Protein kinase A and C regulate leak potassium currents in freshly isolated vascular myocytes from the aorta.

We tested the hypothesis that protein kinase A (PKA) inhibits K2P currents activated by protein kinase C (PKC) in freshly isolated aortic myocytes. PDBu, the PKC agonist, applied extracellularly, increased the amplitude of the K2P currents in the presence of the "cocktail" of K(+) channel blockers. Gö 6976 significantly reduced the increase of the K2P currents by PDBu suggesting the involvement of either α or β isoenzymes of PKC. We found that forskolin, or membrane permeable cAMP, did not inhibit K2P currents activated by the PKC. However, when PKA agonists were added prior to PDBu, they produced a strong decrease in the K2P current amplitudes activated by PKC. Inhibition of PDBu-elicited K2P currents by cAMP agonists was not prevented by the treatment of vascular smooth muscle cells with PKA antagonists (H-89 and Rp-cAMPs). Zn(2+) and Hg(2+) inhibited K2P currents in one population of cells, produced biphasic responses in another population, and increased the amplitude of the PDBu-elicited K(+) currents in a third population of myocytes, suggesting expression of several K2P channel types. We found that cAMP agonists inhibited biphasic responses and increase of amplitude of the PDBu-elicited K2P currents produced by Zn(2+) and Hg(2). 6-Bnz-cAMp produced a significantly altered pH sensitivity of PDBu-elicited K2P-currents, suggesting the inhibition of alkaline-activated K2P-currents. These results indicate that 6-Bnz-cAMP and other cAMP analogs may inhibit K2P currents through a PKA-independent mechanism. cAMP analogs may interact with unidentified proteins involved in K2P channel regulation. This novel cellular mechanism could provide insights into the interplay between PKC and PKA pathways that regulate vascular tone

Dose response inhibition of PDBu-elicited currents produced by zinc and mercury recorded in the presence of the “cocktail” of K⁺-channel blockers.

<p>K⁺ currents were elicited by linear voltage ramps varying from −100 to 100 mV. The area under curve (AUC) was calculated and normalized to membrane capacitance (pF), following which the data sets were normalized to the maximum value. Panel A: dose response curve of the inhibition of PDBu-elicited K⁺ current by mercury was fitted with logistic function. PDBu-elicited K⁺ currents were recorded in control cell (circles) and in cells pretreated with 6-Bnz-cAMp (300 µM) (squares). EC50 calculated for control cells was 7.6±1.2 µM and the slope was 2.3±0.3. EC50 calculated for cells pretreated with 6-Bnz-cAMP was 6.9±1.5 µM and the slope was 1.1±0.2. Panel B: dose response curve of the inhibition of PDBu-elicited K⁺ current by zinc was fitted with logistic function. PDBu-elicited K⁺ currents were recorded in control cell (circles) and in cells pretreated with 6-Bnz-cAMp (300 µM) (squares). EC50 calculated for control cells was 7.4±0.2 µM and the slope was 2.2±0.1. EC50 calculated for cells pretreated with 6-Bnz-cAMP was 7.9±1.4 µM and the slope was 1.2±0.2. Panels C and D: examples of PDBu-elicited K⁺ currents with the cocktail of K⁺ channel blockers in the bath solution. K⁺ currents were elicited by linear voltage ramps varying from −100 to 100 mV. Data are displayed in a concatenated pattern. The application of mercury (Hg) and zinc (Zn) is shown by the horizontal line bars above the panels. 6-Bnz-cAMP did not significantly alter EC50 of the Zn²⁺ and Hg²⁺ inhibited currents (P>0.05, by t-test).</p

Action of cAMP agonists on PDBu-elicited K⁺ currents recorded in freshly isolated myocytes from the mouse aorta.

