Influence of the pattern of jejunal distension on mesenteric afferent sensitivity in the anaesthetized rat.

Vagal, spinal and intestino-fugal fibres all potentially transmit mechanosensory afferent information from the gastrointestinal tract. We aimed to characterize the relative mechanosensitivity of these three different afferent populations supplying the rat jejunum. Afferent nerve discharge was recorded from pentobarbitone-anaesthetized rats during different distension protocols. Saline ramp distension (1 mL min(-1)) and barostat ramp distension (2 mmHg 4 s(-1)) each evoked biphasic responses but with the latter significantly attenuated especially at low distending pressures. Barostat controlled phasic distensions (10-50 mmHg, 25 s) evoked an afferent response with a peak at the onset of distension adapting to a plateau level that was maintained and comparable to the barostat ramp responses at the corresponding pressures. Chronic subdiaphragmatic vagotomy significantly attenuated the low pressure component of the response to balloon ramp distension and both peak and plateau responses to phasic distension. Single unit analysis showed an absence of low threshold afferent activity after vagotomy while the response to fibres with wide-dynamic range and high threshold sensitivity were preserved hexamethonium had no effect on the responses to either ramp or phasic distension. These findings suggest that the nature of the distension stimulus is critical in determining the pattern of response observed from the various subpopulations of afferents supplying the bowel wall.

Cosensitivity of vagal mucosal afferents to histamine and 5-HT in the rat jejunum.

A complex sensitivity of afferent nerves in the mesentery of the rat jejunum to systemic administration of histamine has recently been demonstrated. In the present study, we aimed to characterize subpopulations of mesenteric afferents that mediate this afferent nerve response. Multiunit afferent discharge was recorded from mesenteric nerves supplying the proximal jejunum in anesthetized rats. The majority of mesenteric bundles (84%) exhibited biphasic responses to histamine (8 micromol/kg), and these bundles also responded to 2-methyl-5-HT (2m5HT). In contrast, monophasic responses lacked a short-latency component, and these bundles failed to respond to 2m5HT. Single-unit analysis revealed a population of afferents that possessed cosensitivity for 2m5HT and histamine. This population of afferents was absent in chronically vagotomized animals, whereas mucosal anesthesia with luminal lidocaine reversibly converted the biphasic profile to a monophasic one. Ondansetron (500 microg/kg) blocked the response to 2m5HT with no effect on the profile of the histamine response, whereas pyrilamine (5 mg/kg) blocked the histamine response without affecting the response to 2m5HT. We conclude that histamine-sensitive afferents exist in the rat proximal jejunum that also respond to 5-HT via the 5-HT3 receptor. These fibers appear to be vagal afferents originating in the intestinal mucosa and may be involved in the organization of mast cell-mediated responses.

Vagal afferent responses to fatty acids of different chain length in the rat.

The role of cholecystokinin (CCK) in the effect of dietary lipid on proximal gastrointestinal function and satiety is controversial. Recent work suggests that fatty acid chain length may be a determining factor. We investigated the mechanism by which long- and short-chain fatty acids activate jejunal afferent nerves in rats. Whole mesenteric afferent nerve discharge was recorded in anaesthetized male Wistar rats during luminal perfusion of saline, sodium oleate, and sodium butyrate (both 10 mM). Both fatty acids evoked characteristic afferent nerve responses, distinct from the mechanical response to saline, that were abolished in rats following chronic subdiaphragmatic vagotomy. The effect of oleate was abolished by the CCK-A receptor antagonist Devazepide (0.5 mg/kg), whereas the effect of butyrate persisted despite pretreatment with either Devazepide or a combination of the calcium channel inhibitors nifedipine (1 mg/kg) and the omega-conotoxins GVIA and SVIB (each 25 microg/kg). In summary, long- and short-chain fatty acids activate intestinal vagal afferents by different mechanisms; oleate acts via a CCK-mediated mechanism and butyrate appears to have a direct effect on afferent terminals.

Somatostatin sst(2) receptor-mediated inhibition of mesenteric afferent nerves of the jejunum in the anesthetized rat.

