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A F Brading

Publications and source records attributed to A F Brading.

At least 55 records · Page 3Linked to original sources
PubMed1998

Nitric oxide synthase in the heterogeneous population of intramural striated muscle fibres of the human membranous urethral sphincter.

PURPOSE: Nitric oxide (NO) is known to relax urethral smooth muscle. The role of NO in the control of urethral striated muscle remains unknown. We have investigated the distribution of nitric oxide synthase (NOS) immunoreactivity and its possible relationship with subtypes of intramural striated muscle fibers in the human male membranous urethra. MATERIALS AND METHODS: Whole transverse cryostat sections from seven membranous urethrae were studied using NOS immunohistochemistry and NADPH diaphorase histochemistry. Striated fiber subtypes were demonstrated using immunohistochemistry for troponin T and histochemistry for myofibrillary adenosine triphosphatase (ATPase). Consecutive sections were used to assess the correlation between the distribution of NOS immunoreactivity and the type of striated fibers. RESULTS: NOS immunoreactivity and NADPH diaphorase activity were detected in the sarcolemma of 48.5% of the intramural striated muscle fibers. NOS immunoreactive nerve trunks and fine nerve fibers, a few of which appeared to end on muscle fibers, were present in the striated sphincter. Fast twitch fibers were detected by ATPase staining, and also exhibited positive immunoreactivity for troponin T, constituting 34.6% of the total number of striated fibers. Two populations of slow twitch fibers were identified; one with small diameter (mean: 15.7 microns) and another of larger diameter (mean: 21.7 microns) comparable to that of fast twitch fibers. 86% of the fast twitch fibers and 29% of slow twitch fibers (most of which had larger diameters) exhibited NOS immunoreactivity and NADPH diaphorase activity in the sarcolemma. CONCLUSIONS: The presence of nitrergic nerve fibers in the striated urethral sphincter suggests an involvement in the innervation of urethral striated muscle. Furthermore, the presence of NOS immunoreactivity in the sarcolemma may indicate a role for NO in the regulation of urethral striated muscle metabolism and contraction.

Adolescent↗
PubMed1997

Role of nitric oxide in relaxation of the longitudinal layer of rectal smooth muscle.

PURPOSE: This study was designed to investigate the role of nitric oxide in neurogenic relaxation of the longitudinal layer of human rectal smooth muscle. METHODS: Tissue was obtained from the mid rectum of patients undergoing anterior resection for carcinoma. Adjacent strips of longitudinal muscle were dissected and mounted in organ baths for isometric tension recording. In preliminary experiments to determine the response of strips to cholinergic, adrenergic, and potential excitatory agonists, strips were superfused with standard Krebs solution (37 +/- 0.5 degrees C; pH, 7.4 +/- 0.05). Investigation of inhibitory, nonadrenergic noncholinergic responses required the addition of 3 x 10(-6) M histamine to induce reproducible and stable tension for five-minute "test" periods, during which electrical field stimulation (EFS) and additional drugs were applied. In these experiments, strips were superfused with Krebs solution that contained atropine sulfate (3 x 10(-6) M) and guanethidine (3 x 10(-6) M). RESULTS: The response to cholinergic and adrenergic agonists was typical of nonsphincter specialized gastrointestinal smooth muscle. EFS elicited frequency-dependent, neurogenic (tetrodotoxin-sensitive) relaxations of precontracted strips, which were reduced in dose-dependent fashion by addition of N omega-nitro-L-arginine and restored by addition of 3 x 10(-4) M L-arginine but not by D-arginine. Addition of exogenous nitric oxide (sodium nitroprusside) mimicked the relaxant response induced by EFS. CONCLUSION: Smooth muscle from the longitudinal layer of human rectum receives an intrinsic inhibitory innervation mediated by nitric oxide.

Aged↗
PubMed1997

Activity of glibenclamide-sensitive K+ channels under unstimulated conditions in smooth muscle cells of pig proximal urethra.

