The response of single cardiac sodium channels in neonatal rats to the dihydropyridines CGP 28392 and (−)-Bay K 8644

Summary Cell-attached patch clamp recording of elementary Na⁺ currents were performed at 19°C in neonatal cultured rat heart cells to study Na⁺ channel properties in the presence of dihydropyridines.Bath application of racemic CGP 28392, at 5 gmol/l, activated Na⁺ channels. By increasing the open probability, P _O, and/or the number of functioning Na⁺ channels, peak I _Na in reconstructed macroscopic Na+ currents rose without changes in the decay kinetics. This was accompanied by a prolongation of open time. (–)-Bay K 8644 (1–10 mol/l) had the same effect. In the presence of either agonist, Na⁺ channels retained an uniform open state and, as estimated from the mean number of openings per sequence, their initial tendency to reopen. Rarely appearing ultralong opening sequences are unlikely to be drug-induced as Na⁺ channels can likewise switch into this particular activity mode under drug-free conditions. Racemic CGP 28392, at 50 mol/l, blocked Na⁺ channels in an all-or-none fashion suggesting that one enantiomer acts as agonist and the other enantiomer as blocker. A quite different response consisting of the occurrence of a second open state with a several-fold increased life time and a significantly increased reopening was observed with (–)-Bay K 8644 in damaged cardiocytes with hyperpermeable membranes and after patch excision into drug-containing solution. Evidence was obtained from control inside-out patches that this increased reopening is most probably caused by the solvent, ethanol.It is concluded that cardiac Na⁺- channels affected by agonistic dihydropyridines not only respond with an increased likelihood to open during membrane depolarization but can also preserve their open state. Compared with the response of l-type Ca²⁺ channels (Hess et al. 1984), Na⁺ channel life time is only slightly prolonged by agonistic dihydropyridines. Send offprint requests to M. Kohlhardt at the above address     

Influence of (−)-S-Bay K 8644, (±)-Bay W 5035 and (±)-Bay T 5006 on hemodynamics and FITC-Dextran 3 elution kinetics in isolated rat hearts

• 1.1. We investigated the effects of the new calcium-agonists (±)-Bay W 5035 and (±)-Bay T 5006 in comparison to (-)-S-Bay K 8644 on hemodynamics and epimyocardial perfusion in Langendorff rat hearts.
• 2.2. At equieffective inotropic concentration, vasoconstriction of coronary resistance vessels was significantly less after (±)-Bay W 5035 or (±)-Bay T 5006 than after (-)-S-Bay K 8644 application.
• 3.3. FITC-Dextran 3 elution kinetics indicated that the epimyocardial vascular volume was significantly reduced only by (-)-S-Bay K 8644.
• 4.4. Moreover, (-)-S-Bay K 8644 enhanced transcoronary exchange more markedly than (+-)-Bay W 5035 or (±)-Bay T 5006, reflecting the differences in coronary constrictor activity.
• 5.5. We conclude that in comparison to (-)-S-Bay K 8644 the relation between inotropy and vasoconstriction is more favorable for (±)-Bay W 5035 or (±)-Bay T 5006.

     

BAY K 8644 stimulates glucose-dependent rise of cytoplasmic Ca2+ in hyperpolarized pancreatic β-cells

Summary The effect of BAY K 8644 on the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was studied in pancreatic -cells hyperpolarized by the K⁺ channel-activating agent diazoxide. After 50–60 min preexposure to 0–20 mM glucose in the presence of 400 M diazoxide [Ca²⁺]_i was close to the level in unstimulated -cells. The addition of 5 M BAY K 8644 then triggereed a rise of [Ca²⁺]_i dependent on Ca²⁺ influx. The magnitude of the BAY K 8644 effect increased with the glucose concentration and was almost 10-fold higher in 20 mM than in the absence of the sugar. It is concluded that glucose can modulate Ca²⁺ entry through the voltage-dependent channels by a mechanism additional to depolarization. This action may help to explain why previous exposure to the sugar results in an augmented insulin response to a second challenge.     

