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Induced pluripotent stem cells (iPSCs) are regarded as a promising option for cell-based regenerative medicine. To obtain safe and efficient iPSC-based cell products, it is necessary to selectively eliminate the residual iPSCs prior to in vivo implantation due to the risk of teratoma formation. Bee venom (BV) has long been used in traditional Chinese medicine to treat inflammatory diseases and relieve pain, and has been shown to exhibit anti-cancer, anti-mutagenic, anti-nociceptive, and radioprotective activities. However, the potential benefits of BV in iPSC therapy, particularly its anti-teratoma activity, have not been examined. In this study, we found that BV selectively induced cell death in iPSCs, but not in iPSC-derived differentiated cells (iPSCs-Diff). BV rapidly disrupted cell membrane integrity and focal adhesions, followed by induction of apoptosis and necroptosis in iPSCs. We also found that BV remarkably enhanced intracellular calcium levels, calpain activation, and reactive oxygen speciesgeneration in iPSCs. BV treatment before in ovo grafting efficiently prevented iPSC-derived teratoma formation. In contrast, no DNA damage was observed in iPSCs-Diff following BV treatment, further demonstrating the safety of BV for use with iPSCs-Diff. Taken together, these findings show that BV has potent anti-teratoma activity by eliminating residual iPSCs, and can be used for the development of effective and safe iPSC-based cell therapies.

The electrophysiological properties underlying the automaticity of the guinea pig pulmonary vein myocardium were studied. About 30% of the isolated pulmonary vein tissue preparations showed spontaneous electrical activity, as shown by glass microelectrode recordings from their myocardial layer. The remaining quiescent preparations had a resting membrane potential less negative than that in the left atria. Blockade of the acetylcholine activated potassium current (I K-ACh ) by tertiapin induced a depolarizing shift of the resting membrane potential and automatic electrical activity in the pulmonary vein, but not in the atria. The tertiapin-induced electrical activity, as well as the spontaneous activity, was inhibited by the application of carbachol or by chelation of intracellular Ca 2+ by BAPTA. The isolated pulmonary vein cardiomyocytes had an I K-ACh density similar to that of the atrial cardiomyocytes, but a lower density of the inwardly-rectifying potassium current (I K1 ). Spontaneous Ca 2+ transients were observed in about 30% of the isolated pulmonary vein cardiomyocytes, but not in atrial cardiomyocytes. The Ca 2+ transients in the pulmonary vein cardiomyocytes were induced by tertiapin and inhibited by carbachol. These results indicate that the pulmonary vein cardiomyocytes have a reduced density of the inwardly-rectifying potassium current, which plays a permissive role in their intracellular Ca 2+ -dependent automaticity., (Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Afferent innervation patterns in the vestibular periphery are complex, and vestibular afferents show a large variation in their regularity of firing. Calyx fibers terminate on type I vestibular hair cells and have firing characteristics distinct from the bouton fibers that innervate type II hair cells. Whole-cell patch clamp was used to investigate ionic currents that could influence firing patterns in calyx terminals. Underlying K(Ca) conductances have been described in vestibular ganglion cells, but their presence in afferent terminals has not been investigated previously. Apamin, a selective blocker of SK-type calcium-activated K(+) channels, was tested on calyx afferent terminals isolated from gerbil semicircular canals during postnatal days 1-50. Lowering extracellular calcium or application of apamin (20-500 nM) reduced slowly activating outward currents in voltage clamp. Apamin also reduced the action potential afterhyperpolarization (AHP) in whole-cell current clamp, but only after the first two postnatal weeks. K(+) channel expression increased during the first postnatal month, and SK channels were found to contribute to the AHP, which may in turn influence discharge regularity in calyx vestibular afferents.

