Relationships between nasal potential difference and respiratory function in adults with cystic fibrosis

This study investigated the relations between nasal transepithelial electric potential difference (PD) and the phenotype and genotype of cystic fibrosis (CF) adult patients. Basal nasal PD was measured in 95 adult CF patients who were classified into three groups of nasal PD (expressed as absolute values) according to the 10th and the 90th percentiles (28.3 and 49.2 mV, respectively), which defined group 1 (nasal PD < or =28.3 mV), group 2 (nasal PD 28.3-49.2 mV) and group 3 (nasal PD > or =49.2 mV). Patients from group 1 had a higher forced vital capacity (FVC) than patients from groups 2 and 3 (76.5+/-22.4 versus 57.4+/-21.2 and 55.7+/-21.1% predicted, respectively, p<0.05) and a higher forced expiratory volume in one second (FEV1) (69.3+/-24.0 versus 42.5+/-22.4 and 42.2+/-21.4% pred, respectively, p<0.01). Among patients with severe mutations (deltaF508 homozygotes, or one deltaF508 mutation plus another "severe" mutation, or two "severe" mutations), patients from group 1 had a higher FVC, FEV1 and arterial oxygen tension than patients from groups 2 and 3 (p<0.05 for each comparison). The results show that in adult cystic fibrosis patients a normal basal nasal potential difference is related to milder respiratory disease, irrespective of the severity of the genotype.     

Transcription of ppk from Acinetobacter sp. strain ADP1, encoding a putative polyphosphate kinase, is induced by phosphate starvation

Congenital bilateral absence of the vas deferens (CBAVD) is supposed to be due to defective activity of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in the genital tract. With the aim of studying CFTR activity in vivo we measured nasal potential difference (NPD) in a group of CBAVD subjects, who were then compared with normal control subjects and CF patients. Sodium transport, measured under basal conditions and after amiloride superinfusion, was normal in almost all CBAVD patients, who had NPD values similar to those of normal control subjects. Chloride transport was studied by measuring NPD during perfusion with a chloride-free solution and isoproterenol. Under these circumstances CBAVD patients as a whole showed normal chloride secretion. However, three subjects with CBAVD had abnormal NPD values. They had either elevated sweat chloride concentrations together with symptoms of mild CF, or compound heterozygosity (DeltaF508/R117H). In conclusion the group of CBAVD patients as a whole presented normal bioelectric properties of nasal epithelium, suggesting normal CFTR activity. In a small subgroup NPD was abnormal, suggesting a diagnosis of CF, later confirmed by elevated sweat chloride concentrations or positive DNA testing. We suggest that CBAVD patients with altered NPD should undergo further clinical follow-up in order to detect possible late complications of CF.     

In vivo measurement of chloride and water secretion in the jejunum of cystic fibrosis patients

In the present study, we have investigated the possible consequences of the chloride channel defect in the intestine of cystic fibrosis (CF) patients for electrolyte and water transport in the jejunum in vivo, using a multilumen, double occluding balloon catheter, and an Ag/AgCl intraluminal electrode. During a chloride-free perfusion, to optimize the sensitivity of our measurements, the transmural potential difference (PD) (lumen with reference to serosal side) was found to be significantly higher in the jejunum of CF patients (+8.0 +/- 2.1 mV; n = 5) than in healthy control subjects (-2.2 +/- 2.0 mV; n = 9). The chloride concentration measured in chloride-free jejunal perfusates of CF patients was significantly lower than in controls (10.9 +/- 2.3 and 41.4 +/- 8.2 mM, respectively). Possible differences in net chloride and water secretion did not reach statistical significance (chloride secretion controls: -2.1 +/- 0.9 mmol/10 cm/h; CF: -0.8 +/- 0.2 mmol/10 cm/h; water secretion controls: -0.8 +/- 2.5 mL/10 cm/h; CF: -11.7 +/- 8.9 mL/10 cm/h). In control subjects, intraluminally applied theophylline stimulated the secretion of water (delta 23.4 +/- 4.6 mL/10 cm/h) and chloride (delta 4.1 +/- 1.1 mmol/10 cm/h), but not in CF patients (respectively delta 3.6 +/- 3.3 mL/10 cm/h and delta 1.1 +/- 1.1 mmol/10 cm/h). In controls, theophylline caused a significant increase in lumen negativity (PD -10.2 +/- 2.6 mV), but no change could be seen in CF patient transmural PD. These observations provide in vivo evidence for a decreased chloride permeability in the jejunum in CF, resulting in a significant reduction in net electrolyte and water secretion in the presence, but not in the absence, of an intestinal secretagogue.     

