Selectivity of ATP-activated GTP-dependent Ca2+-permeable channels in rat macrophage plasma membrane

Outside-out configuration of the patch clamp technique was used to test whether an intracellular application of G protein activator (GTPS) affects ATP-activated Ca²⁺-permeable channels in rat macrophages without any agonist in the bath solution. With 145 mm K⁺ (pCa 8.0) in the pipette solution, activity of channels permeable to a variety of divalent cations and Na⁺ was observed and general channel characteristics were found to be identical to those of ATP-activated ones. Absence of extracellular ATP makes it possible to avoid the influence of ATP receptor desensitization and to study the channel selectivity using a number of divalent cations (105 mm) and Na⁺ (145 mm) as the charge carriers. Permeability sequence estimated by extrapolated reversal potential measurements was: Ca²⁺ Ba²⁺ Mn²⁺ Sr²⁺ Na⁺ K⁺ = 68 30 26 10 3.5 1. Slope conductances (in pS) for permeant ions rank as follows: Ca²⁺ Sr²⁺ Na⁺ Mn²⁺ Ba²⁺ = 19 18 14 12 10. Unitary Ca²⁺ currents display a tendency to saturate with the Ca²⁺ concentration increase with apparent dissociation constant (K _d ) of 10 mm. No block of Na⁺ permeation by extracellular Ca²⁺ in millimolar range was found. The data obtained suggest that (i) activation of some G protein is sufficient to gate the channels without the ATP receptor being occupied, (ii) the ATP receptor activation results in the gating of a special channel with the properties that differ markedly from those of the receptoroperated or voltage-gated Ca²⁺-permeable channels on the other cell types.DeceasedThe authors are grateful to K. Kiselyov and A. Mamin for technical assistance. The work was supported by the Russian Basic Research Foundation, Grant N 93-04-21722 and was made possible in part by Grant N R4A000 from the International Science Foundation.     

Single nonselective cation channels and Ca2+-activated K+ channels in aortic endothelial cells

Summary In cultured bovine aortic endothelial cells, elementary K⁺ currents were studied in cell-attached and inside-out patches using the standard patch-clamp technique. Two different cationic channels were found, a large channel with a mean unitary conductance of 150±10 pS and a small channel with a mean unitary conductance of 12.5±1.1 pS. The 150-pS channel proved to be voltag- and Ca²⁺-activatable and seems to be a K⁺ channel. Its open probability increased on membrane depolarization and, at a given membrane potential, was greatly enhanced by elevating the Ca²⁺ concentration at the cytoplasmic side of the membrane from 10^–7 to 10^–4 m. 150-pS channels were not influenced by the patch configuration in that patch excision neither induced rundown nor evoked channel activity in silent cell-attached patches. However, they were only seen in two out of 55 patches. The 12-pS channel was predominant, a nonselective cationic channel with almost the same permeability for K⁺ and Na⁺ whose open probability was minimal near –60 mV but increased on membrane hyperpolarization. An increase in internal Ca²⁺ from 10^–7 to 10^–4 m left the open probability unchanged. Although the K⁺ selectivity of the 150-pS channels remains to be elucidated, it is concluded that they may be involved in controlling Ca²⁺-dependent cellular functions. Under physiological conditions, 12-pS nonselective channels may provide an inward cationic pathway for Na⁺.     

Hyperpolarization-activated Ca2+-permeable Channels in the Plasma Membrane of Tomato Cells

The hyperpolarization of the electrical plasma membrane potential difference has been identified as an early response of plant cells to various signals including fungal elicitors. The hyperpolarization-activated influx of Ca²⁺ into tomato cells was examined by the application of conventional patch clamp techniques. In both whole cell and single-channel recordings, clamped membrane voltages more negative than −120 mV resulted in time- and voltage-dependent current activation. Single-channel currents saturated with increasing activities of Ca²⁺ and Ba²⁺ from 3 to 26 mm and the single channel conductance increased from 4 pS to 11 pS in the presence of 20 mm Ca²⁺ or Ba²⁺, respectively. These channels were 20–25 and 10–13 times more permeable to Ca²⁺ than to K⁺ and to Cl⁻, respectively. Channel currents were strongly inhibited by 10 μm lanthanum and 50% inhibited by 100 μm nifedipine. This evidence suggests that hyperpolarization-activated Ca²⁺-permeable channels provide a mechanism for the influx of Ca²⁺ into tomato cells. Received: 13 February 1996/Revised: 12 August 1996     