The membrane potential in pig proximal urethra was examined by use of the microelectrode technique. In cells 1-2 cm from the bladder neck the membrane potential was quiescent, with a value of -37.2+/-2.5 mV (n = 16). In some cells small spontaneous de- and hyperpolarizations were seen. Glibenclamide (1 microM) caused a small but significant depolarization in tissue strips (12+/-3 mV, n = 3) and also in dispersed cells using whole-cell patch electrodes (13+/-3 mV, n = 5). In the conventional whole-cell voltage-clamp configuration, glibenclamide reduced the noise level of the basal membrane current at -50 mV and inhibited the membrane current in symmetrical 140 mM K+ conditions. In cell-attached patches, brief openings of a glibenclamide-sensitive 43 pS K+ channel (K(GS)-43 pS) were seen even under unstimulated conditions and greater activation occurred in the same membrane patch on subsequent application of 100 microM levcromakalim. These results provide direct evidence that glibenclamide-sensitive K+ channels may play a role in maintaining the resting membrane potential of pig proximal urethra under unstimulated conditions.

Animals↗
PubMed1997

A myogenic basis for the overactive bladder.

OBJECTIVES: This article summarizes evidence supporting the conclusion that the spontaneous rises in pressure that occur in the overactive bladder, particularly in detrusor instability (DI), regardless of etiology, are myogenic. METHODS: The evidence quoted has been obtained by several groups of investigators and includes electrophysiologic studies of detrusor myocytes, tension studies of strips of smooth muscle, in vivo experiments on animal models, and light and electron microscopic studies of the structure of the bladder wall. RESULTS: The results of these studies demonstrate changes in the properties, structure, and innervation of the detrusor. These changes are consistent with the hypothesis that partial denervation of the detrusor may be responsible for altering the properties of the smooth muscle, leading to increased excitability and increased ability of activity to spread between cells, resulting in coordinated myogenic contractions of the whole detrusor. CONCLUSIONS: It is suggested that alterations in the properties of the detrusor myocytes are a necessary prerequisite for the production of the unstable pressure rises seen in DI of any origin.

Animals↗
PubMed1997

Smooth muscle of the bladder in the normal and the diseased state: pathophysiology, diagnosis and treatment.

The smooth muscle of the normal bladder wall must have some specific properties. It must be very compliant and able to reorganise itself during filling and emptying to accommodate the change in volume without generating any intravesical pressure, but whilst maintaining the normal shape of the bladder. It must be capable of synchronous activation to generate intravesical pressure at any length to allow voiding. The cells achieve this through spontaneous electrical activity combined with poor electrical coupling between cells, and a dense excitatory innervation. In the diseased state, alterations of the smooth muscle may lead to failure to store or failure to empty properly. The diseased states discussed are bladder instability and diabetic neuropathy. Bladder instability is characterised urodynamically by uninhibitable rises in pressure during filling, and is seen idiopathically and in association with bladder outflow obstruction and neuropathy. In diabetic neuropathy, many of the smooth muscle changes are a consequence of diuresis, but there is evidence for alterations in the sensory arm of the micturition reflex. In the unstable bladder, additional alterations of the smooth muscle are seen, which are probably caused by the patchy denervation that occurs. The causes of this denervation are not fully established. Nonsurgical treatment of instability is not yet satisfactory; neuromodulation has some promise, but is expensive, and the mechanisms poorly understood. Pharmacological treatment is largely through muscarinic receptor blockade. Drugs to reduce the excitability of the smooth muscle are being sought, since they may represent a better pharmacological option.

Animals↗
PubMed1997

Effects of levcromakalim and nucleoside diphosphates on glibenclamide-sensitive K+ channels in pig urethral myocytes.

