to achieving this goal is greater involvement by the PMDA in drug development through enhancement of scientific consultation and improvement of the review process by reinforcing the operational system, including hiring more reviewers. We discuss here the current projects of the PMDA as well as future challenges.

Ex vivo and in vivo evaluation of the blood compatibility of surface-modified polyurethane catheters.

Catheter model tubes were prepared from a medical-grade polyetherurethane and their outer surfaces modified by surface-graft polymerization of acrylamide and dimethyl acrylamide (DMAA). The surface-graft layer was characterized by means of dry staining, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy, and protein adsorption. Ex vivo evaluation for the blood compatibility of the surface-modified polyurethane was carried out using the polyurethane tube as an arterio-venous shunt between the carotid artery and the jugular vein of rabbits. When the surface density of grafted polymer was in the range of 10-30 microg/cm2, the in vitro adsorption of IgG exhibited a minimum value and platelet adhesion to the grafted polyurethane surface was insignificant, in marked contrast with that to the virgin (nonmodified) surface. The in vivo blood compatibility of polyurethane was evaluated by implanting the catheter tube in the inferior vena cava of rabbits from the femoral vein after ligation of a distal site of the exposed femoral vein. After remaining there for predetermined periods of time, the implanted catheters were taken out together with the veins of the rabbits that had been heparinized and sacrificed just prior to excision of the veins. After exchange of the blood in the veins for saline, the excised veins were opened by cutting longitudinally to inspect for clot formations on the surfaces of the implanted catheters. Occlusion of the inferior vena cava was not observed for any of the catheters, nor was there any apparent damage or microembolizations in the lungs and kidneys. Many small-sized clots were observed on the surfaces of the nonmodified polyurethane tubes after a 2-week implantation whereas the catheter surfaces grafted with DMAA polymer chains had a much smaller number of clots. When the blood compatibility of polyurethane surfaces was graded for relative evaluation from one (marked clotting) to five (no clotting) based on the size and number of the clots, the evaluation results were as follows: 3.1 (virgin, 2 weeks), 4.0 (grafted, 1 week), 4.1 (grafted, 2 weeks), and 3.5 (grafted, 1 month).

DCG-IV, a potent metabotropic glutamate receptor agonist, as an NMDA receptor agonist in the rat cortical slice.

The depolarization induced by DCG-IV, a potent agonist for Group II metabotropic glutamate receptors (Group II mGluRs), was depressed by selective antagonists for NMDA receptors in the rat cortical slice, but was not affected even by a high concentration of a selective antagonist for Group II mGluRs. DCG-IV caused depolarization more effective than NMDA in a dose-dependent manner with a threshold concentration of 3 microM in rat cortical slices, while DCG-IV was less active than NMDA in rat spinal cords. These actions should be carefully considered particularly when DCG-IV is used as an agonist for mGluRs in vivo.

Potent vasoconstrictor actions of cyclopiazonic acid and thapsigargin on femoral arteries from spontaneously hypertensive rats.

