Biomedical subjects

T B Bolton

Publications and source records attributed to T B Bolton.

At least 37 records · Page 2Linked to original sources

PubMed1998

Variability in spontaneous subcellular calcium release in guinea-pig ileum smooth muscle cells.

1. Spontaneous, localized transient increases in [Ca2+]i ('Ca2+ sparks') were observed in about 40 % of fluo-3-loaded myocytes examined using laser scanning confocal microscopy. Ca2+ sparks persisted after application of Cd2+ (200 microM), but were abolished by ryanodine (30 microM) or thapsigargin (0.1 microM), suggesting that they arise from the spontaneous activation of ryanodine receptors (RyR) in the sarcoplasmic reticulum (SR). 2. Ca2+ sparks occurred much more frequently at certain sites (or 'frequent discharge sites', FDSs) within any confocal plane of the cell and line-scan imaging revealed a wide variation in their spatial size, amplitude and time course. Some spontaneous local transients were very similar to 'Ca2+ sparks' observed in heart, i.e. lasting approximately 200 ms with a peak fluorescence ratio of 1.75 +/- 0.23 (mean +/- s.d., n = 33). Other events were faster and smaller, lasting only approximately 40 ms with a peak normalized fluorescence of 1.36 +/- 0.09 (mean +/- s.d., n = 28). 3. Spontaneous Ca2+ waves with a wide range of propagation velocities (between 30 and 260 micron s-1) were also observed. In about 60 % of records (n = 33), Ca2+ sparks could be detected at the sites of wave initiation. Waves of elevated [Ca2+]i propagated with non-constant velocity and in some cases terminated. These observations could be explained by heterogeneity in the distribution of subcellular release sites as well as variability in the contribution of each release site to the wave. 4. Spontaneous [Ca2+]i transients in single dispersed visceral smooth muscle cells have a wide spectrum of behaviour that is likely to be the result of spatio-temporal recruitment of smaller local events, probably via a calcium-induced calcium release (CICR) mechanism. The spatial non-uniformity of SR and RyR distribution within the cell may account for the existence of 'frequent discharge sites' firing the majority of the smooth muscle Ca2+ sparks and the wide variation in the Ca2+ wave propagation velocities observed.

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

PubMed1998

Inwardly rectifying whole cell potassium current in human blood eosinophils.

1. Membrane currents were studied in single human blood eosinophils using the whole cell voltage clamp technique. The whole cell current-voltage relationship exhibited rectification about the membrane potential which followed the potassium equilibrium potential when [K+]o was raised. Elevation of [K+]o considerably potentiated inward current amplitude, and in some cells channel activity was discernible in the whole cell membrane current recordings. The single channel conductance was 24 +/- 1 pS ([K+]o, 100 mM; [K+]i, 140 mM), and eosinophils were found to have as few as three, and on average twenty, inward rectifier channels each. 2. The inward current was inhibited in a voltage-dependent manner by extracellular cations in order of potency Ba2+ > Cs+ > Na+. Intracellular acidification inhibited while alkalization augmented the inward current. Mg2+ contributed to rectification as dialysis with nominally Mg(2+)-free pipette solution was associated with an increase in the outward current during membrane polarization. 3. By reverse transcription-polymerase chain reaction (RT-PCR) using suitable primers on human eosinophils mRNA, an inward rectifier channel, Kir2.1, was identified, which is known from expression studies to have very similar properties to those found in this study. 4. Superoxide anion production or its stimulation by phorbol 12-myristate 13-acetate (PMA) was not significantly affected by depolarization with 140 mM [K+]o, or by 1 mM BaCl2. 5. It is concluded that the single channel currents and the whole cell current rectification observed in human blood eosinophils resulted from the presence of an inwardly rectifying potassium channel, probably Kir2.1.

Action Potentials↗

PubMed1998

Comparative study of effects of isoproterenol and vasoactive intestinal polypeptide on voltage-dependent Ca2+ and Ca(2+)-activated K+ currents in porcine tracheal smooth muscle cells.