1. Effects of levcromakalim and nucleoside diphosphates (NDPs) on both membrane currents and unitary currents in pig proximal urethra were investigated by use of patch clamp techniques (conventional whole-cell configuration, nystatin perforated patch, cell-attached configuration and inside-out patches). 2. Levcromakalim produced a concentration-dependent outward current at a holding potential of -50 mV. The peak current amplitude showed little variation when measured by either conventional whole-cell or nystatin perforated patch configurations. 3. In conventional whole-cell configuration, the levcromakalim (100 microM)-induced outward current decayed by about 90% in 18 min. In contrast, with the nystatin perforated patch, approximately 86% of the levcromakalim-induced outward current still remained after 18 min. 4. The peak amplitude of the levcromakalim (100 microM)-induced outward membrane current recorded by the conventional whole-cell configuration was greatly reduced by inclusion of 5 mM EDTA in the pipette. The much smaller but significant outward membrane current remaining was abolished by glibenclamide. 5. In conventional whole-cell recordings, inclusion of an NDP in the pipette solution induced a small outward current which slowly reached a maximal amplitude (in 2 to 10 min) and was suppressed by glibenclamide. Addition of 100 microM levcromakalim after the NDP-induced current had peaked activated a further outward current which was larger than that recorded in the absence of NDPs. Approximately 50% of this current still remained at 18 min, even when conventional whole-cell configuration was used. 6. In the cell-attached mode in symmetrical 140 mM K+ conditions, glibenclamide inhibited the 100 microM levcromakalim-activated 43 pS K+ channel in a concentration-dependent manner, showing an inhibitory dissociation constant (Ki) of approximately 520 nM. 7. In inside-out patches in which the glibenclamide-sensitive K+ channel had run down after exposure to levcromakalim, both uridine 5'-diphosphate (UDP) and MgATP were capable of reactivating the channel. Further application of Mg2+ to the UDP-reactivated K+ channels enhanced the channel activity reversibly. 8. In inside-out patches UDP was capable of activating the glibenclamide-sensitive K+ channel without levcromakalim, providing that there was free Mg2+ present (either UDP in 5 mM EGTA or UDP in 5 mM EDTA with Mg2+). Additional application of levcromakalim caused a further reversible activation of channel opening. 9. In the presence of levcromakalim, application of adenosine 5'-triphosphate (ATP) to the inner surface of the membrane patch inhibited UDP-reactivated channel opening in a concentration-dependent manner. 10. Addition of an untreated cytosolic extract of pig proximal urethra reactivated the glibenclamide-sensitive K+ channel in the presence of 100 microM levcromakalim in inside-out patches. 11. These results demonstrate the presence in the pig proximal urethra of a glibenclamide-sensitive K+ channel that is blocked by intracellular ATP and can be activated by levcromakalim. Intracellular UDP can reactivate the channel after rundown. Additionally, intracellular Mg2+ may play an important role in regulating the channel activity.

Adenosine Triphosphate↗
PubMed1997

The in vivo and in vitro effects of hypoxia on pig urethral smooth muscle.

OBJECTIVE: To assess the in vitro effects of hypoxia on urethral smooth muscle and the role that these may have in the fall in urethral luminal pressure consequent upon interruption of its arterial blood supply. MATERIALS AND METHODS: In eight anaesthetized female Large White pigs, femoral blood pressure, urethral luminal pressure and lamina propria blood flow were recorded before, during and after aortic occlusion. In three animals, changes in urethral intramural oxygen tension were also recorded. Smooth muscle strips taken from the mid-urethra were studied in an organ bath under normal and hypoxic conditions, and in the presence of cyanide. The generation of spontaneous tone and the response to electrical-field stimulation were also examined. RESULTS: Urethral luminal pressure declined biphasically after aortic occlusion, from a mean of 83.8 to 39.0 cmH2O. The initial rapid phase seemed to be related to a decrease in urethral lamina propria blood flow, whilst the slower phase appeared to mirror the observed decline in intramural oxygen tension. In vitro, the high spontaneous tone of the muscle strips was abolished by hypoxia or metabolic inhibition whilst the contractile response to nerve stimulation was preserved. CONCLUSIONS: The decrease in urethral pressure after aortic occlusion appears to have two components, the first mediated through a decrease in vascular filling of the lamina propria and the second as a result of hypoxia-induced smooth muscle relaxation. The in vitro effects of hypoxia upon urethral smooth muscle concur with the changes observed in vivo.