Effects of norepinephrine and isopentenyladenosine on Na~ /Ca~(2 ) exchangecurrents in isolated guinea pig ventricular myocytes

目的：研究去甲肾上腺素（ＮＥ）和异丙肾上腺素（Ｉｓｏ）对Ｎａ＋／Ｃａ２＋交换电流的影响及受体调控机制．方法：应用全细胞电压钳技术的斜坡脉冲程序，测定离体豚鼠心肌细胞准稳态电流电压关系曲线．结果：ＮＥ０００５，００５和５μｍｏｌ·Ｌ－１分别使膜电位＋５０ｍＶ时的Ｎｉ２＋敏感电流增加２９％±９％，７２％±１１％和１２０％±３１％；Ｉｓｏ１５，１５０和１５００ｎｍｏｌ·Ｌ－１分别使该电流增加２８％±２８％，５６％±１３％和１０２％±１２％．ＮＥ和Ｉｓｏ的这种增强效应能被普萘洛尔１０μｍｏｌ·Ｌ－１完全阻断，而酚妥拉明５０μｍｏｌ·Ｌ－１无此作用．结论：ＮＥ和Ｉｓｏ通过兴奋心脏β肾上腺素受体使Ｎａ＋／Ｃａ２＋交换电流增加．     

Stimulation of adenylate cyclase of guinea pig gastric mucosa by histamine,sodium fluoride and 5′-guanylylimidodiphosphate and inhibition by histamine H1- and H2-receptor antagonists in vitro

Summary There are experimental data indicating that cyclic AMP is involved in the regulation of gastric acid secretion in various mammalian species. In a broken cell preparation of guinea pig gastric mucosa the effects of some stimulants of gastric acid secretion on the activity of adenylate cyclase were studied. The basal adenylate cyclase activity was 483±43 pmoles cyclic AMP/mg proteinx10 min. The activity could be stimulated by histamine maximally 5-fold, by sodium fluoride (NaF) maximally 20-fold and by 5-guanylylimidodiphosphate (GMP-PNP) maximally 10-fold. Neither pentagastrin nor carbachol were able to stimulate the adenylate cyclase. Stimulants of adrenergic - or -receptors (phenylephrine, isoproterenol) were also ineffective.The activation of the adenylate cyclase by histamine was inhibited by the histamine H₁-receptor antagonists diphenhydramine and mepyramine as well as by the histamine H₂-receptor antagonist metiamide. On the other hand, the stimulatory action of NaF or GMP-PNP could be antagonized only by high concentrations of dipenhydramine or mepyramine while metiamide showed no antagonizing effect in this respect. Thus this preparation can be used as a tool to determine the activity and specificity of histamine H₂-receptor antagonists.     

Pertussis toxin abolishes the inhibition of Ca2+ currents and of noradrenaline release via α2-adrenoceptors in chick sympathetic neurons

Summary Effects of ₂-adrenoceptor agonists on whole-cell Ca²⁺ currents and ³H-noradrenaline release were investigated by applying the patch-clamp technique and electrical field stimulation to cultured embryonic chick sympathetic neurons. A 24-h exposure of the sympathetic neurons to pertussis toxin (100 ng/ml) abolished both the 2-adrenoceptor-mediated inhibition of Ca ²⁺ currents and the modulation of noradrenaline release caused by noradrenaline (1 mol/l; in the presence of 10 mol/l cocaine) or the ₂-adrenoceptor agonists 5-bromo-6-(2imidazolin-2-ylamino)quinoxaline (UK 14,304, 10 mol/ l) and clonidine (10 mol/l). These results suggest that the ₂-autoreceptor-mediated inhibition of noradrenaline release from chick sympathetic neurons operates through the modulation of Ca²⁺ channels via pertussistoxin-sensitive GTP-binding-proteins. Send offprint requests to S. Boehm at the above address     

Lack of effect of the α2C-adrenoceptor Del322-325 polymorphism on inhibition of cyclic AMP production in HEK293 cells

Background and purpose:

The α_2C-adrenoceptor has multiple functions, including inhibiting release of noradrenaline from presynaptic nerve terminals. A human α_2C polymorphism, Del322-325, a potential risk factor for heart failure, has been reported to exhibit reduced signalling in CHO cells. To further understand the role of the Del322-325 polymorphism on receptor signalling, we attempted to replicate and further study the reduced signalling in HEK293 cells.

Experimental approach:

Human α_2C wild-type (WT) and Del322-325 adrenoceptors were stably transfected into HEK293 cells. Radioligand binding was performed to determine affinities for both receptors. In intact cells, inhibition of forskolin-stimulated cyclic AMP production by WT and Del322-325 clones with a range of receptor densities (200–2320 fmol·mg⁻¹ protein) was measured following agonist treatment.

Key results:

Noradrenaline, brimonidine and clonidine exhibited similar binding affinities for WT and Del322-325. Brimonidine and clonidine also had similar efficacies and potencies for both receptors for the inhibition of cyclic AMP production at all receptor densities tested. A linear regression analysis comparing efficacy and potency with receptor expression levels showed no differences in slopes between WT and Del322-325.

