Effects of gamma-aminobutyric acid and peripheral benzodiazepine ligands on duodenal bicarbonate secretion

BACKGROUND: The effects of gamma-aminobutyric (GABA(A))-receptor ligands and peripheral-type benzodiazepine (BZ) ligands on duodenal mucosal bicarbonate secretion (DBS) were studied in thiobarbiturate-anesthetized rats. METHODS: A segment of proximal duodenum was perfused, and bicarbonate secretion was continuously titrated by pH-stat. In some experiments the vagus nerves were dissected free and cut at the neck level. RESULTS: Luminal GABA (10(-8) to 10(-6) M) increased (P < 0.01) DBS dose-dependently, and the GABA(A) antagonist (+)-bicuculline (0.1-2.5 mg/kg intravenously) caused a similar increase (P < 0.01) in DBS. Vagotomy abolished the latter response, suggesting a centrally elicited nervous action of bicuculline mediated by the vagal nerves. This was supported by the absence of an effect of bicuculline administered intra-arterially close to the duodenum. The GABA(A) agonist muscimol had no effect on DBS when administered intravenously (0.01-0.25 mg/kg) or into a lateral brain ventricle (2 microg/kg/h) but counteracted any stimulation by subsequent intravenous bicuculline. The 'peripheral-type' BZ agonist 4'-chlorodiazepam increased DBS when infused close intra-arterially but had no effect when administered intravenously. CONCLUSIONS: Inhibition of GABA(A) receptors related to cholinergic excitatory pathways in the central nervous system stimulates duodenal mucosal bicarbonate secretion and may increase the local mucosal defense. The stimulation by luminal GABA may reflect modulation of the local mucosal control of duodenal bicarbonate secretion.     

Gamma-aminobutyric acid up- and downregulates insulin secretion from beta cells in concert with changes in glucose concentration

Aims/hypothesis The role of gamma-aminobutyric acid (GABA) and A-type GABA receptors (GABA_ARs) in modulating islet endocrine function has been actively investigated since the identification of GABA and GABA_ARs in the pancreatic islets. However, the reported effects of GABA_AR activation on insulin secretion from islet beta cells have been controversial. Methods This study examined the hypothesis that the effect of GABA on beta cell insulin secretion is dependent on glucose concentration. Results Perforated patch-clamp recordings in INS-1 cells demonstrated that GABA, at concentrations ranging from 1 to 1,000 μmol/l, induced a transmembrane current (I_GABA) which was sensitive to the GABA_AR antagonist bicuculline. The current–voltage relationship revealed that I_GABA reversed at −42±2.2 mV, independently of glucose concentration. Nevertheless, the glucose concentration critically controlled the membrane potential (V _M), i.e., at low glucose (0 or 2.8 mmol/l) the endogenous V _M of INS-1 cells was below the I_GABA reversal potential and at high glucose (16.7 or 28 mmol/l), the endogenous V _M of INS-1 cells was above the I_GABA reversal potential. Therefore, GABA dose-dependently induced membrane depolarisation at a low glucose concentration, but hyperpolarisation at a high glucose concentration. Consistent with electrophysiological findings, insulin secretion assays demonstrated that at 2.8 mmol/l glucose, GABA increased insulin secretion in a dose-dependent fashion (p<0.05, n=7). This enhancement was blocked by bicuculline (p<0.05, n=4). In contrast, in the presence of 28 mmol/l glucose, GABA suppressed the secretion of insulin (p<0.05, n=5). Conclusions/interpretation These findings indicate that activation of GABA_ARs in beta cells regulates insulin secretion in concert with changes in glucose levels.     