Animals↗
PubMed1997

A case-control study to examine any association between idiopathic detrusor instability and gastrointestinal tract disorder, and between irritable bowel syndrome and urinary tract disorder.

OBJECTIVE: To assess whether there are common malfunctions (e.g. of the autonomic nervous system and smooth muscle) that underlie disorders of the urinary and gastrointestinal tracts by determining whether there is an increased prevalence (i) of urinary symptoms in patients with irritable bowel syndrome (IBS) and (ii) of gastrointestinal symptoms in patients with idiopathic detrusor instability (IDI). PATIENTS AND METHODS: Questionnaires were sent to patients with a diagnosis of IBS or IDI who were seen in the Departments of Gynaecology, Gastroenterology and Urology at the John Radcliffe and Churchill Hospitals, Oxford, during the 3 year period 1993-1995. The questionnaires were also distributed to control patients who were recruited from the day-surgery unit of the Churchill Hospital. Of 236 questionnaires sent out, 168 replies were analysed; 64 from patients with IBS, 49 from patients with detrusor instability and 55 from controls. The questionnaire included questions about micturition and defecatory behaviour (frequency, regularity, urgency, continence, pain, and ease in passing urine and stools). RESULTS: Patients with IBS were more likely to experience certain urinary symptoms than controls (nocturia, urgency and some forms of urinary urge incontinence) and patients with IDI were as likely as patients with IBS to experience gastrointestinal symptoms more frequently than controls. Control patients showed an unexpectedly high probability of experiencing many of the gastrointestinal and urinary symptoms. CONCLUSIONS: The frequent occurrence of symptoms in control patients makes the significance of the results less clear, but the association between certain symptoms of urinary tract disorder and patients with IBS, and of symptoms of gastrointestinal tract disorder with patients with IDI, suggests that they may share some common underlying dysfunction.

Adult↗
PubMed1997

Nicorandil activates glibenclamide-sensitive K+ channels in smooth muscle cells of pig proximal urethra.

The effects of nicorandil on ionic currents recorded from single smooth muscle cells of pig proximal urethra were investigated using patch-clamp techniques. Tension measurement was also performed to study the effects of nicorandil on the resting tone of pig urethra. Nicorandil produced a concentration-dependent sustained outward current that was suppressed by glibenclamide at -50 mV and was carried selectively by K+. In cell-attached configuration, nicorandil activated a 43-pS K+ channel that was reversibly inhibited by 10 microM glibenclamide. This glibenclamide-sensitive 43-pS K+ channel (KGS) "ran down" after excision of the membrane patch. In inside-out configuration, the application of either 1 mM Mg-ATP or 1 mM nucleotide diphosphate reactivated the KGS. In symmetrical 140 mM K+ conditions, 300 microM nicorandil and 300 microM levcromakalim activated a 2.14-pA K+ channel that exhibited the same amplitude and similar channel-opening kinetics. Methylene blue (10-100 microM), a soluble guanylate cyclase inhibitor, did not inhibit the opening of the nicorandil-induced KGS. The KGS was not activated by either sodium nitroprusside (10-100 microM) or 8-bromo guanosine 3':5'-cyclic monophosphate (1 mM). Nicorandil caused a concentration-dependent relaxation of the urethral resting tone but was less potent than levcromakalim. The relaxation induced by 10 microM nicorandil was partially inhibited by glibenclamide (1-10 microM) and also by methylene blue (10-100 microM). These results indicate that two independent nicorandil-induced relaxation mechanisms may be present in pig urethra.

Adenosine Triphosphate↗
PubMed1997

The effect of anoxia and glucose-free solutions on the contractile response of guinea-pig detrusor strips to intrinsic nerve stimulation and the application of excitatory agonists.