BACKGROUND & AIMS: Octreotide inhibits visceral sensations in clinical studies, but the site of action and the receptor type(s) involved are unknown. Our aim was to investigate the effects of octreotide, the selective sst(2) receptor agonist (BIM 23027), and the sst(2) antagonist (Cyanamid154806) on the activity of mesenteric afferent fibers innervating the rat jejunum. Their effects were investigated on baseline discharge, mechanosensitivity, and responses to algesic chemicals. METHODS: Extracellular multiunit recordings of jejunal afferent nerve firing were made in pentobarbitone-anesthetized (60 mg/kg intraperitoneally) male Wistar rats. RESULTS: Octreotide and BIM23027 (0.001-100 microg/kg intravenously) each evoked a long-lasting inhibition of baseline discharge, which was blocked by cyanamid 154806 (3 mg/kg) and absent in chronically vagotomized animals. Afferent responses to bradykinin were also inhibited by an sst(2) receptor-mediated mechanism but were unaffected by vagotomy. Ramp distentions of the jejunum evoked a biphasic activation of afferent nerve discharge, the low threshold component of which was attenuated in vagotomized animals. Sst(2) receptor agonists significantly inhibited the mechanosensitivity of spinal, but not vagal, afferents. CONCLUSIONS: These data suggest that activation of somatostatin sst(2) receptors inhibit populations of mesenteric afferents likely to be involved in nociceptive transmission.

Receptors and transmission in the brain-gut axis: potential for novel therapies. I. Receptors on visceral afferents.

Visceral afferents are the information superhighway from the gut to the central nervous system. These sensory nerves express a wide range of membrane receptors that can modulate their sensitivity. In this themes article, we concentrate on those receptors that enhance the excitability of visceral afferent neurons. Some receptors are part of a modality-specific transduction pathway involved in sensory signaling. Others, which are activated by substances derived from multiple cellular sources during ischemia, injury, or inflammation, act in a synergistic fashion to cause acute or chronic sensitization of the afferent nerves to mechanical and chemical stimuli. Such hypersensitivity is the hallmark of conditions such as irritable bowel syndrome. Accordingly, these receptors represent a rational target for drug treatments aimed at attenuating both the inappropriate visceral sensation and the aberrant reflex activity that are the foundation for alterations in bowel function.

5-HT(3) and histamine H(1) receptors mediate afferent nerve sensitivity to intestinal anaphylaxis in rats.

BACKGROUND & AIMS: The mechanisms underlying brain stem activation during antigen challenge have not been resolved. Our aim was to characterize afferent nerve responses to intestinal anaphylaxis and determine the mediators involved in afferent activation. METHODS: Mesenteric afferent discharge was recorded electrophysiologically after intestinal anaphylaxis in anesthetized rats previously sensitized to chicken egg albumin (EA). RESULTS: Mesenteric afferent nerve discharge increased approximately 1 minute after luminal antigen but not bovine serum albumin (P < 0.001, EA vs. bovine serum albumin). Subsequent administration of antigen had no effect, but systemic EA evoked a marked increase in afferent discharge (P < 0. 05). Afferent responses were unrelated to intestinal motor activity, and the response to luminal antigen was attenuated by luminal anesthetic (1% lidocaine). The 5-HT(3)-receptor antagonist alosetron (30 microg. kg(-1)) and the histamine H(1)-receptor antagonist pyrilamine (5 mg. kg(-1)) markedly attenuated the response to luminal antigen; pretreatment with doxantrazole attenuated responses to both luminal and systemic antigen. CONCLUSIONS: 5-HT(3) and histamine, released from mast cells after intestinal anaphylaxis, stimulate mesenteric afferents via 5-HT(3) and histamine H(1) receptors. Information on intestinal immune status is rapidly relayed to the central nervous system and may play a role in neural reflexes and behavioral responses following activation of the immune system.

Excitatory effect of P2X receptor activation on mesenteric afferent nerves in the anaesthetised rat.

