Effects of ATP on cultured smooth muscle cells from rat aorta.

1. Membrane ionic currents provoked by externally applied ATP were studied by patch-clamp techniques in cultured aortic smooth muscle cells of the rat. 2. Using standard bath and pipette solutions and whole-cell voltage-clamp, ATP evoked an inward current when the cell membrane potential was held at -50 mV and an outward current when the potential was held at 30 mV, with a reversal potential near -10 mV. 3. Application of ATP gamma S gave results similar to those obtained with ATP, while adenosine, AMP and alpha,beta-methylene ATP were ineffective. The ATP-activated current was inhibited by suramin, 100 microM. 4. ATP also induced a biphasic rise in internal free Ca levels as shown directly by Fura-2 measurements and by the increase in Ca-dependent K single-channel activity in cell-attached patches. 5. With outward current through K channels blocked by internal Cs and TEA, modification of the ionic composition of bath and pipette solutions revealed that the reversal potential for the ATP-induced whole-cell current closely followed ECl, the chloride equilibrium potential, and was insensitive to manipulations of the monovalent cation gradient. 6. These results indicate that in rat cultured aortic smooth muscle cells, ATP binding to P2-purinoceptors produces increases of internal free Ca levels and subsequent activation of both Ca-dependent K and Cl currents.

Gap junctional communication in primary culture of cells derived from human aortic intima.

Intercellular communication via gap junctions plays an important role in the regulation and homeostasis. The presence of gap junctions and the efficiency of their function directly correlates with the degree of cell differentiation in a tissue. In the present study, gap junctional communication has been investigated in a primary culture of highly differentiated mesenchymal cells (subendothelial smooth muscle cells isolated from grossly normal and atherosclerotic areas of human aorta) and in poorly differentiated cells of mesenchymal origin (adult human skin fibroblasts as well as skin fibroblasts and aortic smooth muscle cells, derived from human fetus). The fluorescent dye transfer technique was used in this study. In cell cultures isolated from grossly normal and atherosclerotic aorta, the number of cells coupled via gap junctions increased with cell density and reached a plateau at a cell density of 50 to 70 cells/mm2. In cultures of normal aortic cells the number of coupled cells was 23.0 +/- 4.1 per injected cell and was significantly higher than in cultures of atherosclerotic cells (16.4 +/- 2.1, p < 0.05). Gap junctional communication between cells loaded with lipid inclusions was 2.4-fold lower than between cells free of excess intracellular lipids. In cultures of human skin fibroblasts the rate of intercellular communication was lower than in cultures of normal aortic cells and was comparable to that in cultures of atherosclerotic cells. There was practically no cell-to-cell communication in cultures of fetal cells. It is hypothesized that the reduced gap junctional communication in atherosclerotic human aorta is associated with the alterations in the degree of smooth muscle cell differentiation and impairs the function of the intima in atherosclerosis.

Activation and blocking effects of divalent cations on the calcium-dependent potassium channel of high conductance.

Using the patch-clamp technique the action of internal barium- and magnesium-ions on the calcium-dependent potassium channel in the membrane of vascular smooth muscle cells from the media layer of the human aorta was studied. Under symmetrical potassium conditions in excised inside-out patches bathed in the standard solution containing EGTA, the addition of barium as well as magnesium led to a concentration-dependent shift of the activation-curves to negative values of the membrane potential compared with the control, a block of single channel activity by barium, whereas no inhibitory effects by magnesium were registered. Thus, these results suggest that the process of channel activation of the calcium-dependent potassium channel of high conductance depends not merely on calcium but also on other divalent earthalkali ions.

Fatty acid modifies Ca2+-dependent potassium channel activity in smooth muscle cells from the human aorta.

By using the patch-clamp technique the effect of 2-decenoic acid (DA) on Ca2+-activated potassium (K+) channels in the membrane of smooth muscle cells from the human aorta was studied. In the presence of 0.5 microM Ca2+ and 2 mM Mg2+ on the cytoplasmic side of the membrane, a more than tenfold elevation in the probability of the channels being open (po) was observed under the effect of DA. With divalent cation concentrations of less than 1 nM DA caused a more than twofold elevation in po. In the DA-treated membranes Mg2+ ions, which normally fail to activate the channels, brought about a nearly threefold increase in the channel activity when applied to the inner membrane surface. Channel sensitivity to the activating effect of cytoplasmic Ca2+ ions did not increase with the application of DA. Single-channel conductance was unchanged by DA exposure. We suggest that DA alters the Ca2+-binding mechanism of the channel, increasing its sensitivity to Mg2+ ions, presumably owing to membrane fluidization.

Protein kinase C reduces the KCa current of rat tail artery smooth muscle cells.