Effects of (-)-epigallocatechin-3-gallate in Ca2+ -permeable non-selective cation channels and voltage-operated Ca2+ channels in vascular smooth muscle cells

The effects of (-)-epigallocatechin-3-gallate (EGCG), the most abundant catechin of tea, on Ca(2+)-permeable non-selective cation currents (NSCC) and voltage-operated Ca(2+) channels (VOCC) have been investigated in cultured rat aortic smooth muscle cells using the whole-cell voltage-clamp technique. Under the Cs(+)/tetraethylammonium (TEA)-containing internal solution, and in the presence of nifedipine (1 microM), EGCG (30 microM) activated a long-lasting inward current, with a reversal potential (E(rev)) of approximately 0 mV. This current was not significantly altered by the replacement of [Cl(-)](i) or [Cl(-)](o), implying that the inward current was not a chloride channel, but a NSCC. SKF 96365 (30 microM) and Cd(2+) (500 microM) almost completely abolished the EGCG-induced NSCC. A higher dose of EGCG (100 microM) additionally activated a nifedipine-sensitive inward current in the absence of depolarization protocol. EGCG (100 microM) also potentiated a nifedipine-sensitive voltage-dependent Ba(2+)-current during the first 5 min of incubation. However, after > 10 min of incubation with EGCG, this current was significantly inhibited. Our results suggest that EGCG caused a Ca(2+) influx into smooth muscle cells via VOCC (probably L-type) and other SKF-96365- and Cd(2+)-sensitive Ca(2+)-permeable channels. The action described here may be responsible for the contraction induced by EGCG in rat aortic rings and for the rise of the intracellular concentration of Ca(2+) in rat aortic smooth muscle cells evoked by this catechin. On the other hand, the inhibition of VOCC after > 10 min of incubation may be, in part, responsible for the relaxation of rat aorta induced by EGCG.     

Store-operated Ca2+-permeable non-selective cation channels in smooth muscle cells

Over twenty years ago it was shown that depletion of the intracellular Ca2+ store in smooth muscle triggered a Ca2+ influx mechanism. The purpose of this review it to describe recent electrophysiological data which indicate that Ca2+ influx occurs through discrete ion channels in the plasmalemma of smooth muscle cells. The effect of external Ca2+ on the amplitude and reversal potential of whole-cell and single channel currents suggests that there are at least two, and probably more, distinct store-operated channels (SOCs) which have markedly different permeabilities to Ca2+ ions. Two activation mechanisms have been identified which involve Ca2+ influx factor and protein kinase C (PKC) activation via diacylglycerol. In addition, in rabbit portal vein cells there is evidence that stimulation of alpha-adrenoceptors can stimulate SOC opening via PKC in a store-independent manner. There is at present little knowledge on the molecular identity of SOCs but it has been proposed that TRPC1 may be a component of the functional channel. We also summarise the data showing that SOCs may be involved in contraction and cell proliferation of smooth muscle. Finally, we highlight the similarities and differences of SOCs and receptor-operated cation channels that are present in native rabbit portal vein myocytes.     

Identification of putative voltage-dependent Ca2+-permeable channels involved in cryptogein-induced Ca2+ transients and defense responses in tobacco BY-2 cells

Ca(2+) is the pivotal second messenger for induction of defense responses induced by treatment of pathogen-derived elicitor or microbial infection in plants. However, molecular bases for elicitor-induced generation of Ca(2+) signals (Ca(2+) transients) are largely unknown. We here identified cDNAs for putative voltage-dependent Ca(2+)-permeable channels, NtTPC1A and NtTPC1B, that are homologous to TPC1 (two pore channel) from suspension-cultured tobacco BY-2 cells. NtTPC1s complemented the growth of a Saccharomyces cerevisiae mutant defective in CCH1, a putative Ca(2+) channel, in a low Ca(2+) medium, suggesting that both products permeate Ca(2+) through the plasma membrane. Cosuppression of NtTPC1s in apoaequorin-expressing BY-2 cells resulted in inhibition of rise in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in response to sucrose and a fungal elicitor cryptogein, while it did not affect hypoosmotic shock-induced [Ca(2+)](cyt) increase. Cosuppression of NtTPC1s also caused suppression of cryptogein-induced programmed cell death and defense-related gene expression. These results suggest that NtTPC1s are involved in Ca(2+) mobilization induced by the cryptogein and sucrose, and have crucial roles in cryptogein-induced signal transduction pathway.     