1. Ca2+ buffering function of sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) was examined. Differences in the effects of cyclopiazonic acid (CPA) and thapsigargin, agents which inhibit the Ca(2+)-ATPase of SR, on tension and cellular Ca2+ level were assessed in endothelium-denuded strips of femoral arteries from 13-week-old SHR and normotensive Wistar-Kyoto rats (WKY). 2. In resting strips preloaded with fura-PE3, the addition of CPA (10 microM) or thapsigargin (100 nM) caused an elevation of cytosolic Ca2+ level ([Ca2+]i) and a contraction. These responses were significantly greater in SHR than in WKY. 3. The additional of verapamil (3 microM) to the resting strips caused a decrease in resting [Ca2+]i, which was significantly greater in SHR than in WKY. In SHR, but not in WKY, this decrease was accompanied by a relaxation from the resting tone, suggesting the maintenance of myogenic tone in the SHR artery. 4. Verapamil (3 microM) abolished differences between SHR and WKY. The effects of verapamil were much greater on the contraction than on the [Ca2+]i. 5. The resting of Ca2+ influx in arteries measured after a 5 min incubation of the artery with 45Ca was not increased by CPA or thapsigargin in either SHR or WKY. The net Ca2+ entry measured after a 30 min incubation of the artery with 45Ca was decreased by CPA or thapsigargin in both SHR and WKY. The resting Ca2+ influx was significantly higher in SHR than in WKY, and was decreased by nifedipine (100 nM) in the SHR artery, but was unchanged in the WKY artery. 6. The resting 45Ca efflux from the artery was increased during the addition of CPA (10 microM). This increase was less in SHR than in WKY. The resting 45Ca efflux was the same in SHR and WKY. 7. These results suggest that (1) the Ca2+ influx via L-type voltage-dependent Ca2+ channels (VDCCs) was increased in the resting state of the SHR femoral artery, (2) the greater part of the increased Ca2+ influx was buffered by Ca2+ uptake into the SR and some Ca2+ reached the myofilaments resulting in the maintenance of the myogenic tone, and (3) therefore the functional elimination of SR by CPA or thapsigargin caused a large elevation of [Ca2+]i and a potent contraction in this artery. During this process, the contraction was mainly due to the basal Ca2+ influx via L-type VDCCs. The present study also showed the existence of a relatively large compartment of [Ca2+]i which does not contribute to the contraction during the addition of CPA or thapsigargin.

Superficial sarcoplasmic reticulum calcium buffering of resting, voltage-dependent Ca++ influx in rat femoral arterial smooth muscle.

To determine the Ca(+2)-buffering function of sarcoplasmic reticulum (SR) in the resting state of arterial smooth muscle, the effects of cyclopiazonic acid (CPA) and thapsigargin, which inhibit the Ca(+20-ATPase of SR, on tension and cytosolic Ca+2 level ¿[Ca+2]¿ were examined in endothelium-denuded strips of rat femoral artery. In resting strips preloaded with fura-PE3, the addition of CPA (10 microM) or thapsigargin (100 nM) caused an elevation of [Ca+2]i and a contraction. These responses were inhibited by blockers of L-type voltage-dependent Ca+2 channels (verapamil, nifedipine and diltiazem). The inhibition by verapamil was much greater for contraction than for [Ca+2]i Verapamil itself decreased the resting [Ca+2]i without affecting the resting tone. In a Ca(+2)-free solution, CPA caused a small elevation of [Ca+2]i that was not accompanied by a contraction. In the presence of CPA or thapsigargin, the effects of caffeine (20 mM) on [Ca+2]i and tension were greatly inhibited. In strips where the SR Ca+2 was depleted by the addition of caffeine and a Ca(+2)-free solution, the Ca+2 influx from the extracellular space was completely taken up into the SR. This process was inhibited by CPA or thapsigargin, which resulted in a contraction. This contraction was also inhibited by verapamil. However, the basal Ca+2 influx measured by a 5-min incubation of the artery with 45Ca+2 was not increased by CPA or thapsigargin. The net Ca+2 entry measured in a 30-min incubation of the artery with 45Ca+2 was decreased by CPA or thapsigargin. The basal 45Ca+2 efflux from the artery was increased during the addition of CPA. These results suggested that the basal Ca+2 influx was completely buffered by Ca+2 uptake into the SR in the resting state of rat femoral artery and therefore the inhibition of SR Ca+2 uptake by CPA or thapsigargin caused an elevation of [Ca+2]i and a contraction. During this process, the basal Ca+2 influx via L-type voltage-dependent Ca+2 channels mainly contributed to the contraction. The present study also showed the existence of a relatively large compartment of cytosolic Ca+2 that does not contribute to contraction during the addition of CPA or thapsigargin.