1. We examined the effects of P2X purinoceptor agonists and P2 purinoceptor antagonists on mesenteric afferent nerves supplying the jejunum in the pentobarbitone sodium-anaesthetised rat. 2. ATP (0. 01-10 mg kg-1, i.a.) and alpha,beta-methylene-ATP (1-30 microg kg-1, i.a.) each induced dose-dependent increases in afferent nerve discharge and intrajejunal pressure. The effect on afferent nerves comprised an early (< 2 s after administration) intense burst of activity followed by a later increase (> 2 s after administration), less pronounced in comparison, which coincided with elevated intrajejunal pressure. 3. Pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (20 mg kg-1, i.v.) and suramin (80 mg kg-1, i.v. ) each antagonised both the early and later increases in afferent nerve discharge elicited by alpha,beta-methylene-ATP (30 microg kg-1, i.a.). 4. Co-administration of omega-conotoxin MVIIA and omega-conotoxin SVIB (each at 25 microg kg-1, i.v.), or treatment with the selective 5-HT3 receptor antagonist alosetron (30 microg kg-1, i.v.), did not affect the rapid burst of afferent nerve activity elicited by alpha,beta-methylene-ATP (30 microg kg-1, i.a.). However, toxin treatment did attenuate the elevations in intrajejunal pressure and the corresponding later phases of evoked afferent discharge, while alosetron inhibited basal afferent nerve activity. 5. In summary, ATP and alpha,beta-methylene-ATP each evoke excitation of mesenteric afferent nerves in the anaesthetised rat. We propose that the early increase in mesenteric afferent nerve activity represents a direct effect on the nerve ending, mediated by P2X receptors, whereas the later increase reflects activation of mechanosensitive fibres secondary to elevated intrajejunal pressure.

Characterization of adenosine receptors evoking excitation of mesenteric afferents in the rat.

We examined the effects of adenosine receptor agonists and antagonists on the discharge of mesenteric afferent nerves supplying the jejunum in pentobarbitone sodium-anaesthetized rats. Adenosine (0.03-10 mg kg(-1), i.v.), NECA (0.3-300 microg kg(-1), i.v.) and the A1 receptor agonist, GR79236 (0.3-1000 microg kg(-1), i.v.), each induced dose-dependent increases in afferent nerve activity and intrajejunal pressure, hypotension and bradycardia. The A1 receptor antagonist, DPCPX (3 mg kg(-1), i.v.), antagonized all the effects of GR79236 but only the haemodynamic effects of adenosine and NECA. The A2A receptor antagonist, ZM241385 (3 mg kg(-1), i.v.), antagonized the hypotensive effect of NECA but none of the effects of GR79236. The A2A receptor agonist, CGS21680 (0.3-300 microg kg(-1), i.v.), and the A3 receptor agonist, IB-MECA (0.3-300 microg kg(-1), i.v.), each induced only a dose-dependent hypotension. Subsequent administration of adenosine (3 mg kg(-1), i.v.) induced increases in afferent nerve activity and intrajejunal pressure and bradycardia. ZM241385 (3 mg kg(-1), i.v.) antagonized the hypotensive effect of CGS21680 but not the effects of adenosine. Bethanechol (300 microg kg(-1), i.v.) evoked increases in afferent nerve activity and intrajejunal pressure, hypotension and bradycardia. However, adenosine (3 mg kg(-1), i.v.) evoked greater increases in afferent nerve activity than bethanechol despite inducing smaller increases in intrajejunal pressure. In summary, A1 and A2B and/or A2B-like receptors evoke adenosine-induced increases in mesenteric afferent nerve activity and intrajejunal pressure in the anaesthetized rat. Furthermore, elevations in intrajejunal pressure do not wholly account for adenosine-evoked excitation of mesenteric afferent nerves.

Histamine sensitivity of mesenteric afferent nerves in the rat jejunum.

The concept of functional interaction between mast cells and intestinal afferents is gaining support. We have therefore characterized the action of histamine on jejunal afferent discharge in the anesthetized rat. Whole nerve mesenteric afferent discharge was recorded in conjunction with intestinal pressure in response to a range of histamine agonists and antagonists. Histamine at 2, 4, and 8 micromol/kg (iv) evoked a dose-dependent biphasic increase in afferent discharge together with a biphasic rise in intestinal pressure. However, these two events were mediated independently, since nifedipine (1 mg/kg) substantially reduced the intestinal pressure increase but not the afferent discharge. These responses were completely inhibited by pyrilamine (5 mg/kg) but unaffected by ranitidine (5 mg/kg) or thioperamide (2 mg/kg). Neither the selective H2 receptor agonist dimaprit nor the selective H3 receptor agonist R-alpha-methylhistamine caused any modulation of afferent discharge. We conclude that histamine stimulates an H1 receptor-mediated increase in mesenteric afferent discharge that is independent of intestinal motor events. This suggests that histamine potentially acts as a mediator in mast cell-to-afferent nerve communication in the small intestine.