PURPOSE: To study the effects of anoxia and substrate depletion, both separately and combined, on the contractile responses of guinea-pig detrusor smooth muscle strips to activation of intrinsic nerves, application of agonists and depolarization with high potassium solution. MATERIALS AND METHODS: Bladders were opened and the urothelium removed. Strips of detrusor were dissected and mounted for tension recording in small organ baths superfused with warmed solutions of known composition. RESULTS: Anoxia caused a small initial reduction of the contractile responses which then remained constant for several hours. Glucose-free solutions resulted in a slow progressive decline in the responses to field stimulation, carbachol and high potassium solution, with substantial responses still present after five hours. The response to ATP, however, was unaffected. Removal of oxygen and glucose, mimicking ischaemia, caused abolition of all responses within an hour. After reintroduction of normal conditions responses reached their maximal extent of recovery within an hour. Recovery was almost complete for responses to ATP and carbachol, but less so for high K+ solutions. Very little recovery to stimulation of intrinsic nerves was seen. CONCLUSIONS: The results demonstrate that phasic contractions can be fuelled almost fully by oxidative phosphorylation or by anaerobic glycolysis, but that in conditions mimicking ischaemia the intrinsic nerves are more susceptible to ischaemic damage than the detrusor smooth muscle.

Adenosine Triphosphate↗
PubMed1996

Electrical properties of smooth muscle in the guinea-pig urinary bladder.

1. The effects of transmural nerve stimulation were examined on preparations of detrusor smooth muscle from guinea-pig urinary bladder using intracellular recording techniques. Most recordings were made from preparations in which spontaneous and evoked action potentials had been inhibited by nifedipine (10 microM), a dihydropyridine that blocks L-type Ca2+ channels. 2. Supramaximal stimuli evoked excitatory junction potentials (EJPs) which could be divided into three basic types. Type 1 EJPs had short latencies (< 30 ms) and fast rise times (< 60 ms). Type 2 EJPs consisted of two components: a small depolarization that was followed by a second depolarization with a faster rise time. In a third type of cell, at high strengths of stimulation, EJPs resembled type 1 EJPs but at lower strengths of stimulation were similar in time course to type 2 EJPs. 3. All EJPs were abolished by tetrodotoxin (1 microM) and reduced by omega-conotoxin (0.1 microM), but were unaffected by hexamethonium (0.1 mM), suggesting that they result from the release of transmitter from post-ganglionic nerve fibres. All responses persisted in the presence of atropine (1 microM) but were abolished following the desensitization of P2-purinoceptors with alpha, beta-methylene ATP (m-ATP; 10 microM). 4. Spontaneous excitatory junction potentials (SEJPs) were also recorded from most cells. SEJPs were similar in appearance to fast single-component EJPs; however, in general they had a briefer time course. SEJPs persisted in the presence of tetrodotoxin (1 microM). 5. The electrical properties of urinary bladder smooth muscle were also examined. Voltage changes induced by point current injection into cells had fast rates of rise and decay (time constant, 5-20 ms). The input resistance of cells ranged between 12 and 108 M omega. When recordings were taken from cells near the point of current injection, resultant electrotonic potentials could be detected in only a small proportions of cells. 6. The results are discussed in relation to the idea that transmural nerve stimulation in the guinea-pig urinary bladder causes the activation of at least two different membrane conductances. Cells appear to be electrically coupled with one another. However, it is likely that coupling exists within discrete bundles of the smooth muscle.

Adenosine Triphosphate↗
PubMed1996

Nitric oxide and the rectoanal inhibitory reflex: retrograde neuronal tracing reveals a descending nitrergic rectoanal pathway in a guinea-pig model.

Nitric oxide has been implicated as the neurotransmitter mediating internal anal sphincter (IAS) relaxation during the rectoanal inhibitory reflex. However, there has been no direct demonstration of a nitrergic rectoanal neuronal pathway appropriate to mediating the reflex. This study combined retrograde neuronal tracing techniques with enzyme histochemistry in a guinea-pig model. Wheatgerm agglutinin conjugated to horseradish peroxidase was injected into the IAS. Transported tracer was demonstrated in neurones of the myenteric ganglia of the distal rectum and all labelled neurones showed co-localization with nitric oxide synthase as revealed by reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. In vivo anal canal manometry showed that the mean maximal resting pressure was 16 (8-20) cmH2O and confirmed the presence of the rectoanal inhibitory reflex. In vitro organ bath studies showed that strips of IAS developed spontaneous myogenic tone and relaxed in response to intrinsic nerve stimulation. Addition of N omega-nitro-L-arginine (L-NOArg) reduced the relaxant response in a dose-dependent fashion; the relaxant response was maximally reduced by a mean(s.e.m.) 35.2(3.8) per cent (P < 0.001) at a concentration of 3 x 10(-5) mol/l L-NOArg. This study provides direct anatomical evidence of a descending nitrergic rectoanal neuronal pathway in a guinea-pig model. In vivo anal manometry and in vitro organ bath studies provide additional evidence that this pathway is responsible for the inhibitory motor innervation of the rectoanal inhibitory reflex.

