Effect of age on the enteric nervous system of the human colon.

The effect of age on the anatomy and function of the human colon is incompletely understood. The prevalence of disorders in adults such as constipation increase with age but it is unclear if this is due to confounding factors or age-related structural defects. The aim of this study was to determine number and subtypes of enteric neurons and neuronal volumes in the human colon of different ages. Normal colon (descending and sigmoid) from 16 patients (nine male) was studied; ages 33-99. Antibodies to HuC/D, choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), and protein gene product 9.5 were used. Effect of age was determined by testing for linear trends using regression analysis. In the myenteric plexus, number of Hu-positive neurons declined with age (slope = -1.3 neurons/mm/10 years, P = 0.03). The number of ChAT-positive neurons also declined with age (slope = -1.1 neurons/mm/10 years of age, P = 0.02). The number of nNOS-positive neurons did not decline with age. As a result, the ratio of nNOS to Hu increased (slope = 0.03 per 10 years of age, P = 0.01). In the submucosal plexus, the number of neurons did not decline with age (slope = -0.3 neurons/mm/10 years, P = 0.09). Volume of nerve fibres in the circular muscle and volume of neuronal structures in the myenteric plexus did not change with age. In conclusion, the number of neurons in the human colon declines with age with sparing of nNOS-positive neurons. This change was not accompanied by changes in total volume of neuronal structures suggesting compensatory changes in the remaining neurons.

Increased colonic transit in rats produced by a combination of a cholinesterase inhibitor with a 5-HT4 receptor agonist.

Increased cholinergic stimulation and accelerated gastrointestinal (GI) transit may be produced by direct stimulation of the acetylcholine (ACh) receptor with an appropriate agonist by increased release of ACh from cholinergic nerve terminals or by a decreased removal or breakdown of ACh within cholinergic synapses. The acetylcholinesterase inhibitor, neostigmine, and the 5-HT(4) receptor partial agonist tegaserod, are two agents with known prokinetic activity which work by different mechanisms that result in increased levels of ACh at cholinergic synapses innervating intestinal smooth muscle. Here, we aimed to investigate the potential synergistic effect on colonic transit that may occur with concomitant use of these two agents. Colonic transit was indirectly assessed in rats via measurements of fecal pellet output every 30 min for 2.5 h following administration of neostigmine (0.003-0.1 mg kg(-1), i.p.), tegaserod (0.01-1.0 mg kg(-1), i.p.), or a combination of both compounds. When administered alone, neostigmine or tegaserod caused a dose-dependent increase in fecal pellet output. In combination, low doses of the two agents which did not produce statistically significant effects alone, compared to the vehicle, caused a significant increase in fecal pellet output. Combinations of higher doses of neostigmine and tegaserod did not display synergy. In summary, when combined at low doses, neostigmine and tegaserod produce synergistic effects manifested by a statistically significant increase in the expulsion of fecal pellets. A combination of an anticholinesterase agent with a 5-HT(4) receptor agonist may prove to be a useful therapeutic approach to treat conditions associated with slow GI transit.

Inhibition of endothelial cell adhesion molecule expression improves colonic hyperalgaesia.