Effects of isoproterenol (Iso) and vasoactive intestinal polypeptide (VIP) on voltage-dependent Ca2+ channel (ICa) and Ca(2+)-activated K+ channel current (IK-Ca) in porcine tracheal smooth muscle cells were examined. When K+ currents were inhibited using a Cs-rich pipette solution, application of 0.1-1 microM Iso or 1-10 nM VIP increased ICa by 20-30%. On the other hand, IK-Ca elicited upon depolarization and spontaneous transient outward K+ currents (STOCs) recorded at a holding potential of -50 mV were enhanced by 80-100% in the presence of 0.1 microM Iso or 1 nM VIP.

Adrenergic beta-Agonists↗

PubMed1998

Confocal imaging of calcium release events in single smooth muscle cells.

Localized [Ca2+]i transients ('sparks') first directly detected in cardiac myocytes were considered to represent 'elementary' Ca(2+)-release events playing a key role during excitation-contraction coupling (Cheng et al. 1993). In this study we employed confocal [Ca2+]i imaging to characterize subcellular calcium signalling in fluo-3 loaded visceral and vascular smooth muscle cells. In some experiments membrane potential of the myocyte was controlled using whole-cell patch clamp technique and changes in membrane current were recorded simultaneously with [Ca2+]i imaging. Some local [Ca2+]i transients were very similar to 'Ca2+ sparks' observed in heart, i.e. lasting approximately 200 ms with a peak fluorescence ratio of 1.75 +/- 0.23 (mean +/- SD, n = 33). Ca2+ sparks were found to occur in certain preferred locations in the cell, termed frequent discharge sites. Other events were faster and smaller, lasting only approximately 40 ms with a peak normalized fluorescence of 1.36 +/- 0.09 (mean +/- SD, n = 28). A high correlation between spontaneous transient outward currents and spark occurrence was observed. Proliferating waves of elevated [Ca2+]i initiated during membrane depolarization seem to arise from spatio-temporal recruitment of local Ca(2+)-release events. The spatial non-uniformity of sarcoplasmic reticulum and ryanodine receptor distribution within the cell may account for the existence of 'frequent discharge sites' and the wide variation in the Ca2+ wave propagation velocities observed.

Aniline Compounds↗

PubMed1997

Activation of M2 muscarinic receptors in guinea-pig ileum opens cationic channels modulated by M3 muscarinic receptors.

In longitudinal muscle of guinea-pig ileum, activation of muscarinic receptors causes contraction antagonised by M3 receptor subtype antagonists despite a preponderance of M2 receptor subtype binding sites. Experiments on single smooth muscle cells under voltage-clamp described here show that the cationic current evoked by carbachol which normally causes depolarization of the muscle is inhibited competitively by M2 antagonists with affinities typical of antagonism at a M2 receptor. However, M3 antagonists strongly reduced the maximum cationic current which could be evoked by carbachol in a non-competitive manner with affinities typical for an action at M3 receptors. Thus cation channels are gated by M2 receptor activation but strongly modulated by activation of M3 receptors.

Animals↗

PubMed1997

Muscarinic receptor subtypes controlling the cationic current in guinea-pig ileal smooth muscle.

1. The effects of muscarinic antagonists on cationic current evoked by activating muscarinic receptors with the stable agonist carbachol were studied by use of patch-clamp recording techniques in guinea-pig single ileal smooth muscle cells. 2. Ascending concentrations of carbachol (3-300 microM) activated the cationic conductance in a concentration-dependent manner with conductance at a maximally effective carbachol concentration (Gmax) of 27.4+/-1.4 nS and a mean -log EC50 of 5.12+/-0.03 (mean+/-s.e.mean) (n=114). 3. Muscarinic antagonists with higher affinity for the M2 receptor, methoctramine, himbacine and tripitramine, produced a parallel shift of the carbachol concentration-effect curve to the right in a concentration-dependent manner with pA2 values of 8.1, 8.0 and 9.1, respectively. 4. All M3 selective muscarinic antagonists tested, 4-DAMP, p-F-HHSiD and zamifenacin, reduced the maximal response in a concentration-dependent and non-competitive manner. This effect could be observed even at concentrations which did not produce any increase in the EC50 for carbachol. At higher concentrations M3 antagonists shifted the agonist curve to the right, increasing the EC50, and depressed the maximum conductance response. Atropine, a non-selective antagonist, produced both reduction in Gmax (M3 effect) and significant increase in the EC50 (M2 effect) in the same concentration range. 5. The depression of the conductance by 4-DAMP, zamifenacin and atropine could not be explained by channel block as cationic current evoked by adding GTPgammaS to the pipette (without application of carbachol) was unaffected. 6. The results support the hypothesis that carbachol activates M2 muscarinic receptors so initiating the opening of cationic channels which cause depolarization; this effect is potentiated by an unknown mechanism when carbachol activates M3 receptors. As an increasing fraction of M3 receptors are blocked by an antagonist, the effects on cationic current of an increasing proportion of activated M2 receptors are disabled.