Alteration in the Sensitivity of GABAA Receptors to Allosteric Modulatory Drugs in Rat Hippocampus After Chronic Intermittent Ethanol Treatment

Chronic intermittentethanol (CIE)-treated rats exhibited a kindlinglike persistent increase in withdrawal hyperexcitability. The alteration of GABA_A receptor (QABA_AR) function in the hippocampus was suggested as a possible mechanism underlying the hyperexcitability observed in CIE rats, because (1) GABA_AR agonist (muscimol)-evoked ³⁸Cl- efflux was decreased; (2) paired-pulse inhibition in the CA1 area, predominantly due to GABA_AR-mediated recurrent inhibition, was persistently decreased; and (3) GABA_AR subunit expression was altered in the hippocampus from CIE rats. To further characterize the functional alteration of GABA_AR after CIE treatment, their sensitivity to acute ethanol, a steroid anesthetic (alphaxalone), and a benzodiazepine inverse agonist (DMCM; methyl-6, 7-dimethoxy-4-ethyl-β-carboline-3-carboxylate) were studied using either synaptically evoked GABA_AR responses or exogenously applied muscimol-evoked responses in hippocampal slices. Bath application of ethanol (60 mM) enhanced the area of GABA_AR-mediated inhibitory postsynaptic potentials in the hippocampal CA1 region from control and CIE rats, and this potentiation was significantly (p = 0.027) greater in CIE rats (98%) than in control rats (53%). The positive modulatory effect of alphaxalone (1 μM) on GABAAR-inhibitory postsynaptic potentials was not significantly different between control and CIE rats (p = 0.375), whereas alphaxalone allosterically increased [³H]flunitrazepam binding in the CA1 area only in CIE rats (by 20 to 25%, p < 0.01), but not in controls. On the other hand, the negative modulatory effect of DMCM (1 μM) on muscimol-evoked responses was significantly larger in CIE rats (p = 0.002). These results suggest that the sensitization of GABA_AR to acute ethanol and benzodiazepine inverse agonists, and possibly neurosteroids, may underlie ethanol dependence after multiple ethanol withdrawal episodes. These altered pharmacological properties are most consistent with changes in the subunit composition in the CA1 area of this rat model of alcohol dependence.     

Metabotropic glutamate and GABAB receptors contribute to the modulation of glucose-stimulated insulin secretion in pancreatic beta cells

Aims/hypothesis: The neurotransmitters glutamate and γ-aminobutyric acid (GABA) could participate in the regulation of the endocrine functions of islets of Langerhans. We investigated the role of the metabotropic glutamate (mGluRs) and GABA_B (GABA_BRs) receptors in this process. Methods: We studied the expression of mGluRs and GABA _BRs in rat and human islets of Langerhans and in pancreatic α-cell and beta-cell lines using RT-PCR and immunoblot analysis. Effects of mGluR and GABA _B R agonists on insulin secretion were determined by radioimmunoassays and enzyme-linked immunoadsorbent assays (ELISAs). Results: We detected mGluR3 and mGluR5 (but not mGluR1, 6 and 7) mRNAs in all of the samples examined. Trace amount of mGluR2 was found in MIN6 beta cells; mGluR4 was identified in rat islets; and mGluR8 expression was detected in rat islets, RINm5F and MIN6 cells. GABA _BR1 a/b and 2 mRNAs were identified in islets of Langerhans and MIN6 cells. The expression of mGluR3, mGluR5, GABA_BR1 a/b and GABA_BR2 proteins was confirmed using specific antibodies. Group I (mGluR1/5) and group II (mGluR2/3) specific mGluR agonists increased the release of insulin in the presence of 3 to 10 mmol/l or 3 to 25 mmol/l glucose, respectively, whereas a group III (mGluR4/6–8) specific agonist inhibited insulin release at high (10–25 mmol/l) glucose concentrations. Baclofen, a GABA_BR agonist, also inhibited the release of insulin but only in the presence of 25 mmol/l glucose. Conclusion/interpretation: These data suggest that mGluRs and GABA_BRs play a role in the regulation of the endocrine pancreas with mechanisms probably involving direct activation or inhibition of voltage dependent Ca²⁺-channels, cAMP generation and G-protein-mediated modulation of K_ATP channels. [Diabetologia (2002) 45: 242–252] Received: 18 September 2001 and in revised form: 5 November 2001     

Duodenal acidity “sensing” but not epithelial HCO3? supply is critically dependent on carbonic anhydrase II expression