Anal Canal↗
PubMed1996

Nitrergic innervation and relaxant response of rectal circular smooth muscle.

PURPOSE: This study was designed to investigate whether nitric oxide mediates inhibitory innervation in human rectal circular smooth muscle. METHODS: Tissue was obtained from the midrectum of patients undergoing anterior resection for carcinoma. Adjacent strips of circular muscle were dissected and mounted in superfusion organ baths for isometric tension recording and initially loaded with 1 g of weight. Strips were continuously bathed with standard Krebs solution (37 degrees C, bubbled with 97 percent O2/3 percent CO2) containing 3 X X 10(-6) M guanethidine and 3 X 10(-6) M atropine sulfate to block adrenergic and muscarinic cholinergic neurotransmission. After equilibration, strips had no intrinsic tone, and reproducible and stable tension was, therefore, induced by the addition of 3 X 10 M histamine for five-minute "test" periods, during which electrical field stimulation (EFS) and additional drugs were applied. RESULTS: EFS elicited frequency-dependent, neurogenic (tetrodotoxin-sensitive) relaxations of precontracted strips. Addition of N-w-nitro-L-arginine, a powerful competitive inhibitor of nitric oxide synthase, reduced the relaxant response to EFS in a dose-dependent fashion, and effect reversed by addition of s X 10(-4) M L-arginine but not by D-arginine. Addition of exogenous nitric oxide (sodium nitroprusside) mimicked the relaxant response induced by EFT. CONCLUSIONS: Human rectal circular smooth muscle receives an intrinsic inhibitory innervation mediated by nitric oxide. The presennce of a residual response following blockade of the enzyme nitric oxide synthase suggests the involvement of additional neurotransmitters.

Adrenergic Agents↗
PubMed1996

Activation by levcromakalim and metabolic inhibition of glibenclamide-sensitive K channels in smooth muscle cells of pig proximal urethra.

1. The effects of levcromakalim (BRL 38227) on ionic currents recorded from pig proximal urethra were investigated by use of tension measurement and patch clamp techniques (conventional whole-cell configuration, nystatin perforated patch, and cell-attached configuration). 2. Levcromakalim (1 microM) caused a relaxation in the resting tone. This levcromakalim-induced relaxation was inhibited by the pretreatment with 1 microM glibenclamide. 3. The resting membrane potential recorded from single cells in current-clamp mode was-36.1 +/- 4.4 mV (n = 5). 4. Levcromakalim induced a concentration-dependent hyperpolarization with a maximum (at > or = 10 microM) close to the theoretical equilibrium potential of potassium (EK). The membrane hyperpolarization caused by 1 microM levcromakalim (24.7 +/- 5.8 mV, n = 4) was abolished by 1 microM glibenclamide. 5. Levcromakalim (100 microM) caused an outward K current in whole-cell recordings which was unaffected by iberiotoxin (300 nM) but abolished by glibenclamide (10 microM). 6. In cell-attached patches, levcromakalim activated a 43 pS K channel which was inhibited by the application of glibenclamide. 7. The metabolic poison, cyanide (CN), also activated a 43 pS K channel which was suppressed by the application of 10 microM glibenclamide. 8. These results indicate that levcromakalim and metabolic inhibition activate the same 43 pS K channel in pig proximal urethra. The resultant urethral hyperpolarization might reduce the usefulness of K channel openers in the treatment of detrusor instability, but be of value in treating outflow obstruction.