Inhibition by calponin of isometric force in demembranated vascular smooth muscle strips: the critical role of serine-175.

alpha-Calponin is a thin-filament-associated protein which has been implicated in the regulation of smooth muscle contraction. Quantification of the tissue content of rat tail arterial smooth muscle revealed approximately half the amount of alpha-calponin relative to actin compared with chicken gizzard and other smooth muscles, suggesting that this tissue would be particularly suitable for investigation of the effects of exogenous alpha-calponin on the contractile properties of permeabilized muscle strips. Rat tail arterial strips demembranated with Triton X-100 retained approximately 90% of their complement of alpha-calponin, and exogenous chicken gizzard alpha-calponin (which conveniently has a slightly lower molecular mass than the rat arterial protein) bound to the permeabilized muscle, presumably through its high affinity for actin. Exogenous alpha-calponin inhibited force in demembranated muscle strips in a concentration-dependent manner when added at the peak of a submaximal Ca(2+)-induced contraction, with a half-maximal effect at approximately 3 microM alpha-calponin. Pretreatment of demembranated muscle strips with alpha-calponin inhibited subsequent force development at all concentrations of Ca2+ examined over the activation range. The inhibitory effect of alpha-calponin was shown to be Ca(2+)-independent, since exogenous alpha-calponin also inhibited force in the absence of Ca2+ in demembranated muscle strips containing thiophosphorylated myosin. Phosphorylation of alpha-calponin on Ser-175 by protein kinase C has been suggested to alleviate the inhibitory effect of alpha-calponin on smooth muscle contraction. To test this hypothesis, the effects on Ca(2+)-induced and Ca(2+)-independent contractions of demembranated muscle strips of phosphorylated alpha-calponin and three site-specific mutants of alpha-calponin (in which Ser-175 was replaced by Ala, Asp or Thr) were compared with the effects of unphosphorylated tissue-purified and recombinant wild-type alpha-calponins. The recombinant wild-type protein behaved identically to the unphosphorylated tissue-purified protein, as did the S175T mutant, which is known to bind actin with high affinity and to inhibit the actin-activated myosin MgATPase in vitro. On the other hand, phosphorylated alpha-calponin and the S175A and S175D mutants, which bind weakly to actin and have little effect on the actin-activated myosin MgATPase in vitro, failed to cause significant inhibition of force induced by Ca2+ or myosin thiophosphorylation. These results support a role for alpha-calponin in the regulation of smooth muscle contraction and indicate the functional importance of Ser-175 of alpha-calponin as a regulatory site of phosphorylation.

Characteristics of Ca2+ release for activation of K+ current and contractile system in some smooth muscles.

Characteristics of Ca2+ release from stores were investigated in strips from ileum and portal vein and in isolated myocytes from ileum and urinary bladder of the guinea pig with use of caffeine and 9-methyl-7-bromoeudistomin D (MBED), a potent releaser of Ca2+ from skeletal muscle sarcoplasmic reticulum. In skinned strips, 1-30 mM caffeine elicited a transient contraction, but 10-300 microM MBED did not. Pretreatment with 100 microM MBED did not affect the subsequent caffeine-induced contraction. In single cells loaded with indo 1-acetoxymethyl ester, 10 mM caffeine increased cytoplasmic Ca2+ concentration, whereas 100 microM MBED elicited a small or no increase. Under whole cell clamp, spontaneous transient outward currents through Ca(2+)-dependent K+ (BK) channels were first enhanced and then suppressed by 30 microM MBED or 5 mM caffeine. The amplitude of Ca(2+)-dependent transient K+ current on depolarization was reduced by MBED and caffeine (50% inhibitory concentrations = 20 microM and 1 mM, respectively). Other currents and single BK channel activity were not significantly affected by MBED. The Ca2+ release from stores responsible for BK channel activation may be resolved from that for the activation of the contractile system by MBED in these smooth muscle cells.

Possible mechanism of the potent vasoconstrictor actions of ryanodine on femoral arteries from spontaneously hypertensive rats.