Animals↗

PubMed1997

Effects of protons on muscarinic receptor cationic current in single visceral smooth muscle cells.

Effects of extracellular protons on muscarinic receptor-evoked cationic current (Icat) in single guinea pig ileal smooth muscle cells were studied by use of patch-clamp techniques: intracellular pH and pCa were buffered to 7.4 and 7.0, respectively, symmetrical 124 mM Cs+ solutions were used, and divalent cations were removed from the bathing solution. Increasing extracellular pH (pHo) from 7.4 to 8.4 caused a 16-mV parallel negative shift of the activation curve for Icat evoked by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) with an increase in the maximal conductance and slowed Icat relaxation on hyperpolarization; acidification to pHo 6.4 produced equivalent but opposite effects. Carbachol- and GTPgammaS-activated Icat behaved similarly, suggesting that the cationic channel rather than the muscarinic receptor was the major site of action. From 11.4 to 4.4 pHo, maximal cationic conductance was reduced progressively, and the activation curve shifted positively. Na+, K+, Ca2+, and Mg2+ had complex interactions with pHo-induced effects considered to be attributable to interaction of protons with fixed negative surface charges and, at positive potentials, to channel block.

Animals↗

PubMed1996

Voltage-activated proton current in eosinophils from human blood.

1. The resting membrane potential of freshly purified normodense human eosinophils bathed in and dialysed with quasi-physiological solutions was -63 +/- 2 mV (n = 100). 2. In voltage-clamp mode with quasi-physiological internal and external solutions, voltage steps from the holding potential of -60 mV to levels positive to +20 mV resulted in development of a quasi-instantaneous outward current and a slowly developing outward current. The instantaneous current was absent when the cells were bathed in and dialysed with K(+)-free solution. 3. The slow outward current persisted following simultaneous replacement of K+, Na+ and most of the Cl- with largely impermeant ions (tetraethylammonium, N-methyl-D-glucamine and methanesulphonate) and was augmented when the cell was dialysed with a solution of increased buffering capacity for protons. The observed reversal potential of the current closely followed the hydrogen equilibrium potential over a wide range of internal-external pH combinations, indicating that the conductance underlying the slow outward current was highly selective for H+ ions. 4. Acidification of the pipette solution (increasing [H+]i) augmented the outward H+ current and shifted its activation range negatively, whilst acidification of the external solution had the opposite effect. The voltage dependence of the current is modulated by the transmembrane pH gradient so the only outward current could be activated. However, when the outward current was activated by a voltage step, rapid acidification of external solution produced an inward H+ current which rapidly deactivated. 5. The proton current was reversibly inhibited in a voltage-dependent manner by extracellular application of Zn2+. The apparent dissociation constants were 8 nM (at +40 mV), 36 nM (at +70 mV) and 200 nM (at +100 mV). 6. The proton current was augmented by exposure to 10 microM arachidonic acid. This augmentation consisted of a shift of the voltage dependence of activation to more negative potentials and enhancement of maximum conductance (gH,max). The proton current recorded in eosinophils was significantly augmented under conditions of elevated cytosolic free calcium concentration ([Ca2+]i). The threshold level of [Ca2+]i associated with this effect lay between 0.1 and 1 microM and was not measurably affected by cytosolic acidification. 7. Eosinophils from human blood possess a voltage-dependent H+ conductance (gH) which normally allows protons to move outwards only; raising [Ca2+]i was associated with augmentation of gH and intracellular acidification or arachidonate shifted its activation range negatively towards physiological potentials.

Adult↗

PubMed1996

Spontaneous transient outward currents in smooth muscle cells.