Carbonic anhydrase (CA) is strongly expressed in the duodenum and has been implicated in a variety of physiological functions including enterocyte HCO₃⁻ supply for secretion and the “sensing” of luminal acid and CO₂. Here, we report the physiological role of the intracellular CAII isoform involvement in acid-, PGE_2, and forskolin-induced murine duodenal bicarbonate secretion (DBS) in vivo. CAII-deficient and WT littermates were studied in vivo during isoflurane anesthesia. An approximate 10-mm segment of the proximal duodenum with intact blood supply was perfused under different experimental conditions and DBS was titrated by pH immediately. Two-photon confocal microscopy using the pH-sensitive dye SNARF-1F was used to assess duodenocyte pH_i in vivo. After correction of systemic acidosis by infusion of isotonic Na₂CO₃, basal DBS was not significantly different in CAII-deficient mice and WT littermates. The duodenal bicarbonate secretory response to acid was almost abolished in CAII-deficient mice, but normal to forskolin- or 16,16-dimethyl PGE₂ stimulation. The complete inhibition of tissue CAs by luminal methazolamide and i.v. acetazolamide completely blocked the response to acid, but did not significantly alter the response to forskolin. While duodenocytes acidified upon luminal perfusion with acid, no significant pH_i change occurred in CAII-deficient duodenum in vivo. The results suggest that CA II is important for duodenocyte acidification by low luminal pH and for eliciting the acid-mediated HCO₃⁻ secretory response, but is not important in the generation of the secreted HCO₃⁻ ions.     

Gamma-aminobutyric acid (GABAA) receptor-function in a rat model of hepatic encephalopathy

The functional activity of the gamma-aminobutyric acid (GABA_A) receptor-chloride ionophore complex was studied in rats with hepatic encephalopathy (HE) secondary to thioacetamide-induced fulminant hepatic failure (FHF). Muscimol stimulation and benzodiazepine potentiation of GABA receptor-mediated³⁶Cl^– uptake into cerebral cortical synaptoneurosomes was compared in HE and control rats. [³H]Flumazenil binding assays were conducted to determine whether the levels of endogenous benzodiazepine-like ligands in extracts of cortex were increased with stages of encephalopathy in this animal model of HE. In both control and HE rats maximal uptake of³⁶Cl^– via the GABA_A receptor complex occurred at muscimol concentrations of 30M. Potentiation of muscimol-stimulated³⁶Cl^– uptake into synaptoneurosomes by diazepam (5M) was equivalent in both groups. Aqueous extracts of proteolytically digested homogenates of cerebral cortices prepared from control and HE rats were effective in stimulating³⁶Cl^– uptake into synaptoneurosomes. Alkaline organic extracts of proteolytically digested homogenates of cerebral cortices from HE rats were more effective than corresponding extracts from controls at inhibiting the binding of [³H]flumazenil. Inhibition of [³H] fumazenil binding by organic extracts derived from the cerebral cortices of HE rats did not increase with progression of encephalopathy. The results show that muscimol-stimulated³⁶Cl^– uptake into synaptoneurosomes and, consequently, GABA^A receptor-mediated chloride channel function are not significantly altered in the model of HE studied and are consistent with the hypothesis that HE results in an increased availability of one or more endogenous ligands which can augment GABA receptor-gated chloride conductance.     

GABAergic signaling in the rat pineal gland

Pinealocytes secrete melatonin at night in response to norepinephrine released from sympathetic nerve terminals in the pineal gland. The gland also contains many other neurotransmitters whose cellular disposition, activity, and relevance to pineal function are not understood. Here, we clarify sources and demonstrate cellular actions of the neurotransmitter γ‐aminobutyric acid (GABA) using Western blotting and immunohistochemistry of the gland and electrical recording from pinealocytes. GABAergic cells and nerve fibers, defined as containing GABA and the synthetic GAD67, were identified. The cells represent a subset of interstitial cells while the nerve fibers were distinct from the sympathetic innervation. The GABA_A receptor subunit α1 was visualized in close proximity of both GABAergic and sympathetic nerve fibers as well as fine extensions among pinealocytes and blood vessels. The GABA_B1 receptor subunit was localized in the interstitial compartment but not in pinealocytes. Electrophysiology of isolated pinealocytes revealed that GABA and muscimol elicit strong inward chloride currents sensitive to bicuculline and picrotoxin, clear evidence for functional GABA_A receptors on the surface membrane. Applications of elevated potassium solution or the neurotransmitter acetylcholine depolarized the pinealocyte membrane potential enough to open voltage‐gated Ca²⁺ channels leading to intracellular calcium elevations. GABA repolarized the membrane and shut off such calcium rises. In 48–72‐h cultured intact glands, GABA application neither triggered melatonin secretion by itself nor affected norepinephrine‐induced secretion. Thus, strong elements of GABA signaling are present in pineal glands that make large electrical responses in pinealocytes, but physiological roles need to be found.     