Direct and indirect actions of 5-hydroxytryptamine on the discharge of mesenteric afferent fibres innervating the rat jejunum.

1. This study was performed to elucidate the actions of 5-hydroxytryptamine (5-HT) on mesenteric afferent discharge and to determine the receptor-mechanisms responsible for these effects. The activity of mesenteric afferents innervating the mid-jejunum of urethane-anaesthetized rats was recorded with extracellular microelectrodes. The discharge of single nerves within the whole nerve recording was monitored using waveform discriminator software. 2. The intravenous injection of 5-HT produced a complex pattern of afferent activation with two distinct components which could be distinguished both in terms of the response characteristics and the receptors involved. Initially, in 64% of nerve bundles, there was a brief (2.0 +/- 0.1 s) but intense activation of afferent discharge with peak afferent firing increasing with incremental doses of 5-HT. The discharge frequency in seventeen single units from these bundles during the initial response to 10 micrograms 5-HT was 13.0 +/- 1.8 impulses s-1 from a baseline discharge of 1.0 +/- 0.1 impulses s-1. 3. This initial response was mimicked by the 5-HT3 receptor agonist, 2-methyl-5-HT, whereas 5-methoxytryptamine (5-MEOT, 10-100 micrograms) had no comparable effect. Similarly, the initial 4. 5-HT response was completely abolished by the 5-HT3 receptor antagonist, granisetron (0.5 mg kg-1). 5-HT also evoked, in approximately 35% of nerve bundles, a delayed response that single unit analysis showed to be mediated by an entirely different population of afferents from those activated during the initial response. This secondary response to 5-HT was characterized by a more prolonged (> 30 s) but less intense period of afferent activity which was coincident with an increase in intrajejunal pressure, and was mimicked by 5-MEOT (10-100 micrograms). The secondary response to 5-HT and the response to 5-MEOT were significantly attenuated by the 5-HT2A receptor antagonist, ketanserin (0.5 mg kg-1), which had no effect on the initial response. The initial response to 5-HT was unaffected by the L-type calcium channel inhibitor nifedipine (1 mg kg-1) or the N-type calcium channel inhibitor omega-conotoxin GVIA (25 micrograms kg-1). However, the secondary response to 5-HT was significantly reduced after treatment with nifedipine. 5. These results demonstrate that 5-HT activates different populations of afferent fibres innervating the rat jejunum. One population of afferents is activated directly via stimulation of 5-HT3 receptors, while another population responds to 5-HT with a time course consistent with secondary activation of mechanosensitive afferents following 5-HT2A-mediated contractile activity.

Mesenteric afferent sensitivity to cholecystokinin and 5-hydroxytryptamine.

The present electrophysiological investigation examines the effect of CCK and 5-hydroxytryptamine on gastrointestinal afferent fibre discharge. 5-HT markedly stimulated mesenteric afferents. The response was transient (< 10s) and mediated by 5-HT3 receptors as demonstrated by the action of 2-methyl-5-HT and antagonism by granisetron. CCK was also a potent stimulus to mesenteric afferents causing a long-lasting (> 30s) increase in excitability. The response to CCK was mediated via the CCKA receptor as shown by the antagonistic action of devazepide. At doses of granisetron and devazepide which completely block the response to exogenous 5-HT and CCK, the afferent fibres still responded to both mechanical and chemical stimulation of the mucosa. Thus products of enteroendocrine cells can have profound effects on mucosal afferent sensitivity but do not play an obligatory role in afferent signal transduction.

The role of endogenous cholecystokinin in the sensory transduction of luminal nutrient signals in the rat jejunum.

Some vagal afferent fibres are exquisitely sensitive to exogenous administration of cholecystokinin (CCK) but their sensitivity to endogenous CCK released by luminal stimuli has not been demonstrated directly, although implied from reflex and behavioural studies. We have therefore utilised electrophysiological techniques to record afferent discharge in mesenteric nerve bundles supplying the rat jejunum in response to luminal application of casein acid hydrolysate (CAH). CAH stimulated whole nerve afferent discharge in both in vivo and in vitro preparations (P < 0.01) while single unit analysis revealed that fibres sensitive to CAH also responded to exogenous CCK. The responses to both CCK and CAH were abolished by the CCKA antagonist devazepide. This study therefore supports the hypothesis that a functional relationship exists between CCK-containing enteroendocrine cells and the afferent fibres whose terminals lie within close proximity.