Conclusions and implications:

The α_2C WT and Del322-325 adrenoceptors exhibited similar binding properties. Additionally, inhibition of cyclic AMP production by Del322-325 was similar to that of WT over a range of receptor densities. Therefore, in intact HEK293 cells, the α_2C-Del322-325 polymorphism does not exhibit reduced signalling to adenylyl cyclase and may not represent a clinically important phenotype.     

Effects of prostaglandins F2α and E1 on the longitudinal and circular smooth muscle of the guinea pig caecum in relation to the concentration of extracellular calcium

Summary Responses of the longitudinal and circular smooth muscle of the guinea-pig caecum were studied in media with different concentrations of extracellular calcium. A sucrose-gap method was used to record the electrical and mechanical parameters of smooth muscle activity.In the longitudinal smooth muscle no difference in the action of PGF₂ and PGE₁ was seen at low (0.8 mM), normal (2.5 mM) or high (7.5 mM) concentrations of Ca²⁺. Both PG's stimulated the longitudinal strip and the action of acetylcholine at normal Ca²⁺ concentration was slightly inhibited immediately after the superfusion with the PG's and augmented thereafter. In the circular strip no significant difference between the stimulatory effect of PGF₂ and PGE₁ in low and high Ca²⁺ solution was found. However, at 2.5 mM Ca²⁺ PGF₂ evoked much greater stimulation of the circular strip than did PGE₁. After the end of the superfusion with either PG the effect of acetylcholine was inhibited by either PG at low Ca²⁺ level and potentiated at high Ca²⁺ level; at normal Ca²⁺ level the effect of acetylcholine was inhibited by PGE₁ but not by PGF₂.Thus, the effects of PGF₂ and PGE₁ on the circular smooth muscle differed from each other in their dependence on the concentration of extracellular calcium.     

Actions of KMUP‐1, a xanthine and piperazine derivative,on voltage‐gated Na+ and Ca2+‐activated K+ currents in GH3 pituitary tumour cells

Background and Purpose

7‐[2‐[4‐(2‐Chlorophenyl)piperazinyl]ethyl]‐1,3‐dimethylxanthine (KMUP‐1) is a xanthine‐based derivative. It has soluble GC activation and K⁺‐channel opening activity. Effects of this compound on ion currents in pituitary GH₃ cells were investigated in this study.

Experimental Approach

The aim of this study was to evaluate effects of KMUP‐1 on the amplitude and gating of voltage‐gated Na⁺ current (I _Na) in pituitary GH₃ cells and in HEKT293T cells expressing SCN5A. Both the amplitude of Ca²⁺‐activated K⁺ current and the activity of large‐conductance Ca²⁺‐activated K⁺ (BK_Ca) channels were also studied.

Key Results

KMUP‐1 depressed the transient and late components of I _Na with different potencies. The IC₅₀ values required for its inhibitory effect on transient and late I _Na were 22.5 and 1.8 μM respectively. KMUP‐1 (3 μM) shifted the steady‐state inactivation of I _Na to a hyperpolarized potential by −10 mV, despite inability to alter the recovery of I _Na from inactivation. In cell‐attached configuration, KMUP‐1 applied to bath increased BK_Ca‐channel activity; however, in inside‐out patches, this compound applied to the intracellular surface had no effect on it. It prolonged the latency in the generation of action currents elicited by triangular voltage ramps. Additionally, KMUP‐1 decreased the peak I _Na with a concomitant increase of current inactivation in HEKT293T cells expressing SCN5A.

Conclusions and Implications

Apart from activating BK_Ca channels, KMUP‐1 preferentially suppresses late I _Na. The effects of KUMP‐1 on ion currents presented here constitute an underlying ionic mechanism of its actions.