Effects of Leukotriene D4, the Antagonist L-649-923, and Arachidonic Acid on Duodenal Bicarbonate Secretion in the Rat in Vivo

Prostaglandins of the E type stimulate bicarbonate secretion by the duodenal mucosa and inhibit gastric acid secretion, effects that have been related to their anti-ulcer activity. Leukotrienes constitute a more recently discovered branch of the arachidonic acid cascade, and C₄ and D₄ have been suggested to be ulcerogenic in the stomach. We have studied the effects of luminal administration of leukotriene D₄ and the leukotriene C₄/D₄ antagonist L-649–923 on duodenal mucosal alkaline secretion in the anaesthetized rat. Leukotriene D₄ (10^?8-10^?6 M) had no significant effects, but the antagonist dose-dependently increased the bicarbonate secretion and also transiently increased the transmucosal electric potential difference. The precursor arachidonic acid (10^?7-10^?6 M) caused a small increase in secretion. The increase in bicarbonate secretion in response to 10^?3 M of the antagonist was of about the same magnitude as that observed with 10^?5 of prostaglandin E₂, and it was abolished by pretreatment with the cyclooxygenase inhibitor indomethacin. The gastroduodenal protective effects of L-649–923 in vivo may reflect an increase in mucosal prostaglandin production rather than leukotriene antagonism.     

Capsaicin-sensitive sensory neurons are involved in bicarbonate secretion induced by lansoprazole,a proton pump inhibitor,in rats

Lansoprazole, a proton pump inhibitor, exerts prominent antiulcer activityvia both antisecretory and mucosal protective actions. Although the antisecretory action has been explained by inactivation of (H⁺, K⁺)-ATPase in parietal cells, the mode of mucosal protective action remains to be elucidated. In the present study, the effect of lansoprazole on duodenal bicarbonate secretion was studied in anesthetized rats to clarify the mode of the mucosal protective action. Lansoprazole (0.1 mM) applied topically to the duodenum significantly (P<0.01) increased bicarbonate secretion by 0.36±0.11 eq/15 min (21±5%) compared with the value in the vehicle control. Topical administration of capsaicin (10 mg/ml) in the duodenum and intravenous infusion of vasoactive intestinal peptide (10 g/kg/hr) increased bicarbonate secretion. Five-minute perfusion of the duodenal loop with 100 mM HCl increased bicarbonate secretion. Administration of lansoprazole (0.3 and 1 mg/kg, intravenously) 60 min before luminal acidification enhanced the acid-induced bicarbonate secretion dose-dependently and significantly (P<0.01). In the capsaicin-pretreated rats, the effects of lansoprazole on basal and acid-induced bicarbonate secretion were significantly (P<0.05) decreased compared with that of control group. These results indicate that lansoprazole increases basal and acid-induced bicarbonate secretion in the duodenum in rats and that capsaicin-sensitive sensory neurons may be involved in the mode of action for these effects.     