Leucocyte-endothelial cell interactions are prerequisite to leucocyte infiltration and intestinal inflammation. GI270384X is a novel inhibitor of ICAM-1 and E-selectin expression and inhibits leucocyte adhesion and improves experimental colitis. We hypothesized that GI270384X maybe effective in treatment of visceral hyperalgaesia. Visceromotor behavioural responses to colorectal distension (CRD) were obtained in naïve rats or rats treated with zymosan (3 h) or 2,4,6-trinitrobenzene sulphonic acid (TNBS) (4 and 30 days) or rats exposed to acute restraint stress. Studies were also performed in a high-anxiety genetic model of colonic hyperalgaesia using Wistar-Kyoto (WKY) rats. Rats were treated orally with GI270384X or vehicle either prior to or after the administration of sensitizing stimulus. The visceromotor response to CRD was significantly enhanced in all models. GI270384X attenuated the enhanced responses to distension induced by inflammatory stimuli (TNBS and zymosan) and in the high-anxiety WKY rats; however, the drug did not inhibit the hypersensitivity induced by acute restraint stress. GI270384X was most potent in the models of acute inflammatory hyperalgaesia with a minimum efficacious dose (MED) of 0.3 and 1 mg kg(-1) observed in the TNBS and zymosan models respectively. The compound was less potent in the chronic and postinflammatory models with an MED of 10 and 30 mg kg(-1) observed in the WKY and 30-day TNBS models respectively. These findings show for the first time that inhibition of leucocyte-endothelial cell interactions can have a beneficial effect on visceral hyperalgaesia associated with inflammatory and chronic anxiety states, but is less effective against stress-associated visceral hyperalgaesia.

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.

Neurogastroenterology of tegaserod (HTF 919) in the submucosal division of the guinea-pig and human enteric nervous system.

Actions of the 5-HT(4) serotonergic receptor partial agonist, tegaserod, were investigated on mucosal secretion in the guinea-pig and human small intestine and on electrophysiological behaviour of secretomotor neurons in the guinea-pig small intestinal submucosal plexus. Expression of 5-HT(4) receptor protein and immunohistochemical localization of the 5-HT(4) receptor in the submucosal plexus in relation to expression and localization of choline acetyltransferase and the vesicular acetylcholine (ACh) transporter were determined for the enteric nervous system of human and guinea-pig small intestine. Immunoreactivity for the 5-HT(4) receptor was expressed as ring-like fluorescence surrounding the perimeter of the neuronal cell bodies and co-localized with the vesicular ACh transporter. Exposure of mucosal/submucosal preparations to tegaserod in Ussing chambers evoked increases in mucosal secretion reflected by stimulation of short-circuit current. Stimulation of secretion had a relative high EC(50) of 28.1 +/- 1.3 mumol L(-1), was resistant to neural blockade and appeared to be a direct action on the secretory epithelium. Tegaserod acted at presynaptic 5-HT(4) receptors to facilitate the release of ACh at nicotinic synapses on secretomotor neurons in the submucosal plexus. The 5-HT(2B) receptor subtype was not involved in actions at nicotinic synapses or stimulation of secretion.

TRP channels as therapeutic targets: hot property, or time to cool down?

Transient receptor potential (TRP) channels are involved in a wide range of processes ranging from osmoregulation, thermal, chemical and sensory signalling, and potentially in the pathophysiology associated with several diseases. Patents for TRPV1 antagonists alone span diseases ranging across chronic pain, neuropathies, headache, bladder disorders, irritable bowel syndrome (IBS), gastro-oesophageal reflux disease (GORD), and cough amongst others. Most research is currently focused around those TRP channels involved in sensory processes, with the neurogastroenterology and motility field playing a major role, for example, through recent discoveries of differential roles for TRPV receptor subtypes in chemosensitivity and mechanosensitivity of visceral afferents. At this time, however, the understanding of the role of even TRPV1, let alone most of the other TRP channels in disease pathophysiology is only just beginning, and although enthusiasm around the therapeutic potential for modulators of these channels is understandable, based largely upon the experience of the effects of natural ligands, such as capsaicin, the sheer size and complexity of the TRP family as a whole must serve as a warning against expecting too much too soon from drug discovery efforts.

5-HT2B receptors do not modulate sensitivity to colonic distension in rats with acute colorectal hypersensitivity.