Bee venom (BV) has been used in Oriental medicine to treat inflammatory diseases, such as tendonitis, bursitis, and rheumatoid arthritis, despite the sensitivity of the victims and toxicity of the venom. This study examined the mechanisms for the effects of BV on the cardiovascular system in rats. The arterial pressure and heart rate (HR) were measured in anesthetized rats. In addition, the left ventricular development pressure (LVDP) and total magnesium efflux ([Mg]e) in isolated perfused hearts, the vascular tonic responses in the isolated aorta, and the blood ionic and biochemical changes were determined simultaneously. In the anesthetized rats, the mean arterial pressure, systolic pressure, and pulse pressure were reduced by BV in a dose-dependent manner, even though the HR was increased. BV had no effects on the relaxation of phenylephrine- or KCl-induced contraction of the aortic rings. In the isolated hearts, BV generated a reversible decrease in the LVDP and velocity with changes in pressure, which were accompanied by increases in the HR and [Mg]e. BV increased the plasma ionized and total magnesium concentrations, and decreased the total magnesium level in the red blood cells. The ratio of ionized calcium/ionized magnesium was also decreased by the BV treatment. BV caused a detectable increase in blood creatine kinase, glutamic oxaloacetic transaminase, and lactic dehydrogenase, as well as a decrease in the blood total protein albumin and globulin levels. These results suggest that BV induces cardiovascular depression by decreasing the cardiac pressure and increasing the ionized magnesium concentration in the blood.

Background & Aims: Increases in intracellular Ca 2+ are thought to complement cAMP in stimulating Cl - secretion in cholangiocytes, although the site(s) of action and channels involved are unknown. We have identified a Ca 2+ -activated K + channel (SK2) in biliary epithelium that is inhibited by apamin. The purpose of the present studies was to define the role of SK channels in Ca 2+ -dependent cholangiocyte secretion., Methods: Studies were performed in human Mz-Cha-1 cells and normal rat cholangiocytes (NRC). Currents were measured by whole-cell patch clamp technique and transepithelial secretion by Ussing chamber., Results: Ca 2+ -dependent stimuli, including purinergic receptor stimulation, ionomycin, and increases in cell volume, each activated K + -selective currents with a linear IV relation and time-dependent inactivation. Currents were Ca 2+ dependent and were inhibited by apamin and by Ba 2+. In intact liver, immunoflourescence with an antibody to SK2 showed a prominent signal in cholangiocyte plasma membrane. To evaluate the functional significance, NRC monolayers were mounted in a Ussing chamber, and the short-circuit current ( I sc ) was measured. Exposure to ionomycin caused an increase in I sc 2-fold greater than that induced by cAMP. Both the basal and ionomycin-induced I sc were inhibited by basolateral Ba 2+, and approximately 58% of the basolateral K + current was apamin sensitive., Conclusions: These studies demonstrate that cholangiocytes exhibit robust Ca 2+ -stimulated secretion significantly greater in magnitude than that stimulated by cAMP. SK2 plays an important role in mediating the increase in transepithelial secretion due to increases in intracellular Ca 2+. SK2 channels, therefore, may represent a target for pharmacologic modulation of bile flow.

A wasp venom, mastoparan, rapidly increased the cytosolic free Ca2+ concentration [( Ca2+]i) and activated phosphorylase in rat hepatocytes in a concentration-dependent manner. Mastoparan could increase [Ca2+]i even in the absence of extracellular Ca2+, but a larger increase was observed in the presence of extracellular Ca2+. Thus, mastoparan mobilized Ca2+ from intracellular and extracellular Ca2+ stores. It also activated inositol triphosphate (IP3) accumulation, but did not stimulate cAMP production. From these results, we conclude that mastoparan activates rat hepatic glycogenolysis mediated by the accumulation of IP3, which causes an increase of [Ca2+]i but not that mediated by cAMP.

Histamine may be released from rat peritoneal mast cells by compound 48/80 and peptide 401 in the presence and absence of extracellular calcium. The process is non-cytolytic and requires an intact cell metabolism. The release produced under both conditions is inhibited by disodium cromoglycate, theophylline, dibutyryl cyclic AMP and (at high concentrations) quercetin. The efficacy of the drugs in the absence of extracellular calcium cannot be explained in terms of their postulated effect on the calcium-gating mechanism operative in anaphylactic secretion. Alternative modes of action of the compounds are thus considered.