<p>Panel A: Current-voltage relationship recorded in control (up triangles); after application of forskolin (1 µM) (filled squares); and after application of the “cocktail” of K⁺ channel blockers (circles). Forskolin significantly increased the amplitude of the K⁺ currents (*P<0.05, **P<0.01, ***P<0.001 by two-way ANOVA). K⁺ channel blockers significantly inhibited the forskolin-elicited K⁺ current (P<0.001, by two-way ANOVA). Panel B: Current voltage relations recorded in the control (squares); after application of the membrane permeable cAMP analog 6-Bnz-cAMP (300 µM) (circles); after application of the PKC agonist PDBu (up triangles); and after application of the “cocktail” of K⁺ channel blockers (down-triangles). The cocktail of K⁺ channel blockers significantly inhibited the 6-Bnz-cAMP-elicited K⁺ current (*P<0.01, **P<0.001, by two-way ANOVA). Panel C: example of the superimposed families of the currents used to build up the current-voltage relationships. Currents were elicited by voltage steps from −100 mV to 100 mV in increments of 10 mV from holding potential of −60 mV. K⁺ currents were recorded in the control (Contr.); after application of the membrane permeable cAMP analog 6-Bnz-cAMP (cAMP); after application of the PKC agonist PDBu (PDBu); and after application of the cocktail of K⁺ channel blockers (K+-Block.).</p

Effects of mercury and zinc on PDBU-elicited K⁺ currents.

<p>Outward K⁺ currents were elicited by linear voltage ramps varying from −100 to 100 mV. Data are displayed in concatenated pattern. K⁺ currents are shown in the presence of PDBu (1 µM) and the cocktail of K⁺ channel blockers. Application of mercury (10 µM) and zinc (50 µM) is shown by the horizontal line bar above each trace. Panel B and D: Currents recorded from five VSMC representing each type of response were analyzed. The area under curve (AUC) calculated from the K⁺ currents elicited by linear voltage ramps (examples shown in Panel A and C) were normalized to the membrane capacitance (pF) and plotted against time. To illustrate biphasic response VSMC with pronounced two phases were especially chosen.</p

Comparison of the current-voltage relationships obtained by subtraction of the control currents from PDBu-elicited K⁺ currents with the cocktail of K⁺-channel blockers for four experimental conditions (Panel A).

<p>1) 6-Bnz-cAMP (300 µM) was added to the bath solution after the PKC agonist PDBu (squares). 2) Cells were pretreated with 6-Bnz-cAMP (300 µM) for for 15–20 minutes before application of PDBu (diamonds). The two other current voltage relationships show PDBu-elicited K⁺ currents in the presence of cocktail of K⁺ channel blockers recorded from cells pretreated with PKA antagonist H-89 (1 µM). 3) Forskolin (1 µM) was applied to the bath solution before PDBu (circles). 4) Cells were dialyzed with 6-Bnz-cAMP (30 µM) and Rp-cAMPS (300 µM). 6-Bnz-cAMP (300 µM) was also added to the bath solution before application of PKC agonist PDBu (down-triangles). cAMP agonists inhibited significantly PDBu-elicited K⁺ current (P<0.01, by t-test). Panel B. Effects of extracellular pH on the PDBu-elicited K⁺ currents recorded in freshly isolated myocytes from the mouse aorta. Currents were recorded at different pH values and were normalized to membrane capacitance and to the maximum at pH 9. Response curves for control cells (squares) and for cells pretreated with 6-Bn-cAMP (circles). PDBu-elicited K⁺ currents with changes in extracellular pH are shown. 6-Bnz-cAMP significantly shifted the response curve to the low pH values (p<0.01 t-test). Panel C shows representative PDBu-elicited K⁺ currents, as well as the effect of pH = 6 (upper trace) and the effect of pH = 9 lower trace. Currents were elicited by step pulses to 0 mV and to 50 mV from holding potential of −60 mV.</p

Characterization of a charybdotoxin-sensitive intermediate conductance Ca(2+)-activated K(+) channel in porcine coronary endothelium: relevance to EDHF

