Pacemaker activity in urethral interstitial cells is not dependent on capacitative calcium entry

The aim of the present study was to investigate the properties and role of capacitative Ca²⁺ entry (CCE) in interstitial cells (IC) isolated from the rabbit urethra. Ca²⁺ entry in IC was larger in cells with depleted intracellular Ca²⁺ stores compared with controls, consistent with influx via a CCE pathway. The nonselective Ca²⁺ entry blockers Gd³⁺ (10 µM), La³⁺ (10 µM), and Ni²⁺ (100 µM) reduced CCE by 67% (n = 14), 65% (n = 11), and 55% (n = 9), respectively. These agents did not inhibit Ca²⁺ entry when stores were not depleted. Conversely, CCE in IC was resistant to SKF-96365 (10 µM), wortmannin (10 µM), and nifedipine (1 µM). Spontaneous transient inward currents were recorded from IC voltage-clamped at –60 mV. These events were not significantly affected by Gd³⁺ (10 µM) or La³⁺ (10 µM) and were only slightly decreased in amplitude by 100 µM Ni²⁺. The results from this study demonstrate that freshly dispersed IC from the rabbit urethra possess a CCE pathway. However, influx via this pathway does not appear to contribute to spontaneous activity in these cells. smooth muscle; patch clamp; spontaneous transient inward currents     

A P2X7 receptor stimulates plasma membrane trafficking in the FRTL rat thyrocyte cell line

Thyroid cells express a variety of P2Y and P2X purinergic receptor subtypes. G protein-coupled P2Y receptors influence a wide variety of thyrocyte-specific functions; however, functional P2X receptor-gated channels have not been observed. In this study, we used whole cell patch-clamp recording and fluorescence imaging of the plasma membrane marker FM1-43 to examine the effects of extracellular ATP on membrane permeability and trafficking in the Fisher rat thyroid cell line FRTL. We found a cation-selective current that was gated by ATP and 2',3'-O-(4-benzoylbenzoyl)-ATP but not by UTP. The ATP-evoked currents were inhibited by pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, adenosine 5'-triphosphate-2',3'-dialdehyde, 100 µM Zn²⁺, and 50 µM Cu²⁺. Fluorescence imaging revealed pronounced, temperature-sensitive stimulation of exocytosis and membrane internalization by ATP with the same pharmacological profile as observed for activation of current. The EC₅₀ for ATP stimulation of internalization was 440 µM in saline containing 2 mM Ca²⁺ and 2 mM Mg²⁺, and 33 µM in low-Mg²⁺, nominally Ca²⁺-free saline. Overall, the results are most consistent with activation of a P2X₇ receptor by ATP^4–. However, low permeability to N-methyl-D-glucamine⁺ and the propidium cation YO-PRO-1 indicates absence of the cytolytic pore that often accompanies P2X₇ receptor activation. ATP stimulation of internalization occurs in Na⁺-free, Ca²⁺-free, or low-Mg²⁺ saline and therefore does not depend on cation influx through the ATP-gated channel. We conclude that ATP activation of a P2X₇ receptor stimulates membrane internalization in FRTL cells via a transduction pathway that does not depend on cation influx. purinergic receptor; internalization; patch clamp     

Intermediate-conductance Ca2+-activated K+ channel is expressed in C2C12 myoblasts and is downregulated during myogenesis