Cannabinoid CB1 receptor and endothelium-dependent hyperpolarization in guinea-pig carotid, rat mesenteric and porcine coronary arteries

1. The purpose of these experiments was to determine whether or not the endothelium-dependent hyperpolarizations of the vascular smooth muscle cells (observed in the presence of inhibitors of nitric oxide synthase and cyclo-oxygenase) can be attributed to the production of an endogenous cannabinoid. 2. Membrane potential was recorded in the guinea-pig carotid, rat mesenteric and porcine coronary arteries by intracellular microelectrodes. 3. In the rat mesenteric artery, the cannabinoid receptor antagonist, SR 141716 (1 microM), did not modify either the resting membrane potential of smooth muscle cells or the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (17.3 +/- 1.8 mV, n = 4 and 17.8 +/- 2.6 mV, n = 4, in control and presence of SR 141716, respectively). Anandamide (30 microM) induced a hyperpolarization of the smooth muscle cells (12.6 +/- 1.4 mV, n = 13 and 2.0 +/- 3.0 mV, n = 6 in vessels with and without endothelium, respectively) which could not be repeated in the same tissue, whereas acetylcholine was still able to hyperpolarize the preparation. The hyperpolarization induced by anandamide was not significantly influenced by SR 141716 (1 microM). HU-210 (30 microM), a synthetic CB1 receptor agonist, and palmitoylethanolamide (30 microM), a CB2 receptor agonist, did not influence the membrane potential of the vascular smooth muscle cells. 4. In the rat mesenteric artery, the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (19.0 +/- 1.7 mV, n = 6) was not altered by glibenclamide (1 microM; 17.7 +/- 2.3 mV, n = 3). However, the combination of charybdotoxin (0.1 microM) plus apamin (0.5 microM) abolished the acetylcholine-induced hyperpolarization and under these conditions, acetylcholine evoked a depolarization (7.7 +/- 2.7 mV, n = 3). The hyperpolarization induced by anandamide (30 microM) (12.6 +/- 1.4 mV, n = 13) was significantly inhibited by glibenclamide (4.0 +/- 0.4 mV, n = 4) but not significantly affected by the combination of charybdotoxin plus apamin (17.3 +/- 2.3 mV, n = 4). 5. In the guinea-pig carotid artery, acetylcholine (1 microM) evoked endothelium-dependent hyperpolarization (18.8 +/- 0.7 mV, n = 15). SR 141716 (10 nM to 10 microM), caused a direct, concentration-dependent hyperpolarization (up to 10 mV at 10 microM) and a significant inhibition of the acetylcholine-induced hyperpolarization. Anandamide (0.1 to 3 microM) did not influence the membrane potential. At a concentration of 30 microM, the cannabinoid agonist induced a non-reproducible hyperpolarization (5.6 +/- 1.3 mV, n = 10) with a slow onset. SR 141716 (1 microM) did not affect the hyperpolarization induced by 30 microM anandamide (5.3 +/- 1.5 mV, n = 3). 6. In the porcine coronary artery, anandamide up to 30 microM did not hyperpolarize or relax the smooth muscle cells. The endothelium-dependent hyperpolarization and relaxation induced by bradykinin were not influenced by SR 141716 (1 microM). 7. These results indicate that the endothelium-dependent hyperpolarizations, observed in the guinea-pig carotid, rat mesenteric and porcine coronary arteries, are not related to the activation of cannabinoid CB1 receptors.     

