Glenis J. Crane

Small‐ and Intermediate‐Conductance Calcium‐Activated K+ Channels Provide Different Facets of Endothelium‐Dependent Hyperpolarization in Rat Mesenteric Artery

Activation of both small‐conductance (SKCa) and intermediate‐conductance (IKCa) Ca2+‐activated K+ channels in endothelial cells leads to vascular smooth muscle hyperpolarization and relaxation in rat mesenteric arteries. The contribution that each endothelial K+ channel type makes to the smooth muscle hyperpolarization is unknown. In the presence of a nitric oxide (NO) synthase inhibitor, ACh evoked endothelium and concentration‐dependent smooth muscle hyperpolarization, increasing the resting potential (approx. −53 mV) by around 20 mV at 3 μm. Similar hyperpolarization was evoked with cyclopiazonic acid (10 μm, an inhibitor of sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA)) while 1‐EBIO (300 μm, an IKCa activator) only increased the potential by a few millivolts. Hyperpolarization in response to either ACh or CPA was abolished with apamin (50 nm, an SKCa blocker) but was unaltered by 1‐[(2‐chlorophenyl) diphenylmethyl]‐1H‐pyrazole (1 μm TRAM‐34, an IKCa blocker). During depolarization and contraction in response to phenylephrine (PE), ACh still increased the membrane potential to around −70 mV, but with apamin present the membrane potential only increased just beyond the original resting potential (circa−58 mV). TRAM‐34 alone did not affect hyperpolarization to ACh but, in combination with apamin, ACh‐evoked hyperpolarization was completely abolished. These data suggest that true endothelium‐dependent hyperpolarization of smooth muscle cells in response to ACh is attributable to SKCa channels, whereas IKCa channels play an important role during the ACh‐mediated repolarization phase only observed following depolarization.

Activation of endothelial cell IK(Ca) with 1-ethyl-2-benzimidazolinone evokes smooth muscle hyperpolarization in rat isolated mesenteric artery.

In rat small mesenteric arteries contracted with phenylephrine, 1‐ethyl‐2‐benzimidazolinone (1‐EBIO; 3 – 300 μM) evoked concentration‐dependent relaxation that, above 100 μM, was associated with smooth muscle hyperpolarization. 1‐EBIO‐evoked hyperpolarization (maximum 22.1±3.6 mV with 300 μM, n=4) was endothelium‐dependent and inhibited by charybdotoxin (ChTX 100 nM; n=4) but not iberiotoxin (IbTX 100 nM; n=4). In endothelium‐intact arteries, smooth muscle relaxation to 1‐EBIO was not altered by either of the potassium channel blockers ChTX (100 nM; n=7), or IbTX (100 nM; n=4), or raised extracellular K+ (25 mM). Removal of the endothelium shifted the relaxation curve to the right but did not reduce the maximum relaxation. In freshly isolated mesenteric endothelial cells, 1‐EBIO (600 μM) evoked a ChTX‐sensitive outward K‐current. In contrast, 1‐EBIO had no effect on smooth muscle cell conductance whereas NS 1619 (33 μM) stimulated an outward current while having no effect on the endothelial cells. These data show that with concentrations greater than 100 μM, 1‐EBIO selectively activates outward current in endothelial cells, which presumably underlies the smooth muscle hyperpolarization and a component of the relaxation. Sensitivity to block with charybdotoxin but not iberiotoxin indicates this current is due to activation of IKCa. However, 1‐EBIO can also relax the smooth muscle by an undefined mechanism, independent of any change in membrane potential.

Evidence for a Differential Cellular Distribution of Inward Rectifier K Channels in the Rat Isolated Mesenteric Artery

The distribution of functionally active, inwardly rectifying K (KIR) channels was investigated in the rat small mesenteric artery using both freshly isolated smooth muscle and endothelial cells and small arterial segments. In Ca2+-free solution, endothelial cells displayed a KIR current with a maximum amplitude of 190 ± 16 pA at –150 mV and sensitivity to block with 30 µM Ba2+ (n = 7). In smooth muscle cells, outward K current was activated at around –47 ± 3 mV, but there was no evidence of KIR current (n = 6). Furthermore, raising extracellular [K+] to either 60 or 140 mM, or applying the α1-adrenoceptor agonist phenylephrine (PE; 30 µM), failed to reveal an inwardly rectifying current in the smooth muscle cells, although PE did stimulate an iberiotoxin-sensitive outward K current (n = 4). Exogenous K+ (10.8–16.8 mM) both relaxed and repolarized endothelium-denuded segments of the mesenteric artery contracted with PE. These effects were depressed by 100 µM ouabain but unaffected by either 30 µM BaCl2 or 3 µM glibenclamide. These data suggest that functional, inwardly rectifying Ba2+-sensitive channels are restricted to the endothelial cell layer in the rat small mesenteric artery.

Thromboxane receptor stimulation associated with loss of SKCa activity and reduced EDHF responses in the rat isolated mesenteric artery