Spontaneous transient outward currents (STOCs) lasting about 100 ms occur in single smooth muscle cells and represent the simultaneous opening of up to a hundred calcium-activated potassium (BK) channels. The recent observation of brief focal releases of sarcoplasmic reticulum (SR) calcium ('sparks') in smooth muscle cells has provided support for the original suggestion that STOCs arise due to the spontaneous releases of calcium from the SR close to the sarcolemma. However, it is possible that such releases occur in a region of close apposition of SR membrane and sarcolemma about 0.1 microns wide ('junctional space') in which case they would be detectable by endogenous calcium-sensitive molecules such as BK channels but, using present confocal microscopy technique, not by calcium-indicator dyes introduced into the cell; should calcium escape from the junctional space then it may be visualised as 'sparks' by the fluorescent emission from calcium-indicator dyes using confocal microscopy. Some STOCs seem too large to represent the effect of a single 'spark' and some form of calcium-induced calcium release or 'macrospark' may be involved in their generation. Depletion of calcium stores by caffeine, ryanodine, or by activation of receptors linked to the phospholipase C/inositol trisphosphate system abolishes STOCs. However, low concentrations of caffeine or inositol trisphosphate accelerate STOC discharge by an unknown mechanism and often decrease STOC size presumably by depleting store calcium; similar effects are produced by agents such as cyclopiazonic acid and thapsigargin which inhibit calcium storage mechanisms (largely the SR calcium pump).

Animals↗

PubMed1996

Two types of ATP-sensitive potassium channels in rat portal vein smooth muscle cells.

1. Single-channel recordings were made from single, enzymatically isolated smooth muscle cells of rat portal vein by the patch-clamp technique. 2. Unitary potassium currents were identified through two types of K-channels with conductances in 60:130 mM K-gradient of 50 and 22 pS; these are referred to as LK and MK channels respectively. 3. The LK channels became extremely active if isolated patches were created into nucleotide-free solution; activity was inhibited by ATP applied to the inner surface of the patch with a half maximal inhibition (Ki) of 11-23 microM. Channel activity declined and disappeared with time and could be regenerated by a brief application of Mg-ATP or a nucleoside diphosphate such as UDP (in the presence of Mg). LK channel activity was rarely stimulated by levcromakalim and not by pinacidil (K-channel openers, KCOs) but was blocked by glibenclamide. 4. Activity of MK channels declined if isolated patches were created into nucleotide free solution; activity reappeared if UDP or ATP alone (in the presence of Mg) was applied; pinacidil or levcromakalim in the presence of ATP or UDP further increased channel activity which was blocked by glibenclamide. 5. The LK channel inhibited by ATPi is very similar in its conductance and other properties to the KATP channel described in tissues other than smooth muscle, in its conductance and properties the MK channel resembles the KNDP channel we have previous described as present in other smooth muscles and opening in responses to KCOs.

Adenosine Triphosphate↗

PubMed1996

A novel GTP-dependent mechanism of ileal muscarinic metabotropic channel desensitization.

1. Cationic current (Icat) was evoked in single isolated smooth muscle cells either by activating muscarinic receptors with the stable muscarinic agonist, carbachol (CCh), or by dialysing cells with GTP-gamma S. It was studied using patch-clamp recording techniques in cells obtained by enzymatic digestion from the longitudinal muscle layer of the guinea-pig small intestine. 2. Icat appears only when muscarinic receptors or G-proteins are activated, but it is strongly voltage-dependent. Its activation could be described by the Boltzmann equation. During desensitization of Icat evoked by 50 microM CCh, the slope factor, k, remained constant whereas the maximal conductance, Gmax, slowly decreased and the potential of half-maximal activation, V1/2, shifted positively by 32 mV during 4 min. 3. At peak response either to extracellular application of CCh (GTP-free, or 1 mM GTP-containing, pipette solution) or to intracellular application of GTP-gamma S (no CCh), the size and voltage-dependent properties of Icat were similar. However, Icat desensitization was slower in the presence of GTP (CCh applied) in the pipette solution and much slower with GTP-gamma S in the pipette (no CCh) compared to GTP-free pipette solution (CCh applied); the decrease in Gmax with time was much delayed and the positive shift of the activation curve was inhibited. GDP-beta S added to the pipette solution at 2 mM abolished Icat in response to applied CCh; 50 microM did not prevent Icat generation but significantly accelerated desensitization. 4. It was concluded that the rate of desensitization of the carbachol-evoked cationic current was due to a decline in the concentration of activated G-protein in the cell, which reduced the maximum number of channels which could be opened and shifted their activation range to less negative potentials.