1. The Ca2+ buffering function of sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) was examined. Differences in the effects of ryanodine that removes the function of SR, on tension and cellular Ca2+ level were assessed in endothelium-denuded strips of femoral arteries from 13-week-old SHR and normotensive Wistar-Kyoto rats (WKY). 2. The addition of ryanodine to the resting strips caused a concentration-dependent contraction in SHR. This contraction was extremely small in WKY. In the presence of 10(-5) M ryanodine, caffeine (20 mM) failed to cause a further contraction in SHR, but it caused a small contraction in WKY. After washout of the strips with a Krebs solution, the resting tone was greatly elevated in SHR when compared with WKY. 3. The elevated resting tone in SHR strips was abolished by 10(-7) M nifedipine. The ryanodine-induced contraction was also abolished by 10(-7) M nifedipine. Nifedipine itself caused a relaxation from the resting tone of SHR strips, suggesting the maintenance of myogenic tone. 4. In strips preloaded with fura-PE3, the addition of 10(-5) M ryanodine caused a large and moderate elevation of cytosolic Ca2+ level ([Ca2+]i) in SHR and WKY, respectively. After washout, the resting [Ca2+]i was greatly elevated in SHR. The ryanodine-induced elevation of [Ca2+]i was decreased by 5 x 10(-6) M verapamil in SHR. Verapamil itself caused a decrease in resting [Ca2+]i which was significantly greater in SHR than in WKY, and caused a relaxation only in SHR. 5. The resting Ca2+ influx in arteries measured by a 5 min incubation with 45Ca was significantly increased in SHR when compared with WKY. The resting Ca2+ influx was not increased by 10(-5) M ryanodine in both SHR and WKY. The net cellular Ca2+ uptake in arteries measured by a 30 min incubation with 45Ca was decreased by 10(-5) M ryanodine in both strains. 6. The resting Ca2+ influx was decreased by 10(-7) M nifedipine in the SHR artery, but it was unchanged in the WKY artery. 7. These results suggest that (1) the Ca2+ influx via L-type voltage-dependent Ca2+ channels was increased in the resting state of the SHR femoral artery, (2) the greater part of the increased Ca2+ influx was buffered by Ca2+ uptake into the SR and some Ca2+ reached the myofilaments resulting in the maintenance of the myogenic tone, and (3) therefore the functional removal of SR by ryanodine caused a potent contraction in this artery.

Increased function of voltage-dependent Ca++ channels and Ca(++)-activated K+ channels in resting state of femoral arteries from spontaneously hypertensive rats at prehypertensive stage.

The present study examined the possible role of voltage-dependent Ca++ channels (VDCs) and Ca(++)-activated K+ (KCa) channels in the regulation of resting tone of arteries from spontaneously hypertensive rats (SHR) at a prehypertensive stage. Differences in the effects of agents that interact with these channels were assessed in endothelium-denuded strips of femoral arteries isolated from 4-week-old SHR and age-matched normotensive Wistar-Kyoto rats (WKY). Systolic blood pressures at this age were not significantly different between SHR and WKY. The arterial strips from SHR maintained a myogenic tone in the resting state; that is the resting tone in the SHR artery was abolished when either the bathing solution was replaced with a Ca(++)-free solution or 10(-7) M nifedipine was added. Studies using 1- or 5-min pulse labeling of the arteries with 45Ca showed that the resting Ca++ influx was significantly increased in SHR when compared with WKY, and this increase in SHR was abolished by 10(-7) M nifedipine. In strips preloaded with fura-PE3, the addition of 3 x 10(-6) M verapamil to resting muscles decreased the resting cytosolic Ca++ level and caused a relaxation. These effects of verapamil were more evident in SHR than in WKY. The addition to the strips of charybdotoxin and iberiotoxin, blockers of large conductance KCa channels, caused a concentration-dependent contraction, which was significantly greater in SHR than in WKY.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased Ca2+ influx in the resting state maintains the myogenic tone and activates charybdotoxin-sensitive K+ channels in femoral arteries from young SHR.