Action potentials of myotubes in culture prepared from 18-19 day -old mouse embryos have a contractile activity and action potentials that are followed by a long lasting after hyperpolarization (ahp) which is blocked by apamin. Myotubes prepared from embryos of mice with muscular dysgenesis (mdg/mdg) did not contract and had action potentials which were never followed by a.h.p.'s. Voltage-clamp experiments have shown that Na+ channel activity was identical in mutant and control muscles and that the activity of fast and slow Ca2+ channels was nearly absent in the mutant.

Slow outward potassium currents were recorded in isolated frog skeletal muscle fibres using the double mannitol-gap voltage-clamp technique. Detubulated fibres failed to generate a slow outward current, and apamin had no effect on the remaining current. The maximum blocking effect of organic and inorganic Ca2+-channel blockers on the slow outward channels of intact fibres was larger than that of apamin. Apamin failed to induce an additional block when applied after Ca2+-channel blockers. In a low-Ca2+ solution (OCa, EGTA 1 mM) the slow outward current was slightly increased and the blocking effect of apamin was enhanced. A Ca2+-rich solution (Ca2+ X 10) increased the slow outward current and the blocking effect of apamin was drastically reduced. It is concluded that the apamin-sensitive current which is a component of the slow outward K+ current is located in the tubular membrane. Its activation seems barely dependent on the Ca2+ influx via the slow inward Ca2+ current. Apamin-receptor binding appears to be dependent on the extracellular Ca2+ concentration. Blockade of slow outward current by Ca2+-channel blockers is likely to be the result of a direct action on the slow K+ permeability rather than a consequence of Ca2+ channel inhibition.

Action potentials in many excitable cells are followed by a prolonged afterhyperpolarization that modulates repetitive firing. Although it is established that the afterhyperpolarization is produced by Ca-activated K+ currents, the basis of these currents is not known. The large conductance (250 pS) Ca-activated K+ channel (BK channel) is not a major contributor to the afterhyperpolarization in non-innervated skeletal muscle and some nerve cells, because apamin, a neurotoxic component of bee venom, abolishes the afterhyperpolarization but does not block BK channels, and 5 mM extracellular tetraethylammonium ion (TEA) blocks BK channels but does not reduce the afterhyperpolarization. We now report single-channel currents from small conductance (10-14 pS) Ca-activated K+ channels (SK channels) with the necessary properties to account for the afterhyperpolarization. SK channels are blocked by apamin but not by 5 mM external TEA (TEAo). They are also highly Ca-sensitive at the negative membrane potentials associated with the afterhyperpolarization.

The long-lasting after-hyperpolarization(s) (AHP) that follows the action potential in rat myotubes differentiated in culture is due to Ca2+-activated K+ channels. These channels have the property to be specifically blocked by the bee venom toxin apamin at low concentrations. Apamin has been used in this work to analyze, by electrophysiological and biochemical techniques, the role of innervation in expression of these important channels. The main results are as follows: (i) Long-lasting AHP that follows the action potential in rat myotubes in culture disappears when myotubes are cocultured with nerve cells from the spinal cord under the conditions of in vitro innervation. (ii) Extensor digitorum longus muscles from adult rats have action potentials that are not followed by AHP but AHP are systematically recorded after muscle denervation and they are blocked by apamin. (iii) Specific 125I-labeled apamin binding is undetectable in innervated muscle fibers but it becomes detectable 2-4 days after muscle denervation to be maximal 10 days after denervation. (iv) Apamin receptors detected with 125I-labeled apamin are present at fetal stages with biochemical characteristics identical to those found in myotubes in culture. The receptor number decreases as maturation proceeds and 125I-labeled apamin receptors completely disappear after the first week of postnatal life, in parallel with the disappearance of multi-innervation. All these results taken together strongly suggest an all-or-none effect of innervation on the expression of apamin-sensitive Ca2+-activated K+ channels.