Influence of intraduodenal chenodeoxycholic acid on secretory and pressure activity of the stomach and duodenum

The influence of duodenal infusion of bile acid at a concentration similar to that in the common bile-duct (50 mmol/1) on antroduodenal motility, duodenogastric reflux, gastric and duodenal secretion was studied in 10 healthy volunteers. Intraluminal pressures were recorded in the antrum and the first and second parts of the duodenum. Gastric and distal duodenal contents were collected by continuous low pressure sump aspiration during infusion of either saline or chenodeoxycholic acid (CDC) into the second part of the duodenum. Values for duodenogastric reflux and gastric and duodenal secretion were calculated with reference to the recovery of two non-absorbable markers infused into the stomach and second part of the duodenum. Each volunteer received at least 3 h of saline infusion and 2 h of CDC infusion. During saline infusion, duodenogastric reflux varied with the migrating motor complex (MMC), being statistically greater at the end of duodenal phase III activity than at other times (P<0.05). Infusion of CDC abolished the MMC and inhibited antral contractions but the amount of reflux was not increased compared with the saline period. Infusion of CDC also produced marked increases in measured bicarbonate (P<0.001), trypsin (P<0.001), phospholipase A₂ (P<0.05) and endogenous total bile acid (P<0.05) in the duodenum, although gastric acid secretion was unaffected. These findings suggest that bile acid may regulate gastroduodenal motor activity and pancreaticobiliary secretion.     

GABA(A)-current rundown of temporal lobe epilepsy is associated with repetitive activation of GABA(A) "phasic" receptors

A study was made of the “rundown” of GABA_A receptors, microtransplanted to Xenopus oocytes from surgically resected brain tissues of patients afflicted with drug-resistant human mesial temporal lobe epilepsy (mTLE). Cell membranes, isolated from mTLE neocortex specimens, were injected into frog oocytes that rapidly incorporated functional GABA_A receptors. Upon repetitive activation with GABA (1 mM), “epileptic” GABA_A receptors exhibited a GABA_A-current (I_GABA) rundown that was significantly enhanced by Zn²⁺ (≤250 μM), and practically abolished by the high-affinity GABA_A receptor inverse agonist SR95531 (gabazine; 2.5–25 μM). Conversely, I_GABA generated by “control” GABA_A receptors microtransplanted from nonepileptic temporal lobe, lesional TLE, or authoptic disease-free tissues remained stable during repetitive stimulation, even in oocytes treated with Zn²⁺. We conclude that rundown of mTLE epileptic receptors depends on the presence of “phasic GABA_A receptors” that have low sensitivity to antagonism by Zn²⁺. Additionally, we found that GABA_A receptors, microtransplanted from the cerebral cortex of adult rats exhibiting recurrent seizures, caused by pilocarpine-induced status epilepticus, showed greater rundown than control tissue, an event also occurring in patch-clamped rat pyramidal neurons. Rundown of epileptic rat receptors resembled that of human mTLE receptors, being enhanced by Zn²⁺ (40 μM) and sensitive to the antiepileptic agent levetiracetam, the neurotrophin brain-derived neurotrophic factor, and the phosphatase blocker okadaic acid. Our findings point to the rundown of GABA_A receptors as a hallmark of TLE and suggest that modulating tonic and phasic mTLE GABA_A receptor activity may represent a useful therapeutic approach to the disease.     

Influence of hippocampal GABA<Subscript>B</Subscript> receptor inhibition on memory in rats with acute β-amyloid toxicity

The neurotransmitter γ-aminobutyric acid (GABA) is involved in the process of memory. It has been reported that the inhibition of GABA_B receptors has beneficial effects on cognition. The aim of this study was to investigate the role of CGP35348 (a GABA_B receptor antagonist) on dentate gyrus GABA_B receptor inhibition and its effects on learning and memory impairments that had been induced in adult male rats by microinjection of β-amyloid (Aβ). Seventy Wistar male rats were randomly divided into seven groups: control, sham (receiving the Aβ vehicle only), Aβ, Aβ + CGP35348 (1, 10, and 100 μg/μL), and CGP35348 alone (10 μg/μL). Memory impairment was induced by unilateral interventricular microinjection of Aβ (6 μg/6 μL). Rats were cannulated bilaterally in the dentate gyrus, and then, they were treated for 20 consecutive days. Learning and memory were assessed using the novel object recognition and passive avoidance learning tests. The discrimination index and the step-through latency were significantly increased in the Aβ + CGP35348 group in comparison to the Aβ only group (P?<?0.05 and P?<?0.01, respectively). Data showed that the discrimination index was decreased in the Aβ + CGP35348 group in comparison with the control group (P?<?0.05) and sham group (P?<?0.01). Moreover, the step-through latency was significantly decreased in the Aβ + CGP35348 group in comparison to the control and sham groups (P?<?0.01). Data from this study indicated that intra-hippocampal microinjection of the GABA_B receptor antagonist counteracts the learning, memory, and cognitive impairments induced by Aβ. It can be concluded that the GABA_B receptor antagonist is a possible therapeutic agent against the progression of acute Aβ toxicity-induced memory impairment.     

