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A F Brading

Publications and source records attributed to A F Brading.

At least 91 records · Page 5Linked to original sources
PubMed1993

Inactivation of the voltage-dependent Ca2+ channel current in smooth muscle cells isolated from the guinea-pig detrusor.

1. Whole-cell voltage clamp techniques were applied to single smooth muscle cells enzymatically dissociated from guinea-pig urinary bladder. The inactivation and recovery of voltage-dependent Ca2+ channel currents were examined by manipulating the membrane potential over a wide range and by changing the extracellular divalent cation concentrations. 2. After exposing the cells to conditioning potentials (-100 to +80 mV in 20 mV increments), the degree of inactivation was estimated by stepping to a 0 mV test potential. In the presence of 2.5 mM Ca2+, the inactivation of the current was U-shaped with respect to the conditioning potential, with maximum inactivation at 0 mV. The maximal inactivation was 60 and 90% after conditioning durations of 0.8 and 5 s, respectively. The U-shaped curve is characteristic of Ca(2+)-dependent inactivation. When conditioning potentials of +80 mV with either duration were applied, the inward current at the test potential and the subsequent tail current on returning to the holding potential were larger than in control conditions (when the conditioning potential = the holding potential, -60 mV). 3. A U-shaped inactivation curve was also observed in the presence of 2.5 mM Ba2+. The inactivation was maximal with a conditioning potential of about -20 mV, and the inactivation was smaller than seen with Ca2+ entry. 4. Paired-pulse protocols were applied to examine the voltage dependence of recovery of the Ca2+ inward current. After the inward current had been inactivated during a 100 ms depolarization at 0 mV, it took 700 ms at -60 mV for nearly complete recovery of the current. Recovery was also observed at +80 mV. When the potential of the paired pulses was increased to +20 mV, less recovery was seen when the interpulse potential was at +80 mV. When a longer (3 s) depolarization was applied, the peak amplitude of the inward current took much longer to recover, and had not completely recovered after 4 s at either of the interpulse potentials, although recovery was greater with an interpulse potential of -60 mV than with one of +80 mV. Similar recoveries were observed in the presence of Ba2+. 5. During a long depolarization (8 s, 0 mV), the effects of rapid changes in the extracellular solution were examined. Partial recovery of the inward current occurred after a period in which Ca2+ was replaced with Mg2+. This recovery was not observed in the presence of Ba2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Pharmacological evaluation of the alpha-adrenoceptors of the rabbit urethra.

Nerve-mediated responses of the rabbit urethral smooth muscles were studied using electrical stimuli of short duration to selectively stimulate the intramural nerves. The nature of the responses to exogenously applied adrenergic agonists and its antagonists were also examined. The results confirmed that the rabbit urethral smooth muscle contains both subtypes of alpha-adrenoceptors and the nerve-mediated contraction of the rabbit urethra is of adrenergic nature and mediated mainly via alpha 1-adrenoceptors.

Adrenergic alpha-Agonists↗
PubMed1992

The contribution of alpha-adrenoceptors to neurally-mediated contractions of the rabbit urethral smooth muscle.

1. The nature of the nerve-mediated contractions in the urethral smooth muscle from the rabbit was studied in vitro. Field stimulation caused smaller contractile responses than in the detrusor of the rabbit. 2. There was no significant difference in response to field stimulation or exogenous agents acting on adrenoceptors between longitudinal and circular strips from the rabbit urethra. Histological studies showed that the urethral muscle is arranged in three layers, which run circularly and longitudinally. 3. Atropine had very little effect on the response to field stimulation, phentolamine almost abolished the contractile response to nerve stimulation and sometimes unmasked a relaxation. 4. The alpha 1-adrenoceptor blocking agent, prazosin, blocked both the contractile response to the alpha 1-receptor agonist phenylephrine and that to intrinsic nerve stimulation, with similar potencies. The alpha 2-blocking agent yohimbine shifted the dose-response curve of the contractile response to the alpha 2-agonist, clonidine, in a dose-dependent manner, 10(-7) M causing a 10 fold shift. This concentration had no effect on the response to intrinsic nerve stimulation, suggesting that alpha 2-receptors are not involved in the response. Higher concentrations of yohimbine caused a suppression of the nerve-evoked response which is assumed to be non-specific. 5. Noradrenaline, phenylephrine, and clonidine caused dose-dependent contractile responses in the rabbit urethral strips. The contractions induced by clonidine developed more slowly than those induced by noradrenaline and phenylephrine. 6. These results demonstrate that the rabbit urethral smooth muscle contains both alpha 1- and alpha 2-adrenoceptors, and the nerve-mediated contraction of the rabbit urethra is adrenergic in nature and mediated mainly via alpha 1-adrenoceptors.