The 5-HT4 receptor agonist tegaserod is an effective prokinetic agent that increases gastrointestinal secretion and reduces visceral sensitivity. Tegaserod has both 5-HT4 receptor agonist and 5-HT2B receptor antagonist activity, the latter being a less potent effect of the drug. In a rat model of colonic hypersensitivity, selective 5-HT4 receptor antagonists only partially reversed the antihyperalgesic effect of tegaserod suggesting that non-5-HT4 receptor-mediated mechanisms may also be involved in its overall antihyperalgesic action. The objective of this study was to determine whether 5-HT2B receptors play a role in colonic hypersensitivity. A visceromotor response (VMR) in acutely sensitized animals (intracolonic acetic acid, 0.6%, 1.5 mL) quantified colonic hypersensitivity. Acetic acid produced an increase in the VMR at all distension pressures. However, neither the 5-HT2B receptor agonist BW 723C86, the 5-HT2B antagonist SB204741 or the 5-HT2B/2C antagonist SB 206553 caused any significant inhibition of the VMR. In summary, in the same rodent model in which tegaserod has previously been shown to produce a potent antihyperalgesic effect, 5-HT2B receptors do not appear to mediate colonic hypersensitivity. We conclude that 5-HT2B receptor-mediated mechanisms are unlikely to play a role in the antihyperalgesic action of tegaserod in man.

Vagal selective effects of ruthenium red on the jejunal afferent fibre response to ischaemia in the rat.

A variety of inflammatory mediators and local metabolites, have been implicated in the sensitivity of intestinal afferent fibres to brief periods of ischaemia and reperfusion. As yet, the contribution of the vanilloid transient receptor potential (TRPV)1 receptor to the response to intestinal ischaemia remains undetermined. In the present study, the effect of pretreatment with the competitive TRPV1 antagonist capsazepine and the non-selective TRPV channel antagonist ruthenium red, on the mesenteric afferent fibre response to ischaemia was examined. In control animals there was a reproducible biphasic increase in whole nerve afferent fibre activity during two brief periods of ischaemia. Treatment with ruthenium red significantly attenuated the early phase increase in afferent fibre activity during ischaemia. However, capsazepine treatment did not significantly alter the afferent fibre response to either ischaemia or reperfusion. Further experiments in chronically vagotomized animals indicated that the early phase response to ischaemia was mediated via vagal afferent fibres. The mechanism via which ruthenium red selectively inhibited vagal afferent fibres during ischaemia is unknown, but it does not appear to involve blockade of the TRPV1 receptor.

Association between a functional polymorphism in the serotonin transporter gene and diarrhoea predominant irritable bowel syndrome in women.

BACKGROUND AND AIMS: Serotonin (5-hydroxtryptamine, 5-HT) is an important factor in gut function, playing key roles in intestinal peristalsis and secretion, and in sensory signalling in the brain-gut axis. Removal from its sites of action is mediated by a specific protein called the serotonin reuptake transporter (SERT or 5-HTT). Polymorphisms in the promoter region of the SERT gene have effects on transcriptional activity, resulting in altered 5-HT reuptake efficiency. It has been speculated that such functional polymorphisms may underlie disturbance in gut function in individuals suffering with disorders such as irritable bowel syndrome (IBS). The aim of this study was to assess the potential association between SERT polymorphisms and the diarrhoea predominant IBS (dIBS) phenotype. SUBJECTS: A total of 194 North American Caucasian female dIBS patients and 448 female Caucasian controls were subjected to genotyping. METHODS: Leucocyte DNA of all subjects was analysed by polymerase chain reaction based technologies for nine SERT polymorphisms, including the insertion/deletion polymorphism in the promoter (SERT-P) and the variable tandem repeat in intron 2. Statistical analysis was performed to assess association of any SERT polymorphism allele with the dIBS phenotype. RESULTS: A strong genotypic association was observed between the SERT-P deletion/deletion genotype and the dIBS phenotype (p = 3.07x10(-5); n = 194). None of the other polymorphisms analysed was significantly associated with the presence of disease. CONCLUSIONS: Significant association was observed between dIBS and the SERT-P deletion/deletion genotype, suggesting that the serotonin transporter is a potential candidate gene for dIBS in women.