Abbreviations

AC

action current

AP

action potential

BK_Ca

channel large‐conductance Ca²⁺‐activated K⁺ channel

I_K(Ca)

Ca²⁺‐activated K⁺ current

I_Na

voltage‐gated Na⁺ current

I–V

current versus voltage

K_ATP

channel ATP‐sensitive K⁺ channel

ODQ

1H‐[1,2,4]oxadiazolo‐[4,3‐a] quinoxalin‐1‐one

TEA

tetraethylammonium chloride

τ_inact(S)

slow component of inactivation time constant for I _Na

YC‐1

3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzylindazole

     

Effect of K+ channel blockers on the α2-adrenoceptor-coupled regulation of electrically evoked noradrenaline release in hippocampus

Summary The question whether presynaptic ₂-adrenoceptors regulating noradrenaline release in hippocampus directly couple to tetraethylammonium chloride (TEA) or -dendrotoxin (-DTX)-sensitive K⁺ channels was investigated. Hippocampal slices, prelabelled with [³H] noradrenaline, were superfused in the presence of (+)-oxaprotiline and electrically stimulated with 4 pulses delivered at 100 Hz, in order to avoid autoinhibition due to released noradrenaline.TEA enhanced the evoked [³H]noradrenaline release in rabbit hippocampus in a concentration-dependent manner, yielding an approximately 4-fold increase at 30 mmol/l, whereas the spontaneous outflow of tritium was only slightly affected at this concentration. The ₂-adrenoceptor agonist clonidine, at 10–100 nmol/l inhibited the evoked [³H]noradrenaline release between 77% and 96%. The inhibitory effect of the ₂-agonist was distinctly diminished in the presence of 30 mmol/l TEA but was restored in low Ca²⁺/high Mg²⁺ buffer. Therefore, the diminution of the ₂-agonist effect by TEA observed in experiments with normal Ca²⁺ can be explained by an increase of the Ca²⁺ availability for the release process due to the prolongation of action potentials. In rabbit hippocampus -DTX (10–200 nmol/l) did neither affect the evoked release of [³H]noradrenaline nor its ₂-agonist-induced modulation. However, in rat hippocampus -DTX significantly increased the evoked transmitter release and diminished the effect of clonidine.Taken together, the present data for the rabbit hippocampus exclude the possibility that activation of presynaptic ₂-adrenoceptors inhibits depolarization-evoked [³H]noradrenaline release by inducing an outward K⁺ current through TEA- or -DTX-sensitive K⁺ channels. However, there are species differences between the rabbit and the rat so that in the rat the ₂-adrenoceptors could actually be coupled to K⁺ channels — provided that the release-enhancing properties of -DTX do not account for the ₂-antagonism observed.Correspondence to C. Allgaier at the above address     

Differential effects of chronic ethanol exposure on cytochrome P450 2E1 and the hypothalamic–pituitary–adrenal axis in the maternal–fetal unit of the guinea pig

BackgroundEthanol neurobehavioural teratogenicity is a leading cause of developmental mental deficiency, in which the hippocampus is a target site of injury. The multi-faceted mechanism of ethanol teratogenicity is not completely understood. This study tested the hypothesis that chronic ethanol exposure (CEE), via chronic maternal ethanol administration, increases cytochrome P450 2E1 (CYP2E1) expression and alters hypothalamic–pituitary–adrenal (HPA) axis activity in the maternal–fetal unit during the third-trimester-equivalent of gestation.MethodsPregnant Dunkin–Hartley-strain guinea pigs received daily oral administration of ethanol (4 g ethanol/kg maternal body weight) or isocaloric-sucrose/pair-feeding (control) throughout gestation (term, about gestational day (GD) 68). On GD 45, 55 and 65, pregnant animals were euthanized 2 h after the last daily dose. Maternal and fetal body weights and fetal hippocampal brain weight were determined. Maternal and fetal samples were collected for the determination of liver CYP2E1 enzymatic activity and plasma free cortisol and ACTH concentrations.ResultsCEE, with maternal blood ethanol concentration of 108–124 mg/dl at 2 h after the last dose, decreased fetal hippocampal weight only at GD 65 and had no effect on fetal body weight compared with control. CYP2E1 activity increased with gestational age in the fetal liver microsomal and mitochondrial fractions. CEE increased CYP2E1 activity in the microsomal and mitochondrial fractions of maternal liver at the three gestational ages and in both hepatic subcellular fractions of the GD 65 fetus compared with control. There was a gestational-age-dependent increase in maternal and fetal plasma free cortisol concentrations, but no effect of CEE compared with control. Maternal and fetal plasma ACTH concentrations were unaffected by CEE compared with control, and were virtually unchanged during the third-trimester-equivalent that was studied.ConclusionThese data demonstrate that, in the pregnant guinea pig, this CEE regimen increases liver CYP2E1 activity, without affecting HPA axis function, in the maternal–fetal unit during near-term gestation. The CEE-induced increase in liver CYP2E1 activity and potential oxidative stress in the maternal–fetal unit may play a role in the pathogenesis of ethanol teratogenicity.     