1. To determine the possible role of voltage-dependent Ca2+ channels (VDCC) and Ca2+-activated K+ (KCa) channels in the regulation of resting tone of arteries from young spontaneously hypertensive rats (SHR), mechanical responses to the agents which interact with these channels were examined in endothelium-denuded strips of femoral arteries from 4 week old SHR and age-matched normotensive Wistar-Kyoto (WKY) rats. Systolic blood pressures at this age were not significantly different between SHR and WKY. 2. The strips from SHR, but not from WKY, maintained a myogenic tone; that is, the resting tone decreased when nifedipine was added. 3. Studies using 1 or 5 min pulse labelling of the strips with 45Ca showed that the basal Ca2+ influx was increased in SHR when compared with WKY, and this increase in SHR was abolished by nifedipine. Similar results were obtained when the cytoplasmic Ca2+ concentration ([Ca2+]i) in the resting state of the strips was measured by fura-PE3. 4. The addition of charybdotoxin (ChTX, a blocker of large conductance KCa channels) to the resting state caused a concentration-dependent contraction, which was much greater in SHR than in WKY. The ChTX-induced contraction in SHR was abolished by nifedipine. 5. In strips preloaded with 86Rb, the basal 86Rb efflux rate constant was significantly greater in SHR than in WKY. The increase in 86Rb efflux in SHR was abolished by nifedipine. 6. The results suggest that the Ca2+ influx via L-type VDCC was increased in the resting state of the femoral artery from 4 week old SHR, and therefore the myogenic tone was maintained and ChTX-sensitive K+ channels were highly activated.

Tetrahexylammonium ions increase Ca2+ sensitivity of contraction of guinea-pig ileal smooth muscle.

Effects of tetraalkylammonium ions, having tetraalkyl chains of increasing length from ethyl to octyl, on inositol-trisphosphate (InsP3)-induced Ca2+ release and contractile mechanics were examined in guinea-pig skinned ileal smooth muscle longitudinal strips. Although tetrahexylammonium ions (THexA) appeared to be the most potent inhibitor of Ca2+ release among the tetraalkylammonium ions examined, an additional and more prominent effect was found, i.e., the contraction induced by Ca2+ release showed a large sustained component in the presence of THexA. Potentiation of the contraction by THexA (above 30 microM) was also observed in skinned fibers in which the sarcoplasmic reticulum function was destroyed by treatment with A23187. The potentiating effect of THexA was the most potent by far among the tetraalkylammonium ions examined and was elicited by Ca(2+)-dependent and GTP-binding-protein-independent mechanisms. The potentiation was not due to activation of myosin light-chain kinase. The selective inhibitors of myosin light-chain kinase, protein kinase C and calmodulin reduced THexA-induced potentiation of contraction only at concentrations above 30 microM, at which non-specific effects are likely. Furthermore, relaxation induced by changing pCa from 4.5 to 8.5 was not affected by 1 mM THexA, suggesting that the potentiating effect is not mainly due to inhibition of myosin light-chain phosphatase. In conclusion, ThexA sensitizes guinea-pig skinned ileal smooth muscle to Ca2+ in a structure-selective manner. This sensitization appears not to be mediated mainly by a GTP-binding protein, by activation of myosin light-chain kinase or protein kinase C, by enhanced Ca2+ binding to calmodulin, or by inhibition of myosin light-chain phosphatase.

Cyclopiazonic acid, an inhibitor of Ca(2+)-ATPase in sarcoplasmic reticulum, increases excitability in ileal smooth muscle.