Animals↗
PubMed1992

The M3 muscarinic receptor links to three different transduction mechanisms with different efficacies in circular muscle of guinea-pig stomach.

1. In a previous publication, we showed that 10 microM carbachol induced contraction by activating three independent transduction mechanisms in circular smooth muscle of guinea-pig gastric fundus (Parekh & Brading, 1991). These were: inositol trisphosphate-mediated intracellular Ca2+ release, Ca2+ influx through a nifedipine-sensitive route and Ca2+ influx through a receptor operated nifedipine-insensitive pathway. The former two processes contribute to the phasic contraction and the latter two to the tonic contraction. In this paper, we have studied the effects of muscarinic receptor antagonists with known selectivity for different muscarinic receptor subtypes, on the contraction evoked by 10 microM carbachol. 2. Low concentrations of pirenzepine (M1 selective) had little effect on the contraction initiated by carbachol. Higher concentrations (greater than 1 microM) reduced only the phasic component. This concentration of pirenzepine greatly reduced the contraction evoked by 10 microM carbachol in Ca(2+)-free solution, indicating inhibition of intracellular Ca2+ release. 3. In the presence of 10 microM nifedipine, the tonic contraction evoked by 10 microM carbachol (reflecting the receptor-operated nifedipine-insensitive route) was abolished by 10 microM pirenzepine. In the absence of nifedipine pretreatment, however, 10 microM pirenzepine did not abolish the contraction to 10 microM carbachol. This contraction was subsequently abolished by nifedipine. 4. Only high concentrations (greater than 10 microM) of the M2-selective antagonist, gallamine, inhibited the contraction to 10 microM carbachol. Like pirenzepine, gallamine preferentially inhibited the phasic component of the contraction, indicating an effect on intracellular Ca2+ release. 5. The non-selective muscarinic receptor antagonist, atropine, abolished all components of the contraction. At low concentrations, atropine also reduced the phasic component without affecting the tonic one, indicating preferential inhibition of intracellular Ca2+ release.6. It is concluded that (i) the different transduction mechanisms have different sensitivities to the antagonists used and (ii) an M3 receptor activates the three transduction mechanisms with different efficacies.

Animals↗
PubMed1991

Cystometric, physiological and morphological studies after relief of bladder outflow obstruction in the pig.

Experimental bladder outflow obstruction was relieved in 18 pigs between 2 and 15 months after the creation of partial urethral obstruction. Cystometric, physiological and morphological studies were performed 2 to 6 months after relief of the obstruction. An increase in average voiding flow rates from 2.8 +/- 1.0 ml/s to 6.8 +/- 1.2 ml/s was recorded in the Landrace pigs and from 2.2 +/- 0.9 ml/s to 7.4 +/- 1.4 ml/s in the Göttingen mini-pigs. There was a concomitant decrease in the voiding detrusor pressures from 52 +/- 11 cm H2O to 32 +/- 8 cm H2O and from 78 +/- 12 cm H2O to 33 +/- 6 cm H2O respectively. A return towards control values of the physiological responses to exogenously applied agonists (acetylcholine and potassium) and to electrical field stimulation was observed. There was an increase in neuronal innervation in the morphological studies which was more marked in the animals with a shorter period of obstruction. The implications for patient care are discussed.

Acetylcholine↗
PubMed1991

Human, pig and guinea-pig bladder smooth muscle cells generate similar inward currents in response to purinoceptor activation.

The contribution of purinergic neurotransmission to bladder excitation in pigs and man is small. Exogenously-applied adenosine-trisphosphate (ATP) however, elicits large inward currents in dispersed bladder smooth muscle cells in both species. The essential properties of the ATP-induced current in human and pig detrusor are similar and the current intensity is comparable to those in the guinea-pig, which has a powerful purinergic excitatory innervation. This suggests that other features of the tissue such as the closeness of the innervation and the degree of cell-to-cell coupling may be important in determining the effectiveness of purinergic transmission.

Adenosine Triphosphate↗
PubMed1991

The sources of calcium for carbachol-induced contraction in the circular smooth muscle of guinea-pig stomach.

