Biomedical subjects
K Groschner
Publications and source records attributed to K Groschner.
Trp proteins form store-operated cation channels in human vascular endothelial cells.
Members of the Trp protein family have been suggested as the structural basis of store-operated cation conductances. With this study, we provide evidence for the expression of three isoforms of Trp (hTrp1, 3 and 4) in human umbilical vein endothelial cells (HUVEC). The role of Trp proteins in store regulation of endothelial membrane conductances was tested by expression of an N-terminal fragment of hTrp3 (N-TRP) which exerts a dominant negative effect on Trp channel function presumably due to suppression of channel assembly. Depletion of intracellular Ca2+ stores with IP3 (100 microM) or thapsigargin (100 nM) induced a substantial cation conductance in sham-transfected HUVEC as well as in HUVEC transfected with hTrp3. In contrast, HUVEC transfected with N-TRP failed to exhibit store-operated currents. Our results suggest the involvement of Trp related proteins in the store-operated cation conductance of human vascular endothelial cells.
Essential role of the beta subunit in modulation of C-class L-type Ca2+ channels by intracellular pH.
Elevation of intracellular pH (pHi) enhances the activity of native L-type Ca2+ channels in cardiac and smooth muscle. We studied the modulation by pHi of expressed L-type Ca2+ channels comprised of either the alpha1c subunits alone or of alpha1c plus beta2a subunits. Ca2+ channels were expressed in human embryonic kidney cells (HEK 293) and pHi was increased from a basal level of 7.3 to 8.3 by exposure of cells to NH4Cl (20 mM) or by elevation of extracellular pH to 8.5. Elevation of pHi enhanced the activity of Ca2+ channels derived by coexpression of alpah1c and beta2a subunits. This alkalosis-induced stimulation of channel activity was mainly due to an increase in channel availability. Channels derived by expression of alpha1c alone were not affected by intracellular alkalosis. Our results demonstrate that the pHi sensitivity of L-type Ca2+ channels is conferred by the beta subunit of the channel complex.
Divergent effects of extracellular and intracellular alkalosis on Ca2+ entry pathways in vascular endothelial cells.
Modulation by alkalosis of basal leak Ca2+ entry and store-depletion-induced Ca2+ entry was investigated in the vascular endothelial cell line ECV 304. Ca2+ entry was monitored as the increase in the intracellular free Ca2+ concentration ([Ca2+]i) induced by elevation of the extracellular Ca2+ concentration. When ECV 304 cells were challenged with 100 nM thapsigargin in nominally Ca2+-free solution, [Ca2+]i increased transiently, and the increase in [Ca2+]i during a subsequent cumulative elevation of extracellular Ca2+ (from nominally Ca2+-free up to 5 mM) was markedly enhanced compared with non-stimulated cells (i.e. basal Ca2+ leak). Prolonged elevation of the extracellular pH (pHo) from 7.4 to 7.9 did not affect resting [Ca2+]i or the thapsigargin-induced [Ca2+]i transient evoked in nominally Ca2+-free solution, but increased leak Ca2+ entry as well as store-depletion-activated Ca2+ entry significantly. Basal Ca2+ leak and store-depletion-activated Ca2+ entry were enhanced either by acute elevation of pHo from 7.4 to 7.9 or by chronic alkalosis (pHo=7.9). Stimulation of Ca2+ entry by extracellular alkalosis was observed both in normal and in high extracellular K+ (110 mM) solution, suggesting that the effects of alkalosis are independent of membrane potential. The intracellular pH (pHi) increased slightly during both acute and chronic extracellular alkalosis (from 7.22+/-0.01 to 7.37+/-0.04 and 7. 45+/-0.05 respectively). Elevation of pHi to 7.60+/-0.06 at constant pHo by administration of 20 mM NH4Cl failed to stimulate, and in fact inhibited, store-depletion-activated Ca2+ entry. Our results demonstrate that a decrease in the extracellular but not the intracellular proton concentration promotes both basal and stimulated Ca2+ entry into endothelial cells.