We report here the expression in C₂C₁₂ myoblasts of the intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channel. The IK_Ca current, recorded under perforated-patch configuration, had a transient time course when activated by ionomycin (0.5 µM; peak current density 26.2 ± 3.7 pA/pF; n = 10), but ionomycin (0.5 µM) + 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (100 µM) evoked a stable outward current (28.4 ± 8.2 pA/pF; n = 11). The current was fully inhibited by charybdotoxin (200 nM), clotrimazole (2 µM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (300 µM), but not by tetraethylammonium (1 mM) or D-tubocurarine (300 µM). Congruent with the IK_Ca channel, elevation of intracellular Ca²⁺ in inside-out patches resulted in the activation of a voltage-insensitive K⁺ channel with weak inward rectification, a unitary conductance of 38 ± 6 pS (at negative voltages), and an IC₅₀ for Ca²⁺ of 530 nM. The IK_Ca channel was activated metabotropically by external application of ATP (100 µM), an intracellular Ca²⁺ mobilizer. Under current-clamp conditions, ATP application resulted in a membrane hyperpolarization of 35 mV. The IK_Ca current downregulated during myogenesis, ceasing to be detectable 4 days after the myoblasts were placed in differentiating medium. Downregulation was prevented by the myogenic suppressor agent basic FGF (bFGF). We also found that block of the IK_Ca channel by charybdotoxin did not inhibit bFGF-sustained myoblast proliferation. These observations show that in C₂C₁₂ myoblasts the IK_Ca channel expression correlates inversely with differentiation, yet it does not appear to have a role in myoblast proliferation. ATP; cell proliferation     

ATP-dependent regulation of SK4/IK1-like currents in rat submandibular acinar cells: possible role of cAMP-dependent protein kinase

SK4/IK1 encodes an intermediate conductance, Ca²⁺-activated K⁺ channel and fulfills a variety of physiological functions in excitable and nonexcitable cells. Although recent studies have provided evidence for the presence of SK4/IK1 channels in salivary acinar cells, the regulatory mechanisms and the physiological function of the channel remain unknown in these cells. Using molecular and electrophysiological techniques, we examined whether cytosolic ATP-dependent regulation of native SK4/IK1-like channel activity would involve endogenous cAMP-dependent protein kinase (PKA) in rat submandibular acinar (RSA) cells. Electrophysiological properties of tetraethylammonium (TEA) (10 mM)-insensitive, Ca²⁺-dependent K⁺ currents in macropatches excised from RSA cells matched those of whole cell currents recorded from human embryonic kidney-293 cells heterologously expressing rat SK4/IK1 (rSK4/IK1) cloned from RSA cells. In outside-out macropatches, activity of native SK4/IK1-like channels, defined as a charybdotoxin (100 nM)-blockable current in the presence of TEA (10 mM) in the bathing solution, ran down unless both ATP and Mg²⁺ were present in the pipette solution. The nonhydrolyzable ATP analog AMP-PNP failed to support the channel activity as ATP did. The addition of Rp-cAMPS (10 µM), a PKA inhibitor, to the pipette solution containing ATP/Mg²⁺ induced a rundown of the Ca²⁺-dependent K⁺ currents. Inclusion of cAMP (1 mM) into the pipette solution (1 µM free Ca²⁺) containing ATP/Mg²⁺ caused a gradual increase in the currents, the effect being pronounced for the currents induced by 0.1 µM free Ca²⁺. Forskolin (1 µM), an adenylyl cyclase activator, also increased the currents induced by 0.1 µM free Ca²⁺. In inside-out macropatches, cytosolic ATP/Mg²⁺ increased both the maximum current (proportional to the maximum channel activity) and Ca²⁺ sensitivity of current activation. Collectively, these results suggest that ATP-dependent regulation of native SK4/IK1-like channels, at least in part, is mediated by endogenous PKA in RSA cells. Ca²⁺-activated K⁺ channel; patch clamp; human embryonic kidney-293; salivary secretion     

Involvement of Cyclic AMP in ABA- and Ca2--Mediated Signal Transduction of Stomatal Regulation in Vicia faba