Animals↗

PubMed1996

Inositol 1,4,5-trisphosphate-induced Ca(2+)-transient and outward K+ current in single smooth muscle cells of guinea pig small intestine.

The effects of inositol 1,4,5-trisphosphate (InsP3) released from caged InsP3 by flash photolysis on free intracellular Ca2+ concentration, [Ca2+]i, and outward K+ current were simultaneously examined in a single smooth muscle cell of guinea pig small intestine using a patch pipette solution containing Indo-1 (0.1 mM), caged InsP3 (50 microM) and KCl (130 mM). At a holding potential of -50 mV, a depolarizing pulse to +10 mV for 200 msec caused a transient Ca2+ current and an increase in [Ca2+]i. The amplitude of the Ca(2+)-transient was positively correlated with the peak Ca2+ current and negatively correlated with resting [Ca2+]i. InsP3 produced increases in [Ca2+]i and outward K+ current in most of the cells at -50 and -30 mV. The outward K+ current response reached a peak sooner and decayed more quickly than the Indo-1 signal. Both responses to InsP3 were resistant to the removal of extracellular Ca2+. The Ca(2+)-transient and outward K+ current responses to InsP3 at -30 mV were larger than those at -50 mV. The InsP3-induced Ca(2+)-transient was increased by increasing resting [Ca2+]i at -30 mV but not at -50 mV. These results suggest that InsP3-induced Ca2+ release from stores is potentiated by slight increases in [Ca2+]i via membrane depolarization.

Animals↗

PubMed1995

Effects of divalent cations on muscarinic receptor cationic current in smooth muscle from guinea-pig small intestine.

1. Effects of Mg2+ and Ca2+ on muscarinic receptor cationic current (Icat) in guinea-pig ileal smooth muscle cells have been studied using patch-clamp techniques (whole-cell recording). Icat was activated either by externally applied carbachol or, to bypass receptors, by intracellular GTP-gamma-S. 2. Independently of the main permeant cation the current-voltage (I-V) relation for Icat was U-shaped between the reversal potential (usually 0 mV) and very negative potentials such as -120 mV where current could be virtually lost. Adding Ca2+ to Ca(2+)- and Mg(2+)-free external solution reduced inward current and made it less U-shaped whereas adding Mg2+ reduced inward current and shifted more positively the potential at which maximum inward current occurred. 3. Activation of the conductance underlying Icat could be described by the Boltzmann relation which was shifted positively by adding Ca2+ or Mg2+. Extracellular Ca2+ also distorted the relation by increasing the slope factor; maximal conductance was reduced in all cases. Icat relaxation at negative potentials was accelerated by increasing Mg2+ and slowed down by Ca2+. 4. These data suggest the presence of fixed negative surface charges on or near the muscarinic receptor cationic channel, which allow its modulation through alteration of surface potential. Additional more direct ion binding to and blocking of the channel cannot be ruled out. Some additional effects of Ca2+ (if compared with Mg2+) could be explained on the assumption that the Ca(2+)-binding activation site known to be present on the internal side of the channel can be accessible to Ca2+ entering through the open channel during muscarinic receptor stimulation, as Ca2+ ions contribute to a limited extent to Icat. 5. We conclude that voltage-dependent gating of muscarinic receptor cationic channels is an intrinsic channel property and that Ca2+ and Mg2+ have strong modulatory effects.

Animals↗

PubMed1995

Activation and deactivation of membrane currents in human fibroblasts following infection with human cytomegalovirus.