Intracellular Ca2+ inactivates L-type Ca2+ channels with a Hill coefficient of approximately 1 and an inhibition constant of approximately 4 microM by reducing channel's open probability.
The patch-clamp technique was used to characterize the mechanism of Ca2+-induced inactivation of cardiac L-type Ca2+ channel alpha(1C-a) + beta3 subunits stably expressed in CHO cells. Single Ca2+ channel activity was monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)BAYK 8644 and calpastatin plus ATP. This enabled stabilization of channel activity in the inside-out patch and allowed for application of steady-state Ca2+ concentrations to the intracellular face of excised membrane patches in an attempt to provoke Ca2+-induced inactivation. Inactivation was found to occur specifically with Ca2+ since it was not observed upon application of Ba2+. Ca2+-dependent inhibition of mean Ca2+ channel activity was characterized by a Hill coefficient close to 1. Ca2+ binding to open and closed states of the channel obtained during depolarization apparently occurred with similar affinity yielding half-maximal inhibition of Ca2+ channel activity at approximately 4 microM. This inhibition manifested predominantly in a reduction of the channel's open probability whereas availability remained almost unchanged. The reduction in open probability was achieved by an increase in first latencies and a decrease in channel opening frequency as well as channel open times. At high (12-28 microM) Ca2+ concentrations, 72% of inhibition occurred due to a stabilization of the closed state and the remaining 28% by a destabilization of the open state. Our results suggest that binding of one calcium ion to a regulatory domain induces a complex alteration in the kinetic properties of the Ca2+ channel and support the idea of a single EF hand motif as the relevant Ca2+ binding site on the alpha1 subunit.
Estimating the number of channels in patch-clamp recordings: application to kinetic analysis of multichannel data from voltage-operated channels.
Important kinetic information of voltage-operated ion channels can be obtained by estimating the open probability, the availability, and the first latency, and by applying run analysis. In the case of multichannel patches, estimation of the number of available channels is a prerequisite for the above analysis. Here we describe a method for calculation of the a posteriori probability of the number of available channels in each sweep by using the Bayes formula. This probability serves as a measure for the number of channels and allows for first latency determination and run analysis. The methods described were applied to simulated and experimental data obtained from L-type Ca2+ channel recordings.
Intracellular Ca2+ inhibits smooth muscle L-type Ca2+ channels by activation of protein phosphatase type 2B and by direct interaction with the channel.
Modulation of L-type Ca2+ channels by tonic elevation of cytoplasmic Ca2+ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba2+ was used as charge carrier, and run down of Ca2+ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca2+ in intact cells by elevation of extracellular Ca2+ in the presence of the ionophore A23187 inhibited the activity of L-type Ca2+ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca2+ with fura-2 revealed a 50% inhibitory concentration (IC50) of 260 nM and a Hill coefficient close to 4 for Ca2+- dependent inhibition. Ca2+-induced inhibition of Ca2+ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca2+-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 microM) or the cytoskeleton disruptive agent cytochalasin B (20 microM), but prevented by cyclosporin A (1 microg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca2+ at the cytoplasmic side of inside-out patches inhibited Ca2+ channels with an IC50 of 2 microM and a Hill coefficient close to unity. Direct Ca2+-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca2+ concentration of 1 microM inhibited Ca2+ channel open probability and availability. Elevation of cytoplasmic Ca2+ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC50 of approximately 380 nM and a Hill coefficient of approximately 3; i.e., characteristics reminiscent of the Ca2+ sensitivity of Ca2+ channels in intact cells. Our results suggest that L-type Ca2+ channels of smooth muscle are controlled by two Ca2+-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca2+ with a single membrane-associated site that may reside on the channel protein itself.
A type 2A phosphatase-sensitive phosphorylation site controls modal gating of L-type Ca2+ channels in human vascular smooth-muscle cells.