The focus of this study is to investigate possible involvementof cyclic AMP in regulation of Vicia stomatal movements. Thepresence of 0.1 mM 8-Br-cAMP, a membrane-permeable analogueof cAMP, alone in the incubation medium did not affect stomatalopening in the light in leaf epidermal peel experiments. However,addition of 0.1 mM 8-Br-cAMP completely reversed exogenous ABA-and C^a2+-induced inhibition of stomatal opening. Consistentwith these results, patch-clamping experiments showed that intracellularaddition of 0.5 mM or 1 mM cAMP significantly reversed the inhibitionof whole-cell inward K⁺ currents by internally supplied 13 µMCa²⁺ or 10 µM ABA in stomatal guard cell protoplasts,respectively. Furthermore, intracellular addition of either10 µM prostaglandin E₁ (PGE1, an adenylate cyclase activator)or 1 mM 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesteraseinhibitor) mimicked the effect of exogenous cAMP on the removalof ABA- or Ca²⁺ inhibition of inward K⁺-current. These resultssuggest that a cAMP signaling pathway is involved in signaltransduction in stomatal regulation by interacting with ABAand Ca²⁺ signaling cascades. A hypothetical mechanism by whichcAMP may regulate K⁺ in stomatal guard cells is also discussed. (Received May 6, 1999; Accepted August 27, 1999)     

MEK/MAPK as a signaling element in ATP control of endothelial myosin light chain

Phosphorylation of endothelial myosin light chains (MLC) is a key mechanism in control of endothelial contractile machinery. Extracellular ATP influences endothelial MLC phosphorylation by either activation of Ca²⁺-dependent MLC kinase or Ca²⁺-independent MLC phosphatase. Here, the role of the MEK/MAPK pathway in this signaling was investigated in porcine aortic endothelial cells. Phosphorylation of ERK2 and phosphorylation of MLC were analyzed in cultured aortic endothelial cells. ATP (10 µM) increased ERK2 phosphorylation from basal 17 ± 3 to 53 ± 4%, an effect suppressed in the presence of the MEK inhibitors PD-98059 (20 µM) or U0126 (10 µM). Phosphorylation of ERK2 was not dependent on the ATP-induced cytosolic Ca²⁺ rise, because it was unaltered when this was suppressed by the Ca²⁺ chelator BAPTA (10 µM) or xestospongin C (3 µM), an inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release mechanism of the endoplasmic reticulum. Phosphorylation of ERK2 was neither induced by the adenosine analog 5'-(N-ethylcarboxamido)adenosine (1 µM) nor inhibited in the presence of the adenosine receptor antagonist 8-phenyltheophylline (10 µM). ATP increased MLC kinase activity, and this was blocked in presence of PD-98059. ATP also increased MLC phosphatase activity, which was not inhibited by PD-98059. The MEK/MAPK pathway is a Ca²⁺-independent part of ATP signaling toward MLC kinase but not of ATP signaling toward MLC phosphatase. mitogen-activated protein kinase; contractile machinery; myosin light chain kinase; myosin light chain phosphatase     

Changes in intracellular Ca2+ concentration induced by L-type Ca2+ channel current in guinea pig gastric myocytes

We investigatedthe relationship between voltage-operatedCa²⁺ channel current and thecorresponding intracellular Ca²⁺concentration([Ca²⁺]_i)change (Ca²⁺ transient) in guineapig gastric myocytes. Fluorescence microspectroscopy was combined withconventional whole cell patch-clamp technique, and fura 2 (80 µM) wasadded to CsCl-rich pipette solution. Step depolarization to 0 mVinduced inward Ca²⁺ current(I_Ca) andconcomitantly raised[Ca²⁺]_i.Both responses were suppressed by nicardipine, an L-typeCa²⁺ channel blocker, and thevoltage dependence of Ca²⁺transient was similar to the current-voltage relation ofI_Ca. When pulseduration was increased by up to 900 ms, peakCa²⁺ transient increased andreached a steady state when stimulation was for longer. The calculatedfast Ca²⁺ buffering capacity(B value), determined as the ratio ofthe time integral ofI_Ca divided bythe amplitude of Ca²⁺ transient,was not significantly increased after depletion of Ca²⁺ stores by the cyclicapplication of caffeine (10 mM) in the presence of ryanodine (4 µM).The addition of cyclopiazonic acid (CPA, 10 µM), a sarco(endo)plasmicreticulum Ca²⁺-ATPase inhibitor,decreased B value by ~20% in areversible manner. When KCl pipette solution was used,Ca²⁺-activatedK⁺ current[I_K(Ca)]was also recorded during step depolarization. CPA sensitivelysuppressed the initial peak and oscillations of I_K(Ca) withirregular effects on Ca²⁺transients. The above results suggest that, in guinea pig gastric myocyte, Ca²⁺ transient is tightlycoupled to I_Caduring depolarization, and global[Ca²⁺]_iis not significantly affected byCa²⁺-inducedCa²⁺ release from sarcoplasmicreticulum during depolarization.

