Search PubMed⌕ Search

Biomedical subjects

D J Beech

Publications and source records attributed to D J Beech.

At least 73 records · Page 4Linked to original sources
PubMed1995

Surgical management of primary soft tissue sarcoma.

Few areas in oncology more clearly illustrate the need for multidisciplinary management than the soft tissue sarcoma problem. Using this approach has led to limb preservation with survival results at least equivalent to those for amputations. This achievement has dramatically improved the quality of life and overall clinical outcome for patients with this disease. In the future, improved understanding of the biology of this disease may broaden our therapeutic options. Meanwhile, a detailed understanding of the surgical possibilities will enhance the multimodality care that can be offered by the teams of clinicians managing patients suffering from this disease.

Amputation, Surgical↗
PubMed1993

K channel activation by nucleotide diphosphates and its inhibition by glibenclamide in vascular smooth muscle cells.

1. Whole-cell and inside-out patch recordings were made from single smooth muscle cells that had been isolated enzymatically and mechanically from the rabbit portal vein. 2. In whole-cells the inclusion in the recording pipette solution of nucleotide diphosphates (NDPs), but not tri- or monophosphates, induced a K-current that developed gradually over 5 to 15 min. Intracellular 1 mM guanosine 5'-diphosphate (GDP) induced a slowly developing outward K-current at -37 mV that reached a maximum on average of 72 +/- 4 pA (n = 40). Half maximal effect was estimated to occur with about 0.2 mM GDP. Except for ADP, other NDPs had comparable effects. At 0.1 mM, ADP was equivalent to GDP but at higher concentration ADP was less effective. ADP induced its maximum effect at 1 mM but had almost no effect at 10 mM. 3. In 14% of inside-out patches exposed to 1 mM GDP at the intracellular surface, characteristic K channel activity was observed which showed long (> 1 s) bursts of openings separated by longer closed periods. The current-voltage relationship for the channel was linear in a 60 mM:130 mM K-gradient and the unitary conductance was 24 pS. 4. Glibenclamide applied via the extracellular solution was found to be a potent inhibitor of GDP-induced K-current (IK(GDP)) in the whole-cell. The Kd was 25 nM and the inhibition was fully reversible on wash-out. 5. IK(GDP) was not evoked if Mg ions were absent from the pipette solution. In contrast the omission of extracellular Mg ions had no effect on outward or inward IK(GDP). 6. Inclusion of 1 mM ATP in the recording pipette solution reduced IK(GDP) and also attenuated its decline during long (25 min) recordings. 7. When perforated-patch whole-cell recording was used, metabolic poisoning with cyanide and 2-deoxy-D-glucose induced a glibenclamide-sensitive K-current. This current was not observed when conventional whole-cell recording was used. Possible reasons for this difference are discussed. 8. These K channels appear similar to ATP-sensitive K channels but we refer to them as nucleotide diphosphate-dependent K channels (KNDP) to emphasise what seems to be a primary role for nucleotide diphosphates in their regulation.

Adenosine Triphosphate↗
PubMed1993

Single channel and whole-cell K-currents evoked by levcromakalim in smooth muscle cells from the rabbit portal vein.

1. Single channel and whole-cell current recordings were made from single smooth muscle cells isolated from the rabbit portal vein. 2. Application of 10 microM levcromakalim ((-)-Ckm) to single cells held with pipettes containing 1 mM GDP induced a K-current (IK(Ckm)) which occurred in addition to the current caused by GDP alone (IK(GDP)) and averaged 135 pA at -37 mV. We have investigated whether the same K channels underlie the GDP- and Ckm-induced K-currents. 3. If 1 mM GDP was in the pipette but Mg ions were omitted the effect of GDP was absent and IK(Ckm) averaged only 10 pA, suggesting that the action of (-)-Ckm was Mg-dependent. 4. Intracellular ATP was not observed to have much effect on IK(-Ckm). Loading of cells with 10 mM ATP from the recording pipette had no significant effect and flash photolysis of caged-ATP loaded into cells from the pipette, estimated to release about 1 mM free ATP, also had no effect on IK(-Ckm). 5. Bath-applied glibenclamide inhibited IK(-Ckm) with an IC50 of 200 nM, a value 8 times higher than that found for inhibition of IK(GDP). The delayed rectifier K-current (IK(DR)) was also inhibited by glibenclamide but at higher concentrations (IC50 100 microM). Bath-applied tetraethylammonium ions (TEA) inhibited IK(-Ckm) and IK(GDP) to the same extent (IC50 about 7 mM). 6. In inside-out patch recordings (- )-Ckm (10 microM) applied to the intracellular surface of the membrane potentiated the opening of K channels already stimulated by I mM GDP and all of the channel activity was abolished by 10 microM glibenclamide. The unitary conductance of the channels was 24lpS in a 60 mM: 130 mM K-gradient.7. We suggest that (-)-Ckm may hyperpolarize and relax smooth muscle cells by opening KNDP, a class of small conductance K channels that are related to the ATP-sensitive K channels seen in other tissues.