Peripheral opiate action on afferent fibres supplying the rat intestine.

The aim of the present study was to examine the sensitivity of mesenteric afferents supplying the rat small intestine to mu-opioid receptor ligands. Mesenteric afferent discharge was recorded electrophysiologically in response to [D-ALA2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO; 100 mug kg(-1) i.v.), before and after treatment with the mu-receptor antagonist alvimopan (1 mg kg(-1) i.v.). DAMGO markedly stimulated whole nerve mesenteric afferent discharge (P < 0.05), an effect completely blocked by alvimopan. The response of mesenteric afferents to 2-methyl-5-hydroxytryptamine (30 microg kg(-1) i.v.), bradykinin (0.1-1 microg kg(-1) i.a.) and both low- and high-threshold distension (0-60 mmHg) was unaffected by alvimopan. In chronically vagotomized animals, the low-threshold response to distension was attenuated while the remaining high-threshold response was unaffected by alvimopan. In conclusion, mesenteric afferent fibres are markedly stimulated by mu-opioid receptor agonists, an effect blocked by alvimopan, which may contribute to the gastrointestinal reflex and behavioural responses to opiate treatment or abuse. However, alvimopan did not influence the normal sensitivity of intestinal afferents to chemical and mechanical stimuli that activate different subpopulations of vagal and spinal afferents. Thus, alvimopan may be useful for the treatment of gastrointestinal sequelae following opiate treatment for postoperative or chronic pain.

Preclinical studies of opioids and opioid antagonists on gastrointestinal function.

Opioid receptors in the gastrointestinal (GI) tract mediate the effects of endogenous opioid peptides and exogenously administered opioid analgesics, on a variety of physiological functions associated with motility, secretion and visceral pain. The studies reviewed or reported here describe a range of in vivo activities of opioid receptor antagonists upon GI function in rodents, focusing on mu receptors. Naloxone, and the peripherally acting mu-opioid receptor antagonists alvimopan and methylnaltrexone, reverse morphine-induced inhibition of GI transit in mice and rats, and morphine- or loperamide-induced inhibition of castor oil-induced diarrhoea in mice. At doses producing maximal reversal of morphine-induced effects upon GI transit, only the central nervous system (CNS) penetrant antagonist naloxone was able to reverse morphine-induced analgesia. Both central and peripheral opioid antagonists may affect GI function and/or visceromotor sensitivity in the absence of exogenous opioid analgesics, suggesting a constitutive role for endogenous opioid peptides in the control of GI physiology. Furthermore, in contrast to naloxone, alvimopan does not produce hypersensitivity to the visceromotor response induced by nociceptive levels of colorectal distension in a rodent model of post-inflammatory colonic hypersensitivity, suggesting that in the periphery endogenous mu-opioid receptor-mediated mechanisms do not regulate colonic sensitivity. The data support the hypothesis that peripherally acting opioid antagonists may be able to selectively block opioid receptors in the GI tract, thereby preserving normal GI physiology, while not blocking the effects of endogenous opioid peptides or exogenous opioid analgesics in the CNS. These findings suggest that the primary sites of action of mu-opioid agonists with respect to inhibition of GI function are in the periphery, whereas analgesic activity resides primarily in the CNS.

Characterization of the primary spinal afferent innervation of the mouse colon using retrograde labelling.