Participation of GABAA-mediated inhibition in ictallike discharges in the rat entorhinal cortex

The spontaneous, synchronous activity induced by 4-aminopyridine (4AP, 50 microM) in the adult rat entorhinal cortex was analyzed with simultaneous field potential and intracellular recordings in an in vitro slice preparation. Four-AP induced isolated negative-going field potentials (interval of occurrence = 27.6 +/- 9.9 (SD) s; n = 27 slices) that corresponded to intracellular long-lasting depolarizations (LLDs), and ictallike epileptiform discharges (interval of occurrence = 10.4 +/- 5.7 min; n = 27 slices) that were initiated by the negative field potentials. LLDs recorded with K-acetate-filled microelectrodes triggered few action potentials of variable amplitude and had a duration of 1.7 +/- 0.8 s (n = 26 neurons), a peak amplitude of 11.8 +/- 5.0 mV (n = 26 neurons) and a reversal potential of -66.2 +/- 3.9 mV (n = 17 neurons). The ictal discharges studied with K-acetate microelectrodes consisted of prolonged depolarizations (duration = 72.9 +/- 44.3 s; peak amplitude = 29.2 +/- 11.4 mV; n = 25 neurons) with action-potential firing during both the tonic and the clonic phase. These depolarizations had a reversal potential of -45.3 +/- 3.8 mV (n = 4 neurons). Intracellular Cl- diffusion from KCl-filled microelectrodes made both LLDs and ictal depolarizations increase in amplitude (30.5 +/- 8.2 mV, n = 8 and 41.8 +/- 9.8 mV, n = 6 neurons, respectively). LLDs recorded with KCl and 2-(trimethyl-amino)N-(2, 6-dimethylphenyl)-acetamide (QX-314) microelectrodesreached an amplitude of 36.3 +/- 5.2 mV, lasted 12.5 +/- 6.5 s, and had a reversal potential of -31.3 +/- 2.5 mV (n = 4 neurons); under these recording procedures the ictal discharge amplitude was 41.5 +/- 5.0 mV and the reversal potential -24.0 +/- 7.0 mV (n = 4 neurons). The N-methyl-D-aspartate (NMDA) receptor antagonist 3,3-(2-carboxy-piperazine-4-yl)-pro-pyl-l-phosphonate (10 microM, n = 5 neurons) alone or concomitant with the nonNMDA receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (10 microM, n = 4 neurons) abolished ictal discharges, without influencing LLDs. LLDs were blocked by the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline methiodide (BMI, 10 microM, n = 6 neurons) or the mu-opioid receptor agonist (-Ala2-N-Me-Phe, Gly-ol) enkephalin (DAGO, 10 microM, n = 2 neurons). Application of BMI (n = 4 neurons) or DAGO (n = 2 neurons) to control the medium abolished LLDs and ictal discharges but disclosed a novel type of epileptiform depolarization that lasted 3.5 +/- 1.2 s and occurred every 5.2 +/- 2.6 s (n = 6 neurons). Our data indicate that 4AP induces in the rat entorhinal cortex a synchronous, GABA-mediated potential that is instrumental in initiating NMDA-dependent, ictal discharges. Moreover we present evidence for an active role played by GABAA-mediated potentials in the maintenance and termination of these prolonged epileptiform events.     

Electrogenic glucose absorption in untreated and treated coeliac disease

Using a method for measuring changes in transmural potential difference across the human jejunum in vivo, the operational kinetic parameters of 'Apparent Km' and PD max for the active electrogenic component of glucose absorption were estimated in a group of healthy volunteers and in patients with coeliac disease. Both the 'Apparent Km' (17+/2mM; mean +/SEM) and the PD max (8.6+/0.7 mV) in nine patients with untreated coeliac disease were significantly lower (p less than 0.005) than in the control group ('Apparent Km' = 74+/5mM; PD max 12.8+/0.9mV, n=20). Treatment of five coeliac patients by gluten withdrawal for less than three months increased significantly the values of both the "Apparent Km (35+/6mM) and the TPD max (11.4+/1.2mV). Treatment of five patients for more than six months caused a further increase in the values of both kinetic parameters ('Apparent Km' = 108+/13mM; PD max =15.6+/2.7mV) to levels which exceeded those in healthy subjects. The possible interpretations of the differences in the kinetic characteristics of electrogenic glucose transport between coeliac patients and healthy subjects are discussed.     