Adenosine Triphosphate↗
PubMed1993

Inhibitory effects of histamine and bradykinin on calcium current in smooth muscle cells isolated from guinea-pig ileum.

1. Single smooth muscle cells were isolated from the longitudinal muscle layer of the guinea-pig ileum and within 10 h Ca(2+)-currents (ICa) were recorded using the whole-cell patch clamp technique. 2. Histamine (10 microMs) and bradykinin (BK, 1 microM) suppressed ICa; the effect had two phases: a rapid and transient suppression of ICa followed by a sustained suppression. Acetylcholine and substance P appeared to have similar effects but these were not investigated in detail. 3. The effects of histamine and BK on ICa were established by high intracellular concentrations of the Ca2+ buffer EGTA (30 mM) or 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (5 mM) in the absence of Ca2+ added to the pipette solution. When [Ca2+]i was strongly buffered to 125 or 190 nM by BAPTA-Ca2+ mixtures in the pipette the transient suppression of ICa was blocked but the sustained effect still occurred. This indicated that the transient effect was caused by a rise in [Ca2+]i. The sustained effect, in contrast, did not seem to be caused by a rise in [Ca2+]i but did show Ca2+ dependence because it did not occur if [Ca2+]i was abnormally low. 4. Application of caffeine (10 mM) to deplete stored Ca2+ or intracellular heparin (1 mM) to block the action of D-myo-inositol 1,4,5-trisphosphate (IP3) to release stored Ca2+ prevented the transient but not the sustained suppression of ICa. Heparin also blocked the transient Ca(2+)-activated K+ current in response to histamine or BK. Both transient and sustained suppressions of Ca2+ channel activity were observed in the absence of extracellular Ca2+ when current was carried mostly by Na+ ions. 5. Intracellular guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S; 10 or 100 microM) induced a gradual decline of ICa upon which transient decreases of current were superimposed. Histamine caused a larger than normal inhibition of ICa and no recovery occurred on wash-out. Intracellular guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S; 1 mM) abolished the effects of histamine and BK on ICa.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Pertussis toxin and voltage dependence distinguish multiple pathways modulating calcium channels of rat sympathetic neurons.

Agonist-induced suppression of current in voltage-gated Ca2+ channels was studied in rat sympathetic neurons. We have previously distinguished two intracellular signaling pathways used by muscarinic agonists to suppress neuronal Ca2+ current-one fast and membrane delimited, the other slow and acting via a diffusible second messenger. We now show that the fast pathway is sensitive mainly to pertussis toxin and shifts the gating of Ca2+ channels to more positive voltages (voltage dependent). The slow pathway is pertussis toxin insensitive and depresses currents at all test potentials (voltage independent). Muscarinic agonists may also activate a pertussis toxin-insensitive fast pathway. alpha-Adrenergic agonists use the fast pertussis toxin-sensitive and the fast insensitive pathways, but not the slow one.

Adrenergic alpha-Agonists↗
PubMed1991

Intracellular Ca2+ buffers disrupt muscarinic suppression of Ca2+ current and M current in rat sympathetic neurons.

The role of intracellular Ca2+ concentration ([Ca2+]i) in the muscarinic suppression of Ca2+ current and M-type K+ current has been investigated in isolated rat sympathetic neurons using the whole-cell patch-clamp technique and fura-2 fluorescence measurements. Muscarinic stimulation suppressed currents without raising [Ca2+]i. Nonetheless, intracellular bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) (11-12 mM), a Ca2+ chelator, reduced Ca2(+)-current suppression from 82 to 15%. For the latter, we explain the BAPTA action by a requirement for a certain minimum [Ca2+]i for continued operation of the pathway coupling muscarinic receptors to M-type K+ channels. The pathway coupling muscarinic receptors to Ca channels also showed some dependence on [Ca2+]i, but there may also be a blocking action of BAPTA that is independent of Ca2+ chelation.

Animals↗
PubMed1991

A diffusible second messenger mediates one of the pathways coupling receptors to calcium channels in rat sympathetic neurons.