Animals↗
PubMed1996

Role of intracellular Ca2+ in the K channel opener action of CGRP in the guinea-pig ureter.

1. The aim of this study was to assess the role of sarcoplasmic reticulum (SR) calcium (Ca2+) in the smooth muscle relaxant and hyperpolarizing actions of calcitonin gene-related peptide (CGRP) in the guinea-pig ureter. 2. CGRP (0.1 microM) rapidly and transiently reduced myogenic phasic contractions (twitches) produced by electrical field stimulation (EFS). Approximately 70% of the response to CGRP was antagonized by glibenclamide (1 microM). 3. Cyclopiazonic acid (CPA, 10 microM), ryanodine (100 microM) and thapsigargin (1 microM) reduced only the glibenclamide-sensitive component of the response to CGRP (0.1 microM) but did not modify the mechano-inhibitory effect of cromakalim (3 microM). A low concentration of CPA (1 microM), assumed to produce a limited impairment of Ca2+ uptake from the stores, prolonged the duration of the inhibitory response to CGRP. Pre-exposure to caffeine (5 mM) inhibited the suppression of twitches by CGRP or cromakalim. 4. When the frequency of EFS was increased, the suppression of twitches by CGRP was reduced. Under these conditions, CPA (1 microM) again prolonged the duration of the inhibitory response to CGRP. 5. CGRP (0.1 microM) and cromakalim (3 microM) markedly depressed the phasic component of contractions to 80 mM KCl. CPA (10 microM) antagonized the inhibitory effect of CGRP but not that of cromakalim. Inhibition of the tonic contraction to 80 mM KCl by CGRP was insensitive to CPA. 6. In sucrose gap experiments, a 5 min exposure to CGRP (0.1 microM) or cromakalim (3 microM) produced a sustained membrane hyperpolarization. Caffeine (5 mM) produced a glibenclamide-sensitive transient hyperpolarization followed by a sustained depolarization. When tested in a Ca(2+)-free medium the hyperpolarization produced by CGRP, cromakalim or caffeine was reduced. In normal Krebs, pre-exposure to CPA (10 microM, 60 min) only abolished the hyperpolarization induced by CGRP. In contrast, 5 min after a caffeine challenge (5 mM) the hyperpolarizations induced by CGRP or cromakalim were reduced. The CGRP-induced hyperpolarization was insensitive to apamin (0.1 microM) or charybdotoxin (0.1 microM). 7. We conclude that the K channel-opening action of CGRP in the guinea-pig ureter requires the mobilization of intracellular Ca2+ from a caffeine- and CPA-sensitive store, leading to transient activation of glibenclamide-sensitive K channels. The K channel-opening action of caffeine appears to involve Ca2+ mobilization from a store which is insensitive to depletion by CPA.

Animals↗
PubMed1996

Long Ca2+ channel opening induced by large depolarization and Bay K 8644 in smooth muscle cells isolated from guinea-pig detrusor.

1. In smooth muscle cells enzymatically isolated from guinea-pig urinary bladder, Ca2+ channel currents were recorded by conventional cell-attached patch clamp techniques. In most recordings Bay K 8644 (2 microM) was contained in the patch pipette. 2. Closure of Ca2+ channels observed during the repolarizing steps was significantly slowed by preconditioning with large depolarizations (+80 and 100 mV), with or without Bay K 8644 in the pipette. 3. The sum of the unitary Ca2+ channel current traces obtained after large conditioning depolarizations (in the presence of Bay K 8644) showed a slowly deactivating tail current. 4. By use of this slow deactivating feature, the current-voltage relationship of the unitary Ca2+ channel current was continuously measured with a ramp pulse after large depolarization. The slope conductance ranged from 22 to 30 pS, compatible with that of L-type Ca2+ channels. 5. It is concluded that L-type Ca2+ channels in guinea-pig detrusor cells are open for much longer after large depolarizations consistent with their being two channel open states, and that Bay K 8644 prolongs the lifetime of both open states. The underlying mechanisms are discussed.

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