Cyclic Nucleotide-gated Channels Mediate Membrane Depolarization following Activation of Store-operated Calcium Entry in Endothelial Cells
2000; Elsevier BV; Volume: 275; Issue: 25 Linguagem: Inglês
10.1074/jbc.m002795200
ISSN1083-351X
AutoresSongwei Wu, Timothy M. Moore, George H. Brough, Sherry R. Whitt, Michael Chinkers, Ming Li, Troy Stevens,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoCalcium agonists induce membrane depolarization in endothelial cells through an unknown mechanism. Present studies tested the hypothesis that pulmonary artery endothelial cells express a cyclic nucleotide-gated (CNG) cation channel activated by store-operated calcium entry to produce membrane depolarization. In the whole-cell configuration, voltage-clamped cells revealed a large non-inactivating, outwardly rectifying cationic current in the absence of extra- or intracellular Ca2+ that was reduced upon replenishment of Ca2+. The inward current was non-selective for K+, Na+, Cs+, and Rb+ and was not inhibited by high tetraethylammonium concentrations. cAMP and cGMP stimulated the current and changed the cation permeability to favor Na+. Moreover, 8-bromo-cAMP stimulated the current in voltage-clamped cells in the perforated patch mode. The cationic current was inhibited by the CNG channel blocker LY83,583, and reverse transcriptase-polymerase chain reaction cloning identified expression of a CNG channel resembling that seen in olfactory neurons. Activation of store-operated calcium entry using thapsigargin increased a current through the CNG channel. Stimulation of the current paralleled pulmonary artery endothelial cell membrane depolarization, and both the current and membrane depolarization were abolished using LY83,583. Taken together, these data demonstrate activation of store-operated calcium entry stimulates a CNG channel producing membrane depolarization. Such membrane depolarization may contribute to slow feedback inhibition of store-operated calcium entry. Calcium agonists induce membrane depolarization in endothelial cells through an unknown mechanism. Present studies tested the hypothesis that pulmonary artery endothelial cells express a cyclic nucleotide-gated (CNG) cation channel activated by store-operated calcium entry to produce membrane depolarization. In the whole-cell configuration, voltage-clamped cells revealed a large non-inactivating, outwardly rectifying cationic current in the absence of extra- or intracellular Ca2+ that was reduced upon replenishment of Ca2+. The inward current was non-selective for K+, Na+, Cs+, and Rb+ and was not inhibited by high tetraethylammonium concentrations. cAMP and cGMP stimulated the current and changed the cation permeability to favor Na+. Moreover, 8-bromo-cAMP stimulated the current in voltage-clamped cells in the perforated patch mode. The cationic current was inhibited by the CNG channel blocker LY83,583, and reverse transcriptase-polymerase chain reaction cloning identified expression of a CNG channel resembling that seen in olfactory neurons. Activation of store-operated calcium entry using thapsigargin increased a current through the CNG channel. Stimulation of the current paralleled pulmonary artery endothelial cell membrane depolarization, and both the current and membrane depolarization were abolished using LY83,583. Taken together, these data demonstrate activation of store-operated calcium entry stimulates a CNG channel producing membrane depolarization. Such membrane depolarization may contribute to slow feedback inhibition of store-operated calcium entry. cyclic nucleotide-gated pulmonary artery endothelial cells tetraethylammonium polymerase chain reaction reverse transcriptase-PCR isobutylmethylxanthine 8-bromo-cAMP soluble guanylyl cyclase Endothelial cells form a semi-permeable barrier that compartmentalizes circulating blood elements from underlying tissue. Neuro-humoral mediators target endothelium to regulate both production of vasoactive autacoids important for control of blood pressure and cell shape important for control of fluid balance, migration, and angiogenesis (1.Moore T.M. Chetham P.M. Kelly J.J. Stevens T. Am. J. Physiol. 