Muscarinic and alpha-adrenergic suppression of current through Ca2+ channels was studied in adult rat superior cervical ganglion neurons using whole-cell and cell-attached configurations of the patch-clamp technique. Oxotremorine methiodide suppressed ICa by both a rapid (much less than 1 s) and a slow (greater than 4 s) process, whereas norepinephrine suppressed ICa only by a rapid process. The slow muscarinic suppression could be prevented by adding 20 mM BAPTA, a Ca2+ chelator, to the recording pipette, whereas the adrenergic suppression was not affected. Muscarinic, but not alpha-adrenergic, receptors can couple to Ca2+ channels by a second messenger capable of diffusing into an on-cell patch. This signal seems not to be carried by intracellular Ca2+, cGMP, cAMP, or protein kinase C.

Animals↗
PubMed1990

Effects of pinaverium on voltage-activated calcium channel currents of single smooth muscle cells isolated from the longitudinal muscle of the rabbit jejunum.

1. Smooth muscle cells of the longitudinal muscle of the rabbit jejunum were dispersed by enzyme treatment and recordings of membrane current were made in the whole-cell mode by patch clamp technique. The action of pinaverium bromide on the voltage-dependent inward current of single isolated smooth muscle cells was studied in solutions containing normal concentrations of calcium or high concentrations of barium at room temperature. 2. Pinaverium reduced the voltage-dependent inward current with an IC50 of 1.5 microM. This IC50 is similar to those of verapamil, diltiazem and flunarizine on these cells as described by others. Occasionally evidence of a potentiating action of pinaverium on the inward current was seen. 3. Repetitive stimulation of the cells did not increase blockade of inward current by pinaverium unlike the use-dependent blockade seen with verapamil, methoxyverapamil, and diltiazem in these and in other smooth muscle cells. 4. The inactivation of inward current was studied by holding at various potentials for 2 or 10 s before evoking inward current. The voltage at which current was 50% available was changed very little by pinaverium although other calcium entry blockers, for example the dihydropyridines, have been reported to produce appreciable negative shifts which indicate considerable voltage-dependence of their blockade. This may indicate that pinaverium has similar affinities for the closed available and inactivated calcium channel states so that blockade is not appreciably voltage-dependent.

Animals↗
PubMed1989

Characterization of a voltage-gated K+ channel that accelerates the rod response to dim light.

In this study a K+ current, IKx, in isolated salamander rod photoreceptors was characterized and its role in shaping small photovoltages was examined. IKx is a standing outward current of about 40 pA at -30 mV that deactivates slowly when the cell is hyperpolarized (tau max = 0.25 s). The voltage and time dependence of IKx are similar to that of M-current, but IKx can be distinguished from M-current because it is not suppressed by acetylcholine and is "blocked" by external Ba2+ in a surprising manner: the activation range of IKx is shifted strongly in the positive direction. Using current-clamp recordings and a computer simulation of the photo-response, we show that IKx figures prominently in setting the dark resting potential and accelerates the voltage response to small photocurrents.

Animals↗
PubMed1989

Effects of potassium channel toxins from Leiurus quinquestriatus hebraeus venom on responses to cromakalim in rabbit blood vessels.

1. The effects of fractionated Leiurus quinquestriatus hebraeus venom on cromakalim-induced 86Rb+ efflux in rabbit aortic smooth muscle were examined. 2. Crude venom (0.1-30 micrograms ml-1) produced a concentration-dependent decrease of 1 microM cromakalim-induced 86Rb+ response. The maximum blocking activity attainable was approximately 60%. 3. Fractionation of crude venom by gel permeation chromatography and subsequent chromatography on a cation ion-exchange column, produced two fractions (X and XI), active in the 86Rb+ blocking assay. 4. Fraction XII contained charybdotoxin (approximately 85% pure). After a final high performance liquid chromatography (h.p.l.c.) purification step, the purified toxin failed to inhibit the cromakalim-stimulated 86Rb+ efflux although it was a potent inhibitor of A23187-induced K+ flux in human erythrocytes and the large conductance calcium-activated potassium channel in rabbit portal vein smooth muscle. 5. Subsequent purification of fraction X by h.p.l.c. yielded a minor peak which contained 86Rb+ blocking activity. This subfraction was also capable of inhibiting apamin-sensitive, angiotensin II-stimulated K+ flux in guinea-pig hepatocytes. 6. It is concluded that the potassium channel opened by cromakalim in rabbit aortic smooth muscle is not blocked by charybdotoxin but by another distinct toxin in the venom of Leiurus quinquestriatus hebraeus.

Amino Acid Sequence↗
PubMed1989

Properties of the cromakalim-induced potassium conductance in smooth muscle cells isolated from the rabbit portal vein.

