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

1. The possibility that thromboxane (TXA(2)) receptor stimulation causes differential block of the SK(Ca) and IK(Ca) channels which underlie EDHF-mediated vascular smooth muscle hyperpolarization and relaxation was investigated in the rat isolated mesenteric artery. 2. Acetylcholine (30 nm-3 microm ACh) or cyclopiazonic acid (10 microm 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 microm), but not the alpha(1)-adrenoceptor agonist phenylephrine (PE). 3. The ability of U46619 to abolish EDHF-evoked smooth muscle hyperpolarization was prevented by prior exposure of mesenteric arteries to the TXA(2) receptor antagonist 1 microm SQ29548. 4. Similar-sized smooth muscle hyperpolarization evoked with the SK(Ca) activator 100 microm riluzole was also abolished by prior stimulation with U46619, while direct muscle hyperpolarization in response to either levcromakalim (1 microm, K(ATP) activator) or NS1619 (40 microm, BK(Ca) activator) was unaffected. 5. 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 microm 1-EBIO (IK(Ca) activator), an action not modified by exposure to U46619. 6. An early consequence of TXA(2) receptor stimulation is a reduction in the arterial hyperpolarization and relaxation attributed to EDHF. This effect appears to reflect a loss of SK(Ca) activity.

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 microM. Similar hyperpolarization was evoked with cyclopiazonic acid (10 microM, an inhibitor of sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA)) while 1-EBIO (300 microM, 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 microM 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.

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 (K(IR)) channels was investigated in the rat small mesenteric artery using both freshly isolated smooth muscle and endothelial cells and small arterial segments. In Ca(2+)-free solution, endothelial cells displayed a K(IR) current with a maximum amplitude of 190 +/- 16 pA at -150 mV and sensitivity to block with 30 microM Ba(2+) (n = 7). In smooth muscle cells, outward K current was activated at around -47 +/- 3 mV, but there was no evidence of K(IR) current (n = 6). Furthermore, raising extracellular [K(+)] to either 60 or 140 mM, or applying the alpha(1)-adrenoceptor agonist phenylephrine (PE; 30 microM), 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 microM ouabain but unaffected by either 30 microM BaCl(2) or 3 microM glibenclamide. These data suggest that functional, inwardly rectifying Ba(2+)-sensitive channels are restricted to the endothelial cell layer in the rat small mesenteric artery.

Cellular coupling and conducted vasomotor responses.

1. The present brief review examines the concept of spreading vasodilator responses in arteriolar trees, its physiological relevance and possible mechanisms. 2. The most likely mechanisms involve spread of hyperpolarization through tissues in the vessel wall, made possible by electrical coupling between the cells. It is generally agreed that there is coupling between cells within the muscle and endothelial layers, but coupling between the two layers is not always present. 3. The passive electrical properties of arterioles can be modelled, using different techniques depending on the complexity of branching of the arteriolar tree. Comparison of experimental results with the model indicates that hyperpolarization can spread further than expected from passive properties alone, implying that spreading vasodilatation may be an active process.

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

1. In rat small mesenteric arteries contracted with phenylephrine, 1-ethyl-2-benzimidazolinone (1-EBIO; 3-300 microM) evoked concentration-dependent relaxation that, above 100 microM, was associated with smooth muscle hyperpolarization. 2. 1-EBIO-evoked hyperpolarization (maximum 22.1+/-3.6 mV with 300 microM, n=4) was endothelium-dependent and inhibited by charybdotoxin (ChTX 100 nM; n=4) but not iberiotoxin (IbTX 100 nM; n=4). 3. 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. 4. In freshly isolated mesenteric endothelial cells, 1-EBIO (600 microM) evoked a ChTX-sensitive outward K-current. In contrast, 1-EBIO had no effect on smooth muscle cell conductance whereas NS 1619 (33 microM) stimulated an outward current while having no effect on the endothelial cells. 5. These data show that with concentrations greater than 100 microM, 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 IK(Ca). However, 1-EBIO can also relax the smooth muscle by an undefined mechanism, independent of any change in membrane potential.

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.

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. METHODS: A two-layered, cable-like model of an arteriole was used, and the assumptions involved in the approach explicitly stated. Several common experimental situations that involve the passive spread of membrane potential changes in microvascular trees were simulated. The calculations were performed using NEURON, a well-established computer simulation program that we have modified for use with vascular trees. RESULTS: Simulated results show that membrane potential changes would probably not spread as far in the endothelium as they would in the smooth muscle of arterioles. Where feed arteries are connected to larger distributing arteries, passive spread alone may not explain the physiologically observed spread of diameter changes. CONCLUSIONS: Simulated results suggest that the morphology of an arteriole, in which the muscle layer is much thicker than the endothelium, favors electrical conduction along smooth muscle rather than the endothelium. However, it seems that passive electrical spread is insufficient to explain the apparent spread of membrane potential changes in experimental situations. Active responses involving voltage-dependent conductances may be involved, and these can also be included in our simulation.