The whole cell patch clamp technique was used to study the effects on membrane currents of infection of cultured human embryonic lung (HEL) fibroblasts with human cytomegalovirus (CMV). Four types of membrane currents were found in uninfected HEL cells, namely: Ca(2+)-activated potassium current, inward rectifier potassium current, delayed rectifier potassium current and voltage-dependent CMV. Voltage-dependent sodium current was detected in 30% of uninfected HEL cells whenever they were examined up to 72 h after seeding; however this current had completely disappeared by 18 h after infection with CMV. The delayed rectifier potassium current was detectable in 8% of uninfected HEL cells but, after infection, the proportion of cells expressing this current gradually increased from 20% at 18-24 h post-infection to 100% at 48 h and 72 h. Pharmacological agents known to regulate the activity of ion channels, via cellular secondary messengers, did not alter the frequency at which either current was detected in uninfected and infected cells. Phosphonoformate, an inhibitor of CMV DNA polymerase, caused 95% block of expression of CMV 'late' proteins in infected cells but did not prevent the switching off of the sodium current or the increased expression of the potassium current. The results indicate an association between the expression of CMV 'immediate-early' or 'early' proteins and the down-regulation of the sodium current and up-regulation of the potassium current.

Cells, Cultured↗

PubMed1995

G-protein involvement in muscarinic receptor-stimulation of inositol phosphates in longitudinal smooth muscle from the small intestine of the guinea-pig.

1. Aluminium fluoride (AlF), pertussis toxin (PTX) and cholera toxin (ChTX) have been used to examine the involvement of G-proteins during muscarinic acetylcholine receptor (AChR) stimulation of inositol phospholipid hydrolysis in fragments of longitudinal smooth muscle from the small intestine of the guinea-pig. 2. Carbachol (CCh) induced time- and concentration-dependent increases in [3H]-inositol monophosphates, [3H]-inositol (1,4) bisphosphate, [3H]-inositol (1,3,4) trisphosphate, [3H]-inositol (1,4,5) trisphosphate ([3H]-Ins (1,4,5)P3) and [3H]-inositol tetrakisphosphates measured by h.p.l.c. These increases were inhibited > 95% in the presence of the muscarinic AChR antagonist atropine (0.5 microM). 3. AlF transiently increased the basal levels of [3H]-Ins (1,4,5)P3 but increases in the levels of the other [3H]-inositol phosphates occurred more slowly. CCh-induced increases in the levels of all the [3H]-inositol phosphates were strongly inhibited in the presence of AlF. 4. PTX had no effect on basal levels of any of the [3H]-inositol phosphates but reduced the effects of CCh on these; ChTX had no effects on either basal or CCh-stimulated levels. 5. It was concluded that muscarinic AChR-stimulated increases in the levels of [3H]-inositol phosphates occur via both a PTX-sensitive G-protein and a PTX-insensitive mechanism. The actions of AlF may suggest the involvement of an inhibitory G-protein in the regulation of muscarinic AChR-stimulated inositol phospholipid turnover.

Aluminum↗

PubMed1995

Effect of UK-84149 on voltage-activated calcium currents of single smooth muscle cells from guinea-pig and rabbit jejunum and rabbit coronary artery.

1. Smooth muscle cells of the longitudinal muscle of the guinea-pig and the rabbit jejunum and rabbit coronary artery were dispersed by enzyme treatment and recordings of membrane current were made in the standard whole cell voltage-clamp mode and by perforated patch technique. The effect of UK-84149 on the voltage-dependent calcium inward current of single isolated smooth muscle was studied at room temperature. 2. When inward and outward currents were evoked by stepping to 0 mV or + 10 mV from -80 mV every 20 s, UK-84149 reduced the voltage-dependent inward current in guinea-pig jejunum smooth muscle cells with a ID50 of 2.3 microM, and also reduced voltage-dependent outward current with a ID50 of 7.2 microM. The effect of UK-84149 was difficult to wash out. 3. The inactivation of the inward current was studied by holding at various potentials for 10 s before evoking inward current at a test potential of 0 mV. The voltage-inactivation curve was shifted to the left by 3 microM UK-84149. The voltage at which current was 50% available (Vh) was changed from -37 +/- 2 mV to -50 mV +/- 2.2 (n = 6, means +/- s.e. mean). 4. When inward current was evoked by stepping to 0 mV from -80 mV for 50 ms, every 20 s, nicardipine immediately, and verapamil and UK-84149 slowly, reduced the peak amplitude. Inhibition by the latter two drugs depended on the number of stimulations applied. So, like verapamil, UK-84149 had a use-dependent action. 5. The effect of UK-84149 on inward current from rabbit jejunum and coronary artery smooth muscle cells was compared with the effect of nicardipine and D600. When inward current was evoked by stepping to 0 mV from - 80 mV every 20 s, UK-84149 inhibited inward currents from both types of smooth cell with an ID50 of 1.7 microM for jejunum and an ID50 of 3.2 microM for coronary artery. ID50 values of D600 were 8.1 microM for jejunum and 3.9 microM for coronary artery. ID50 values of nicardipine were 24 nM for jejunum and 12 nM for coronary artery.6. The results of the present studies indicate that UK-84149 has a calcium-antagonist action that can explain its inhibitory action on bowel motility. Its bowel selectivity may arise due to its use-dependent,and persistent, action.