1. The action of carbachol on the mechanical activity of circular muscle from guinea-pig upper stomach was studied. High concentrations of carbachol (e.g. 10(-4) M) produced a rapid phasic contraction followed by a smaller, sustained tonic contraction. Low concentrations (e.g. 10(-7) M) caused a contraction which did not generally show marked distinction between phasic and tonic components. 2. The response to 10(-7) M carbachol was very sensitive to 10(-5) M nifedipine as was the phasic response to 10(-4) M carbachol. The tonic contraction to the latter, however, was only slightly reduced by nifedipine. 3. The carbachol-induced contractions remaining in the presence of nifedipine were dose-related and very dependent on the presence of external calcium. 4. Carbachol, 10(-7) M, did not produce a contraction after 4 min exposure to calcium-free solution whereas 10(-4) M carbachol did and this was phasic in nature but much reduced relative to the control in normal Ca. 5. A phasic followed by a small tonic contraction to 10(-4) M carbachol was seen superimposed on the K contracture in tissues depolarized with 100 mM K, whereas only a small tonic response occurred for 10(-7) M carbachol. 6. In the absence of a functional carbachol-sensitive intracellular store, 10(-4) M carbachol was unable to trigger a contraction in calcium-free solution. However, when calcium was simultaneously readmitted with carbachol after exposure to calcium-free solution, a contraction occurred. 7. Carbachol, 10(-7) M, did not significantly increase inositol polyphosphate levels, whereas 10(-4) M carbachol did. The increase with 10-4M occurred rapidly peaking within 2min and was undetectable after 5 min, in the absence of lithium. 8. It is concluded that low concentrations of muscarinic agonist trigger a contraction predominantly through a nifedipine-sensitive route whereas higher concentrations further utilize intracellular calcium release and a receptor-operated extracellular calcium-dependent pathway. The former is probably associated with the phasic component and the latter with the tonic one.

Animals↗
PubMed1991

Ion channels and excitatory transmission in the smooth muscle of the urinary bladder.

The bladder of most non-human species receives dual purinergic and cholinergic excitatory innervation. Activation of P2x-purinoceptors depolarizes the cells, increases the spike frequency and causes contraction. Addition of agonists rapidly activates non-selective cation channels, which underly the excitatory junction potentials seen on simulation of the intrinsic nerves. In all species studied including humans the detrusor possesses well developed post-junctional purinergic mechanisms, and interspecies differences here cannot account for the large difference in the potency of the purinergic innervation seen. Muscarinic receptor activation causes contraction through pharmacomechanical coupling, using inositol trisphosphate as a second messenger. Increase in intracellular calcium results in outward currents through Ca activated K channels. In most species little depolarization or increase in spike frequency is seen, although in some, a slowly developing inward current mechanism is activated, which might account for the small delayed muscarinic depolarization seen following the purinergic excitatory junction potential.

Adenosine Triphosphate↗
PubMed1990

An animal model of non-obstructive bladder instability.

Eight minipigs with proven normal bladder function have undergone a circumferential supratrigonal bladder transection. Urodynamic studies were performed before, and at various times up to eight months after operation. Over the same period biopsies were taken for physiological and histological studies. By three weeks after transection all animals had developed bladder instability, the abnormal contractions being maintained for the duration of the study. Concomitant with this change was the development, in vitro, of a non-specific detrusor smooth muscle supersensitivity similar to that which occurs in obstructed animals. By contrast with the latter group there was, however, no loss of cholinergic nerve from the transected bladder muscle. The transected (decentralised) minipig may be a useful model for the study of bladder instability not associated with outflow obstruction. The findings suggest that anatomical denervation is not a prerequisite for unstable contractions although a functional loss of nerve may be of importance. Supersensitivity, however, appears to occur in both obstructed and nonobstructed instability.

Animals↗
PubMed1990

Contractile responses of smooth muscle strips from rat and guinea-pig urinary bladder to transmural stimulation: effects of atropine and alpha,beta-methylene ATP.