The patch-clamp technique was employed to investigate phosphorylation/dephosphorylation-dependent modulation of L-type Ca2+ channels in smooth-muscle cells isolated from human umbilical vein. Okadaic acid, an inhibitor of phosphoprotein phosphatases type 1 (PP1) and 2A (PP2A), increased the probability of channels being in the open state (Po) in intact cells. This increase in Po was due mainly to promotion of long-lasting channel openings, i.e. promotion of 'mode 2' gating behaviour. Exposure of the cytoplasmic side of excised patches of membrane to the purified catalytic subunit of PP2A (PP2Ac) resulted in the opposite modulation of channel function. PP2Ac (0.2 unit/ml) reduced the Po of Ca2+ channels mainly via suppression of 'mode 2' gating. This effect of PP2Ac was completely prevented by 1 microM okadaic acid. The catalytic subunit of PPI (0.2 unit/ml), however, barely affected channel activity. Our results provide evidence for a PP2A-sensitive regulatory site that controls modal gating of L-type Ca2+ channels in smooth muscle.
Evidence for a direct inhibitory effect of extracellular H+ on store depletion-activated Ca2+ entry in vascular endothelial cells.
Modulation of store depletion-activated Ca2+ entry by acidosis was investigated in ECV304 endothelial cells. Lowering extracellular pH from 7.4 to 6.9 markedly suppressed Ca2+ entry elicited by direct depletion of Ca2+ stores with thapsigargin (100 nM), but did not significantly affect leak Ca2+ entry. Acidosis diminished thapsigargin-induced Ca2+ entry by 53.7 +/- 7.8% at 2.5 mM extracellular Ca2+. A similar degree of inhibition was observed in cells depolarized by high extracellular K+ (100 mM). Reduction of extracellular pH from 7.4 to 6.9 was associated with a decrease in intracellular pH from 7.23 +/- 0.01 to 7.01 +/- 0.03. Propionate (20 mM) caused a reduction of intracellular pH to 6.97 +/- 0.02, but failed to suppress store depletion-activated Ca2+ entry at 2.5 mM extracellular Ca2+ significantly. Our results suggest that an increase in extracellular proton concentration inhibits store depletion-activated Ca2+ entry through a direct, membrane potential-independent effect on the plasmalemmal Ca2+ channel.
NH4Cl-induced contraction of porcine coronary artery involves activation of dihydropyridine-sensitive Ca2+ entry.
The role of voltage-dependent, dihydropyridine-sensitive Ca2+ channels in NH4Cl-induced vasoconstriction was investigated in isolated porcine coronary arteries by measuring in parallel isometric tone and 45Ca2+ uptake. NH4Cl (10-80 mM) concentration dependently induced tonic contractions which were preceded by a time lag of several minutes. Contractile responses to high (60 mM) as well as low (25 mM) concentrations of NH4Cl were markedly inhibited by 1 microM nifedipine or removal of extracellular Ca2+. The contractile effect of 25 mM NH4Cl was substantially enhanced by increasing extracellular K+ to 14.7 mM or by pretreatment of coronary arteries with either 5 mM tetraethylammonium chloride or 0.1 microM 1,4-dihydro- 2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester (BAY K8644). NH4Cl (60 mM) significantly increased 45Ca2+ uptake with a lag time of more than 5 min. The increase in 45Ca2+ uptake induced by 60 mM NH4Cl was abolished in the presence of 1 microM nifedipine. Although NH4Cl (25 mM) did not detectably stimulate 45Ca2+ uptake in normal K+ solution, it significantly augmented 45Ca2+ uptake when extracellular K+ was increased to 14.7 mM. Furthermore, NH4Cl (20 mM) potentiated histamine-induced contraction of coronary arteries. This potentiating effect of NH4Cl was completely antagonized by nifedipine. Our results suggest an involvement of nifedipine-sensitive Ca2+ channels in NH4Cl-induced vasoconstriction of porcine coronary artery.
Inhibition of a store-operated Ca2+ entry pathway in human endothelial cells by the isoquinoline derivative LOE 908.