The venom of the wasp Habrobracon hebetor presynaptically blocks excitatory but not inhibitory neuromuscular transmission at locust skeletal muscle. Its mode of action on excitatory motor nerve terminals has been studied at the retractor unguis muscle of Schistocerca by means of ultrastructural stereology. paralysed and unparalysed preparations, either resting or stimulated for 7 min at 20 Hz, were compared. Paralysis does not cause structural damage to the nerve terminals but prevents the depletion of vesicles occurring upon nerve stimulation in the controls. Prolonged paralysis leads to an increase in the number and the size of vesicles resulting in an increase of total membrane per terminal cross-section by about 33% after 2 days. Stimulation causes swelling of mitochondria both in controls and in paralysed preparations, resulting from a rise of intraterminal [Ca2+] as is indicated by the absence of the swelling if extracellular Ca2+ is replaced by Mg2+. In addition, stimulation leads to a reduction of vesicle size, an increase in the area of axolemma and in the number of cisternae and of profiles of the smooth endoplasmic reticulum in controls but not in paralysed preparations. However, neither in controls nor in paralysed preparations is the total amount of membrane per terminal cross-section affected by stimulation. Under paralysis, vesicles tend to stick to the presynaptic membrane. It is concluded that Habrobracon venom does not block the depolarizing-dependent Ca2+-influx into the nerve terminal and that it is unlikely to interfere with some transmitter-related process. Rather, the venom seems to block vesicle exocytosis itself. The results lend further support to the view that in insect neuromuscular synapses exocytosis is the mechanism whereby transmitter quanta are released.

Mono[125I]iodoapamin bound to specific sites on cultured rat embryonic neurons. The dissociation constant for the receptor-neurotoxin complex measured at equilibrium was 60-120 pM at pH 7.2 and 4 degrees C, with a maximal binding capacity of 3-8 fmol/mg of cell protein. Apamin inhibited calcium ionophore-induced 86Rb+ release from cell cultures. The dose effect curve for this pharmacological test corresponded closely to the displacement of 125I-apamin by native apamin in binding experiments. Formation of the 125I-apamin receptor complex requires exogenous K+. Reduced binding in the absence of K+ was due to diminished binding capacity rather than a lower affinity. The apamin receptor seems to be associated with a cell surface K+ site which shows 50% occupancy at 1.6 mM, and which could be involved in the regulation of channel activity. Apamin sites were present at the earliest developmental stage tested and their number did not evolve during 8 days in culture. In the same period, however, alpha-scorpion toxin binding increased by a factor of 10. The ontogenesis of Ca2+-activated K+ channels does not seem to occur in parallel with that of voltage-sensitive Na+ channels.

The bee venom neurotoxin apamin failed to affect 86Rb outflow and insulin release from rat pancreatic islets stimulated by D-glucose or the Ca2+-ionophore A23187. Apamin, in contrast to quinine or A23187, also failed to affect bioelectrical activity in mouse islet cells. These findings suggest that, like in erythrocytes, and at variance with the situation found in smooth muscle, liver or neuroblastoma cells, the Ca2+-activated K+ permeability in the pancreatic B-cell is resistant to apamin.