Inositol 1,4,5-trisphosphate activates two types of Ca2(+)-permeable channels in human carcinoma cells

Ca2(+)-permeable channels in human carcinoma A431 cells were studied using the patch clamp technique. We have found two types of Ca2(+)-permeable channels which are activated by inositol 1,4,5-trisphosphate (IP3) applied to the intracellular side of the plasma membrane. Unitary conductances of these channels are 3.7 and 13 pS (105 mM Ca2+ in recording pipette, 30-33 degrees C). From the extracellular side of the membrane the channels are activated by EGF. It is assumed that extracellular agonists open both channel types by stimulating the release of IP3 from the membrane.     

Paramecium Na+ channels activated by Ca2+-calmodulin: Calmodulin is the Ca2+ sensor in the channel gating mechanism

Paramecium Na⁺ channels, which were Ca²⁺-calmodulin activated, were studied in the inside-out mode of patch clamp. After excision of the membrane patch, they were active in the presence of 10^–5 to 10^–3 m Ca²⁺ in the bath. They became much less active in the presence of 10^–6 m Ca²⁺, and their activity subsided completely at 10^–8 m Ca²⁺. A Hill plot showed a dissociation constant of 6 m for Ca²⁺ binding. This dissociation constant shifted to a submicromolar range in the presence of 1 mm Mg²⁺. The channels also exhibited a mild voltage dependence. When exposed to 10^–8 m Ca²⁺ for an extended period of 2–4 min, channels were further inactivated even after bath Ca²⁺ was restored to 10^–4 m. Whereas neither high voltage (+100 mV) nor high Ca²⁺ (10^–3 m) was effective in reactivation of the inactive channels, addition of Paramecium wild-type calmodulin together with high Ca²⁺ to the bath restored channel activity without a requirement of additional Mg²⁺ and metabolites such as ATP. The channels reactivated by calmodulin had the same ion conductance, ion selectivity and Ca²⁺ sensitivity as those prior to inactivation. These inactivation and reactivation of the channels could be repeated, indicating that the direct calmodulin effect on the Na⁺ channel was reversible. Thus, calmodulin is a physiological factor critically required for Na⁺ channel activation, and is the Ca²⁺ sensor of the Na⁺-channel gating machinery.We thank C. Kung for his kind support, and A. Boileau for critical reading. Supported by grants from National Institutes of Health GM 22714-20 and 36386-09.     

Prenatal stress on the kinetic properties of Ca2+ and K+ channels in offspring hippocampal CA3 pyramidal neurons

Prenatal stress is known to cause neuronal loss and oxidative damage in the hippocampus of offspring rats. To further understand the mechanisms, the present study was undertaken to investigate the effects of prenatal stress on the kinetic properties of high-voltage-activated (HVA) Ca(2+) and K(+) channels in freshly isolated hippocampal CA3 pyramidal neurons of offspring rats. Pregnant rats in the prenatal stress group were exposed to restraint stress on days 14-20 of pregnancy three times daily for 45 min. The patch clamp technique was employed to record HVA Ca(2+) and K(+) channel currents. Prenatal stress significantly increased HVA Ca(2+) channel disturbance including the maximal average HVA calcium peak current amplitude (-576.52+/-7.03 pA in control group and -702.05+/-6.82 pA in prenatal stress group, p<0.01), the maximal average HVA Ca(2+) current density (-40.89+/-0.31 pA/pF in control group and -49.44+/-0.37 pA/pF in prenatal stress group, p<0.01), and the maximal average integral current of the HVA Ca(2+) channel (106.81+/-4.20 nA ms in control group and 133.49+/-4.59 nA ms in prenatal stress group, p<0.01). The current-voltage relationship and conductance--voltage relationship of HVA Ca(2+) channels and potassium channels in offspring CA3 neurons were not affected by prenatal stress. These data suggest that exposure of animals to stressful experience during pregnancy can exert effects on calcium ion channels of offspring hippocampal neurons and that the calcium channel disturbance may play a role in prenatal stress-induced neuronal loss and oxidative damage in offspring brain.     