     

Mitochondrial transport in processes of cortical neurons is independent of intracellular calcium

Mitochondria show extensive movement along neuronal processes, but the mechanisms and function of this movement are not clearly understood. We have used high-resolution confocal microscopy to simultaneously monitor movement of mitochondria and changes in intracellular [Ca²⁺] ([Ca²⁺]_i) in rat cortical neurons. A significant percentage (27%) of the total mitochondria in cortical neuronal processes showed movement over distances of >2 µM. The average velocity was 0.52 µm/s. The velocity, direction, and pattern of mitochondrial movement were not affected by transient increases in [Ca²⁺]_i associated with spontaneous firing of action potentials. Stimulation of Ca²⁺ transients with forskolin (10 µM) or bicuculline (10 µM), or sustained elevations of [Ca²⁺]_i evoked by glutamate (10 µM) also had no effect on mitochondrial transit. Neither removal of extracellular Ca²⁺, depletion of intracellular Ca²⁺ stores with thapsigargin, or inhibition of synaptic activity with TTX (1 µM) or a cocktail of CNQX (10 µM) and MK801 (10 µM) affected mitochondrial movement. These results indicate that movement of mitochondria along processes is a fundamental activity in neurons that occurs independently of physiological changes in [Ca²⁺]_i associated with action potential firing, synaptic activity, or release of Ca²⁺ from intracellular stores. calcium transient; dendrites     

Small-conductance Ca2+-dependent K+ channels activated by ATP in murine colonic smooth muscle

The patch-clamptechnique was used to determine the ionic conductances activated by ATPin murine colonic smooth muscle cells. Extracellular ATP, UTP, and2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) increasedoutward currents in cells with amphotericin B-perforated patches. ATP(0.5-1 mM) did not affect whole cell currents of cells dialyzedwith solutions containing ethylene glycol-bis(-aminoethylether)-N,N,N',N'-tetraaceticacid. Apamin (3 × 10⁷M) reduced the outward current activated by ATP by 32 ± 5%. Single channel recordings from cell-attached patches showed that ATP, UTP, and2-MeS-ATP increased the open probability of small-conductance, Ca²⁺-dependentK⁺ channels with a slopeconductance of 5.3 ± 0.02 pS. Caffeine (500 µM) enhanced the openprobability of the small-conductance K⁺ channels, and ATP had no effectafter caffeine. Pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS,10⁴ M), a nonselectiveP₂ receptor antagonist, preventedthe increase in open probability caused by ATP and 2-MeS-ATP. PPADS hadno effect on the response to caffeine. ATP-induced hyperpolarization inthe murine colon may be mediated byP_2y-induced release of Ca²⁺ from intracellular stores andactivation of the 5.3-pSCa²⁺-activatedK⁺ channels.