1998; 275: L203-L222Crossref PubMed Google Scholar). Gq-coupled agonists accomplish these diverse functions partly through generation of inositol 1,4,5-trisphosphate which, upon binding its internal receptor, depletes intracellular calcium stores and activates a membrane calcium entry channel (2.Berridge M.J. J. Physiol. (Lond.). 1997; 499: 291-306Crossref Scopus (916) Google Scholar, 3.Berridge M. Lipp P. Bootman M. Curr. Biol. 1999; 9: R157-R159Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 4.Putney Jr., J.W. Bird G.S. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (393) Google Scholar, 5.Putney Jr., J.W. Bird G.S. Endocr. 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Physiol. 1991; 261: H1246-H1254PubMed Google Scholar, 15.He P. Curry F.E. J. Appl. Physiol. 1994; 76: 2288-2297Crossref PubMed Scopus (54) Google Scholar) responses. Whereas hyperpolarization promotes calcium entry and permeability, depolarization reduces calcium entry and permeability. Activation of store-operated calcium entry by Gq-coupled agonists including bradykinin or Ca2+-ATPase inhibitors like thapsigargin cause an initial hyperpolarization attributed to activation of maxi- or intermediate KCa channels (16.Vaca L. Licea A. Possani L.D. Am. J. Physiol. 1996; 270: C819-C824Crossref PubMed Google Scholar). This hyperpolarization is transient but further promotes Ca2+entry by increasing the electrochemical driving force. A large, sustained depolarization occurs subsequently that reduces calcium entry. Mechanism(s) underlying this sustained depolarization are unknown with the exception that it is caused by neither KIRnor KCa channel activity and is La3+-sensitive, suggesting a Ca2+ dependence. Either Ca2+ or Na+ entry could produce membrane depolarization, and although several cationic conductances have been described in endothelial cells, putative channels mediating Ca2+ or Na+ entry are poorly understood (1.Moore T.M. Chetham P.M. Kelly J.J. Stevens T. Am. J. Physiol. 1998; 275: L203-L222Crossref PubMed Google Scholar,17.Nilius B. Verh. K Acad. Geneeskd Belg. 1998; 60: 215-250PubMed Google Scholar). Cyclic nucleotide-gated (CNG)1 cation channels are permeable to both Ca2+ and Na+ and mediate membrane depolarization in neurons (18.Zufall F. Firestein S. Shepherd G.M. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 577-607Crossref PubMed Scopus (172) Google Scholar, 19.Zagotta W.N. Siegelbaum S.A. Annu. Rev. 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Non-excitable endothelial cells have been shown to express CNG1 channels, and whereas their activation by calcium-elevating agents would be predicted to cause membrane depolarization, a functional role for CNG channels in endothelium has not been established (37.Yao X. Leung P.S. Kwan H.Y. Wong T.P. Fong M.W. Cardiovasc. Res. 1999; 41: 282-290Crossref PubMed Scopus (25) Google Scholar). Thus, studies were undertaken to determine whether endothelial cells express an endogenous CNG channel that mediates membrane depolarization following activation of store-operated calcium entry. Male Harlan Sprague-Dawley rats (CD strain, 350–400 g; Charles River) were euthanized by an intraperitoneal injection of 50 mg of pentobarbital sodium (Nembutal, Abbott). After sternotomy, the heart and lungs were removed en bloc, and the pulmonary arterial segment between the heart and lung hili was dissected, split, and fixed onto a 35-mm plastic dish. Pulmonary artery endothelial cells (PAECs) were obtained from the intima by gentle scraping with a plastic cell lifter and were seeded onto a 100-mm Petri dish containing 10 ml of seeding medium (∼1:1 Dulbecco's modified Eagle's medium/Ham's F-12 + 10% fetal bovine serum). Cells were verified as endothelial by positive factor VIII staining and uptake of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate-labeled acetylated low density lipoprotein. When the primary culture reached confluence, cells were passaged by trypsin digestion into 75-cm2 culture flasks (Corning), and standard tissue culture techniques were followed until the cells were used for experiments. Cells were studied between passages 6 and 20. Confluent rat PAECs were enzyme-dispersed, seeded onto 35-mm plastic culture dishes, and then allowed to re-attach for at least 24 h before patch clamp experiments were performed. Whole-cell and perforated patch clamp recordings were obtained from single (electrically isolated) rat PAECs, exhibiting a flat, polyhedral morphology. These cells were chosen for study because their morphology was consistent with rat PAECs from a confluent monolayer (38.Moore T.M. Brough G.H. Babal P. Kelly J.J. Li M. Stevens T. Am. J. Physiol. 