Mastoparan, a tetradecapeptide toxin from wasp venom stimulates secretion in mast cells and enhances GTPase activity of several purified guanine nucleotide regulatory proteins (G proteins). This suggests that this toxin may effect cellular functions through activation of G proteins. In this report, we probed the effects of mastoparan on cytosolic calcium concentration ([Ca2+]i) and inositol trisphosphate (IP3) formation in human polymorphonuclear leukocytes (PMN). At noncytotoxic concentrations up to 35 microM, mastoparan induced a dose-dependent elevation in [Ca2+]i in PMN, as determined by the fluoroprobe Fura 2. The increase in [Ca2+]i was attained through two discrete processes involving an initial rapid and transient calcium rise followed by a slower sustained increase. The initial but not the second [Ca2+]i increase was absent in PMN pretreated with pertussis toxin. The second but not the first [Ca2+]i rise required external calcium. The kinetics of [Ca2+]i changes and dependency on extracellular calcium induced by mastoparan correlated with the production of IP3. Pertussis toxin inhibited only the initial phase of IP3 production. The ability of pertussis toxin to ADP-ribosylate Gi-like proteins in PMN membrane was potentiated in the presence of mastoparan. Thus, mastoparan activates phospholipase C in PMN through two independent mechanisms. The first pathway is similar to that induced by chemoattractant receptors in that the rapid and transient activation of phospholipase C is dependent on a pertussis toxin-sensitive Gi protein. The second pathway is delayed, sustained, insensitive to pertussis toxin, and requires extracellular calcium.

Effects of apamin on rat sympathetic neurones were investigated by means of intracellular and extracellular recording. Apamin (50 nM) significantly shortened the after-hyperpolarization (AH) following the spike evoked by current injection and slightly decreased its peak amplitude without affecting the time course of the spike. The AH following the synaptically-evoked spike was also blocked by apamin. This effect was dose- and time-dependent (ID50 estimated by extracellular recording approximately 15 nM, 20 min after application) and poorly reversible. Transmission of a single volley was not affected by 50 nM apamin. Though a long depolarizing current caused one or two spikes in the cell, greater repetitive firing was observed in the presence of apamin. Spontaneous repetitive firing, however, was not observed except for anodal-break spikes. Resting potential and input membrane resistance were essentially unchanged by apamin. The maximum rate of rise of the Ca spike was not decreased by 50 nM apamin but the duration of the spike was lengthened by 60%. The AH following the Ca spike was also blocked by apamin. These results suggest that apamin suppressed the slow AH without any inhibition of the Ca flux into the cell and is useful as a blocker of GK(Ca) in the rat sympathetic neurone.

The interaction between calmodulin and its target protein is a key step in many calcium-regulated cellular functions. Melittin binds tightly to calmodulin in the presence of calcium and is a competitive inhibitor of calmodulin function. Using melittin as a model for the target peptide of calmodulin, we have found a large Ca2+-dependent conformational change of calmodulin in solution induced by peptide binding. Mg2+ does not substitute for Ca2+ in producing the conformation change. Small-angle x-ray scattering has shown that calmodulin exists as a dumbbell in solution, similar to that observed in the crystalline state. Our present measurements reveal that the overall structure of the Ca2+-calmodulin-melittin complex is not a dumbbell but a globular shape. Upon binding melittin, the radius of gyration decreases from 20.9 to 18.0 A and the largest dimension decreases from 60 to 47.5 A. In the absence of calcium, however, melittin has little effect on the solution structure of calmodulin.

Apamin is a toxic polypeptide extracted from bee venom. It has been considered as a neurotoxin with central action, but its low concentrations (10(-8)-10(-7) M) were shown to reversibly block the nonadrenergic inhibition and effects of externally applied ATP, noradrenaline and caffeine in smooth muscles of the gastrointestinal tract. All these processes are related to the activation of Ca-dependent potassium permeability. Current-clamp, voltage-clamp and patch-clamp experiments have also shown that apamin blocks specifically some types of these conductances in other tissues: skeletal muscles, mammalian neurons and neuroblastoma, hepatocytes. Nowadays apamin is the most specific but not a universal blocker of the Ca-activated potassium conductance.