1. The novel cation channel blocker, LOE 908, was tested for its effects on Ca2+ entry and membrane currents activated by depletion of intracellular Ca2+ stores in human endothelial cells. 2. LOE 908 inhibited store-operated Ca2+ entry induced by direct depletion of Ca2+ stores with 100 nM thapsigargin or 100 nM ionomycin with an EC50 of 2 microM and 4 microM, respectively. 3. LOE 908 did not affect thapsigargin- or ionomycin-induced Ca2+ release from intracellular stores up to concentrations of 3 microM. 4. LOE 908 reversibly suppressed thapsigargin- as well as ionomycin-induced whole-cell membrane currents. 5. The LOE 908-sensitive membrane conductance corresponded to a cation permeability of 5.5 and 6.9 fold selectivity for Ca2+ over K+ in the presence of thapsigargin and ionomycin, respectively. 6. Our results suggest that the isoquinoline, LOE 908 is a novel, potent inhibitor of the store-operated (capacitive) Ca2+ entry pathway in endothelial cells.
Basal dephosphorylation controls slow gating of L-type Ca2+ channels in human vascular smooth muscle.
The role of cellular phosphatase activity in regulation of smooth muscle L-type Ca2+ channels was investigated using tautomycin, a potent and specific inhibitor of serin/threonin phosphatases type 1 and 2A. Tautomycin (1-100 nM) inhibited Ca2+ channel activity in smooth muscle cells isolated from human umbilical vein. Tautomycin-induced inhibition of Ca2+ channel activity was due to a reduction of channel availability which originated mainly from prolongation of the lifetime of unavailable states of the channel. Pretreatment of smooth muscle cells with the protein kinase inhibitor H-7 (10 microM) prevented the inhibitory effect of tautomycin. Our results suggest modulation of slow gating between available and unavailable states as a mechanism of phosphorylation-dependent down-regulation of Ca2+ channels in vascular smooth muscle.
Trypsin increases availability and open probability of cardiac L-type Ca2+ channels without affecting inactivation induced by Ca2+.
The patch-clamp technique was employed to investigate the response of single L-type Ca2+ channels to the protease trypsin applied to the intracellular face of excised membrane patches from guinea pig ventricular myocytes. Calpastatin and ATP were used to prevent run-down of Ca2+ channel activity monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)-BAYK 8644. Upon application of trypsin (100 micrograms/ml) channel activity was enhanced fourfold and remained elevated upon removal of trypsin, as expected of a proteolytic, irreversible modification. The trypsin effect was not mediated by a proteolytic activation of protein kinases, as evidenced by the insensitivity of this effect to protein kinase inhibitors. Trypsin-modified Ca2+ channels exhibited the usual run-down phanomenon upon removal of calpastatin and ATP. In ensemble average currents trypsin-induced changes of channel function are apparent as a threefold increase in peak current and a reduction in current inactivation. At the single channel level these effects were based on about a twofold increase in both Ca2+ channels' availability and open probability. Neither the actual number of channels in the patch nor their unitary conductance as well as reversal potential was changed by trypsin. The Ca(2+)-induced inactivation was not impaired, as judged by a comparable sensitivity of trypsin-modified Ca2+ channels to intracellular Ca2+. Similarly, trypsin treatment did not affect the sensitivity of Ca2+ channels to phenylalkylmine inhibition. The observed alterations in channel function are discussed in terms of possible structural correlates.
Protein kinase-C mediates dual modulation of L-type Ca2+ channels in human vascular smooth muscle.
The role of protein kinase C (PKC) in cellular regulation of L-type Ca2+ channels was investigated in human umbilical vein smooth muscle. Activation of PKC, by low concentrations (< 30 nM) of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) caused inhibition of Ca2+ channels, while higher concentrations of TPA (> 100 nM) elicited a transient rise, followed by sustained inhibition of Ca2+ channel activity in cell-attached patches. Low TPA concentrations predominantly reduced channel availability, while high concentrations of TPA (100 nM) transiently increased channel availability and, in addition, prolonged mean open time. The inactive 4-alpha-phorbol-12,13- didecanoate failed to affect channel activity, and pretreatment of the cells with PKC inhibitors (H-7, chelerythrine) antagonized inhibitory and stimulatory effects of TPA. Our results provide evidence for two distinct PKC-dependent mechanisms of L-type Ca2+ channel regulation in smooth muscle.