1. Strength-duration curves for threshold mechanical responses to single transmural stimuli were identical for rat and guinea-pig detrusor. In both species atropine had no effect on the curves, but the curves were shifted to the right by nerve blockade with tetrodotoxin (TTX), and by blockade of P2-purinoceptors with alpha,beta-methylene ATP (alpha,beta-MeATP). 2. With short duration pulses of 50 V and less, the responses were nerve-mediated. Increase in either the strength or duration of the stimulus caused direct muscle stimulation, resistant to blockade with atropine, TTX and alpha-beta-MeATP. 3. The shape of the contractile response to a single nerve stimulus varied from tissue to tissue. The responses could be mono-, bi-, or multiphasic. Bi- or multiphasic responses were normally seen in tissues which were spontaneously active. The multiphasic nature of the response was enhanced by factors which increased the excitability of the cells and was reduced by factors which decreased the excitability. 4. The frequency-response curves in the rat are similar to those previously obtained in the guinea-pig. Atropine suppresses the high frequency response by 25%, with little effect at low frequencies, whereas desensitization of P2-purinoceptors with alpha,beta-MeATP suppresses the responses maximally at low frequencies but still by 75% at high frequencies. A combination of both drugs eliminates the nerve-mediated responses. 5. It is concluded that the response to a single nerve stimulus is mediated by a non-cholinergic transmitter, through activation of P2-purinoceptors. The possibility that simultaneous release of acetylcholine can modify the excitability of cells and thus the configuration of the response to a single stimulus is discussed.

Acetylcholine↗
PubMed1990

Potassium channel blockers and the effects of cromakalim on the smooth muscle of the guinea-pig bladder.

1. The K+ channel blocking drugs tetraethylammonium Cl (TEA), procaine, 4-aminopyridine (4AP) and quinidine all produced concentration-dependent contractions of strips of smooth muscle from the guinea-pig urinary bladder. Apamin and glibenclamide caused little increase in the mechanical activity, and tolbutamide inhibited it. 2. TEA, procaine, 4AP, quinidine and apamin all increased the frequencies of spontaneous action potentials recorded with microelectrodes. TEA, quinidine and procaine all caused prolongation of the falling phase of the spike, and procaine and apamin completely abolished the after-hyperpolarization. 3. TEA and procaine increased K+ efflux from the tissue, an effect blocked by nifedipine. TEA and apamin increased, whereas quinidine, procaine and 4AP decreased K+ uptake. 4. Cromakalim caused a concentration-dependent hyperpolarization of the membrane, abolished spike activity, increased K+ fluxes and relaxed the smooth muscle. The relaxant effect of cromakalim was unaffected by apamin, and in its presence the effects of cromakalim on membrane potential and K+ fluxes were unchanged. Procaine abolished all the effects of cromakalim, and TEA at high concentrations reduced but did not abolish these effects. Quinidine reduced the effects of cromakalim on tension and membrane potential, but its actions were surmounted by higher concentrations of cromakalim. The effects of 4AP on tension and membrane properties were transitory, but it had some effects on the actions of cromakalim. Glibenclamide and tolbutamide reversed the relaxant effects of submaximal cromakalim concentrations, tolbutamide only transiently. 5. It is concluded that the channels opened by cromakalim are not those involved in generating the spike after-hyperpolarization. They have properties similar to the delayed rectifier K+ channels responsible for spike repolarization, and also are similar to the ATP-dependent K channels in vascular smooth muscle.

4-Aminopyridine↗
PubMed1990

The properties of the ATP-induced depolarization and current in single cells isolated from the guinea-pig urinary bladder.

1. The actions of exogenously applied ATP were investigated with the whole-cell patch clamp method in single cells isolated from guinea-pig urinary bladder with a modified concentration jump technique. 2. Rapid application of ATP (threshold ca. 100 nM) depolarized the cell membrane with superimposition of action potentials which was followed by transient hyperpolarization. In the presence of D600, the amplitude of the ATP-induced depolarization was a function of the ATP concentration (EC50: 0.5-1 microM). 3. ATP activated a dose-dependent inward current with a short latency (18 ms with 10 microM ATP; measured as the time between the start of application and 10% of the peak). The relationship of the peak current versus ATP concentration was well fitted by a Michaelis-Menten equation with a Hill coefficient (n) of 1.7 and a dissociation constant (Kd) of 2.3 microM. The current desensitized rapidly and the time course of desensitization was a function of the ATP concentration and could be fitted by two exponentials. 4. The reversal potential of the ATP-activated current was near 0 mV. Replacement of extracellular Na by other monovalent or divalent cations indicated that the current flows through nonselective cation channels. 5. alpha,beta-Methylene ATP also produced a dose-dependent inward current but was less potent than ATP (n: 1.6, Kd: 10.4 microM). alpha,beta-Methylene ATP blocked the response to ATP by desensitization of the receptor. 6. alpha,beta-Methylene ATP was 50-100 times more potent than ATP at eliciting a contractile response of strips of detrusor smooth muscle. 7. The relevance of the above results to the possible role of ATP as the fast excitatory transmitter is discussed.