1998; 275: L574-L582Crossref PubMed Google Scholar). Conventional whole-cell and nystatin-perforated voltage-clamp configurations were performed to measure transmembrane currents in single rat PAEC by the standard giga-seal patch clamp technique. Perforated patch technique (39.Mistry D.K. Hablitz J.J. Brain Res. 1990; 535: 318-322Crossref PubMed Scopus (12) Google Scholar) was applied to avoid disturbing the intracellular milieu of the cell, in particular resting cytosolic Ca2+. Conventional whole-cell recordings were used to dialyze the cell with our artificial "intracellular" solutions. For nystatin-perforated patch recording, the pipette was filled with nystatin containing intracellular solution and gentle suction applied to achieve giga-ohm resistance. The access resistance gradually decreased within 5 min after the giga-ohm seal was formed, and then the transmembrane current was recorded in the voltage-clamp mode when a steady value was achieved. Recording pipettes were manufactured from glass capillary tubes (Warner Instrument Corp., Hamden, CT), pulled by a two-stage puller (PC-10, Narishige Co., Ltd., Tokyo, Japan) and heat-polished before use. Pipette resistance was in the range of 2–5 megohms when filled with our intracellular solution. All experiments were performed at room temperature (22–25 °C). An EPC-9 patch clamps amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) was used to acquire data with Pulse/PulseFit software (HEKA) and filtered at 2.9 kHz. Patch clamp electrophysiological experiments were performed using two solutions, asymmetrical and symmetrical extra- and intracellular (pipette) solutions. Asymmetrical solutions contained (in mm) the following: for extracellular, 120 glutamic acid, 20 HEPES, and 1N-phenylanthranilic acid and the pH was adjusted to 7.4 with tetraethylammonium (TEA) hydroxide; for intracellular, 145 potassium glutamate, 10 HEPES, 6 MgCl2 (pH 7.2, titrated with KOH). Symmetrical solutions contained (in mm) the following: for extracellular, 100 potassium methanesulfonate (KCH3SO3), 20 HEPES, 1N-phenylanthranilic acid, (pH 7.4 titrated with methane sulfonic acid); for intracellular, 100 potassium glutamate, 10 HEPES, 1 MgCl2, 5 EGTA (pH 7.2 titrated with KOH). The osmolality in all solutions was adjusted with sucrose to 290–300 mosm. The above asymmetrical extra- and intracellular solutions were used for both whole-cell and perforated patch recordings; however, in perforated patch recordings the intracellular solution was also supplemented with nystatin (100 μg ml−1). LY83,583 [6-(phenylamino)-5.8-quinolinedione] (Research Biochemicals International, Natick, MA) was prepared in ethanol. Working solutions were made fresh each use with final ethanol concentrations of less than 1% (v/v). Thapsigargin (Sigma) was prepared in Me2SO. Dilutions were made with final Me2SO concentrations of less than 0.1% (v/v). These concentrations of ethanol and Me2SO did not alter the electrophysiological characteristics of endothelial cells. Stock solutions of cAMP and 8-Br-cAMP were dissolved fresh in the extracellular solution. N-Phenylanthranilic acid was purchased from Fluka (Switzerland). Unless otherwise stated, all chemicals were purchased from Sigma. Total RNA was isolated from a confluent, early passage 75-cm2 flask of rat PAECs by the RNeasy Total RNA (Qiagen, Inc., Chatsworth, CA) method. Approximately 1 μg of RNA was reverse-transcribed with or without 200 units of Superscript II (Life Technologies, Inc.) reverse transcriptase for 1 h at 42 °C. The first strand cDNA synthesis reactions were primed with an adapter primer (Life Technologies, Inc.) with the following sequence: 5′-GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT T-3′. PCR was performed with consequent reverse transcriptase products for 30 cycles (Profile: 94 °C, 30 s; 55 °C, 45 s; 72 °C, 1 min and 45 s; final extension 72 °C, 10 min) with the following primer set: (sense) 5′-TGA GTT CTT TGA CCG CAC