Effect of mycophenolic acid on TNF alpha-induced expression of cell adhesion molecules in human venous endothelial cells in vitro

1. The characteristic features of the endothelium-mediated regulation of the electrical and mechanical activity of the smooth muscle cells of cerebral arteries were studied by measuring membrane potential and isometric force in endothelium-intact and -denuded strips taken from the rabbit middle cerebral artery (MCA). 2. In endothelium-intact strips, histamine (His, 3-10 microM) and high K+ (20-80 mM) concentration-dependently produced a transient contraction followed by a sustained contraction. Noradrenaline (10 microM), 5-hydroxytryptamine (10 microM) and 9,11-epithio-11, 12-methano-thromboxane A2 (10 nM) each produced only a small contraction (less than 5% of the maximum K+-induced contraction). 3. N(G)-nitro-L-arginine (L-NOARG, 100 microM), but not indomethacin (10 microM), greatly enhanced the phasic and the tonic contractions induced by His (1-10 microM) in endothelium-intact, but not in endothelium-denuded strips, suggesting that spontaneous or basal release of nitric oxide (NO) from endothelial cells potently attenuates the His-induced contractions. Acetylcholine (ACh, 0.3-3 microM) caused concentration-dependent relaxation (maximum relaxation by 89.7 +/- 7.5%, n=4, P<0.05) when applied to endothelium-intact strips precontracted with His. L-NOARG had little effect on this ACh-induced relaxation (n=4; P<0.05). Apamin (0.1 microM), but not glibenclamide (3 microM), abolished the relaxation induced by ACh (0.3-3 microM) in L-NOARG-treated strips (n=4, P<0.05). 4. In endothelium-intact tissues, His (3 microM) depolarized the smooth muscle membrane potential (by 4.4 +/- 1.8 mV, n = 12, P < 0.05) whereas ACh (3 microM) caused membrane hyperpolarization (-20.9 +/- 3.0 mV, n = 25, P< 0.05). The ACh-induced membrane hypepolarization persisted after application of L-NOARG (-23.5 +/- 5.9 mV, n=8, P<0.05) or glibenclamide (-20.6 +/- 5.4 mV, n=5, P<0.05) but was greatly diminished by apamin (reduced to - 5.8 +/- 3.2 mV, n = 3, P< 0.05). 5. Sodium nitroprusside (0.1-10 microM) did not hyperpolarize the smooth muscle cell membrane potential (0.2 +/- 0.3 mV, n=4, P>0.05) but it greatly attenuated the His-induced contraction in endothelium-denuded strips (n-4, P<0.05). 6. These results suggest that, under the present experimental conditions: (i) spontaneous or basal release of NO from endothelial cells exerts a significant negative effect on agonist-induced contractions in rabbit MCA, and (ii) ACh primarily activates the release of endothelium-derived hyperpolarizing factor (EDHF) in rabbit MCA.     

Decreased sodium ion absorption across nasal epithelium of very premature infants with respiratory distress syndrome

OBJECTIVE AND STUDY DESIGN: Successful adaptation to air breathing at birth depends on rapid absorption of fetal lung liquid that is mediated by activation of amiloride-sensitive sodium ion channels. To test the relationship between respiratory epithelial Na+ transport and development of respiratory distress syndrome (RDS), we measured nasal transepithelial potential difference (PD) in 31 very premature (< or = 30 weeks of gestation) newborn infants. Infants were retrospectively assigned to RDS (22 infants) and non-RDS (9 infants) groups on the basis of clinical and chest x-ray criteria. RESULTS: Maximal nasal epithelial PD increased with birth weight (-1.2 mV/100 gm) and was lower in infants with RDS (-16.5 +/- 0.6 mV) than in those without RDS (-22.0 +/- 1.3 mV). Infants without RDS had PD values similar to normal fullterm infants. Amiloride inhibition of PD, an index of Na+ absorption, was significantly lower, within the first 24 hours of life, in infants in whom RDS developed (3.8 +/- 0.2 mV; 29.5% +/- 0.8% inhibition) than in those without RDS (6.1 +/- 0.6 mV; 38.6% +/- 0.5% inhibition). Maximal and amiloride-sensitive PD returned to normal during the recovery phase of RDS. CONCLUSIONS: We conclude that Na+ absorption across nasal epithelium increases with increasing birth weight and that impairment of Na+ absorption across the respiratory epithelia of very premature infants may contribute to the pathogenesis of RDS.     