1. Single smooth muscle cells were isolated freshly from the rabbit portal vein and membrane currents were recorded by the whole-cell or excised patch configurations of the patch-clamp technique at room temperature. 2. Cromakalim (Ckm, 10 microM) induced a potassium current (ICkm) that showed no pronounced voltage-dependence and had low current noise. 3. This current, ICkm, was inhibited by (in order of potency): phencyclidine greater than quinidine greater than 4-aminopyridine greater than tetraethylammonium ions (TEA). These drugs inhibited the delayed rectifier current, IdK, which is activated by depolarization of the cell, with the same order of potency. 4. Large conductance calcium-activated potassium channels (LKCa) in isolated membrane patches were blocked by (in order of potency) quinidine greater than TEA approximately phencyclidine. 4-Aminopyridine was ineffective. A similar order of potency was found for block of spontaneous transient outward currents thought to represent bursts of openings of LKCa channels. 5. The low current noise of ICkm at positive potentials, and its susceptibility to inhibitors indicated that it was not carried by LKCa channels, and that it may be carried by channels which underlie IdK. It was observed that when ICkm was activated, IdK was reduced. However, in two experiments, ICkm was much more susceptible to glibenclamide than IdK; possible reasons for this are discussed.

Animals↗
PubMed1989

A voltage-dependent outward current with fast kinetics in single smooth muscle cells isolated from rabbit portal vein.

1. Single smooth muscle cells were isolated enzymatically from the rabbit portal vein. They were voltage-clamped at room temperature using the whole-cell configuration of the patch-clamp technique. 2. When cells were bathed in physiological salt solution, depolarization from a holding potential of -70 mV elicited a time-dependent outward current which reached a maximum within 0.2-0.5 s, but when a more negative holding potential was used, an additional outward current could be activated. The current (Ifo) developed rapidly, was transient and seemed to be carried by potassium ions (K+). 3. The steady-state inactivation plot for Ifo was steeply voltage-dependent between -90 and -60 mV, current being 50% inactivated at -78 mV. The activation threshold was around -65 mV. The activation and inactivation kinetics were fast and voltage-dependent. When the test potential was -35 mV, peak current occurred after about 15 ms and the decay was complete within 250 ms. Recovery from inactivation was maximal after 1 s at -100 mV but was about five times slower at -70 mV. 4. The outward current Ifo was blocked completely by 4-aminopyridine (5 mM) or phencyclidine (0.1 mM), but was insensitive to tetraethylammonium ions (32 mM), apamin (0.1 microM), charybdotoxin from the venom of Leiurus quinquestriatus (0.1 microM), toxin-I from the venom of Dendroaspis polylepis (1 microM) or the putative K+ channel opener, cromakalim (10 microM). 5. The steady-state inactivation range and activation threshold, kinetics of activation and inactivation all showed a marked dependence on the concentration of divalent cations in the bathing solution. This effect was consistent with the hypothesis that Ifo was affected by membrane surface potential. The current did not seem to be Ca2+-activated. 6. Ifo closely resembled the A-current which has been described previously in neurones but not in smooth muscle.

Animals↗
PubMed1989

Two components of potassium current activated by depolarization of single smooth muscle cells from the rabbit portal vein.

1. Using the patch-clamp technique at 20-23 degrees C membrane currents were recorded from single smooth muscle cells enzymatically isolated from the rabbit portal vein. Single-channel currents were observed in outside-out patches excised from these. 2. Outward current elicited upon depolarization from -70 mV was not activated as a result of Ca2+ influx. It could be divided into two components: an inactivating, 4-aminopyridine- and phencyclidine-sensitive low-noise current (IdK), and a non-inactivating, tetraethylammonium (TEA)- and charybdotoxin-sensitive high-noise current (IcK). 3. IdK activated with a threshold around -40 mV and was carried by K+. It was substantially inhibited by 4-aminopyridine (5 mM) or phencyclidine (0.1 mM) but was insensitive to TEA+ (4 mM), charybdotoxin (0.1 microM) or apamin (0.1 microM). Upon stepping to 0 mV it reached a maximum within about 0.2 s. The time course of its activation could be described by a fourth-order single exponential; the time constants of these exponentials changed e-fold every 56 mV. It inactivated in a time- and voltage-dependent manner with a fast and slow component, and was about 50% available at -30 mV. From single-channel recordings in isolated patches single channels underlying this current have a small unitary conductance (around 5 pS). 4. IcK did not inactivate significantly over 6 s. It activated with a less negative threshold than IdK, usually near 0 mV when the pipette solution contained 0.8 mM-EGTA with no added calcium. It was blocked by TEA (4 mM) or charybdotoxin (0.1 microM), but not by 4-aminopyridine (5 mM), phencyclidine (0.1 mM) or apamin (0.1 microM). Estimates of the single-channel conductance from the noise variance of the whole-cell current IcK indicated a value at +80 mV of 115 pS, very similar to that of the large-conductance Ca2(+)-activated K+ channels studied in these cells using single-channel recording. 5. The results suggest that outward current evoked by depolarization from the resting potential can be carried by 100 pS Ca2(+)-activated K+ channels and by small-conductance delayed-rectifier K+ channels. It is likely that opening of both types of channel contributes to the repolarization phase of the action potential in this smooth muscle.