TG-3′; (antisense) 5′-TTG ACA GCA TCA ATC TTG GC-3′. Following gel purification of PCR products, a second "nested" primer set was used: (sense) 5′-GGT CCT TTA CAT CTT GGT CAT C-3′ and (antisense) 5′-GAG GAC ACC AAT CAA GAA G-3′. The PCR profile was exactly as described above. PCR products were ligated directly into pCR2.1®-TOPO vector (Invitrogen, Carlsbad, CA) and transformed into chemically competent Escherichia coli. Plasmids were isolated from positive clones (verified by PCR analysis) using the Qiaprep spin miniprep method (Qiagen, Inc.) and submitted to the Biopolymer Laboratory at the University of South Alabama for automated fluorescence sequence analysis (AB373XL DNA stretch sequencer). Sequence accuracy was confirmed by sequencing in both directions using double-stranded plasmids as templates with universal primers. Nucleotide and amino acid alignments were performed with the assistance of BLAST (NCBI) and DNASIS version 2.0 (Hitachi Software) programs. Confluent rat PAECs were loaded with 1 μm of the anionic potentially sensitive fluorescent dye, bis(1,3-dibutylbarbituric acid) trimethine oxonol, according to methods previously described (40.Stevens T. Cornfield D.N. McMurtry I.F. Rodman D.M. Am. J. Physiol. 1994; 266: H1416-H1421PubMed Google Scholar). Cells were studied with an Olympus IX70 inverted microscope at × 400 using a xenon arc lamp photomultiplier system (Photon Technologies Inc., Monmouth Junction, NJ), and data were acquired and analyzed with PTI Felix software. Cells (3.Berridge M. Lipp P. Bootman M. Curr. Biol. 1999; 9: R157-R159Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 4.Putney Jr., J.W. Bird G.S. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (393) Google Scholar) were excited by the xenon arc lamp (490 nm wavelength), and emission of epifluorescence at 520 nm (signal averaged) was measured. Total fluorescence intensity was adjusted by reducing the arc lamp illumination intensity to minimize photobleaching of the dye, although some degree of photobleaching still occurred during the experiment. Data were corrected for the calculated rates of photobleaching for each individual experiment. An estimation of the relationship between fluorescence intensity and change inE m was performed by exchanging the experimental physiologic salt solution (in mm, 11 d-glucose, 0.6 MgSO4, 1 KH2PO4, 4.7 KCl, 118 NaCl, 25 HEPES, 2 CaCl2, titrated to pH 7.35–7.45 using 11 NaOH) for a high [K+]-low [Na+] solution (in mm, 11 d-glucose, 0.6 MgSO4, 1 KH2PO4, 50 KCl, 68 NaCl, 25 HEPES, 2 CaCl2, titrated to pH 7.35–7.45 using 11 NaOH) and recording the resulting increase in fluorescence (depolarization). Confluent PAECs in 12-well plates were treated as indicated in Dulbecco's modified Eagle's medium containing 10 mm HEPES. Cyclic GMP in cell extracts was then determined by radioimmunoassay as described previously (41.Chinkers M. Wilson E.M. J. Biol. Chem. 1992; 267: 18589-18597Abstract Full Text PDF PubMed Google Scholar). Initial experiments were performed using voltage-clamped cells to test for the presence of cationic currents in PAECs. Fig.1 A shows membrane current traces and current-voltage (I-V) relationships recorded using symmetrical K+ methylsulfonate solutions, since CNG channels poorly discriminate between K+ and Na+(25.Frings S. Lynch J.W. Lindemann B. J. Gen. Physiol. 1992; 100: 45-67Crossref PubMed Scopus (160) Google Scholar, 42.Kolesnikov S.S. Zhainazarov A.B. Kosolapov A.V. FEBS Lett. 1990; 266: 96-98Crossref PubMed Scopus (53) Google Scholar, 43.Sesti F. Straforini M. Lamb T.D. Torre V. J. Physiol. (Lond.). 1994; 474: 203-222Crossref Scopus (22) Google Scholar, 44.Picones A. Korenbrot J.I. J. Gen. Physiol. 1992; 100: 647-673Crossref PubMed Scopus (54) Google Scholar, 45.Menini A. J. Physiol. (Lond.). 1990; 424: 167-185Crossref Scopus (52) Google Scholar, 46.Furman R.E. Tanaka J.C. J. Gen. Physiol. 