The antiepileptic effect of Centella asiatica on the activities of Na+/K+, Mg2+ and Ca2+-ATPases in rat brain during pentylenetetrazol�Cinduced epilepsy

Background:

To study the anticonvulsant effect of different extracts of Centella asiatica (CA) in male albino rats with reference to Na⁺/K⁺, Mg²⁺ and Ca²⁺-ATPase activities.

Materials and Methods:

Male Wistar rats (150±25 g b.w.) were divided into seven groups of six each i.e. (a) control rats treated with saline, (b) pentylenetetrazol (PTZ)-induced epileptic group (60 mg/kg, i.p.), (c) epileptic group pretreated with n-hexane extract (n-HE), (d) epileptic group pretreated with chloroform extract (CE), (e) epileptic group pretreated with ethyl acetate extract (EAE), (f) epileptic group pretreated with n-butanol extract (n-BE), and (g) epileptic group pretreated with aqueous extract (AE).

Results:

The activities of three ATPases were decreased in different regions of brain during PTZ-induced epilepsy and were increased in epileptic rats pretreated with different extracts of CA except AE.

Conclusion:

The extracts of C. asiatica, except AE, possess anticonvulsant and neuroprotective activity and thus can be used for effective management in treatment of epileptic seizures.     

Correlation between the inhibitory activities of calcium entry blockers on vascular smooth muscle constriction in vitro after K+-depolarisation and in vivo afterα 2-adrenoceptor stimulation

Summary The acetylcholine (ACh) release was studied in superfused, electrically-stimulated slices of guinea-pig cerebral cortex.Muscimol and 4,5,6,7-tetrahydroisoxazolo (5-4-c)-pyridin-3-ol (THIP), as well as exogenous GABA, reduced the electrically-evoked ACh release and enhanced its spontaneous outflow. Picrotoxin antagonized these effects.In addition, picrotoxin and ethanolamine-O-sulphate (EOS) caused opposite changes in transmitter outflow, suggesting the existence of an endogenous GABAergic control on the cholinergic nerve endings. Neither flurazepam 6.6×10^–6–3.3×10^–5 mol/l nor diazepam 3.3×10^–6–3.3×10^–5 mol/l by themselves affected ACh release.Diazepam prevented GABA-, muscimol- and EOS-induced changes in spontaneous and 1 Hz-evoked outflow. Ro 15-1788 3.3×10^–6 mol/l abolished diazepam antagonism vs exogenous GABA.The ineffectiveness of flurazepam and diazepam on normal release (i.e. the lack of potentiation vs the endogenous GABAergic control) supports the view that synaptic GABA receptors acting upon the cholinergic nerve endings are not coupled with Benzodiazepine receptors.The unexpected diazepam antagonism vs exogenous GABA and GABA-like compounds can be explained with an unusual Diazepam negative cooperation with extrasynaptic GABA receptors, possibly present on the cholinergic terminals.Thus, the rule of benzodiazepine-GABA synergism does not seem always tenable, at least at certain pre-synaptic sites.     

Effect of β-naphthoflavone on AhR-regulated genes (CYP1A1, 1A2, 1B1, 2S1, Nrf2, and GST) and antioxidant enzymes in various brain regions of pig

The constitutive and inducible expression of aryl hydrocarbon receptor (AhR) and of the AhR-regulated genes coding for CYP1A1, CYP1A2, CYP1B1, CYP2S1, and Nrf2 was investigated by real-time or traditional PCR in cerebral areas (cortex, cerebellum, midbrain, and hippocampus), blood–brain interfaces (meninges and brain microvessels) and liver obtained from control pigs and from pigs treated with β-naphthoflavone (βNF), a potent AhR agonist. The enzymatic activities of ethoxyresorufin-O-deethylase (EROD), and methoxyresorufin-O-deethylase (MEROD), marker for CYP1A1 and CYP1A2, the GST and various antioxidant enzymes (catalase, superoxide dismutase, GSSG-reductase, and GSH-peroxidase) were also determined in the same CNS regions. The AhR, CYP1A1, CYP1A2, CYP1B1, Nrf2 mRNAs were detected, although at different extent, in all the CNS regions, while CYP2S1 mRNA was detected only in midbrain. In the blood–brain interfaces, the constitutive basal expression of AhR and CYP1A1 was comparable to the hepatic one and even higher for CYP1B1 and Nrf2. The treatment with βNF determined the induction of CYP1A1 and 1B1 (but not of AhR, CYP1A2, and Nrf2) mRNA levels in various CNS areas; notably, CYP1A1 mRNA was increased to about 300-fold in the microvessels. The analysis of enzymatic activities revealed that EROD, but not MEROD, was induced in microsomes but not in mitochondria of all the CNS areas. However, the mitochondrial EROD activities were comparable (in midbrain, meninges) or higher (in cortex, cerebellum, hippocampus) than the microsomal ones, suggesting an important metabolic function of CYP1A1 in this subcellular localization. The activities of GST and antioxidant enzymes were detected in all CNS tissues, with levels lower than the hepatic ones, but found quite evenly distributed and marginally affected by βNF treatment. The high expression of metabolic enzymes found in blood–brain interfaces could represent a very important defence toward toxins of CNS.     