The hypothesis that protein kinase C (PKC) is able to regulate the whole cell Ca-activated K (KCa) current independently of PKC effects on local Ca release events was tested using the patch-clamp technique and freshly isolated rat tail artery smooth muscle cells dialyzed with a strongly buffered low-Ca solution. The active diacylglycerol analog 1,2-dioctanoyl-sn-glycerol (DOG) at 10 microM attenuated the current-voltage (I-V) relationship of the KCa current significantly and reduced the KCa current at +70 mV by 70 +/- 4% (n = 14). In contrast, 10 microM DOG after pretreatment of the cells with 1 microM calphostin C or 1 microM PKC inhibitor peptide, selective PKC inhibitors, and 10 microM 1,3-dioctanoyl-sn-glycerol, an inactive diacylglycerol analog, did not significantly alter the KCa current. Furthermore, the catalytic subunit of PKC (PKCC) at 0.1 U/ml attenuated the I-V relationship of the KCa current significantly, reduced the KCa current at +70 mV by 44 +/- 3% (n = 17), and inhibited the activity of single KCa channels at 0 mV by 79 +/- 9% (n = 6). In contrast, 0.1 U/ml heat-inactivated PKCC did not significantly alter the KCa current or the activity of single KCa channels. Thus these results suggest that PKC is able to considerably attenuate the KCa current of freshly isolated rat tail artery smooth muscle cells independently of effects of PKC on local Ca release events, most likely by a direct effect on the KCa channel.

Iloprost activates KCa channels of vascular smooth muscle cells: role of cAMP-dependent protein kinase.

The patch-clamp technique was used to investigate the effect of iloprost on activity of calcium-activated potassium (KCa) channels of freshly isolated rat tail artery smooth muscle cells. In the whole cell configuration, outward current, determined largely by KCa channels, was enhanced 1.73 +/- 0.11-fold by 5 x 10(-7) M iloprost, 1.80 +/- 0.12-fold by 10(-4) M 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-cyclic monophosphothioate (Sp-5,6-DCl-cBIMPS), a specific activator of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), and 2.78 +/- 0.95-fold by 10 U/ml of the catalytic subunit of PKA + 10(-4) M MgATP, whereas the heat-inactivated catalytic subunit of PKA + MgATP was without effect. Iloprost at 5 x 10(-7) M increased this current 1.70 +/- 0.27-fold after pretreatment of cells with 10(-6) M okadaic acid, a specific phosphatase inhibitor, but did not alter this current after pretreatment of cells with 2 x 10(-4) M Rp-8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphorothioate, a specific PKA inhibitor. In the cell-attached configuration, activity of KCa channels was enhanced 2.48 +/- 0.44-fold by 5 x 10(-7) M iloprost and 2.09 +/- 0.07-fold by 10(-4) M Sp-5,6-DCl-cBIMPS. Iloprost at 5 x 10(-7) M did not alter intracellular calcium concentration in these cells measured using indo 1. In the inside-out configuration, activity of KCa channels was increased 87.12 +/- 45.04-fold by 10 U/ml of the catalytic subunit of PKA together with 10(-4) M MgATP, whereas no effect was observed after application of the catalytic subunit of PKA together with its regulatory subunit and MgATP, MgATP, cAMP, or the catalytic subunit of PKA alone also did not change KCa channel activity. Thus these results show that iloprost is able to activate KCa channels of freshly isolated rat tail artery smooth muscle cells and suggest that this effect is mediated by a PKA-induced phosphorylation of the channel.

Iloprost dilates rat small arteries: role of K(ATP)- and K(Ca)-channel activation by cAMP-dependent protein kinase.

The effect of the stable prostacyclin analog iloprost and its mechanism of action were investigated with the use of pressurized rat tail small arteries with a spontaneous myogenic tone. Iloprost concentration dependently dilated these vessels with a half-maximal effective dose of 5.0 +/- 0.5 x 10(-8) M. Application of 10(-7)-10(-6) M glibenclamide, a blocker of ATP-sensitive potassium (K(ATP)) channels, inhibited the iloprost-induced dilation. Glibenclamide did not affect the basal vessel diameter. The application of 5 x 10(-5)-10(-3) M tetraethylammonium (TEA) and 5 x 10(-9)-10(-7) M iberiotoxin, blockers of calcium-activated potassium (K(Ca)) channels, decreased vessel diameter in the presence of iloprost. Both TEA and iberiotoxin reduced the basal vessel diameter. Glibenclamide at 10(-6) M inhibited the dilation produced by 5 x 10(-5) M Sp-5,6-DCl-cBIMPS, an activator of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. Iberiotoxin at 10(-7) M decreased vessel diameter in the presence of Sp-5,6-DCl-cBIMPS. H-89 and Rp-8-CPT-cAMPS, blockers of cAMP-dependent protein kinase A (PKA), inhibited the iloprost-induced dilation of these vessels. With use of the whole cell configuration of the patch-clamp technique, it was observed that 5 x 10(-7) M iloprost enhanced an outward current, determined largely by K(Ca) channels, 1.79 +/- 0.17-fold in freshly isolated smooth muscle cells from rat tail small artery. These data show that iloprost dilates rat tail small arteries with a spontaneous myogenic tone and suggest that K(ATP) as well as K(Ca) channels are involved in this effect, which is mediated, at least partly, by PKA.