Visceral pain is the most common form of pain produced by disease and is thus of interest in the study of gastrointestinal (GI) complaints such as irritable bowel syndrome, in which sensory signals perceived as GI pain travel in extrinsic afferent neurones with cell bodies in the dorsal root ganglia (DRG). The DRG from which the primary spinal afferent innervation of the mouse descending colon arises are not well defined. This study has combined retrograde labelling and immunohistochemistry to identify and characterize these neurones. Small to medium-sized retrogradely labelled cell bodies were found in the DRG at levels T8-L1 and L6-S1. Calcitonin gene-related peptide (CGRP)- and P2X3-like immunoreactivity (LI) was seen in 81 and 32%, respectively, of retrogradely labelled cells, and 20% bound the Griffonia simplicifolia-derived isolectin IB4. CGRP-LI and IB4 were co-localized in 22% of retrogradely labelled cells, whilst P2X3-LI and IB4 were co-localized in 7% (vs 34% seen in the whole DRG population). Eighty-two per cent of retrogradely labelled cells exhibited vanilloid receptor 1-like immunoreactivity (VR1-LI). These data suggest that mouse colonic spinal primary afferent neurones are mostly peptidergic CGRP-containing, VR1-LI, C fibre afferents. In contrast to the general DRG population, a subset of neurones exist that are P2X3 receptor-LI but do not bind IB4.

Neuronal location of 5-hydroxytryptamine3 receptor-like immunoreactivity in the rat colon.

The presence of 5-hydroxytryptamine(3) receptors on enteric neurons is known from pharmacological data that date back more than 40 years. However, an adequate account of which neurons bear these receptors has not been made because suitable antisera have not been available. We have found that the majority of antisera that have been raised against sequences from the 5-hydroxytryptamine(3) receptor also recognize pre-prosomatostatin. We report that this source of false labeling can be eliminated by pre-incubating the antisera with a peptide designed for this purpose. We have used the pre-absorbed antiserum to localize 5-hydroxytryptamine(3) receptors in the rat colon. Immunoreactive nerve cell bodies occurred in the myenteric and submucosal ganglia. The majority had smooth cell bodies and long, smooth processes, that is, Dogiel type II morphology. The initial segments of the long processes of the Dogiel type II neurons were strongly immunoreactive. About 12% of immunoreactive myenteric nerve cells were of the same or smaller size, and had multiple short filamentous processes. Some of the immunoreactive Dogiel type II neurons were also immunoreactive for calretinin in both plexuses, and the majority were immunoreactive for calbindin in submucosal ganglia. Specific immunoreactivity occurred in non-varicose, but not in varicose, fibers in the myenteric and submucosal ganglia, and in fiber bundles that traversed the longitudinal and circular muscle layers. Immunoreactive varicose fibers were observed only in the mucosa. It is concluded that 5-hydroxytryptamine(3) receptors occur on intrinsic sensory neurons in the rat colon, and on extrinsic sensory nerve fibers that innervate the colon.

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.

Evidence that P2X purinoceptors mediate the excitatory effects of alphabetamethylene-ADP in rat locus coeruleus neurones.

Extracellular and whole-cell patch clamp recordings were used to study the excitatory responses elicited by purine nucleotides in pontine slices of the rat brain containing the locus coeruleus (LC). The P2 purinoceptor agonists, alphabeta-methyleneadenosine 5'-triphosphate (alphabetameATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPalphabetaS), and a novel purinoceptor agonist, alphabeta-methyleneadenosine 5'-diphosphate (alphabetameADP), elicited concentration-dependent increases in the spontaneous firing rate over the concentration range (1-300 microM). On vagus nerve or dorsal root preparations alphabetameADP (100 microM) had no agonist activity. In the presence of both alphabetameATP (300 microM), ADPbetaS (300 microM) elicited a further and significant increase in the firing rate of the LC neurones, whilst neither alphabetameATP nor alphabetameADP (300 microM) elicited a further response. The P2 purinoceptor antagonists, suramin (100 microM) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 30 microM), markedly attenuated responses to all three agonists. Whole-cell recording of membrane current showed that, at - 60 mV, alphabetameATP and alphabetameADP (both 100 microM) elicited inward currents of a similar magnitude, whilst the inward currents elicited by a lower concentration of ADPbetaS (30 microM) were larger and faded in the presence of this agonist. In the presence of tetrodotoxin and a combination of other neurotransmission blockers, both alphabetameATP and alphabetameADP still produced inward currents. Based on the known selectivity of the agonists used in this study, there appear to be two distinct P2 purinoceptor types present on neurones in the LC, which correspond to the P2X and P2Y types. The responses elicited by alphabetameADP appear to be mediated through a putative P2X purinoceptor, although further work is required to determine which P2X receptor subtype(s) are involved.