cAMP-dependent protein kinase enhances inositol 1,4,5-trisphosphate-induced Ca2+ release in AR4-2J cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP(3)R), a ligand-gated Ca(2+) channel, plays an important role in the control of intracellular Ca(2+). There are three subtypes of IP(3)R that are differentially distributed among cell types. AR4-2J cells express almost exclusively the IP(3)R-2 subtype. The purpose of this study was to investigate the effect of cAMP-dependent protein kinase (PKA) on the activity of IP(3)R-2 in AR4-2J cells. We showed that immunoprecipitated IP(3)R-2 is a good substrate for PKA. Using a back-phosphorylation approach, we showed that endogenous PKA phosphorylates IP(3)R-2 in intact AR4-2J cells. Pretreatment with PKA enhanced IP(3)-induced Ca(2+) release in permeabilized AR4-2J cells. Pretreatment with the cAMP generating agent's forskolin and vasoactive intestinal peptide (VIP) enhanced carbachol (Cch)-induced and epidermal growth factor (EGF)-induced Ca(2+) responses in intact AR4-2J cells. Our results are consistent with an enhancing effect of PKA on IP(3)R-2 activity. This conclusion supports the emerging concept of crosstalk between Ca(2+) signaling and cAMP pathways and thus provides another way by which Ca(2+) signals are finely encoded within non-excitable cells.     

ATP-inhibited and Ca2+-dependent K+ channels in the soma membrane of cultured leech Retzius neurons

The properties of one ATP-inhibited and one Ca²⁺-dependent K⁺ channel were investigated by the patch-clamp technique in the soma membrane of leech Retzius neurons in primary culture. Both channels rectify at negative potentials. The ATP-inhibited K⁺ channel with a mean conductance of 112 pS is reversibly blocked by ATP (K _i = 100 m), TEA (K _i =0.8 mm) and 10 mm Ba²⁺ and irreversibly blocked by 10 nm glibenclamide and 10 m tolbutamide. It is Ca²⁺ and voltage independent. Its open state probability (P _o) decreases significantly when the pH at the cytoplasmic face of inside-out patches is altered from physiological to acid pH values. The Ca²⁺-dependent K⁺ channel with a mean conductance of 114 pS shows a bell-shaped Ca²⁺ dependence of P _o with a maximum at pCa 7–8 at the cytoplasmic face of the membrane. The P _o is voltage independent at the physiologically relevant V range. Ba²⁺ (10 mm) reduces the single channel amplitude by around 25% (ATP, TEA, glibenclamide, tolbutamide, and Ba²⁺ were applied to the cytoplasmic face of the membrane).We conclude that the ATP-dependent K⁺ channel may play a role in maintaining the membrane potential constant—independently from the energy state of the cell. The Ca²⁺-dependent K⁺ channel may play a role in generating the resting membrane potential of leech Retzius neurons as it shows maximum activity at the physiological intracellular Ca²⁺ concentration.This study was supported by the Deutsche Forschungsgemeinschaft (W.-R. Schlue) and by a fellowship of the Konrad-Adenauer-Stiftung (G. Frey). We thank Dr. Draeger (Hoechst AG) for the gift of glibenclamide. The data are part of a future Ph.D. thesis of G. Frey.     

The store-operated calcium entry pathways in human carcinoma A431 cells: functional properties and activation mechanisms

Activation of phospholipase C (PLC)-mediated signaling pathways in nonexcitable cells causes the release of Ca2+ from intracellular Ca2+ stores and activation of Ca2+ influx across the plasma membrane. Two types of Ca2+ channels, highly Ca2+-selective ICRAC and moderately Ca2+-selective ISOC, support store-operated Ca2+ entry process. In previous patch-clamp experiments with a human carcinoma A431 cell line we described store-operated Imin/ICRACL plasma membrane Ca2+ influx channels. In the present paper we use whole-cell and single-channel recordings to further characterize store-operated Ca2+ influx pathways in A431 cells. We discovered that (a) ICRAC and ISOC are present in A431 cells; (b) ICRAC currents are highly selective for divalent cations and fully activate within 150 s after initiation of Ca2+ store depletion; (c) ISOC currents are moderately selective for divalent cations (PBa/PCs = 14.5) and require at least 300 s for full activation; (d) ICRAC and ISOC currents are activated by PLC-coupled receptor agonists; (e) ISOC currents are supported by Imin/ICRACL channels that display 8.5-10 pS conductance for sodium; (f) ICRAC single channel conductance for sodium is estimated at 0.9 pS by the noise analysis; (g) Imin/ICRACL channels are activated in excised patches by an amino-terminal fragment of InsP3R1 (InsP3R1N); and (h) InsP3 binding to InsP3R1N is necessary for activation of Imin/ICRACL channels. Our findings provide novel information about store-operated Ca2+ influx pathways in A431 cells.     