Undifferentiated PC12 cell produce high levels of apamin receptors (measured with 125I-apamin) after 7 days in culture. These levels are at least 50 times higher than those found in other cellular types which are also known to have apamin receptors and apamin-sensitive Ca2+-activated K+ channels in their membranes. Treatment of undifferentiated PC12 cells with nerve growth factor maintains these cells in a state having a low level (10 times less after 7 days of culture) of apamin receptors. Ca2+ injection into PC12 cells with the calcium ionophore A23187 has been used to monitor the activity of the Ca2+-activated K+ channel following 86Rb+ efflux. A large component of this Ca2+-activated 86Rb+ efflux is inhibited by apamin. Half-maximum inhibition by apamin of both 86Rb+ efflux and 125I-apamin binding was observed at 240 pM apamin. Another component of 86Rb+ efflux is due to another type of Ca2+-activated K+ channel which is resistant to apamin and sensitive to tetraethylammonium. The Ca2+ channel activator Bay K8644 also triggers an apamin-sensitive Ca2+-dependent 86Rb+ efflux. Bay K8644 has been used to analyze the internal Ca2+ concentration dependence of the apamin-sensitive channel activity. Under normal conditions, the internal Ca2+ concentration is 109 +/- 17 nM, and the apamin-sensitive channel is not activated. The channel is fully activated at an internal Ca2+ concentration of 320 +/- 20 nM.

Nanomolar concentrations of charybdotoxin or dendrotoxin increase the cytoplasmic free Ca2+ concentration in isolated central nerve terminals. The effects of the two toxins, normally considered to be blockers of K+ channels controlled by voltage in a Ca2+-sensitive or -insensitive manner, respectively, show only marginal additivity. Apamin, and inhibitor of low conductance Ca2+-activated K+ channels, was without effect in either the absence or presence of dendrotoxin. The effect of charybdotoxin on polarized, isolated central nerve terminals seems to be mediated largely by a block of K+ channels sensitive to dendrotoxin. Apparently, these voltage-operated K+ channels make a more significant contribution to maintaining the polarized potential of synaptosomes than do those activated by Ca2+.

Intracellular Ca2+ levels in human erythrocytes were increased by incubating them with variable concentrations of Ca2+ in the presence of ionophore A23187. Experiments were done to confirm that the Ca2+ loading did induce changes in the cell shape and membrane protein composition. The effect of the increased cytoplasmic Ca2+ levels on the membrane phospholipid organization was analysed using bee venom and pancreatic phospholipases A2, Merocyanine 540 and fluorescamine as the external membrane probes. About 20% phosphatidylethanolamine (PE) and 0% phosphatidylserine (PS) were hydrolysed by the phospholipases in intact control cells, whereas in identical conditions these enzymes readily degraded, 20-30% PE and 7-30% PS, in Ca2+-loaded erythrocytes, depending on the cytoplasmic Ca2+ concentration. Also, Merocyanine 540 failed to stain the fresh or control erythrocytes, but it labeled the cells loaded with Ca2+. Furthermore, fluorescamine labeled approx. 20% PE in fresh or control erythrocytes while in identical conditions, significantly higher amounts of PE were modified in intact Ca2+-loaded cells. These results demonstrate that Ca2+ loading in human erythrocytes leads to loss of the transbilayer phospholipid asymmetry, and suggest that, together with spectrin, polypeptides 2.1 and 4.1 may also play an important role in maintaining the asymmetric distribution of various phospholipids across the erythrocyte membrane bilayer.