1990; 96: 57-82Crossref PubMed Scopus (36) Google Scholar). The holding potential was 0 mV. Current was measured for 200 ms at voltages ranging from −100 to +100 mV. PAECs exhibited a sustained (non-inactivating) current that was outwardly rectifying at positive voltages. As expected in symmetrical solutions, the reversal potential was 0 mV. Inclusion of tetraethylammonium in the patch pipette did not alter the K+ current (Fig. 1 B). Similarly, 4-aminopyridine did not inhibit the current (data not shown), indicating the K+ conductance was not due to KIR or KCa channel activity. We next examined the permeability ratio to monovalent cations. Ion replacement studies were performed using Rb+ and Cs+. In these experiments, intracellular K+methylsulfonate (100 mm) was replaced with either 100 mm Rb+ nitrate or 100 mmCs+ methylsulfonate. These replacements resulted in a 22 and 60% decrease in conductance, respectively, indicating the permeability ratio was K+ (1.Moore T.M. Chetham P.M. Kelly J.J. Stevens T. Am. J. Physiol. 1998; 275: L203-L222Crossref PubMed Google Scholar) > Rb+(0.8) > Cs+ (0.4). Because the anionic carrier for Rb+ was different from Cs+, we performed studies to examine the anionic contribution to the current. Studies were conducted using both 100 mm K+ glutamate and methylsulfonate. The magnitude of the K+ current was slightly associated with its predominant anion. For example, the outward K+ conductance at +100 mV was 12.3 ± 1.04 pA/picofarads when glutamate was the anion and 15.0 ± 2.47 when methylsulfonate was the anion, suggesting the presence of an anionic conductance that contributed to the current. We performed ion replacement studies using symmetrical K+, Cs+, and Na+ in a glutamate solution (Fig.2). Under these conditions the inward permeability ratio was K+ (1.Moore T.M. Chetham P.M. Kelly J.J. Stevens T. Am. J. Physiol. 1998; 275: L203-L222Crossref PubMed Google Scholar) > Na+(0.6) > Cs+ (0.5), and the outward permeability ratio was K+ (1.Moore T.M. Chetham P.M. Kelly J.J. Stevens T. Am. J. Physiol. 1998; 275: L203-L222Crossref PubMed Google Scholar) > Cs+ (0.4) > Na+ (0.1). Taken together, these findings suggest the presence of a current conducted through a non-selective cation channel. Conductance of monovalent cations through some non-selective cation channels is inhibited by Ca2+ and Mg2+. We tested whether Ca2+ and Mg2+ regulate the K+ current observed presently. Fig.3 shows the K+ current magnitude was similarly inhibited by either of the divalent cations tested, irrespective of whether Ca2+ or Mg2+were placed in the intra- or extracellular pipette solutions. Thus, the K+-conducting channel exhibits features of divalent cation block. To address putative non-selective channel(s) that may mediate the observed cationic current, we investigated the effect of cAMP on the I-Vprofile, within a physiologically relevant range of voltages. Inclusion of cAMP in the internal solution increased the cation current (Fig.4 A) and altered the ion permeability where Na+ conductance was favored over K+ and Cs+ (Fig. 4 B; TableI). This change in ion permeability induced by cAMP resembles the slip-mode conductance observed in tetrodotoxin-sensitive Na+ channels (47.Santana L.F. Gomez A.M. Lederer W.J. Science. 1998; 279: 1027-1033Crossref PubMed Scopus (153) Google Scholar). The outwardly rectifying nature of the I-V plot illustrated in Figs. 1 and2 and the permeability ratio favoring Na+ in the presence of cAMP generally resemble the electrophysiological profile previously described in both endogenous CNG channels and overexpressed CNG2 channels (18.Zufall F. Firestein S. Shepherd G.M. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 577-607Crossref PubMed Scopus (172) Google Scholar). Importantly, cyclic nucleotide increased the current within a physiologically relevant range of voltages.Table IK = 1 inward current ± cAMP at −100 mV testing potentialcAMPK+Cs+Na++10.511.69−10.450.63 Open table in a new tab CNG channels are non-selective cation channels recently shown to be expressed in diverse cell types (30.Weyand I. Godde M. Frings S. Weiner J. Muller F. Altenhofen W. Hatt H. Kaupp U.B. Nature. 1994; 368: 859-863Crossref PubMed Scopus (232) Google Scholar, 31.Kingston P.A. Zufall F. Barnstable C.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10440-10445Crossref PubMed Scopus (95) Google Scholar, 32.Ruiz M.L. London B. Nadal-Ginard B. J. Mol. Cell. Cardiol. 1996; 28: 1453-1461Abstract Full Text PDF PubMed Scopus (25) Google Scholar, 33.Ding C. Potter E.D. Qiu W. Coon S.L. Levine M.A. Guggino S.E. Am. J. Physiol. 