Expression of Ca2+-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells

The second messenger NAADP triggers Ca²⁺ release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2^−/−), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca²⁺ responses as assessed by single-cell Ca²⁺ imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P₂ were only partially dependent on TPCs. In Tpcn1/2^−/− cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca²⁺-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca²⁺ release. High-affinity [³²P]NAADP binding still occurs in Tpcn1/2^−/− tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca²⁺-permeable channels indispensable for NAADP signalling.     

Mechanosensitive Ca2+ permeant cation channels in human prostate tumor cells

The acquisition of cell motility plays a critical role in the spread of prostate cancer (PC), therefore, identifying a sensitive step that regulates PC cell migration should provide a promising target to block PC metastasis. Here, we report that a mechanosensitive Ca²⁺-permeable cation channel (MscCa) is expressed in the highly migratory/invasive human PC cell line, PC-3 and that inhibition of MscCa by Gd³⁺ or GsMTx-4 blocks PC-3 cell migration and associated elevations in [Ca²⁺]_i. Genetic suppression or overexpression of specific members of the canonical transient receptor potential Ca²⁺ channel family (TRPC1 and TRPC3) also inhibit PC-3 cell migration, but they do so by mechanisms other that altering MscCa activity. Although LNCaP cells are nonmigratory, they also express relatively large MscCa currents, indicating that MscCa expression alone cannot confer motility on PC cells. MscCa in both cell lines show similar conductance and ion selectivity and both are functionally coupled via Ca²⁺ influx to a small Ca²⁺-activated K⁺ channel. However, MscCa in PC-3 and LNCaP cell patches show markedly different gating dynamics—while PC-3 cells typically express a sustained, non-inactivating MscCa current, LNCaP cells express a mechanically-fragile, rapidly inactivating MscCa current. Moreover, mechanical forces applied to the patch, can induce an irreversible transition from the transient to the sustained MscCa gating mode. Given that cancer cells experience increasing compressive and shear forces within a growing tumor, a similar shift in channel gating in situ would have significant effects on Ca²⁺ signaling that may play a role in tumor progression.     

Mechanosensitive Ca2+ permeant cation channels in human prostate tumor cells

The acquisition of cell motility plays a critical role in the spread of prostate cancer (PC), therefore, identifying a sensitive step that regulates PC cell migration should provide a promising target to block PC metastasis. Here, we report that a mechanosensitive Ca²⁺-permeable cation channel (MscCa) is expressed in the highly migratory/invasive human PC cell line, PC-3 and that inhibition of MscCa by Gd³⁺ or GsMTx-4 blocks PC-3 cell migration and associated elevations in [Ca²⁺]_i. Genetic suppression or overexpression of specific members of the canonical transient receptor potential Ca²⁺ channel family (TRPC1 and TRPC3) also inhibit PC-3 cell migration, but they do so by mechanisms other that altering MscCa activity. Although LNCaP cells are nonmigratory, they also express relatively large MscCa currents, indicating that MscCa expression alone cannot confer motility on PC cells. MscCa in both cell lines show similar conductance and ion selectivity and both are functionally coupled via Ca²⁺ influx to a small Ca²⁺-activated K⁺ channel. However, MscCa in PC-3 and LNCaP cell patches show markedly different gating dynamics—while PC-3 cells typically express a sustained, non-inactivating MscCa current, LNCaP cells express a mechanically-fragile, rapidly inactivating MscCa current. Moreover, mechanical forces applied to the patch, can induce an irreversible transition from the transient to the sustained MscCa gating mode. Given that cancer cells experience increasing compressive and shear forces within a growing tumor, a similar shift in channel gating in situ would have significant effects on Ca²⁺ signaling that may play a role in tumor progression.     

Bupivacaine inhibits large conductance,voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K⁺ channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca²⁺-activated K⁺ channels (BK_Ca). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K⁺ currents carried by BK_Ca channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC₅₀ 324 µM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 µM) can also block whole-cell K⁺ currents (~45% blockage) in which, under our working conditions, BK_Ca is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK_Ca channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.     

Bupivacaine inhibits large conductance, voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K(+) channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca(2+) -activated K(+) channels (BK(Ca)). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K(+) currents carried by BK(Ca) channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC(50) 324 μM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 μM) can also block whole-cell K(+) currents (~45% blockage) in which, under our working conditions, BK(Ca) is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK(Ca) channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.     

