Blockade of chloride channels reveals relaxations of rat small mesenteric arteries to raised potassium

Abstract

1. Raised extracellular K(+) relaxes some arteries, and has been proposed as Endothelium-Derived Hyperpolarizing Factor (EDHF). However, relaxation of rat small mesenteric arteries to K(+) is highly variable. We have investigated the mechanism of K(+)-induced dilatation and relaxation of pressurized arteries and arteries mounted for measurement of isometric force. 2. Raising [K(+)](o) from 5.88 - 10.58 mM did not dilate or relax pressurized or isometric arteries. Relaxation to raised [K(+)](o) was revealed in the presence of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB); this effect of NPPB was concentration-dependent (IC(50): 1.16 microM). 3. Relaxations to raised [K(+)](o) in the presence of NPPB, were abolished by 30 microM Ba(2+) or endothelial-denudation. Acetycholine (10 microM) relaxed endothelium-intact arteries in presence of raised [K(+)](o) NPPB and Ba(2+). 4. Relaxations to raised [K(+)](o) were revealed in hyperosmotic superfusate (+60 mM sucrose). These relaxations were abolished by 30 microM Ba(2+). In the presence of raised [K(+)](o), 60 mM sucrose and 30 microM Ba(2+), 10 microM acetycholine still relaxed all arteries. 5. Fifty microM 18 alpha-glycyrrhetinic acid (18 alpha-GA), a gap junction inhibitor, depressed relaxations to both 10 microM acetylcholine and raised [K(+)](o), in the presence of 10 microM NPPB. 6. In summary, blockade of a volume-sensitive Cl(-) conductance in small rat mesenteric arteries, using NPPB or hyperosmotic superfusion, reveals a endothelium-dependent, Ba(2+) sensitive dilatation or relaxation of rat mesenteric arteries to raised [K(+)](o). We conclude that inwardly rectifying potassium channels on the endothelium underlie relaxations to raised [K(+)](o) in rat small mesenteric arteries.

Figure 1

The effect of NPPB on dilatations to K⁺ in pressurized arteries. (a) In arteries where stepping [K⁺]_o from 5.88 to 10.58 mM failed to produce dilatation, dilatation to K⁺ was induced in the presence of 20 μM NPPB. The effect of NPPB on the ability of K⁺ to dilate was fully reversible on washout. The gap in the data trace indicates a period of removal of the endothelium with an air bubble. In the absence of an endothelium, the effects of NPPB were abolished. (b) Meaned data (+E, n=4±s.e.mean; −E; n=3±s.e.mean). These data are not paired, and therefore P values are for an unpaired Student's t-test. ^* Shows significance (P<0.05).

Figure 2

The effect of NPPB on dilatations to K⁺ in arteries mounted for isometric measurement of force. (a) In arteries where stepping [K⁺]_o from 5.88 to 10.58 mM failed to produce relaxation, relaxation to K⁺ was induced in the presence of 1 μM NPPB. This effect was reversed by 30 μM Ba²⁺. Ten μM ACh was still able to relax fully in the presence of K⁺, NPPB and Ba²⁺. (b) Mean data (n=8±s.e.mean).

Figure 3

The concentration-effect curve for NPPB in arteries mounted for isometric measurement of force. In endothelium-intact arteries, NPPB relaxed with an IC₅₀ of 7.20 μM (n=6±s.e.mean). In the presence of 10.58 mM K⁺, the concentration-effect curve for relaxation of arteries by NPPB was shifted left (IC₅₀: 1.16 μM) (n=10±s.e.mean). This leftward shift was inhibited in the presence of 30 μM Ba²⁺ (IC₅₀: 4.69 μM) (n=6±s.e.mean). P values are for an unpaired Student's t-test. ^* Show a significant difference from the control curve (P<0.05). In endothelium-denuded arteries, NPPB relaxed with an IC₅₀ of 7.43 μM (n=4±s.e.mean). In the presence of 10.58 mM K⁺, the concentration-effect curve for relaxation of arteries by NPPB was not significantly affected (IC₅₀: 8.22 μM) (n=5±s.e.mean).

Figure 4

Hyper-osmotic stress mimics the effects of NPPB to reveal relaxation of isometric arteries to K⁺. (a) In arteries where raising [K⁺]_o from 5.88 to 10.58 mM did not produce relaxation, relaxation to K⁺ was induced in the presence of a hyper-osmotic solution (60 mM sucrose added). This effect was reversed by 30 μM Ba²⁺. (b) Mean data (n=6±s.e.mean). 10 μM ACh was still able to relax fully in the presence of Ba²⁺.

Figure 5

Hypo-osmotic stress prevents relaxation of isometric arteries to K⁺. In arteries where raising [K⁺]_o from 5.88 to 10.58 mM produced relaxation, relaxation to K⁺ was abolished in the presence of 30 μM Ba²⁺ or a hypo-osmotic solution (40 mM NaCl omitted). (b) Mean data (n=2±s.e.mean).

Figure 6

After a transient relaxation to K⁺, sustained relaxation to K⁺ was induced in the presence of 10 μM NPPB. This effect was reversed by 30 μM Ba²⁺. Relaxation to 10 μM ACh, and sustained relaxation to K⁺ was reduced in the presence of 50 μM 18α-glycyrrhetinic acid (b) Mean data (n=8±s.e.mean). P values are for a paired Student's t-test. ^* Show a significant difference between the control data, and in the presence of 18α-glycyrrhetinic acid (P<0.05).

Figure 7

Model. (a) Inwardly rectifying potassium channels (K_ir) and volume-sensitive chloride channels (Cl_swell) are the major determinants of the endothelial cell membrane potential. Potassium efflux through calcium-activated potassium channels (K_v) increases outward current through inwardly rectifying potassium channels (K_ir) if membrane potential is close to E_K, thus hyperpolarizing the endothelium. Hyperpolarization will be transmitted to smooth muscle through myoendothelial gap junctions. (b) The membrane potential of most endothelial cells is depolarized towards E_Cl, and these arteries do not relax/hyperpolarize to K⁺. NPPB or hyperosmotic stress can be used to block chloride channels, shifting the membrane potential towards E_K, such that arteries relax/hyperpolarize to K⁺. If membrane potential close to E_K, such that K⁺ relaxes/hyperpolarizes the artery, hypo-osmotic stress, which activates chloride channels, or Ba²⁺, which blocks K_ir, will depolarize the endothelial membrane potential towards E_Cl and block relaxation.