     

ATP induces dephosphorylation of myosin light chain in endothelial cells

In cultured porcine aortic endothelial monolayers, theeffect of ATP on myosin light chain (MLC) phosphorylation, whichcontrols the endothelial contractile machinery, was studied. ATP (10 µM) reduced MLC phosphorylation but increased cytosolicCa²⁺ concentration ([Ca²⁺]_i).Inhibition of the ATP-evoked [Ca²⁺]_i rise byxestospongin C (10 µM), an inhibitor of the inositol trisphosphate-dependent Ca²⁺ release from endoplasmicreticulum, did not affect the ATP-induced dephosphorylation of MLC. MLCdephosphorylation was prevented in the presence of calyculin A (10 nM),an inhibitor of protein phosphatases PP-1 and PP-2A. Thus ATP activatesMLC dephosphorylation in a Ca²⁺-independent manner. In thepresence of calyculin A, MLC phosphorylation was incremented afteraddition of ATP, an effect that could be abolished when cellswere loaded with the Ca²⁺ chelator1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester (10 µM). Thus ATP also activates aCa²⁺-dependent kinase acting on MLC. In summary, ATPsimultaneously stimulates a functional antagonism toward bothphosphorylation and dephosphorylation of MLC in which thedephosphorylation prevails. In endothelial cells, ATP is the firstphysiological mediator identified to activate MLC dephosphorylation bya Ca²⁺-independent mechanism.

     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by miconazole

The inhibition of sarcoplasmic reticulumCa²⁺-ATPase activity by miconazole was dependent on theconcentration of ATP and membrane protein. Half-maximal inhibition wasobserved at 12 µM miconazole when the ATP concentration was 50 µMand the membrane protein was 0.05 mg/ml. When ATP was 1 mM, a lowmicromolar concentration of miconazole activated the enzyme, whereashigher concentrations inhibited it. A qualitatively similar responsewas observed when Ca²⁺ transport was measured. Likewise,the half-maximal inhibition value was higher when the membraneconcentration was raised. Phosphorylation studies carried out aftersample preequilibration in different experimental settings shed lighton key partial reactions such as Ca²⁺ binding and ATPphosphorylation. The miconazole effect on Ca²⁺-ATPaseactivity can be attributed to stabilization of theCa²⁺-free enzyme conformation giving rise to a decrease inthe rate of the Ca²⁺ binding transition. The phosphoryltransfer reaction was not affected by miconazole.

     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

Ca(2+) mobilization through dorsal root ganglion Ca(2+)-sensing receptor stably expressed in HEK293 cells

The rat dorsal root ganglion (DRG) Ca²⁺-sensing receptor (CaR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168–175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [Ca²⁺] ([Ca²⁺]_e) from 0.5 to 1 mM resulted in increases in [Ca²⁺]_i levels, which were blocked by 30 µM 2-aminoethyldiphenyl borate. [Ca²⁺]_e-response studies indicate a Ca²⁺ sensitivity with an EC₅₀ of 1.75 ± 0.10 mM. NPS R-467 and Gd³⁺ activated the CaR. When [Ca²⁺]_e was successively raised from 0.25 to 4 mM, peak [Ca²⁺]_i, attained with 0.5 mM, was reduced by 50%. Similar reductions were observed with repeated applications of 10 mM Ca²⁺, 1 and 10 µM NPS R-467, or 50 and 100 µM Gd³⁺, indicating desensitization of the response. Furthermore, Ca²⁺ mobilization increased phosphorylated protein kinase C (PKC) levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca²⁺ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Ca_e²⁺. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca²⁺]_i transients by 49.9 ± 5.2% (at 1 mM Ca²⁺) and 40.5 ± 6.5% (at 2 mM Ca²⁺), compared with controls. The findings suggest involvement of PKC in the pathway for Ca²⁺ mobilization following CaR activation. desensitization; protein kinase C     

Differential modulation of caffeine- and IP3-induced calcium release in cultured arterial tissue