NMDA receptors regulate GABAA receptor lateral mobility and clustering at inhibitory synapses through serine 327 on the γ2 subunit

Modification of the number of GABA_A receptors (GABA_ARs) clustered at inhibitory synapses can regulate inhibitory synapse strength with important implications for information processing and nervous system plasticity and pathology. Currently, however, the mechanisms that regulate the number of GABA_ARs at synapses remain poorly understood. By imaging superecliptic pHluorin tagged GABA_AR subunits we show that synaptic GABA_AR clusters are normally stable, but that increased neuronal activity upon glutamate receptor (GluR) activation results in their rapid and reversible dispersal. This dispersal correlates with increases in the mobility of single GABA_ARs within the clusters as determined using single-particle tracking of GABA_ARs labeled with quantum dots. GluR-dependent dispersal of GABA_AR clusters requires Ca²⁺ influx via NMDA receptors (NMDARs) and activation of the phosphatase calcineurin. Moreover, the dispersal of GABA_AR clusters and increased mobility of individual GABA_ARs are dependent on serine 327 within the intracellular loop of the GABA_AR γ2 subunit. Thus, NMDAR signaling, via calcineurin and a key GABA_AR phosphorylation site, controls the stability of synaptic GABA_ARs, with important implications for activity-dependent control of synaptic inhibition and neuronal plasticity.     

Indomethacin Decreases Gastroduodenal Mucosal Bicarbonate Secretion in Humans

Background: Cyclooxygenase inhibitors reduce mucosal bicarbonate secretion in the duodenum, but the evidence for their effect on bicarbonate secretion in the stomach remains controversial. We have, therefore, studied how indomethacin influences gastroduodenal bicarbonate secretion and luminal release of prostaglandin (PG) E₂ by means of a method that enables simultaneous measurements in the stomach and the duodenum.

Methods: Gastric and duodenal perfusions were performed twice in random order during control conditions or after pretreatment with indomethacin (100 mg intravenously) in eight healthy volunteers. Bicarbonate and PGE₂ were measured in the gastroduodenal effluents by back-titration and radioimmunoassay, respectively.

Results: Vagal stimulation and duodenal luminal acidification (0.1 M HCI; 20 ml; 5 min) increased gastroduodenal bicarbonate secretion (p < 0.05). Indomethacin markedly inhibited both basal and stimulated gastric and duodenal mucosal bicarbonate secretion, and this reduction was similar to the degree of cyclooxygenase inhibition estimated by the luminal release of PGE₂ (p < 0.05).

Conclusion: These results unequivocally demonstrate that endogenous PG modulates basal and stimulated bicarbonate secretion, both in the stomach and in the duodenum.     

γ-Aminobutyric acid (GABA) signalling in human pancreatic islets is altered in type 2 diabetes

Aims/hypothesis

γ-Aminobutyric acid (GABA) is a signalling molecule in the interstitial space in pancreatic islets. We examined the expression and function of the GABA signalling system components in human pancreatic islets from normoglycaemic and type 2 diabetic individuals.

Methods

Expression of GABA signalling system components was studied by microarray, quantitative PCR analysis, immunohistochemistry and patch-clamp experiments on cells in intact islets. Hormone release was measured from intact islets.

Results

The GABA signalling system was compromised in islets from type 2 diabetic individuals, where the expression of the genes encoding the α1, α2, β2 and β3 GABA_A channel subunits was downregulated. GABA originating within the islets evoked tonic currents in the cells. The currents were enhanced by pentobarbital and inhibited by the GABA_A receptor antagonist, SR95531. The effects of SR95531 on hormone release revealed that activation of GABA_A channels (GABA_A receptors) decreased both insulin and glucagon secretion. The GABA_B receptor antagonist, CPG55845, increased insulin release in islets (16.7?mmol/l glucose) from normoglycaemic and type 2 diabetic individuals.