The role of myoendothelial cell contact in non-nitric oxide-, non-prostanoid-mediated endothelium-dependent relaxation of porcine coronary artery.
1. Experiments were designed to analyse the requirement of myoendothelial junctions by bradykinin-induced endothelium-dependent relaxations resistant to NG-nitro-L-arginine (L-NOARG) and indomethacin porcine coronary arteries. 2. Rings of porcine coronary arteries were contracted with the thromboxane receptor agonist, U46619 and relaxations to bradykinin recorded isometrically. All experiments were performed in the presence of indomethacin. Nitric oxide (NO)-mediated effects were blocked by the NO synthase inhibitor L-NOARG (250 microM) and myoendothelial contacts inhibited by treatment with hypertonic solution containing D-mannitol or sucrose (each 180 mM) or the gap junctional uncoupling agent 1-heptanol (2 mM). High [K+] solutions (40 mM) were used to probe a possible contribution of endothelium-derived hyperpolarizing factor (EDHF). 3. In the presence of endothelium, bradykinin induced concentration-dependent relaxations with a mean EC50 of 3.2 nM and a maximum response of 95 +/- 1% of papaverine-induced relaxation (control curve). 4. In the absence of endothelium, bradykinin failed to induce relaxations. Addition of cultured porcine aortic endothelial cells to the organ bath resulted in some relaxation and restored in part the relaxant effect of bradykinin. This endothelial cell-mediated relaxant effect was completely abolished in the presence of 250 microM L-NOARG. 5. Bradykinin-induced relaxations in endothelium-preserved rings were only slightly suppressed by L-NOARG (86% of control). In vessels partially depolarized by high extracellular [K+] (40 mM) relaxation was reduced to 72% of control. In the presence of L-NOARG, bradykinin failed to relax partially depolarized vessels. 6. In the presence of 2 mM -heptanol, 180 mM mannitol or 180 mM sucrose maximum relaxation to bradykinin was reduced to ~70%, i.e. to the same extent as in the presence of high [K+]. The remaining relaxation was sensitive to blockade by L-NOARG.7. Tissue cyclic GMP content which reflects NO activity, was increased about 4 fold by bradykinin(300 nM). This increase was unaffected by high [K+], heptanol or sucrose but blocked by L-NOARG.8 Our results suggest that non-nitric oxide- and non-prostanoid-mediated endothelium-dependent relaxation of porcine coronary artery requires functionally intact myoendothelial junctions.
Histamine induces K+, Ca2+, and Cl- currents in human vascular endothelial cells. Role of ionic currents in stimulation of nitric oxide biosynthesis.
The nature of the membrane currents mediating agonist-induced Ca2+ entry and enhanced nitric oxide (NO) production in endothelial cells is still unclear. Using both perforated-patch and conventional whole-cell clamp technique, we have studied the membrane response associated with histamine stimulation of human vascular endothelial cells. In perforated-patch experiments, the initial histamine (10 mumol/L)-induced current reversed close to the K+ equilibrium potential and was blocked by tetrabutylammonium ions (TBA, 10 mmol/L). In addition, a TBA-insensitive current that developed slowly in the presence of histamine was recorded. This delayed histamine-induced current reversed close to neutral potential and was inhibited by SK&F 96365 (25 mumol/L), a putative blocker of receptor-operated Ca2+ channels. Similar histamine effects were observed in conventional whole-cell experiments using pipette solutions with low Ca(2+)-buffering capacity. Strong buffering of intracellular free Ca2+ suppressed the initial, but not the delayed, current response. The delayed component of histamine-induced current was substantially inhibited by the Cl- channel blocker N-phenylanthranilic acid (NPA, 100 mumol/L), and an eightfold change in the Cl- gradient shifted the reversal potential of this current by 30 mV. In Cl(-)-free solutions, histamine induced an SK&F 96365-sensitive NPA-resistant current, which, according to reversal potential measurements in 20 mmol/L extracellular Ca2+, corresponded to a cation conductance with 13- to 25-fold selectivity for Ca2+ over K+. Both SK&F 96365 and TBA strongly suppressed histamine-induced rises in intracellular free Ca2+ and cellular cGMP levels, whereas NPA did not. Our results provide the first demonstration that three distinct ionic conductances contribute to the histamine-induced membrane response of endothelial cells. It is suggested that histamine induces a Cl- conductance that is apparently not involved in Ca2+ homeostasis and regulation of NO biosynthesis, while, in parallel, joint activation of a rapidly induced K+ permeability and a slowly developing cation permeability mediate Ca2+ entry and stimulation of endothelial NO production.