To investigatethe Ca²⁺-dependent plasticity ofsarcoplasmic reticulum (SR) function in vascular smooth muscle,transient responses to agents releasing intracellularCa²⁺ by either ryanodine(caffeine) orD-myo-inositol1,4,5-trisphosphate [IP₃;produced in response to norepinephrine (NE),5-hydroxytryptamine (5-HT), arginine vasopressin (AVP)] receptorsin rat tail arterial rings were evaluated after 4 days of organculture. Force transients induced by all agents were increased comparedwith those induced in fresh rings. Stimulation by 10% FCSduring culture further potentiated the force andCa²⁺ responses to caffeine (20 mM)but not to NE (10 µM), 5-HT (10 µM), or AVP (0.1 µM). The effectwas persistent, and SR capacity was not altered after reversibledepletion of stores with cyclopiazonic acid. The effects of serum couldbe mimicked by culture in depolarizing medium (30 mMK⁺) and blocked by the additionof verapamil (1 µM) or EGTA (1 mM) to the medium, loweringintracellular Ca²⁺ concentration([Ca²⁺]_i)during culture. These results show that modulation of SR function canoccur in vitro by a mechanism dependent on long-term levels of basal[Ca²⁺]_iand involving ryanodine- but notIP₃ receptor-mediatedCa²⁺release.     

Regulation of intracellular calcium in N1E-115 neuroblastoma cells: the role of Na(+)/Ca(2+) exchange

In fura 2-loaded N1E-115 cells, regulationof intracellular Ca²⁺ concentration([Ca²⁺]_i) following a Ca²⁺ loadinduced by 1 µM thapsigargin and 10 µM carbonylcyanidep-trifluoromethyoxyphenylhydrazone (FCCP) wasNa⁺ dependent and inhibited by 5 mM Ni²⁺. Incells with normal intracellular Na⁺ concentration([Na⁺]_i), removal of bath Na⁺,which should result in reversal of Na⁺/Ca²⁺exchange, did not increase [Ca²⁺]_i unlesscell Ca²⁺ buffer capacity was reduced. When N1E-115 cellswere Na⁺ loaded using 100 µM veratridine and 4 µg/mlscorpion venom, the rate of the reverse mode of theNa⁺/Ca²⁺ exchanger was apparently enhanced,since an ~4- to 6-fold increase in [Ca²⁺]_ioccurred despite normal cell Ca²⁺ buffering. In SBFI-loadedcells, we were able to demonstrate forward operation of theNa⁺/Ca²⁺ exchanger (net efflux ofCa²⁺) by observing increases (~ 6 mM) in[Na⁺]_i. These Ni²⁺ (5 mM)-inhibited increases in [Na⁺]_i could onlybe observed when a continuous ionomycin-induced influx ofCa²⁺ occurred. The voltage-sensitive dyebis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used tomeasure changes in membrane potential. Ionomycin (1 µM) depolarizedN1E-115 cells (~25 mV). This depolarization was Na⁺dependent and blocked by 5 mM Ni²⁺ and 250-500 µMbenzamil. These data provide evidence for the presence of anelectrogenic Na⁺/Ca²⁺ exchanger that is capableof regulating [Ca²⁺]_i after release ofCa²⁺ from cell stores.

     

Calmodulin reverses rundown of L-type Ca(2+) channels in guinea pig ventricular myocytes