Conclusions/interpretation

Interstitial GABA activates GABA_A channels and GABA_B receptors and effectively modulates hormone release in islets from type 2 diabetic and normoglycaemic individuals.     

Interdigestive canine pancreatic juice composition and pancreatic reflux and pancreatic sphincter anatomy

Canine and human exocrine pancreatic secretion into the duodenum during fasting is cyclical and related to intestinal motility. To characterize the composition of pure pancreatic juice during the cyclically recurring sequence of propagated motor events (interdigestive motor complex) and to establish whether pancreatic reflux occurs, dogs were prepared with three permanent indwelling duodenal catheters and a pancreatodochal cutaneous catheter. The duodenal catheters were used to record duodenal pressures and measure pancreatic secretion of trypsin, lipase, and bicarbonate, based on the recovery of a constantly perfused marker, [¹⁴C]PEG. Pancreatic duct pressures or pancreatic juice concentrations of [¹⁴C]PEG, trypsin, lipase, or bicarbonate (done separately in each of five dogs throughout one interdigestive cycle on 4 different days) were related to duodenal motor activity. Finally, the pancreatic duct orifice of freshly sacrificed dogs was examined by light and electron microscopy. During fasting, (1) pancreatic volume secretion increased 10-fold during phases II, III, and IV (P<0.001), and bicarbonate concentration increased during phases III and IV (P<0.05) compared with phase I, while trypsin and lipase concentrations did not change; (2) reflux of duodenally perfused [¹⁴C]PEG into the pancreatic duct occurred in two of five dogs and was minimal (<0.1%); and (3) a positive mean pressure gradient from duodenum to pancreatic duct occurred only during phase III (7.4±4.1 cm H₂O). Anatomic studies of the pancreatic duct opening showed a specialized papillary mucosa and an independent crescentic sphincter muscle. We conclude that during fasting, pancreatic juice composition is intimately linked to the different phases of interdigestive intestinal motor activity and that an efficient antireflux mechanism exists.Supported in part by contract CP 55660 and grant CA 25064 from the National Institutes of Health, U.S. Public Health Service, Bethesda, Maryland.     

Adenosine receptor antagonists alter the stability of human epileptic GABAA receptors

We examined how the endogenous anticonvulsant adenosine might influence γ-aminobutyric acid type A (GABA_A) receptor stability and which adenosine receptors (ARs) were involved. Upon repetitive activation (GABA 500 μM), GABA_A receptors, microtransplanted into Xenopus oocytes from neurosurgically resected epileptic human nervous tissues, exhibited an obvious GABA_A-current (I_GABA) run-down, which was consistently and significantly reduced by treatment with the nonselective adenosine receptor antagonist CGS15943 (100 nM) or with adenosine deaminase (ADA) (1 units/ml), that inactivates adenosine. It was also found that selective antagonists of A2B (MRS1706, 10 nM) or A3 (MRS1334, 30 nM) receptors reduced I_GABA run-down, whereas treatment with the specific A1 receptor antagonist DPCPX (10 nM) was ineffective. The selective A2A receptor antagonist SCH58261 (10 nM) reduced or potentiated I_GABA run-down in ≈40% and ≈20% of tested oocytes, respectively. The ADA-resistant, AR agonist 2-chloroadenosine (2-CA) (10 μM) potentiated I_GABA run-down but only in ≈20% of tested oocytes. CGS15943 administration again decreased I_GABA run-down in patch-clamped neurons from either human or rat neocortex slices. I_GABA run-down in pyramidal neurons was equivalent in A1 receptor-deficient and wt neurons but much larger in neurons from A2A receptor-deficient mice, indicating that, in mouse cortex, GABA_A-receptor stability is tonically influenced by A2A but not by A1 receptors. I_GABA run-down from wt mice was not affected by 2-CA, suggesting maximal ARs activity by endogenous adenosine. Our findings strongly suggest that cortical A2–A3 receptors alter the stability of GABA_A receptors, which could offer therapeutic opportunities.     