Electrophysiological basis of arteriolar vasomotion in vivo.

We tested the hypothesis that cyclic changes in membrane potential (E(m)) underlie spontaneous vasomotion in cheek pouch arterioles of anesthetized hamsters. Diameter oscillations (approximately 3 min(-1)) were preceded (approximately 3 s) by oscillations in E(m) of smooth muscle cells (SMC) and endothelial cells (EC). Oscillations in E(m) were resolved into six phases: (1) a period (6 +/- 2 s) at the most negative E(m) 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 microm); (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 microm) during vasomotion; (6) a slow repolarization (2.5 +/- 0.2 mV s(-1)) and relaxation. The absolute change in E(m) correlated (r = 0.60, p < 0.01) with the most negative E(m). Sodium nitroprusside or nifedipine caused sustained hyperpolarization and dilation, whereas tetraethylammonium or elevated PO(2) caused sustained depolarization and constriction. We suggest that vasomotion in vivo reflects spontaneous, cyclic changes in E(m) of SMC and EC corresponding with cation fluxes across plasma membranes.

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.

Clinical trials and clinical practice: do doctors use the same criteria to judge outcome?

AIMS: The results of clinical trials often seem to have little influence on the practice of individual doctors. This could be because trial information is presented in the style of a scientific experiment which cannot often be clearly related to the context of everyday patient care. We tested the hypothesis that such framing effects would cause doctors to assess the clinical significance of treatment outcomes differently when presented as clinical trial results rather than as individual patient data. METHODS: Fourteen rheumatologists independently reviewed the same 50 sets of data obtained from patients with rheumatoid arthritis. The data consisted of 10 commonly used clinical and laboratory variables measured before and after a period of treatment. The same data were presented in two formats on two separate occasions. The patient data format was a collection of typed sheets attributing each set of results to an individual patient. The clinical trial format was a professionally printed and bound booklet in which each set of results was laid out as summary results of a small uncontrolled clinical trial. Doctors judged the degree of improvement or deterioration and its clinical importance for each data set for both formats. These changes were converted into units of 'Clinical Importance'. RESULTS: Although some statistically significant differences emerged in the individual doctors' judgements between the formats none of these was of a clinically important size. The median of the mean trial--patient difference between the formats for all 14 doctors was 0.035 units of clinical importance [95% CI -0.244 to 0.074]. CONCLUSIONS: This evidence does not support the hypothesis that framing effects are a major cause of the failure of clinical trials to influence clinical practice.

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.

Age-related changes in femoral trabecular bone in arthrosis.

Age-related changes in the cancellous bone in selected regions of the proximal femur and iliac crest were assessed. An arthrosis group and a control cadaver group, partitioned into subjects younger and older than aged 50 years, were compared. The control group comprised 69 heads of femur and iliac crest samples. The arthrosis group comprised 28 consecutive heads of femur affected by primary arthrosis and an iliac crest biopsy taken during hip arthroplasty. Cancellous bone was sampled from four selected regions in the proximal femur. Histomorphometric estimates of percentage bone volume were determined using image analysis. In the young controls the bone volume was higher than that in the old controls for all the regions. In the arthrosis group the bone volume was higher than that of the old controls except for the subchondral principal tensile and medial to the greater trochanter regions. The old controls had regression of bone volume on age for the subchondral and medial principal compressive regions and the iliac crest. The arthrosis group had a minimal dependence of bone volume on age. Our study showed that primary arthrosis modulates the age dependence of bone volume in the proximal femur.

How can we design trials to detect clinically important changes in disease severity?

1. Forty-eight British rheumatologists judged the change in disease activity in 50 sets of patient data drawn from life and presented as 'paper patients'. Each set comprised two values, recorded a year apart, for 10 commonly measured clinical variables. Doctors recorded the size of improvement or deterioration on a visual analogue scale (VAS) and whether the change was clinically important or not. 2. Clinical judgement policies were modelled using linear regression of the clinical variables on the VAS score. 3. Doctors showed little agreement over which patients had improved and which had not. Possible reasons could be discovered by inspecting their judgement policies. 4. The weights attributed to the clinical variables differed considerably between doctors. Furthermore weights the doctors believed they attached to the variables frequently differed from the weights in the regression models. 5. These models could be used to calculate the smallest change required in any clinical variable before it would be considered clinically important. However, the size of such changes was often outside the observed clinical range suggesting that the use of single outcome variables is unrealistic. 6. The modelling procedure described can be applied during the planning stage of the trial to participating physicians, patients, health economists or any other group having an interest in the results. The models themselves can then be used to reach a consensus policy for judging what is a successful outcome. This may be expressed as a linear combination of specific outcome measures. Its use may improve the power of clinical trials and the relevance of their results.