1. This study characterizes the K(+) channel(s) underlying charybdotoxin-sensitive hyperpolarization of porcine coronary artery endothelium. 2. Two forms of current-voltage (I/V) relationship were evident in whole-cell patch-clamp recordings of freshly-isolated endothelial cells. In both cell types, iberiotoxin (100 nM) inhibited a current active only at potentials over +50 mV. In the presence of iberiotoxin, charybdotoxin (100 nM) produced a large inhibition in 38% of cells and altered the form of the I/V relationship. In the remaining cells, charybdotoxin also inhibited a current but did not alter the form. 3. Single-channel, outside-out patch recordings revealed a 17.1±0.4 pS conductance. Pipette solutions containing 100, 250 and 500 nM free Ca(2+) demonstrated that the open probability was increased by Ca(2+). This channel was blocked by charybdotoxin but not by iberiotoxin or apamin. 4. Hyperpolarizations of intact endothelium elicited by substance P (100 nM; 26.1±0.7 mV) were reduced by apamin (100 nM; 17.0±1.8 mV) whereas those to 1-ethyl-2-benzimidazolinone (1-EBIO, 600 μM, 21.0±0.3 mV) were unaffected (21.7±0.8 mV). Substance P, bradykinin (100 nM) and 1-EBIO evoked charybdotoxin-sensitive, iberiotoxin-insensitive whole-cell perforated-patch currents. 5. A porcine homologue of the intermediate-conductance Ca(2+)-activated K(+) channel (IK1) was identified in endothelial cells. 6. In conclusion, porcine coronary artery endothelial cells express an intermediate-conductance Ca(2+)-activated K(+) channel and the IK1 gene product. This channel is opened by activation of the EDHF pathway and likely mediates the charybdotoxin-sensitive component of the EDHF response

A delayed ATP-elicited K(+) current in freshly isolated smooth muscle cells from mouse aorta

1. Adenosine 5′-triphosphate (ATP) activated two sequential responses in freshly isolated mouse aortic smooth muscle cells. In the first phase, ATP activated Ca(2+)-dependent K(+) or Cl(−) currents and the second phase was the activation of a delayed outward current with a reversal potential of −75.9±1.4 mV. 2. A high concentration of extracellular K(+) (130 mM) shifted the reversal potential of the delayed ATP-elicited current to −3.5±1.3 mV. The known K(+)-channel blockers, iberiotoxin, charybdotoxin, glibenclamide, apamin, 4-aminopyridine, Ba(2+) and tetraethylammonium chloride all failed to inhibit the delayed ATP-elicited K(+) current. Removal of ATP did not decrease the amplitude of the ATP-elicited current back to the control values. 3. The simultaneous recording of cytosolic free Ca(2+) and membrane currents revealed that the first phase of the ATP-elicited response is associated with an increase in intracellular Ca(2+), while the second delayed phase develops after the return of cytosolic free Ca(2+) to control levels. 4. ATP did not activate Ca(2+)-dependent K(+) currents, but did elicit Ca(2+)-independent K(+) currents, in cells dialyzed with ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). The delay of activation of Ca(2+)-independent currents decreased from 10.5+3.4 to 1.27±0.33 min in the cells dialyzed with 2 mM EGTA. 5. Adenosine alone failed to elicit a Ca(2+)-independent K(+) current but simultaneous application of ATP and adenosine activated the delayed K(+) current. Intracellular dialysis of cells with guanosine 5′-O-(2-thiodiphosphate) transformed the Ca(2+)-independent ATP-elicited response from a sustained to a transient one. A phospholipase C inhibitor, U73122 (1 μM), was shown to abolish the delayed ATP-elicited response. 6. These results indicate that the second phase of the ATP-elicited response was a delayed Ca(2+)-independent K(+) current activated by exogenous ATP. This phase might represent a new vasoregulatory pathway in vascular smooth muscle cells