Effect of low-density lipoproteins on Ca(2+)-dependent K+ channels in the membrane of smooth muscle cells isolated from human fetal aorta.

The effect of low-density lipoproteins (LDL) on the kinetics of Ca(2+)-dependent K+ channels was investigated in patch-clamp experiments with fetal smooth muscle cells (SMC) from human aorta cultured in normal medium (control SMC), on cells cultured in LDL-free medium for 24-48 h (LDL-SMC), and on cells from the preceding group that had been cultured for a further 24-48 h in a medium containing 50 micrograms ml-1 of LDL (LDL + SMC). Under identical conditions (at 50 mV, in 5 x 10(-7) M [Ca2+]i), the channel kinetics of control SMC and of LDL-SMC were the same, but for LDL + SMC the average time the channels were open (To) was either shorter or longer than in the control cells: To was about 30 ms in control SMC and around either 10 or 50 ms in LDL + SMC. For each group, a plot of log(To) versus membrane potential gave parallel lines, indicating that the voltage dependence remained unchanged. The average time the channels of LDL + SMC were closed (Tc) also bracketed control values. Single-channel conductance was not changed with LDL treatment. Thus LDL-enriched medium induces changes in the kinetics of Ca(2+)-dependent K+ channels. Because cholesterol and its esters carried by LDL can be incorporated into the plasma membrane, the effects seen with LDL may be due to this.

cAMP-dependent protein kinase is in an active state in rat small arteries possessing a myogenic tone.

The hypothesis that cAMP-dependent protein kinase (protein kinase A; PKA) is in an active state in small arteries possessing a myogenic tone was investigated in pressurized rat tail small arteries. At a pressure of 80 mmHg, these vessels constricted to 71.6 +/- 1.0% (n = 32) of the diameter of the fully relaxed state. The PKA inhibitors Rp-8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphothioate (Rp-CPT-cAMPS) and N-(2-([3-(4-bromophenyl)-2-propenyl]amino)-ethyl)-5- isoquinolinesulfonamide HCl (H-89) constricted these vessels dose dependently. For example, 300 microM Rp-CPT-cAMPS and 9 microM H-89 reduced vessel diameter by 11.0 +/- 1.2% (n = 8) and 14.3 +/- 3.6% (n = 5), respectively. The cGMP-dependent protein kinase (protein kinase G; PKG) inhibitor Rp-8-bromo-beta-phenyl-1,N(2)-etheno-guanosine 3', 5'-cyclic monophosphothioate (Rp-8-Br-PET-cGMPS) did not alter vessel diameter up to a concentration of 10 microM. Neither endothelium removal nor inhibition of neural transmission affected the action of Rp-CPT-cAMPS. The effect of 300 microM Rp-CPT-cAMPS was reduced by 82% after pretreatment of the vessel with 100 nM iberiotoxin, a blocker of calcium-activated potassium (K(Ca)) channels. However, the effect of 300 microM Rp-CPT-cAMPS was not altered after pretreatment with 1 mM 4-aminopyridine, a blocker of delayed rectifier potassium channels, or 10 microM ryanodine, a blocker of ryanodine receptor-generated calcium sparks. In inside-out patch-clamp experiments on cells isolated from rat tail small arteries, 10 U/ml of the catalytic subunit of PKA together with 100 microM MgATP increased K(Ca) channel activity 30.1 +/- 9. 8-fold (n = 9). Additionally, neither inhibition of PKA or PKG nor moderate activation of PKA or PKG altered the vessel response to a pressure step from 80 to 120 mmHg. These results suggest that in rat tail small arteries possessing a myogenic tone 1) PKA is in an active state modulating the level of the myogenic tone, and 2) K(Ca) channels mediate, at least partly, this effect of PKA.

Tertiary ammonium ion induces contraction of rat aorta segments and increases the mobility of lipids in the membrane of rat aorta smooth muscle cells.

The effects of tertiary ammonium ion--IEM-1194 (decyldiisopropylammonium bromide)--on smooth muscle cells (SMC) isolated from rat aorta were investigated. Using the patch-clamp technique in the cell-attached configuration, two types of effects on the Ca(2+)-activated K+ channels were recorded after application of IEM-1194: an increase in the number of fast transition bursts in open channels and the prolongation of the burst. The removal of the agonist led to the activation of these channels. Mechanical responses of aortic rings were recorded using the method of isometric tension measurements. IEM-1194 (100-400 microM) caused contractions consisting of two phases. The first phase emerged immediately after application of the agonist. The second phase was observed 10-15 min after application. The contraction of the aorta was followed by the relaxation for over 1.5 h. The same results were obtained in the Ca(2+)-free external solution. Measurements of SMC membrane fluidity by the EPR method showed that IEM-1194 induced an increase in the mobility of membrane lipids. Based on these results, we suggest a mechanism of IEM-1194 action on the Ca(2+)-activated K+ channels.