The possibility that thromboxane (TXA2) receptor stimulation causes differential block of the SKCa and IKCa channels which underlie EDHF‐mediated vascular smooth muscle hyperpolarization and relaxation was investigated in the rat isolated mesenteric artery. Acetylcholine (30 nM–3 μM ACh) or cyclopiazonic acid (10 μM CPA, SERCA inhibitor) were used to stimulate EDHF‐evoked smooth muscle hyperpolarization. In each case, this led to maximal hyperpolarization of around 20 mV, which was sensitive to block with 50 nM apamin and abolished by repeated stimulation of mesenteric arteries with the thromboxane mimetic, U46619 (30 nM–0.1 μM), but not the α1‐adrenoceptor agonist phenylephrine (PE). The ability of U46619 to abolish EDHF‐evoked smooth muscle hyperpolarization was prevented by prior exposure of mesenteric arteries to the TXA2 receptor antagonist 1 μM SQ29548. Similar‐sized smooth muscle hyperpolarization evoked with the SKCa activator 100 μM riluzole was also abolished by prior stimulation with U46619, while direct muscle hyperpolarization in response to either levcromakalim (1 μM, KATP activator) or NS1619 (40 μM, BKCa activator) was unaffected. During smooth muscle contraction and depolarization to either PE or U46619, ACh evoked concentration‐dependent hyperpolarization (to −67 mV) and complete relaxation. These responses were well maintained during repeated stimulation with PE, but with U46619 there was a progressive decline, so that during a third exposure to U46619 maximum hyperpolarization only reached –52 mV and relaxation was reduced by 20%. This relaxation could now be blocked with charybdotoxin alone. The latter responses could be mimicked with 300 μM 1‐EBIO (IKCa activator), an action not modified by exposure to U46619. An early consequence of TXA2 receptor stimulation is a reduction in the arterial hyperpolarization and relaxation attributed to EDHF. This effect appears to reflect a loss of SKCa activity.

A modified Luria-Delbrück fluctuation assay for estimating and comparing mutation rates.

We have investigated the accuracy with which mutation rates may be estimated using a modification of the Luria and Delbrück fluctuation experiment protocol. The modification involves growing a larger-than-usual culture, and plating out a small aliquot of it. Monte Carlo simulations of the experiments confirm that the modification leads to a decrease in the coefficient of variation of the estimated mutation rate where this is based on the median number of mutants detected in a number of cultures grown in parallel. If sets of experimental and control cultures are compared using the Mann-Whitney U-test, then fractional increases in mutation rate can be reliably detected using relatively small numbers of cultures. The modified protocols promise better estimates of mutation rates, offer a powerful test of differences in mutation rates, and are easier to implement in practice.

Electrophysiological basis of arteriolar vasomotion in vivo.

We tested the hypothesis that cyclic changes in membrane potential (Em) underlie spontaneous vasomotion in cheek pouch arterioles of anesthetized hamsters. Diameter oscillations (∼3 min–1) were preceded (∼3 s) by oscillations in Em of smooth muscle cells (SMC) and endothelial cells (EC). Oscillations in Em were resolved into six phases: (1) a period (6 ± 2 s) at the most negative Em observed during vasomotion (–46 ± 2 mV) correlating (r = 0.87, p < 0.01) with time (8 ± 2 s) at the largest diameter observed during vasomotion (41 ± 2 µm); (2) a slow depolarization (1.8 ± 0.2 mV s–1) with no diameter change; (3) a fast (9.1 ± 0.8 mV s–1) depolarization (to –28 ± 2 mV) and constriction; (4) a transient partial repolarization (3–4 mV); (5) a sustained (5 ± 1 s) depolarization (–28 ± 2 mV) correlating (r = 0.78, p < 0.01) with time (3 ± 1 s) at the smallest diameter (27 ± 2 µm) during vasomotion; (6) a slow repolarization (2.5 ± 0.2 mV s–1) and relaxation. The absolute change in Em correlated (r = 0.60, p < 0.01) with the most negative Em. Sodium nitroprusside or nifedipine caused sustained hyperpolarization and dilation, whereas tetraethylammonium or elevated PO2 caused sustained depolarization and constriction. We suggest that vasomotion in vivo reflects spontaneous, cyclic changes in Em of SMC and EC corresponding with cation fluxes across plasma membranes.

Contribution of Active Membrane Processes to Conducted Hyperpolarization in Arterioles of Hamster Cheek Pouch

Objective: Conduction of vasodilation triggered by acetylcholine (ACh) in arteriolar networks reflects hyperpolarization and its spread from cell to cell along the vessel wall. The amplitude and distance of the vasomotor response appear greater than can be explained by simple passive decay of the electrical signal. The authors tested the hypothesis that the conduction of hyperpolarization involves active membrane processes as the signal travels along the arteriolar wall.

Simulating the spread of membrane potential changes in arteriolar networks.

Objective: Our aim was to simulate the spread of membrane potential changes in microvascular trees and then make the simulation programs accessible to other researchers. We have applied our simulations to demonstrate the implications of electrical coupling between arteriolar smooth muscle and endothelium.

An equation describing spread of membrane potential changes in a short segment of blood vessel

The spread of membrane potential changes throughout certain cells and tissues plays an important role in their physiology. The attenuation of such changes in any tissue is usually characterized by the cable length constant lambda, which can be determined experimentally if the equations describing membrane potential spread in the tissue are known. Here we derive an equation describing spread of membrane potential changes in a short cable, which is an appropriate model for short segments of blood vessels. This equation is more general than those already published in that the positions of both the current source that gives rise to a potential change, and the point at which the change is measured, can be anywhere along the cable.

Electrical coupling between smooth muscle and endothelium in arterioles of the guinea-pig small intestine

Equations describing the steady-state passive electrical properties of arterioles have been derived. The arteriole was modelled as having two thin layers of cells (muscle and endothelium) with strong electrical coupling between cells within a layer and variable coupling between the layers. The model indicated that spread of membrane potential changes was highly dependent on the thickness of cells within the layers. The model was also used to identify the optimal experimental strategy for detecting coupling between the two layers, and experiments were carried out on arterioles from the guinea-pig small intestine. Thickness of the endothelial layer was measured using electron microscopy and was found to be around 0.5 microm. Electrical input resistance was measured in intact arterioles and compared to input resistance of arterioles from which the endothelium had been removed. The experiments confirmed that there was a strong electrical coupling between the muscle and endothelium in these vessels.