Studies on the effects of anandamide in rat hepatic artery.

1. The effects of anandamide on K+ currents and membrane potential have been examined in freshly-isolated smooth muscle cells from rat hepatic artery and the results compared with the effects of this arachidonic acid derivative on tension and membrane potential changes in segments of whole artery. 2. In the presence of 0.3 mM L-NOARG and 10 microM indomethacin, anandamide (0.1-100 microM) and endothelium-derived hyperpolarizing factor (EDHF; liberated by acetylcholine, 0.01-10 microM) each relaxed endothelium-intact segments of hepatic artery precontracted with phenylephrine. These effects of anandamide, but not those of EDHF, were antagonized by the cannabinoid receptor antagonist, SR141716A (3 microM). 3. The relaxant effects of anandamide were unaffected by a toxin combination (apamin plus charybdotoxin, each 0.3 microM) which abolishes EDHF relaxations and were essentially unchanged in endothelium-denuded arteries. The relaxant effects of anandamide in endothelium-intact arteries were significantly reduced in a physiological salt solution containing 30 mM KCl and abolished when the K+ concentration was raised to 60 mM. 4. Anandamide (10 microM), acetylcholine (1 microM, via release of EDHF) and levcromakalim (10 microM) each markedly hyperpolarized the membrane potential of the smooth muscle cells of endothelium-intact arteries. However, when the endothelium was removed, the hyperpolarizing effects of both anandamide (10 microM) and acetylcholine were essentially abolished whereas those of levcromakalim (10 microM) were unaffected. 5. Under voltage-clamp conditions, anandamide (10 microM) abolished spontaneous transient outward currents (STOCs) in freshly-isolated single hepatic artery cells held at 0 mV but had no effect on the holding current at this potential. In current-clamp mode, the spontaneous hyperpolarizing potentials which corresponded to the STOCs were abolished with no significant change in basal membrane potential. 6. Anandamide (10 microM) abolished the iberiotoxin-sensitive K+ current (IBK(Ca)) produced by caffeine and the corresponding hyperpolarizations generated by this xanthine derivative in current-clamp mode. In contrast, anandamide had no effect on IBK(Ca) generated on exposure to NS1619 (30 microM). 7. It was concluded that anandamide is not EDHF in the rat hepatic artery. Anandamide-induced hyperpolarization is exerted indirectly and requires the presence of the endothelium. Anandamide also acts on the smooth muscle cells to inhibit processes which require functional intracellular calcium stores. This direct action seems more important than membrane hyperpolarization in relaxing phenylephrine-contracted vessels.

Modulation of membrane currents and mechanical activity by niflumic acid in rat vascular smooth muscle.

The effects of niflumic acid on whole-cell membrane currents and mechanical activity were examined in the rat portal vein. In freshly dispersed portal vein cells clamped at -60 mV in caesium (Cs+)-containing solutions, niflumic acid (1-100 microM) inhibited calcium (Ca2+)-activated chloride currents (IC1(Ca)) induced by caffeine (10 mM) and by noradrenaline (10 microM). In a potassium (K+)-containing solution and at a holding potential of - 10 mV, niflumic acid (10-100 microM) induced an outward K+ current (IK(ATP)) which was sensitive to glibenclamide (10-30 microM). At concentrations < 30 microM and at a holding potential of -2 mV, niflumic acid had no effect on the magnitude of the caffeine- or noradrenaline-stimulated current (IBK(Ca)) carried by the large conductance, Ca(2+)-sensitive K+ channel (BKCa). However, at a concentration of 100 microM, niflumic acid significantly inhibited IBK(Ca)) evoked by caffeine (10 mM) but not by NS1619 (1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3 H) benzimidazolone; 20 microM). In Cs(+)-containing solutions, niflumic acid (10-100 microM) did not inhibit voltage-sensitive Ca2+ currents. In intact portal veins, niflumic acid (1-300 microM) inhibited spontaneous mechanical activity, an action which was partially antagonised by glibenclamide (1-10 microM), and contractions produced by noradrenaline (10 microM), an effect which was glibenclamide-insensitive. It is concluded that inhibition of ICl(Ca) and stimulation of IK(ATP) both contribute to the mechano-inhibitory actions of niflumic acid in the rat portal vein.