Adenosine Triphosphate↗
PubMed1990

Effect of Na+ and K+ on Cl- distribution in guinea-pig vas deferens smooth muscle: evidence for Na+, K+, Cl- co-transport.

1. Smooth muscle cells of the guinea-pig vas deferens after Cl- depletion actively reaccumulate ions to a level many times higher than that predicted by a passive distribution, even when anion exchange (largely responsible for Cl- movements in this preparation) is inhibited by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid). The cells therefore must possess a second mechanism for Cl- accumulation. We have now investigated the ionic requirement of this mechanism using a combination of ion analysis, 36Cl fluxes and direct measurement of the intracellular Cl- activity (aiCl). 2. In the steady state, the Cl- content of tissues was 12-16% less in Na(+)-free solution than in normal Krebs solution. 3. Loss of 36Cl into Cl(-)-free solution was slowed by the absence of Na+ and accelerated on its readdition. Uptake of 36Cl by Cl(-)-depleted tissues was also reduced in the absence of extracellular Na+, particularly at longer time intervals as uptake reached completion. These effects occurred in the presence and absence of CO2-HCO3- and in the presence of DIDS. 4. The initial rate of rise of aiCl on readdition of Cl- to Cl(-)-depleted cells was not significantly affected by the absence of Na+ in the presence of a functional anion exchange, but aiCl stabilized at a lower value than in normal solution. Readdition of Na+ stimulated a rise in aiCl to the control level. Removal and readdition of K+ under these conditions had negligible effects. 5. When anion exchange was inhibited by the presence of DIDS, removal and readdition of Na+ caused, respectively, a marked inhibition and stimulation of the rise in aiCl during Cl- reaccumulation. Under these conditions removal and readdition of K+ had similar effects. 6. The results suggest that Na+, K+, Cl- co-transport is involved in transmembrane movements of Cl- at least when the anion exchange mechanism is blocked. 7. The possibility that the marked effects of changes in external Na+ on transmembrane Cl- movements in the presence of a functional anion exchange mechanism are caused by secondary effects due to changes in intracellular pH as well as to suppression of Na+, K+, Cl- co-transport is discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1990

The effect of loop diuretics on Cl- transport in smooth muscle of the guinea-pig vas deferens and taenia from the caecum.

1. The role of Na+, K+, Cl- co-transport, identified in the previous paper (Aickin & Brading, 1990), has been characterized further by investigation of the effects of loop diuretics on Cl- movements in the smooth muscle cells of guinea-pig vas deferens measured by 36Cl fluxes and Cl(-)-sensitive microelectrodes. Some flux experiments were also repeated in the taenia from the guinea-pig caecum. 2. Frusemide (2 mM) reduced the steady-state Cl- content, slowed 36Cl loss into Cl(-)-free solution and both slowed and reduced Cl- accumulation by Cl(-)-depleted cells of the vas deferens. When anion exchange was inhibited by the presence of DIDS, (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid), frusemide further slowed the loss of Cl- into Cl(-)-free solution, further reduced Cl- accumulation such that Cl- uptake amounted to a level consistent with a passive distribution and halted the rise in the intracellular Cl- activity (aiCl) at levels above about 10 mM. 3. Application of the higher-affinity loop diuretics bumetanide and piretanide in vas deferns had no significant effect on 36Cl efflux into Cl(-)-free solution or on the initial rate of rise of aiCl but reduced the final level attained. In the presence of DIDS, however, both agents further slowed efflux into Cl(-)-free solution, and halted the rise in aiCl at levels above about 10 mM. Measurement of greatly slowed intracellular pH transients on removal and readdition of external Cl- (Clo-) in the presence of frusemide suggests that the larger effects of this drug are mediated by inhibition of anion exchange as well as of co-transport. 4. The relative potency of the loop diuretics, investigated in the presence of DIDS was: bumetanide greater than piretanide greater than frusemide. This sequence was found in both vas deferens, using direct measurement of aiCl, and taenia, using 36Cl uptake. 5. Comparison of data from the vas and taenia showed that 36Cl efflux into Cl(-)-free, HCO3(-)-free solution was about twice as fast in the taenia, and that bumetanide or piretanide reduced this efflux to about the same rate as that observed in the vas with or without the loop diuretic. DIDS caused a similar absolute reduction of efflux in both preparations. 6. Stimulation of 36Cl efflux on readdition, and inhibition on removal of Nao+ in the presence of DIDS, was much greater in the taenia than in vas and in both preparations was blocked by bumetanide or piretanide.(ABSTRACT TRUNCATED AT 400 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