1997; 272: C1335-C1344Crossref PubMed Google Scholar, 34.Feng L. Subbaraya I. Yamamoto N. Baehr W. Kraus-Friedmann N. FEBS Lett. 1996; 395: 77-81Crossref PubMed Scopus (22) Google Scholar, 35.Biel M. Zong X. Distler M. Bosse E. Klugbauer N. Murakami M. Flockerzi V. Hofmann F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3505-3509Crossref PubMed Scopus (124) Google Scholar, 36.Bradley J. Zhang Y. Bakin R. Lester H.A. Ronnett G.V. Zinn K. J. Neurosci. 1997; 17: 1993-2005Crossref PubMed Google Scholar), although expression in non-excitable endothelial cells is not fully resolved (37.Yao X. Leung P.S. Kwan H.Y. Wong T.P. Fong M.W. Cardiovasc. Res. 1999; 41: 282-290Crossref PubMed Scopus (25) Google Scholar). Since cAMP stimulated the cationic current similar to CNG channels of the olfactory neuron, RT-PCR cloning was performed to address whether PAECs express CNG2 channels. Primers were designed to isolate the pore region of CNG channels based upon the rat olfactory neuron sequence (48.Dhallan R.S. Yau K.W. Schrader K.A. Reed R.R. Nature. 1990; 347: 184-187Crossref PubMed Scopus (514) Google Scholar) (Fig.5 A). RT-PCR revealed a single product of predicted size (253-base pair fragment) from both rat PAEC and rat kidney total RNA. The cloned PAEC product was 100% identical to the rat olfactory (CNG2) channel. Deduced amino acid sequence from PAECs was compared with rat olfactory (CNG2), gustatory (CNG1), and rod (CNG3) α subunits (Fig. 5 B). By using physiological salt solutions, we examined the function of endogenously expressed CNG channels in PAECs. Voltage-clamped cells exhibited a limited degree of run-down over 10 min (data not shown). Inclusion of thapsigargin in the patch pipette increased the current 300% above control values and right-shifted the reversal potential, consistent with stimulation of either a Na+ or Ca2+ current (Fig. 6). The thapsigargin-stimulated current progressively increased over time until its peak was reached 3 min after establishing a seal; this peak increase in current was stable until completion of the experiment (data not shown). We next examined whether the cationic current was inhibited by LY83,583, a potent CNG channel blocker (49.Leinders-Zufall T. Rand M.N. Shepherd G.M. Greer C.A. Zufall F. J. Neurosci. 1997; 17: 4136-4148Crossref PubMed Google Scholar, 50.Leinders-Zufall T. Zufall F. J. Neurophysiol. 1995; 74: 2759-2762Crossref PubMed Scopus (52) Google Scholar), by using physiological salt solutions. PAECs were incubated for 15 min in the presence of 40 μm extracellular LY83,583 prior to establishing a seal. Cells incubated with LY83,583 exhibited a 54% lower K+conductance at +100 mV than did the untreated cells (Fig.7). Moreover, LY83,583 inhibited the current induced by both cAMP and thapsigargin, to the level obtained in control experiments, suggesting that cAMP and thapsigargin activate a similar CNG channel (Fig. 8). To confirm this idea further, maximal concentrations of cAMP and thapsigargin were applied together in the patch pipette. The combined application of cAMP and thapsigargin did not produce an additive increase in current (Fig.8 C). Taken together, these results support the idea that PAECs possess a CNG channel that regulates cationic conductance.Figure 8Inhibition of CNG channels prevents cAMP and thapsigargin from activating an inward cationic current.Current-voltage relationships recorded 10 min after the whole-cell configuration was established in the absence (closed circles) and presence (open circles) of 40 μm LY83,583. PAECs were pretreated with LY83,583 for 15 min before establishing the whole-cell configuration. A,inclusion of 400 μm cAMP in the patch pipette stimulated an inward cationic current. The current stimulated by cAMP was abolished by LY83,583 (p < 0.05). B,thapsigargin (1 μm) stimulated an inward cationic current that was also abolished by LY83,583 (p < 0.05).C, inclusion of both cAMP (400 μm) and thapsigargin (1 μm) to the patch pipette did not produce an additive increase in current density. Data were normalized to membrane capacitance to yield current density and plotted as mean ± S.E. against voltage. pF, picofarad.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We performed experiments using a perforated voltage-clamp
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