Pharmacological characterization of the inwardly-rectifying current in the smooth muscle cells of the rat bladder.

1. In freshly-isolated single cells of the rat bladder detrusor, outwardly-rectifying and inwardly-rectifying membrane currents were identified by the whole-cell voltage-clamp technique. 2. The inwardly-rectifying current (IIR) exhibited features of a cation current permeable to both K+ and Na+ but it was unaffected by changes in extracellular Ca2+. It had an activation threshold close to -60 mV and an estimated reversal potential of -29 mV. 3. IIR activated slowly with a voltage-sensitive time-constant of 69 ms at -140 mV and 209 ms at -100 mV but did not exhibit time-dependent inactivation. 4. IIR was unaffected by tetraethylammonium (up to 20 mM) but it was reduced by extracellular Ba2+ (1 mM) and by extracellular Cs+ (1 mM). 5. IIR was reduced by terikalant (100 microM) and markedly inhibited by ciclazindol (100 microM) although at these concentrations, both agents also reduced outward currents. 6. IIR was inhibited by ZD7288 (10-100 microM) in a concentration-dependent manner. At concentrations up to 30 microM, ZD7288 did not reduce the magnitude of outward currents but these were inhibited by 100 microM ZD7288. 7. In strips of bladder detrusor, spontaneous mechanical activity was increased by ZD7288 (0.3-100 microM) and by ciclazindol (0.3-100 microM) but was unaffected by glibenclamide (1-10 microM). 8. It is concluded that IIR closely resembles the hyperpolarization-activated current Ih, previously described in the smooth muscle of rabbit jejunum and in a variety of other cell types. This current may play an important role in modulating detrusor excitability but this could not be confirmed using the inhibitors ZD7288 and ciclazindol.

Investigation of the effects of 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) on membrane currents in rat portal vein.

1. The effects of 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) were investigated on evoked and spontaneous currents in freshly-isolated cells from the rat portal vein by use of conventional whole-cell recording and perforated-patch techniques. 2. At a holding potential of -60 mV in potassium-free, caesium-containing solutions, NPPB (10 microM) inhibited calcium (Ca)-sensitive chloride currents (ICl(Ca)) evoked by caffeine (10 mM) and by noradrenaline (10 microM) by 58% and 96%, respectively. 3. At a holding potential of -2 mV in potassium (K)-containing solutions, NPPB (10 microM) inhibited charybdotoxin-sensitive K-currents (IBK(Ca)) induced by noradrenaline (10 microM) and acetylcholine (10 microM) by approximately 90%. In contrast, IBK(Ca) induced by caffeine (10 mM) was unaffected in the presence of NPPB (10 microM). Conversely, IBK(Ca) elicited by caffeine (2 mM) was reduced by approximately 50% whereas IBK(Ca) evoked by noradrenaline (50 microM) was not significantly inhibited by NPPB. 4. In K-containing solutions, NPPB (10 microM) abolished spontaneous transient outward currents (STOCs) and induced a slowly-developing outward K-current. Bath application of glibenclamide (10 microM) abolished the outward current but did not antagonize the inhibitory effects of NPPB on STOCs or on IBK(Ca) evoked by noradrenaline. 5. In caesium-containing solutions, NPPB (30 microM) inhibited voltage-sensitive Ca-currents. 6. In Ca-free, K-containing solutions and in the presence of glibenclamide (5 microM), IBK(Ca) induced by 20 microM NS1619 was enhanced by NPPB (10 microM). 7. It is concluded that NPPB inhibits agonist-induced ICl(Ca) in rat portal vein smooth muscle. However, this agent also inhibits agonist-evoked IBK(Ca) and STOCs. Moreover, NPPB inhibits voltage-sensitive Ca-currents and stimulates a glibenclamide-sensitive K-current and IBK(Ca). The effects of this agent on evoked ICl(Ca) and IBK(Ca) and on STOCs probably involves an inhibitory action on intracellular Ca-stores.