Cyclic AMP enhances agonist-induced Ca2+ entry into endothelial cells by activation of potassium channels and membrane hyperpolarization.
The mechanism underlying cyclic AMP (cAMP)-mediated amplification of agonist-induced Ca2+ responses in endothelial cells was investigated in pig endothelial cells. Forskolin, adenosine and isoprenaline, as well as the membrane-permeant cAMP analogue dibutyryl cAMP, enhanced bradykinin-induced rises in intracellular free Ca2+ as well as bradykinin-induced Mn2+ entry. These agents were also found to hyperpolarize endothelial cells without increasing intracellular Ca2+ by itself, i.e. in the absence of bradykinin. Both amplification of bradykinin effects and the hyperpolarizing action was blocked by the protein kinase inhibitor H-8. The involvement of K+ channels in the hyperpolarizing effects of forskolin was consequently studied in perforated outside-out vesicles. Two different types of K+ channels were recorded, one of which had a large conductance (170 pS) and was activated by forskolin. We suggest that stimulation of endothelial adenylate cyclase results in activation of large-conductance K+ channels and consequently in membrane hyperpolarization, which in turn enhances bradykinin-induced entry of Ca2+ by increasing its electrochemical gradient.
Cromakalim inhibits multiple mechanisms of smooth muscle activation with similar stereoselectivity.
Purified cromakalim trans enantiomers were tested for their ability to antagonize three specific mechanisms of smooth muscle activation, i.e., depolarization-induced Ca2+ entry through voltage-gated channels, agonist-induced Ca2+ entry, and agonist-induced Ca2+ release. Cromakalim effects were studied in rabbit aortic rings contracted by stimuli corresponding to the above mechanisms. First, aortic rings were contracted by increase in extracellular [K+] (to 27 mM), which causes partial membrane depolarization. Under these conditions, (-)-cromakalim exhibited an EC50 of 0.18 microM and a 150-fold higher relaxing potency than the (+)-enantiomer. Second, in aortic rings tonically contracted by 1 microM norepinephrine (NE) in the presence of 1 microM nifedipine, i.e., in rings contracted mainly owing to NE-stimulated Ca2+ entry through receptor-operated channels, (-)-cromakalim induced relaxation with an EC50 of 0.68 microM and exhibited a 191-fold higher potency than the (+)-enantiomer. Third, phasic, NE-induced contractions of rabbit aortic rings in the absence of extracellular Ca2+, i.e., contractions that reflect release of Ca2+ from intracellular stores, were antagonized with an EC50 of 0.29 microM and a 144-fold higher potency than the (+)-enantiomer. All effects of (-)-cromakalim were blocked by either completely depolarizing the vessels with high extracellular [K+] (40 mM) or by addition of the K+ channel blocker glibenclamide (10 microM). Cromakalim relaxed rabbit aorta independent of the mechanism underlying smooth muscle tone. Cromakalim effects were equivalent with respect to dose dependence, stereoselectivity, and sensitivity to extracellular [K+] and glibenclamide.(ABSTRACT TRUNCATED AT 250 WORDS)