Outward current produced by somatostatin (SRIF) in rat anterior cingulate pyramidal cells in vitro.

1. A high density of receptors for somatostatin (SRIF) exists in the anterior cingulate cortex but their function is unknown. Whole-cell patch clamp recordings were made from visualized deep layer pyramidal cells of the rat anterior cingulate cortex contained in isolated brain slices to investigate the putative effects of SRIF and to identify the receptor subtype(s) involved. 2. SRIF (1-1000 nM) produced a concentration-dependent outward current which was associated with an increased membrane conductance, was sensitive to Ba2+ (300 microM - 1 mM), and was absent in the presence of a maximal concentration of the GABA(B) receptor agonist, baclofen (100 microM). These observations suggest the outward current was carried by K+ ions. 3. SRIF analogues also elicited outward currents with a rank potency order of (EC50, nM): octreotide (1.8)>BIM-23027 (3.7)>SRIF (20)=L-362,855 (20). BIM-23056 was without agonist or antagonist activity. Responses to L-362,855 were unlike those to the other agonists since they were sustained for the duration of the application. 4. The sst2 receptor antagonist, L-Tyr8Cyanamid 154806 (1 microM), had no effect alone but partially reversed responses to submaximal concentrations of SRIF (100 nM, 44+/-6% reversal) and L-362,855 (100 nM, 70+/-6% reversal) and fully reversed the response to BIM-23027 (10 nM). In contrast, L-Tyr8Cyanamid 154806 did not antagonize the response to baclofen (10 microM). 5. We conclude that SRIF activates a K+ conductance in anterior cingulate pyramidal neurones via an action predominantly at sst2 receptors.

Ca2+-dependent inactivation of large conductance Ca2+-activated K+ (BK) channels in rat hippocampal neurones produced by pore block from an associated particle.

1. Recordings of the activity of the large conductance Ca2+-activated K+ (BK) channel from over 90 % of inside-out patches excised from acutely dissociated hippocampal CA1 neurones revealed an inactivation process dependent upon the presence of at least 1 microM intracellular Ca2+. Inactivation was characterized by a sudden switch from sustained high open probability (Po) long open time behaviour to extremely low Po, short open time channel activity. The low Po state (mean Po, 0.001) consisted of very short openings (time constant (tau), approximately 0.14 ms) and rare longer duration openings (tau, approximately 3.0 ms). 2. Channel inactivation occurred with a highly variable time course being observed either prior to or immediately upon patch excision, or after up to 2 min of inside-out recording. Inactivation persisted whilst recording conditions were constant. 3. Inactivation was reversed by membrane hyperpolarization, the rate of recovery increasing with further hyperpolarization and higher extracellular K+. Inactivation was also reversed when the intracellular Ca2+ concentration was lowered to 100 nM and was permanently removed by application of trypsin to the inner patch surface. In addition, inactivation was perturbed by application of either tetraethylammonium ions or the Shaker (Sh)B peptide to the inner membrane face. 4. During inactivation, channel Po was greater at hyperpolarized rather than depolarized potentials, which was partly the result of a greater number of longer duration openings. Depolarizing voltage steps (-40 to +40 mV) applied during longer duration openings produced only short duration events at the depolarized potential, yielding a transient ensemble average current with a rapid decay (tau, approximately 3.8 ms). 5. These data suggest that hippocampal BK channels exhibit a Ca2+-dependent inactivation that is proposed to result from block of the channel by an associated particle. The findings that inactivation was removed by trypsin and prolonged by decreasing extracellular potassium suggest that the blocking particle may act at the intracellular side of the channel.