Inhibition by mibefradil, a novel calcium channel antagonist, of Ca(2+)- and volume-activated Cl- channels in macrovascular endothelial cells

1. We have studied the effects of mibefradil, a novel calcium antagonist, on the resting potential and ion channel activity of macrovascular endothelial cells (calf pulmonary artery endothelial cells, CPAE). The patch clamp technique was used to measure ionic currents and the Fura-II microfluorescence technique to monitor changes in the intracellular Ca2+ concentration, [Ca2+]i. 2. Mibefradil (10 microM) hyperpolarized the membrane potential of CPAE cells from its mean control value of -26.6 +/- 0.6 mV (n = 7) to -59.8 +/- 1.7 mV (n = 6). A depolarizing effect was observed at higher concentrations (-13.7 +/- 0.6 mV, n = 4, 30 microM mibefradil). 3. Mibefradil inhibited Ca(2+)-activated Cl- currents, ICl,Ca, activated by loading CPAE cells via the patch pipette with 500 nM free Ca2+ (Ki = 4.7 +/- 0.18 microM, n = 8). 4. Mibefradil also inhibited volume-sensitive Cl- currents, ICl,vol, activated by challenging CPAE cells with a 27% hypotonic solution (Ki = 5.4 +/- 0.22 microM, n = 6). 5. The inwardly rectifying K+ channel, IRK, was not affected by mibefradil at concentrations up to 30 microM. 6. Ca2+ entry activated by store depletion, as assessed by the rate of [Ca2+]i-increase upon reapplication of 10 mM extracellular Ca2+ to store-depleted cells, was inhibited by 17.6 +/- 6.5% (n = 8) in the presence of 10 microM mibefradil. 7. Mibefradil inhibited proliferation of CPAE cells. Half-maximal inhibition was found at 1.7 +/- 0.12 microM (n = 3), which is similar to the concentration for half-maximal block of Cl- channels. 8. These actions of mibefradil on Cl- channels and the concomitant changes in resting potential might, in addition to its effect on T-type Ca2+ channels, be an important target for modulation of cardiovascular function under normal and pathological conditions.     

Intraneuronal [Ca2+] changes induced by 2-deoxy-D-glucose in rat hippocampal slices

Temporary replacement of glucose by 2-deoxyglucose (2-DG; but not sucrose) is followed by long-term potentiation of CA1 synaptic transmission (2-DG LTP), which is Ca2+-dependent and is prevented by dantrolene or N-methyl--aspartate (NMDA) antagonists. To clarify the mechanism of action of 2-DG, we monitored [Ca2+]i while replacing glucose with 2-DG or sucrose. In slices (from Wistar rats) kept submerged at 30 degreesC, pyramidal neurons were loaded with [Ca2+]-sensitive fluo-3 or Fura Red. The fluorescence was measured with a confocal microscope. Bath applications of 10 mM 2-DG (replacing glucose for 15 +/- 0.38 min, means +/- SE) led to a rapid but reversible rise in fluo-3 fluorescence (or drop of Fura Red fluorescence); the peak increase of fluo-3 fluorescence (DeltaF/F0), measured near the end of 2-DG applications, was by 245 +/- 50% (n = 32). Isosmolar sucrose (for 15-40 min) had a smaller but significant effect (DeltaF/F0 = 94 +/- 14%, n = 10). The 2-DG-induced DeltaF/F0 was greatly reduced (to 35 +/- 15%, n = 16) by,-aminophosphono-valerate (50-100 microM) and abolished by 10 microM dantrolene (-4.0 +/- 2.9%, n = 11). A substantial, although smaller effect, of 2-DG persisted in Ca2+-free 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA) medium. Two adenosine antagonists, which do not prevent 2-DG LTP, were also tested; 2-DG-induced DeltaF/F0 (fluo-3) was not affected by the A1 antagonist 8-cyclopentyl-3, 7-dihydro-1,3-dipropyl-1H-purine-2,6-dione (DPCPX 50 nM; 287 +/- 38%; n = 20), but it was abolished by the A1/A2 antagonist 8-SPT; 25 +/- 29%, n = 19). These observations suggest that 2-DG releases glutamate and adenosine and that the rise in [Ca2+] may be triggered by a synergistic action of glutamate (acting via NMDA receptors) and adenosine (acting via A2b receptors) resulting in Ca2+ release from a dantrolene-sensitive store. The discrepant effects of sucrose and 8-SPT on DeltaF/F0, on the one hand, and 2-DG LTP, on the other, support other evidence that increases in postsynaptic [Ca2+]i are not essential for 2-DG LTP.     