1. Effects of cyclopiazonic acid (CPA), a specific inhibitor of Ca(2+)-ATPase in endo- and sarcoplasmic reticulum (ER/SR), on contractile responses, cytosolic Ca2+ concentration and spontaneous electrical activity were examined in ileal longitudinal smooth muscle strips. 2. After intracellular stored Ca2+ in intact ileal strips was depleted by application of 25 mM caffeine in Ca(2+)-free solution, Ca(2+)-loading was performed in the absence or presence of 10 microns CPA in a standard solution containing 2.2 mM Ca2+. Subsequent application of caffeine in Ca(2+)-free solution induced a phasic contraction which was significantly smaller in the strip pretreated with CPA than that in the control. 3. Spontaneous and 20 mM K(+)-induced contractions in the presence of 1 microM atropine were markedly enhanced by 1-30 microM CPA, whereas that induced by 80 mM K+ was not. The magnitude of repetitive transient elevation of cytosolic Ca2+ concentration ([Ca2+])i) and concomitant phasic contractions were markedly enhanced by CPA. The effects were abolished by 10 microM verapamil and restored by 10 microM Bay K 8644. 4. Application of 10 microM CPA depolarized the cell by about 5 mV, decreased the action potential (AP) afterhyperpolarization and markedly increased the frequency of spontaneous AP. These effects were mimicked by 100 nM charybdotoxin. 5. The rate of decay of [Ca2+]i and tension after the bathing solution was changed from one containing 140 mM K+ and 2.2 mM Ca2+ to one containing 5.9 mM K+ and 0 mM Ca2+ was significantly slowed when 10 microM CPA was added to the latter solution. 6. These results indicate that CPA enhances ileal smooth muscle excitability and increases Ca2+-influx through voltage-dependent Ca2+ channels. The effect may be consistent with the hypothesis that CPA-induced decrease in stored Ca due to Ca-pump inhibition reduces the Ca2+-dependent K+ current and indirectly enhances Ca2+-influx through membrane activity resulting from the increased excitability.Direct evidence for the regulation of Ca2+ channel activity by intracellular Ca storage sites was not obtained in the present study.

Effects of cyclopiazonic acid, a novel Ca(2+)-ATPase inhibitor, on contractile responses in skinned ileal smooth muscle.

1. Effects of cyclopiazonic acid (CPA), a specific inhibitor of the Ca(2+)-ATPase in sarcoplasmic reticulum (SR) of skeletal and cardiac muscles, on contractile responses induced by Ca(2+)-release from intracellular storage sites were examined in the longitudinal smooth muscle strip of the guinea-pig ileum skinned with beta-escin. 2. Ca(2+)-loading of storage sites (Ca(2+)-uptake) was performed in pCa 6.3 solution. The amount of Ca2+ taken up was monitored by use of the amplitude of contraction following application of 25 mM caffeine or 25 microM inositol 1,4,5-trisphosphate (IP3). 3. Contractile responses to caffeine or IP3 were reduced or abolished when the preceding Ca(2+)-uptake was performed in the presence of 0.1-10 microM CPA. The dose of CPA required to inhibit the contraction induced by caffeine or IP3 by 50% was approximately 0.6 microM. The CPA-sensitive Ca(2+)-uptake completely depended upon the presence of ATP in the solution during Ca(2+)-uptake. 4. When 1 microM CPA was added after Ca(2+)-uptake, the subsequent caffeine- or IP3-induced contraction was not significantly affected by the presence of CPA. 5. Acetylcholine-induced contraction was also almost abolished when the preceding Ca(2+)-uptake was performed in the presence of 10 microM CPA. 6. The relationship between pCa and contraction was not affected by the presence of 10 microM CPA in skinned fibres where Ca2+ storage sites had been destroyed by treatment with A23187. The enhancement of contraction in pCa 6.0 solution by calmodulin was not affected by 10 microM CPA.7. These results suggest that CPA selectively inhibits ATP-dependent Ca2"-uptake into intracellular storage sites in skinned ileal smooth muscle strips. CPA appears to be a potent, reversible, and very specific inhibitor of the Ca2+-pump in the storage sites of smooth muscle, and is an extremely valuable pharmacological tool.