Extracellular ATP induces fast and transient non-selective cationic currents and cytosolic Ca2+ changes in human umbilical artery smooth muscle cells

Ionotropic purinergic receptors (P2X) are expressed in endothelial and smooth muscle cells of blood vessels. ATP acting on smooth muscle P2X receptors is able to induce vasoconstriction in different kind of vessels. However, to our knowledge, there are no reports that directly show the activity of these purinergic receptors in native human vascular smooth muscle cells. In this work, we describe for the first time an ATP-induced current in freshly isolated human umbilical artery (HUA) smooth muscle cells. The current was measured by patch-clamp technique in whole-cell condition on cells clamped at -50 mV. At 100 μM of ATP the current showed a rapid activation and desensitization, and was carried by both Na(+) and Ca(2+). The current was completely blocked by suramin (300 μM) and partially blocked by 100 μM of Zn(2+) without affecting the kinetic of desensitization. All these properties suggest that the ATP-induced ionic currents are mediated through P2X(1)-like receptors. Moreover, we show that ATP transiently increased cytosolic Ca(2+) in "in situ" smooth muscle cells of intact HUA segments and that this response is dependent of extracellular and intracellular Ca(2+). These data expand the knowledge of purinergic receptors properties in vascular smooth muscle cells and the probable role of ATP as a paracrine modulator of contractile tone in a human artery which is fundamental for feto-placental blood flow.     

Effects of Ca2+ and Mg2+ on ATP-dependent 45Ca2+ influx in A-431 human epidermoidal carcinoma cells

Upon stimulation with 10(-6) -10(-3) M ATP, A-431 human epidermoidal carcinoma cells incorporated radioactive calcium from their medium in a temperature-dependent manner. The rate of incorporation of 45Ca2+ was rapid for the initial 5 min, but decreased immediately thereafter. The preincubation of cells for 2 h in medium depleted of both Ca2+ and Mg2+ abolished the ATP-dependent 45Ca2+ incorporation, irrespective of whether or not the subsequent incubation medium contained Mg2+ ions. ATP-dependent 45Ca2+ incorporation could be restored by a second preincubation (1 h) in medium containing 1 mM Mg2+, but no Ca2+. The Mg2+ ions in the second preincubation medium could be replaced by Ca2+, Co2+, or Cu2+ for restoration of such activity. Elevation of inositol trisphosphate (InsP3) was observed in cells depleted of either Ca2+ or Mg2+, but not in cells depleted of both ions. A parallel effect was observed in changes in [Ca2+]i. Since the concentration of cytosolic calcium ions does not change by incubation of cells in medium depleted of and (or) restored with calcium ions, we conclude that either calcium or magnesium ions associated with some cellular component(s) are responsible for production of InsP3, which then supposedly mobilizes Ca2+ and provokes 45Ca2+ influx.     

Modulation of small conductance calcium-activated potassium channels in C6 glioma cells

Using the patch clamp technique, we have characterized a small conductance, calcium-activated potassium (SK) channel in the C6 glioma cell line. Elevation of cytosolic Ca²⁺ concentration ([Ca²⁺] _i ) by applications of serotonin or ionomycin induced bursts of channel openings recorded in the cell-attached configuration. These channels underlie the serotonin-induced, [Ca²⁺] _i -activated whole-cell K⁺ conductance described previously. [Ca²⁺] _i directly activated SK channels in inside-out patches with a biphasic concentration dependence. Submicromolar [Ca²⁺] _i induced bursts of channel openings with a unitary conductance of about 25 pS, similar to that of the serotonin-induced channels. Supramicromolar [Ca²⁺] _i caused prolonged openings with a unitary conductance of about 35 pS, resulting in a pronounced increase of the average current in patches exposed to [Ca²⁺] _i above 100 m. The two modes of opening reflect the activity of the same SK channel. The channel conductance depended on external K⁺ concentration with K _Dof 5 m. The channel was slightly permeable to cations other than K⁺, with a permeability ratio for K⁺Ca²⁺Na⁺ of 10.0400.030, respectively. ATP was required to maintain channel activity in outside-out patches but was not essential in inside-out patches. The modulation of SK channels in C6 cells by components in their microenvironment may be related to the role of glial cells in controlling the extracellular milieu in the CNS.The authors are grateful to Dr. M. Segal for continuous support, stimulating discussions and criticism throughout the course of this work, to Dr. I. Steinberg for helpful suggestions and to Dr. H. Jarosch, for helping with the Fortran application. N.M.'s research was supported in part by BARD, the U.S.-Israel Binational Agricultural Research and Development Fund, grant no. IS-1670-89RC.