NMDA receptor dependence of mGlu-mediated depression of synaptic transmission in the CA1 region of the rat hippocampus

1. The depression of synaptic transmission by the specific metabotropic glutamate receptor (mGlu) agonist (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylate ((1S,3R)-ACPD) was investigated in area CA1 of the hippocampus of 4-10 week old rats, by use of grease-gap and intracellular recording techniques. 2. In the presence of 1 mM Mg2+, (1S,3R)-ACPD was a weak synaptic depressant. In contrast, in the absence of added Mg2+, (1S,3R)-ACPD was much more effective in depressing both the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptor-mediated components of synaptic transmission. At 100 microM, (1S,3R)-ACPD depressed the slope of the field excitatory postsynaptic potential (e.p.s.p.) by 96 +/- 1% (mean +/- s.e.mean; n = 7) compared with 23 +/- 4% in 1 mM Mg(2+)-containing medium (n = 17). 3. The depressant action of 100 microM (1S,3R)-ACPD in Mg(2+)-free medium was reduced from 96 +/- 1 to 46 +/- 6% (n = 7) by the specific NMDA receptor antagonist (R)-2-amino-5-phosphonopentanoate (AP5; 100 microM). 4. Blocking both components of GABA receptor-mediated synaptic transmission with picrotoxin (50 microM) and CGP 55845A (1 microM) in the presence of 1 mM Mg2+ also enhanced the depressant action of (1S,3R)-ACPD (100 microM) from 29 +/- 5 to 67 +/- 6% (n = 6). 5. The actions of (1S,3R)-ACPD, recorded in Mg(2+)-free medium, were antagonized by the mGlu antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG). Thus, depressions induced by 30 microM (1S,3R)-ACPD were reversed from 48 +/- 4 to 8 +/- 6% (n = 4) by 1 mM (+)-MCPG. 6. In Mg(2+)-free medium, a group I mGlu agonist, (RS)-3, 5-dihydroxyphenylglycine (DHPG; 100 microM) depressed synaptic responses by 74 +/- 2% (n = 18). In contrast, neither the group II agonists ((2S,1'S,2'S)-2-(2'-carboxycyclopropyl)glycine; L-CCG-1; 10 microM; n = 4) and ((2S,1'R,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine; DCG-IV; 100 nM; n = 3) nor the group III agonist ((S)-2-amino-4-phosphonobutanoic acid; L-AP4; 10 microM; n = 4) had any effect. 7. The depolarizing action of (1S,3R)-ACPD, recorded intracellularly, was similar in the presence and absence of Mg(2+)-AP5 did not affect the (1S,3R)-ACPD-induced depolarization in Mg(2+)-free medium. Thus, 50 microM (1S,3R)-ACPD induced depolarizations of 9 +/- 3 mV (n = 5), 10 +/- 2 mV (n = 4) and 8 +/- 2 mV (n = 5) in the three respective conditions. 8. On resetting the membrane potential in the presence of 50 microM (1S,3R)-ACPD to its initial level, the e.p.s.p. amplitude was enhanced by 8 +/- 3% in 1 mM Mg2+ (n = 5) compared with a depression of 37 +/- 11% in the absence of Mg2+ (n = 4). Addition of AP5 prevented the (1S,3R)-ACPD-induced depression of the e.p.s.p. (depression of 4 +/- 5% (n = 5)). 9. It is concluded that activation by group 1 mGlu agonists results in a depression of excitatory synaptic transmission in an NMDA receptor-dependent manner.     