Putative pre- and postsynaptic ATP-sensitive potassium channels in the rat substantia nigra in vitro.

Pre- and postsynaptic adenosine 5'-triphosphate-sensitive potassium (ATP-K+) currents were studied using whole-cell recordings from substantia nigra zona compacta "principal" neurons in midbrain slices. The GABAA and GABAB receptor-mediated synaptic potentials were unaffected by the ATP-K+ channel inhibitor glibenclamide (30 microM) or by the opener diazoxide (500 microM), indicating that ATP-K+ channels on GABA-ergic terminals are not active, nor can they be activated pharmacologically, under control conditions. However, application of a glucose-free solution to reduce intracellular ATP levels caused a reduction of the GABAB IPSP in all neurons. This was substantially reversed by the sulfonylurea inhibitor tolbutamide (300 microM) in 50% of the neurons tested. The reduction of the GABAB IPSP was a presynaptic effect since postsynaptic hyperpolarizations induced by the GABAB receptor agonist baclofen (10 microM) were unaffected by glucose-free solutions. Diazoxide (500 microM) induced a slowly developing hyperpolarization or outward current in 64% of principal neurons, which was tolbutamide- (100-300 microM) or glibenclamide- (30 microM) sensitive. In contrast, the GABAB receptor agonist baclofen (30 microM) induced a rapid hyperpolarization or outward current in all neurons tested that was unaffected by tolbutamide (300 microM). Although both the diazoxide-induced current and the baclofen-induced current were inhibited by Ba2+ (300 microM), the currents elicited by diazoxide and baclofen summated. The reversal potential for the diazoxide-induced current was also less negative than that for baclofen, which was close to EK. In the presence of intracellular cesium, diazoxide induced a tolbutamide-sensitive inward current in a proportion of neurons, indicating that it has other actions in addition to activating a potassium current. Our results suggest that functional ATP-K+ channels exist both pre- and postsynaptically in the SN, where they modulate the activity of principal neurons. They are different to the potassium channels activated by the GABAB receptor agonist baclofen.

Localization of high affinity [3H]glibenclamide binding sites within the substantia nigra zona reticulata of the rat brain.

The rat substantia nigra zona reticulata contains a high density of binding sites for glibenclamide, an adenosine triphosphate-sensitive potassium channel inhibitor, but the precise location of glibenclamide binding sites within this area has not previously been examined. By combining neurochemical lesion and autoradiographical studies we have shown that high affinity [3H]glibenclamide binding sites are located on striatonigral terminals. Unilateral injections of 6-hydroxydopamine into the medial forebrain bundle or of quinolinic acid into the striatum were performed in anaesthetized adult rats to lesion the nigrostriatal and striatonigral pathways respectively. Autoradiography was performed on coronal sections of midbrain with [3H]glibenclamide, [3H]YM-09151-2 (dopamine D2 receptor antagonist) and [3H]SCH 23390 (dopamine D1 receptor antagonist) at three rostrocaudal levels of the substantia nigra. Under the conditions of the incubation [3H]glibenclamide binds primarily to the high affinity site. Following the 6-hydroxydopamine nigrostriatal lesion, D2 receptor binding was reduced (by up to 67%) on the lesioned side at all three levels of the substantia nigra whereas D1 receptor and glibenclamide binding were not significantly affected. In contrast, following striatonigral pathway lesion with quinolinic acid D2 receptor binding was unchanged on the lesioned side, but both D1 receptor and glibenclamide binding were reduced at all three levels (by up to 85% and 63% in the area of maximum lesion, respectively). In adjacent sections, the pattern of D1 binding loss was closely paralleled by the loss of glibenclamide binding. These results demonstrate that the high affinity glibenclamide binding sites of the substantia nigra zona reticulata are, at least in part, located on the terminals of striatonigral projection neurons.