Effects of melittin, an amphipathic polypeptide, on various species of protein kinases were investigated. It was found that melittin inhibited the newly identified phospholipid-sensitive Ca2+-dependent protein kinase (from heart, brain, spleen and neutrophils) and the cardiac myosin light-chain kinase, a calmodulin-sensitive Ca2+-dependent enzyme. In contrast, melittin had little or no effect on either the holoenzymes of the cardiac cyclic AMP-dependent and cyclic GMP-dependent protein kinases or the catalytic subunit of the former. Kinetic analysis indicated that melittin inhibited phospholipid-sensitive Ca2+-dependent protein kinase non-competitively with respect to ATP (Ki = 1.3 microM); although exhibiting complex kinetics, its inhibition of the enzyme was overcome by phosphatidylserine (a phospholipid cofactor), but not by protein substrate (histone H1) or Ca2+. On the other hand, melittin inhibited myosin light-chain kinase non-competitively with respect to ATP (Ki = 1.4 microM) or Ca2+ (Ki = 1.9 microM), and competitively with respect to calmodulin (Ki = 0.08 microM); although exhibiting complex kinetics, its inhibition of the enzyme was reversed by myosin light chains (substrate protein). The present findings indicate the presence of functionally important hydrophobic or hydrophilic loci on the Ca2+-dependent protein kinases, but not on the cyclic nucleotide-dependent class of protein kinase, with which melittin can interact. Moreover, the kinetic data suggest that melittin inhibited myosin light-chain kinase by interacting with a site on the enzyme the same as, or proximal to, the calmodulin-binding site, thus interfering with the formation of active enzyme-calmodulin-Ca2+ complex.

In B-type neurones of bullfrog sympathetic ganglia, apamin (10 nM) suppressed the Ca2+-dependent K+ current (IAH) involved in the afterhyperpolarization of an action potential, while it did not affect the Ca2+-dependent K+ current (Ic) underlying the spike repolarization. IAH was further separated into two exponential components which were differentially affected by apamin, voltage and alterations in Ca2+ influx, suggesting the existence of 3 different types of Ca2+-dependent K+ channel in bullfrog sympathetic neurones.

Apamin, which blocks Ca2+-dependent increases in K+ permeability, antagonizes ATP-induced relaxation of several smooth muscles. The ATP photoaffinity label arylazido aminopropionyl ATP (ANAPP3), following its photolysis in the presence of the guinea-pig vas deferens, antagonizes contractile responses to ATP. This study was conducted to determine whether apamin antagonizes ATP-induced responses in the guinea-pig vas deferens, and also to evaluate whether ANAPP3 antagonizes responses to ATP by interfering with Ca2+-dependent K+ permeability changes. Apamin (10(-6) M) potentiated ATP-induced contractions. This potentiation was nonspecific in that responses to norepinephrine, histamine and acetylcholine also were enhanced; responses to KCl were unaffected. To evaluate the possible interactions between the two agents at the same cellular site, the effect of apamin was examined in ANAPP3-treated tissues. In such tissues apamin did not potentiate the residual responses to ATP; however, apamin was nevertheless able to potentiate responses of ANAPP3-treated tissues to norepinephrine, histamine and acetylcholine, and responses to KCl remained unaffected. These studies provide additional support for the view that ANAPP3 antagonizes ATP-induced responses of the guinea-pig vas deferens by blocking P2-purinergic receptors. The antagonism by ANAPP3 is not attributable to a blockade of Ca2+-dependent K+ permeability changes.

Apamin is a bee venom neurotoxin of 18 amino-acids containing two disulfide bridges. Current clamp and voltage clamp experiments have shown that externally applied apamin blocks specifically at low concentration (0.1 microM) the Ca2+-dependent slow K+ conductance which mediates the long-lasting after-hyperpolarization in neuroblastoma cells and rat muscle cells in culture. The apamin-sensitive Ca2+-dependent slow K+ conductance is voltage-dependent and tetraethylammonium (TEA) insensitive. It is distinct from the high conductance Ca2+-dependent K+ channel revealed by patch clamp experiments. Biochemical characterization of the apamin receptor in rat striated muscle, neuroblastoma cells, rat synaptosomes, smooth muscles and hepatocytes was carried out with the use of a radiolabelled monoiodo-apamin derivative (125I-apamin) of high specific radioactivity (2 000 Ci/mmol). The dissociation constant of the apamin-receptor complex is between 15 and 60 pM for all tissue preparations. The density of binding sites is very low; it varied between 1 and 40 fmol/mg of protein. Radiation inactivation analysis indicates a molecular weight for the apamin receptor of 250 000 daltons whereas affinity labelling with 125I-apamin results in covalent labelling of a single polypeptide chain with a molecular weight of about 30 000 daltons. We conclude that the apamin-sensitive Ca2+-dependent K+ channel is probably a large oligomeric structure containing one subunit of 30 000 daltons.