Voltage- and use-dependent inhibition of Na+ channels in rat sensory neurones by 4030W92, a new antihyperalgesic agent

1. Whole cell patch clamp techniques were used to study the effects of 4030W92 (2,4-diamino-5-(2,3-dichlorophenyl)-6-fluoromethylpyrimidine), a new antihyperalgesic agent, on rat dorsal root ganglion (DRG) neurones. 2. In small diameter, presumably nociceptive DRG neurones under voltage-clamp, 4030W92 (1-100 microM) produced a concentration-related inhibition of slow tetrodotoxin-resistant Na+ currents (TTXR). From a holding potential (Vh) of -90 mV, currents evoked by test pulses to 0 mV were inhibited by 4030W92 with a mean IC50 value of approximately 103 microM. 3. The inhibitory effect of 4030W92 on TTX(R) was both voltage- and use-dependent. Currents evoked from a Vh of -60 mV were inhibited by 4030W92 with a mean IC50 value of 22 microM, which was 5 fold less than the value obtained at -90 mV. Repeated activation of TTX(R) by a train of depolarizing pulses (5 Hz, 20 ms duration) enhanced the inhibitory effects of 4030W92. These data could be explained by a preferential interaction of the drug with inactivation states of the channel. In support of this hypothesis 4030W92 (30 microM) produced a significant hyperpolarizing shift of 10 mV in the slow inactivation curve for TTX(R) and markedly slowed the recovery from channel inactivation. 4. Fast TTX-sensitive Na+ currents (TTXs) were also inhibited by 4030W92 in a voltage-dependent manner. The IC50 values obtained from Vhs of -90 mV and -70 mV were 37 microM and 5 microM, respectively. 4030W92 (30 microM) produced a 13 mV hyperpolarizing shift in the steady-state inactivation curve of TTXs. 5. High threshold voltage-gated Ca2+ currents were only weakly inhibited by 4030W92. The reduction in peak Ca2+ current amplitude produced by 100 microM 4030W92 was 20+/-6% (n=6). Low threshold T-type Ca2+ currents were inhibited by 17+/-8% and 43+/-3% by concentrations of 4030W92 of 30 microM and 100 microM, respectively (n=6). 6. Under current clamp, some cells exhibited broad TTX-resistant action potentials whilst others showed fast TTX-sensitive action potentials in response to a depolarizing current injection. In most cells a long duration (800 ms) supramaximal current injection evoked a train of action potentials. 4030W92 (10-30 microM) had little effect on the first spike in the train but produced a concentration-related inhibition of the later spikes. The number of spikes per train was significantly reduced from 9.7+/-1.5 to 4.2+/-1.0 and 2.6+/-1.1 in the presence of 10 microM and 30 microM 4030W92, respectively (n=5). 7. Thus, 4030W92 is a potent voltage- and use-dependent inhibitor of Na+ channels in sensory neurones. This profile can be explained by a preferential action of the drug on a slow inactivation state of the channel that results in a delayed recovery to the resting state. This state-dependent modulation by 4030W92 of Na+ channels that are important in sensory neurone function may underlie or contribute to the antihyperalgesic profile of this compound observed in vivo.     

Electrophysiological characterization of 5-HT receptors on rat petrosal neurons in dissociated cell culture