Animals↗
PubMed1989

Mechanism of calcium channel block by D600 in single smooth muscle cells from rabbit ear artery.

This study investigated the action of the calcium antagonist D600 on calcium channel currents recorded in high barium solution from single, enzymatically isolated smooth muscle cells from the rabbit ear artery using the whole cell configuration of the patch-clamp technique. D600 (1-100 microM) was applied by path perfusion or by a new technique that allowed a concentration jump during the current. Application of D600 at rest (holding potential, -60 mV) did not alter the peak inward current elicited on depolarization, but the activation of the channels led to a marked block and an acceleration of the current decay. The mechanism of block of calcium channels by D600 was studied by using pulse protocols with different pulse length and different interpulse intervals. The results were consistent with the hypothesis that D600 has a low affinity for the calcium channels in the resting state and that they have to pass to the open state before the drug affects the calcium channel current. A fast onset of the calcium channel block by D600 (time constant, 502 msec) could be shown by rapid application of D600 during the sustained current component of the barium inward current. However, experiments did not definitely distinguish whether binding occurred to the open or to the inactivated state (although there was some evidence of a long-lasting binding to an inactivated state).

Animals↗
PubMed1988

Action of nifedipine or BAY K8644 is dependent on calcium channel state in single smooth muscle cells from rabbit ear artery.

The actions of nifedipine or BAY K 8644 were studied on barium currents recorded from single, collagenase- and elastase-dispersed, smooth muscle cells from the rabbit ear artery using the whole-cell configuration of the patch-clamp technique. Nifedipine (3 microM) caused a reduction in the barium current (IBa) evoked by steps to potentials positive of -10 mV. This was characterized by a pronounced 'initial' block, an increase in the rate of current decay during the voltage-clamp step, but by no increase in block if pulses were repeated every 600 ms. Rapid extracellular application of nifedipine (1 microM) during the sustained current component (using a new concentration-jump technique) was found to have no effect on IBa over 4s at +20 mV, but after returning to the holding potential (-60 mV) for 10s, sustained IBa was subsequently abolished. BAY K 8644 (1 microM) increased IBa at all potentials, and on rapid application during the sustained current component markedly potentiated IBa. The results suggest that nifedipine binds with high affinity to the closed, available state of the Ca++ channels but they do not suggest binding to the open or inactivated states. The effect of BAY K 8644 is consistent with high affinity binding to the open or inactivated and to the closed, available states.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1988

Calcium channels and calcium-mediated effects in smooth muscle cells.

In single smooth muscle cells held at depolarized potentials under voltage-clamp, spontaneous transient outward currents (STOCs) of about 100 ms in duration and 100-300 pA in size are observed. These behave as if they are caused by the cyclical release of calcium from storage sites within the cell and can therefore be used as a method of monitoring the release of calcium from these stores by various agents. Muscarinic receptor activation or caffeine applied to single smooth muscle cells from rabbit jejunum produced outward current, probably due to the release of calcium from stores, and STOCs were subsequently abolished. Levels of inositol 1,4,5 trisphosphate in fragments of the muscle were increased within 5 s by carbachol application. STOCs and outward current due to calcium store release were probably due to the opening of calcium-activated K-channels since they were abolished by 4 mM tetraethylammonium. Receptor activation also opens channels that admit cations, including calcium, which may also contribute to tension generation.

Animals↗
PubMed1987

A simple method of fast extracellular solution exchange for the study of whole-cell or single channel currents using patch-clamp technique.

A new concentration-jump technique was devised for the rapid application of drugs to single, isolated cells attached to the base of the experimental chamber while recording from them with patch-clamp technique. Cells were placed in a micro-drop (less than 0.1 microliter) in a small inner bath which was separated from an outer bath by a ring of "Sylgard" polymer. Stable whole-cell recordings were made in the micro-drop and rapid solution exchange took place when a much larger volume of test solution from the outer bath was flooded over the Sylgard ring and mixed with the micro-drop. Complete equilibration occurred within less than 10 ms.

Adenosine Triphosphate↗