Modulation of GABAA Receptor Function by Alcohols: Effects of Subunit Composition and Differential Effects of Ethanol

We sought to test the hypotheses that closely related alcohols would have effects on GABA_A receptor function that were not predicted by differences in lipid solubility, and that the subunit structure of the GABA_A receptor would significantly affect the actions of different alcohols. Cloned subunits of human GABA_A receptors were expressed in Xenopus oocytes, and two-electrode voltage-clamp recording was used to quantify the membrane current response to GABA_A in the presence and absence of different alcohols. 1-Butanol and 2-butanol differentially potentiated the response to 20 μM GABA in oocytes expressing the α₁β₂γ_2L and α₂β₂γ_2L, receptor isoforms. In the α₁β₂γ_2L receptor construct, 1-butanol was more potent than 2-butanol to potentiate GABA, receptor function, but 2-butanol had a greater efficacy. In the α₂β₂ receptor construct, 1-butanol and 2-butanol were equipotent, but 2-butanol again had a greater efficacy. In the a2p2 receptor construct, both 1-butanol and 2-butanol produced large potentiations of the current response to 3 μM GABA. The efficacy for butanol potentiation of GABA responses in the absence of a γ_2L subunit was greater, but the potency was greatly reduced. Low concentrations (20 mM) of ethanol potentiated GABA responses in the α₁β₂γ_2L receptor construct. Ethanol potentiation of GABA_A receptor function was completely blocked by the benrodiazepine receptor partial inverse agonist R015-4513 at a concentration (0.5 μM) that did not alter the control GABA response. In contrast, R015-4513 did not block potentiation of GABA_A receptor activity induced by npropanol, 1-butanol, 2-butanol, 1-heptanol, or propofol (2,0-diisopropylphenol). These results suggest that alcohols have specific interactions with GABA_A receptors, and that ethanol may have unique effects not shared by other longer chain alcohols.     

Measurement of Gastric Bicarbonate Secretion in the Human Stomach: Different Methods Produce Discordant Results

Odes HS, Hogan DL, Steinbach JH, Ballesteros MA, Koss MA, Isenberg JI. Measurement of gastric bicarbonate secretion in the human stomach: different methods produce discordant results. Scand J Gastroenterol 1992;27:829-836.

Human gastric bicarbonate secretion has been measured by back-titration, from pH and pressure of carbon dioxide (Pco₂) determinations (using the Henderson-Hasselbalch formula), and from equations based on gastric juice osmolality and [H⁺] (osmolality-[H⁺] method). Since these methods show large quantitative differences in their estimations of gastric bicarbonate secretion, we examined each to define the reasons for these discrepancies and establish guidelines for future work in this area. Bicarbonate recovery from ‘non-parieta' secretions (0 to 80 mM HCO₃) reacting with ‘pure parietal secretion' (160mM HC1) was studied both in vitro and in the pylorus-occluded healthy human stomach during acid suppression, exogenous acidification, and pentagastrin stimulation. The pH/Pco₂ method estimated HC0₃ accurately under anaerobic conditions in vitro, whereas the osmolality-[H⁺] method (with correction factors for osmolality incorporated by us) was accurate under aerobic conditions. In the acid-suppressed stomach back-titration was significantly more accurate than the pH/Pco₂ method. In the exogenously acidified and pentagastrin-stimulated stomachs the pH/Pco₂ method underestimated bicarbonates, and the osmolality-[H⁺] method was spuriously elevated in the low range and diminished at high bicarbonate concentrations. Estimates of'basa' bicarbonate secretion (at zero added bicarbonate) were severalfold higher by the osmolality-[H⁺] method (5.26 ± 0.33 mmol/h) than by the pH/Pco₂ method (1.20 ± 0.23 mmol/h) or back-titration (0.65 ± 0.14 mmol/h). In conclusion, gastric bicarbonate was determined most correctly by back-titration in the acid-suppressed stomach, whereas measurement of bicarbonate in the acid-secreting stomach was not accurate with any method.