Intracellular free Ca+2 concentration was measured in cultured human fibroblasts (HSWP) utilizing the Ca+2-sensitive fluorescent probe quin2. The addition of peptide growth factors to serum-deprived HSWP cells induced an immediate rise in intracellular Ca+2 concentration. This mitogen-induced rise in Ca+2 concentration could be blocked by the addition of the intracellular Ca+2 antagonist TMB-8. Addition of the phospholipase activator, melittin, to cells in the absence of growth factors also caused a dramatic rise in intracellular Ca+2 concentration which was blocked by TMB-8.

Calcium dissociation from the C-terminal and N-terminal halves of calmodulin, intact bovine brain calmodulin and the respective phenoxybenzamine complexes or melittin complexes was measured directly by stopped-flow fluorescence with the calcium chelator Quin 2 and, when possible, also by protein fluorescence using endogenous tyrosine fluorescence by mixing with EGTA. Calcium dissociation from the C-terminal half of calmodulin, which contains only the two high-affinity calcium-binding sites, and from intact calmodulin was monophasic, with good correlation of the rates of calcium dissociation obtained by the two methods. The apparent rates with Quin 2 and endogenous tyrosine fluorescence were 13.4 s-1 and 12.8 s-1, respectively, in the C-terminal half and 10.5 s-1 and 10.8 s-1, respectively, in intact calmodulin (pH 7.0, 25 degrees C, 100 mM KCl). Alkylation of the C-terminal half resulted in a biphasic calcium dissociation (Quin 2: kobs 1.90 s-1 and 0.73 s-1 respectively; tyrosine: kobs 1.65 s-1 and 0.61 s-1 respectively). Alkylation of intact calmodulin resulted in a four-phase calcium dissociation measured with Quin 2 (kobs 85.3 s-1, 11.1 s-1, 1.92 s-1 and 0.59 s-1); the latter two phases are assumed to represent calcium release from high-affinity sites since they correspond to the biphasic tyrosine fluorescence change in intact alkylated calmodulin (kobs 2.04 s-1 and 0.53 s-1 respectively) and the rate parameters determined in the C-terminal half. Evidently perturbation of the calcium-binding sites by alkylation reduces the rate of calcium dissociation and allows a distinction to be made between dissociation from each of the two high-affinity sites as well as the distinct conformational change on dissociation of each calcium. Alkylation of the N-terminal half resulted in biphasic calcium release with rates (kobs 153 s-1 and 10.9 s-1 respectively) similar to those observed in intact alkylated calmodulin. The rates of calcium dissociation from calmodulin-melittin or fragment-melittin complexes, measured with Quin 2, were slower and monophasic in the C-terminal half (kobs 1.12 s-1), biphasic in the N-terminal half (kobs 140 s-1 and 26.8 s-1 respectively) and triphasic in intact calmodulin (kobs 126 s-1, 12.1 s-1 and 1.38 s-1). Calmodulin antagonists thus increase the apparent calcium affinity of high and low-affinity sites mainly due to a reduced calcium 'off rate', presumably because of conformation restrictions.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of a number of polypeptide cytotoxins and neurotoxins on various protein kinases were examined. It was found that cobra cytotoxin I and marine worm cytotoxin A-IV effectively and specifically inhibited phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK) relative to myosin light chain kinase and cyclic nucleotide-dependent protein kinases. Inhibition of PL-Ca-PK by these cytotoxins could be overcome by phosphatidylserine. Neurotoxins, in comparison, were much less effective inhibitors. The present findings indicated that these polypeptide cytotoxins, unlike other agents reported to date, were selective inhibitors of PL-Ca-PK and could be used to differentiate Ca2+-dependent events regulated by phospholipid or calmodulin.