Calmodulin (CaM) is implicated in regulation of Ca²⁺ channels as a Ca²⁺ sensor. The effect of CaM on rundown of L-type Ca²⁺ channels in inside-out patch form was investigated in guinea pig ventricular myocytes. Ca²⁺ channel activity disappeared within 1–3 min and did not reappear when the patch was excised and exposed to an artificial intracellular solution. However, application of CaM (0.03, 0.3, 3 µM) + 3 mM ATP to the intracellular solution within 1 min after patch excision resulted in dose-dependent activation of channel activity. Channel activity averaged 11.2%, 94.7%, and 292.9%, respectively, of that in cell-attached mode. Channel activity in inside-out patch mode was induced by CaM + ATP at nanomolar Ca²⁺ concentrations ([Ca²⁺]); however, increase to micromolar [Ca²⁺] rapidly inactivated the channel activity induced, revealing that the effect of CaM on the channel was Ca²⁺ dependent. At the 2nd, 4th, 6th, 8th, and 10th minutes after patch excision, CaM (0.75 µM) + ATP induced Ca²⁺ channel activity to 150%, 100%, 96.9%, 29.3%, and 16.6%, respectively, revealing a time-dependent action of CaM on the channel. CaM added with adenosine 5'-(,-imido)triphosphate (AMP-PNP) also induced channel activity, although with much lower potency and shorter duration. Protein kinase inhibitors KN-62, CaM-dependent protein kinase (CaMK)II 281-309, autocamtide-related CaMKII inhibitor peptide, and K252a (each 1–10 µM) did not block the effect of CaM, indicating that the effect of CaM on the Ca²⁺ channel was phosphorylation independent. Neither CaM nor ATP alone induced Ca²⁺ channel activity, showing a cooperative effect of CaM and ATP on the Ca²⁺ channel. These results suggest that CaM is a crucial regulatory factor of Ca²⁺ channel basal activity. cardiac myocyte; calcium channel; patch clamp     

Cytoplasmic Ca2+ has No Effect on the Excitability of Chara Plasmalemma

Internodal cells of Chara australis were subjected to two consecutiveintracellular perfusions with a Ca²⁺-free EGTA medium whichdisintegrated the tonoplast within about 10 minutes and thenwith a Ca²⁺-buffered medium. All perfusion media usually contained1 mM ATP. To stop the electrogenic pump, the internode was depletedof intracellular ATP. The excitability of the plasmalemma wasnot significantly influenced by intracellular free Ca²⁺ concentrationsup to 10^–4 M. To trigger action potentials, minimum currentdensities of 1 to 2 µA cm^–2 had to be applied atall tested Ca²⁺ concentrations. In the absence of cytoplasmicATP, excitability was completely lost at all Ca²⁺ concentrations. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, D-5300 Bonn, FRG. (Received September 22, 1984; Accepted March 6, 1985)     

Effects of estradiol on phenylephrine contractility associated with intracellular calcium release in rat aorta

The ability of estradiol to affect phenylephrine-induced contraction and the subsequent increase in resting tone, associated with capacitative Ca²⁺ entry across the plasma membrane, was evaluated in rat aortic rings incubated in Ca²⁺-free solution. The incubation with estradiol (1–100 nM, 5 min) inhibited both the phenylephrine-induced contraction and the IRT. Neither cycloheximide (1 µM; inhibitor of protein synthesis) nor tamoxifen (1 µM; blocker of estrogenic receptors) modified the effects of estradiol. Estradiol (100 µM) also blocked the contractile response to serotonin (10 µM) but not to caffeine (10 mM). In addition, estradiol (100 µM) inhibited the contractile responses to cyclopiazonic acid (1 µM; selective Ca²⁺-ATPase inhibitor) associated with capacitative Ca²⁺ influx through non-L-type Ca²⁺ channels. Finally, estradiol inhibited the Ca²⁺-induced increases in intracellular free Ca²⁺ (after pretreatment with phenylephrine) in cultured rat aorta smooth muscle cells incubated in Ca²⁺-free solution. In conclusion, estradiol interfered in a concentration-dependent manner with Ca²⁺-dependent contractile effects mediated by the stimuli of ₁-adrenergic and serotonergic receptors and inhibited the capacitative Ca²⁺ influx through both L-type and non-L-type Ca²⁺ channels. Such effects are in essence nongenomic and not mediated by the intracellular estrogenic receptor. estrogen; ₁-adrenergic agonists     

Intracellular Ca(2+) stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception

Thenotion that intracellular Ca²⁺ (Ca_i²⁺)stores play a significant role in the chemoreception process inchemoreceptor cells of the carotid body (CB) appears in the literaturein a recurrent manner. However, the structural identity of theCa²⁺ stores and their real significance in the function ofchemoreceptor cells are unknown. To assess the functional significanceof Ca_i²⁺ stores in chemoreceptor cells, we havemonitored 1) the release of catecholamines (CA) from thecells using an in vitro preparation of intact rabbit CB and2) the intracellular Ca²⁺ concentration([Ca²⁺]_i) using isolated chemoreceptor cells;both parameters were measured in the absence or the presence of agentsinterfering with the storage of Ca²⁺. We found thatthreshold [Ca²⁺]_i for high extracellularK⁺ (K_e⁺) to elicit a release response is250 nM. Caffeine (10-40 mM), ryanodine (0.5 µM), thapsigargin(0.05-1 µM), and cyclopiazonic acid (10 µM) did not alter thebasal or the stimulus (hypoxia, high K_e⁺)-inducedrelease of CA. The same agents produced Ca_i²⁺transients of amplitude below secretory threshold; ryanodine (0.5 µM), thapsigargin (1 µM), and cyclopiazonic acid (10 µM) did notalter the magnitude or time course of the Ca_i²⁺responses elicited by high K_e⁺. Several potentialactivators of the phospholipase C system (bethanechol, ATP, andbradykinin), and thereby of inositol 1,4,5-trisphosphate receptors,produced minimal or no changes in [Ca²⁺]_i anddid not affect the basal release of CA. It is concluded that, in therabbit CB chemoreceptor cells, Ca_i²⁺ stores do not playa significant role in the instant-to-instant chemoreception process.

     

Extracellular ATP stimulates volume decrease in Necturus red blood cells

This study examined whether extracellular ATP stimulatesregulatory volume decrease (RVD) in Necturusmaculosus (mudpuppy) red blood cells (RBCs). Thehemolytic index (a measure of osmotic fragility) decreased withextracellular ATP (50 µM). In contrast, the ATP scavenger hexokinase(2.5 U/ml, 1 mM glucose) increased osmotic fragility. In addition, theATP-dependent K⁺ channelantagonist glibenclamide (100 µM) increased the hemolytic index, andthis inhibition was reversed with ATP (50 µM). We also measured cellvolume recovery in response to hypotonic shock electronically with aCoulter counter. Extracellular ATP (50 µM) enhanced cell volumedecrease in a hypotonic (0.5×) Ringer solution. In contrast, hexokinase (2.5 U/ml) and apyrase (an ATP diphosphohydrolase, 2.5 U/ml)inhibited cell volume recovery. The inhibitory effect of hexokinase wasreversed with the Ca²⁺ ionophoreA-23187 (1 µM); it also was reversed with the cationophore gramicidin(5 µM in a choline-Ringer solution), indicating that ATP was linkedto K⁺ efflux. In addition,glibenclamide (100 µM) and gadolinium (10 µM) inhibited cell volumedecrease, and the effect of these agents was reversed with ATP (50 µM) and A-23187 (1 µM). Using the whole cell patch-clamp technique,we found that ATP (50 µM) stimulated a whole cell current underisosmotic conditions. In addition, apyrase (2.5 U/ml), glibenclamide(100 µM), and gadolinium (10 µM) inhibited whole cell currents thatwere activated during hypotonic swelling. The inhibitory effect ofapyrase was reversed with the nonhydrolyzable analog adenosine5'-O-(3-thiotriphosphate) (50 µM), and the effect of glibenclamide or gadolinium was reversed withATP (50 µM). Finally, anionic whole cell currents were activated withhypotonic swelling when ATP was the only significant charge carrier,suggesting that increases in cell volume led to ATP efflux through aconductive pathway. Taken together, these results indicate thatextracellular ATP stimulated cell volume decrease via aCa²⁺-dependent step that led toK⁺ efflux.