Multiple effects of 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca2+ signaling in MDCK cells: depletion of thapsigargin-sensitive Ca2+ store followed by capacitative Ca2+ entry, activation of a direct Ca2+ entry, and inhibition of thapsigargin-induced capacitative Ca2+ entry

The effect of 1-[β-[3-(4-methoxyphenyl)pro- poxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca²⁺ signaling in Madin Darby canine kidney (MDCK) cells was examined. SKF 96365 at 25–100 μM evoked a robust [Ca²⁺]_i transient in a dose-dependent manner, measured by fura-2 fluorimetry. A concentration of 10 μM SKF 96365 did not have an effect. The transient consisted of a slow rise, a gradual decay, and a sustained plateau in physiological Ca²⁺ medium. Removal of extracellular Ca²⁺ reduced the Ca²⁺ signals evoked by 50–100 μM SKF 96365 by nearly half in the area under the curve, suggesting that SKF 96365 induced intracellular Ca²⁺ release and also extracellular Ca²⁺ influx. A concentration of 100 μM SKF 96365 caused significant Mn²⁺ quench of fura-2 fluorescence, which was partly inhibited by La³⁺ (1 mM) or Gd³⁺ (0.1 mM), indicating that the SKF 96365-induced Ca²⁺ influx had two components: one is sensitive to La³⁺ (1 mM) or Gd³⁺ (0.1 mM), the other is not. The internal Ca²⁺ source for the SKF 96365-induced [Ca²⁺]_i transient was the endoplasmic reticulum Ca²⁺ store because, pretreatment with thapsigargin and cyclopiazonic acid, two inhibitors of the endoplasmic reticulum Ca²⁺ pump nearly abolished the SKF 96365-induced [Ca²⁺]_i increase in Ca²⁺-free medium. In contrast, pretreatment with 100 μM SKF 96365 only partly depleted the thapsigargin-sensitive Ca²⁺ store. Addition of 10 mM Ca²⁺ induced a significant [Ca²⁺]_i increase after prior incubation with 100 μM SKF 96365 in Ca²⁺-free medium, demonstrating that SKF 96365 induced capacitative Ca²⁺ entry. This capacitative Ca²⁺ entry was about 40% of that induced by 1 μM thapsigargin. Additional to inducing its own capacitative Ca²⁺ entry, 100 μM SKF 96365 partly inhibited thapsigargin- or uridine trisphos-phate (UTP)-induced capacitative Ca²⁺ entry. We also investigated the mechanisms underlying the decay of the SKF 96365-induced [Ca²⁺]_i transient. Inhibition of the plasma membrane Ca²⁺ pump with La³⁺ or Gd³⁺, or lowering extracellular Na⁺ level to 0.35 mM, significantly increased the SKF 96365-induced [Ca²⁺]_i transient. In contrast, the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone had little effect. In Ca²⁺-free medium, the thapsigargin-induced [Ca²⁺]_i increase was greatly reduced by pretreatment with SKF 96365. Collectively, we have found that besides its well-known inhibitory action on capacitative Ca²⁺ entry in many cell types, in MDCK cells SKF 96365 exerted multiple and complex effects on Ca²⁺ signaling. It induced a considerable increase in [Ca²⁺]_i by releasing Ca²⁺ from the endoplasmic reticulum store followed by capacitative Ca²⁺ entry. It also caused a direct Ca²⁺ entry. The decay of the SKF 96365 response was significantly governed by efflux via the plasma membrane Ca²⁺ pump or Na⁺/Ca²⁺ exchange. Sequestration by mitochondria or the endoplasmic reticulum played a minor role. We caution use of SKF 96365 as an inhibitor of capacitative Ca²⁺ entry. Received: 21 September 1998 / Accepted: 2 December 1998