Animals↗

PubMed1995

Effects of GTP gamma S on muscarinic receptor-stimulated inositol phospholipid hydrolysis in permeabilized smooth muscle from the small intestine.

1. Smooth muscle fragments from the longitudinal layer of the small intestine of the guinea-pig were permeabilized with Staphylococcus aureus alpha toxin (alpha-toxin) and used to investigate the role of G-protein activation in the regulation of muscarinic acetylcholine receptor (AChR)-stimulated inositol phospholipid hydrolysis. 2. The efficiency of alpha-toxin permeabilization was estimated by the release of [3H]-2-deoxyglucose ([3H]-2DG) after prior loading or lactate dehydrogenase (LDH) enzyme release from the smooth muscle fragments. 3. In alpha-toxin-permeabilized smooth muscle, but not in non-permeabilized muscle, GTP gamma S induced time- and concentration-dependent increases in labelled inositol phosphates. Carbachol (CCh) increased labelled inositol phosphates in both permeabilized and non-permeabilized muscle, although the increases were greater in non-permeabilized smooth muscle. The response to 100 microM CCh was severely reduced by 0.5 microM atropine. 4. In permeabilized muscle the effects of GTP gamma S or CCh on inositol phosphate levels were reduced by treatment with pertussis toxin (PTX) and completely inhibited by GDP beta S. 5. GTP gamma S caused a concentration-dependent inhibition of the CCh-induced increases in the levels of labelled inositol phosphates. Dibutyryl cyclic AMP or Sp-cAMPs (adenosine-3',5'-cyclic phosphorothiolate-Sp) reduced the effects of CCh on inositol phosphate levels. 6. The results suggest that muscarinic AChR activation induces inositol phospholipid hydrolysis via more than one G-protein in this smooth muscle and that several mechanisms may contribute to the modulation of both stimulatory and inhibitory responses observed.

Animals↗

PubMed1995

Action of angiotensin II, 5-hydroxytryptamine and adenosine triphosphate on ionic currents in single ear artery cells of the rabbit.

1. Angiotensin II, 5-hydroxytryptamine (5-HT) and adenosine triphosphate (ATP) evoked a transient inward current in isolated single car artery cells of rabbit held at -60 mV by whole cell voltage clamp in physiological saline using a KCL-containing pipette solution. Under these conditions agonist did not activate a calcium-dependent potassium current. 2. Responses to each agonist were transient and desensitized rapidly. Inward current at -60 mV holding potential was not abolished by blockade of voltage-dependent calcium channels or by buffering intracellular calcium with BAPTA, a calcium chelator, or following depletion of intracellular calcium stores with ryanodine. 3. The shape of the current-voltage relationships and the reversal potentials of the current induced by angiotensin II, 5-HT and ATP were similar under a variety of ionic conditions. Agonist-induced current was unaffected by replacing intracellular chloride with citrate ions or by replacing intracellular sodium with caesium or extracellular sodium with barium or calcium. Replacement of extracellular sodium with Tris shifted the reversal potential in all cases by around 30 mV negatively. 4. These data suggest that angiotensin II, 5-HT and ATP activate similar cationic conductances which are relatively non-selective allowing mono- and divalent cations to cross the smooth muscle cell membrane. These channels may allow the influx of calcium under physiological conditions.

Adenosine Triphosphate↗