The petrosal ganglion supplies chemoafferent pathways via the glossopharyngeal (IXth) nerve to peripheral targets which release various neurotransmitters including serotonin (5-HT). Here, we combined rapid 5-HT application with patch clamp, whole-cell recording to investigate whether 5-HT receptors are expressed on isolated petrosal neurons (PN), cultured from 7-12 day-old rat pups. In responsive cells, the dominant effect of 5-HT was a rapid depolarization associated with a conductance increase in approximately 43% of the neurons (53/123); however, in a minority population ( approximately 6%; 8/123), 5-HT caused membrane depolarization associated with a conductance decrease. In the former group, 5-HT produced a transient inward current (I5-HT) in neurons voltage-clamped near the resting potential ( approximately -60 mV); the effect was mimicked by the 5-HT3 receptor-specific agonist, 2-methyl-5-HT, suggesting it was mediated by 5-HT3 receptors. Further, I5-HT was selectively inhibited by the 5-HT3 receptor-specific antagonist MDL72222 (1-10 microM), but was unaffected by either 5-HT1/5-HT2 receptor antagonist, spiperone, or by 5-HT2 receptor-specific antagonist, ketanserin (50-100 microM). I5-HT displayed moderate inward rectification and had a mean reversal potential (+/-S.E.M.) of -4.3+/-6.6 mV (n=6). Application of 5-HT (dose range: 0.1-100 microM) produced a dose-response curve that was fitted by the Hill equation with EC50= approximately 3.4 microM and Hill coefficient= approximately 1.6 (n=8). The activation phase of I5-HT (10 microM 5-HT at -60 mV) was well fitted by a single exponential with mean (+/-S.E.M.) time constant of 45+/-30 ms (n=6). The desensitization phase of I5-HT was best fitted by a single exponential with mean (+/-S.E.M.) time constant of 660+/-167 ms (n=6). Fluctuation analysis yielded an apparent mean single-channel conductance (+/-S.E.M) of 2.7+/-1.5 pS (n=4) at -60 mV. In the minority ( approximately 6%) population of neurons which responded to 5-HT with a conductance decrease, the depolarization was blocked by the 5-HT2 receptor antagonist, ketanserin (50 microM). Taken together, these results suggest that 5-HT3 receptors are the major subtype expressed by rat petrosal neurons, and therefore are candidates for facilitating chemoafferent excitation in response to 5-HT released from peripheral targets.     

Contribution of phosphodiesterase isozymes to the regulation of the L-type calcium current in human cardiac myocytes

1. To determine the contribution of the various phosphodiesterase (PDE) isozymes to the regulation of the L-type calcium current (ICa(L)) in the human myocardium, we investigated the effect of selective and non-selective PDE inhibitors on ICa(L) in single human atrial cells by use of the whole-cell patch-clamp method. We repeated some experiments in rabbit atrial myocytes, to make a species comparison. 2. In human atrial cells, 100 microM pimobendan increased ICa(L) (evoked by depolarization to +10 mV from a holding potential of -40 mV) by 250.4 +/- 45.0% (n = 15), with the concentration for half-maximal stimulation (EC50) being 1.13 microM. ICa(L) was increased by 100 microM UD-CG 212 by 174.5 +/- 30.2% (n = 10) with an EC50 value of 1.78 microM in human atrial cells. These two agents inhibit PDE III selectively. 3. A selective PDE IV inhibitor, rolipram (1-100 microM), did not itself affect ICa(L) in human atrial cells. However, 100 microM rolipram significantly enhanced the effect of 100 microM UD-CG 212 on ICa(L) (increase with UD-CG 212 alone, 167.9 +/- 33.9, n = 5; increase with the two agents together, 270.0 +/- 52.2%; n = 5, P < 0.05). Rolipram also enhanced isoprenaline (5 nM)-stimulated ICa(L) by 52.9 +/- 9.3% (n = 5) in human atrial cells. 4. In rabbit atrial cells, ICa(L) at +10 mV was increased by 22.1 +/- 9.0% by UD-CG 212 (n = 10) and by 67.4 +/- 12.0% (n = 10) by pimobendan (each at 100 microM). These values were significantly lower than those obtained in human atrial cells (P < 0.0001). Rolipram (1-100 microM) did not itself affect ICa(L) in rabbit atrial cells. However, ICa(L) was increased by 215.7 +/- 65.2% (n = 10) by the combination of 100 microM UD-CG 212 and 100 microM rolipram. This value was almost 10 times larger than that obtained for the effect of 100 microM UD-CG 212 alone. 5. These results imply a species difference: in the human atrium, the PDE III isoform seems dominant, whereas PDE IV may be more important in the rabbit atrium for regulating ICa(L). However, PDE IV might contribute significantly to the regulation of intracellular cyclic AMP in human myocardium when PDE III is already inhibited or when the myocardium is under beta-adrenoceptor-mediated stimulation.     

Increased sensitivity of the cough reflex in progressive systemic sclerosis patients with interstitial lung disease

Cough is a common presenting symptom of interstitial lung disease (ILD). The aim of this study was to examine the cough reflex in patients with progressive systemic sclerosis (PSS), with and without associated ILD. The cough reflex to inhalation of chloride deficient solutions and capsaicin was determined in patients with PSS with associated ILD (n=12), compared to patients with PSS without ILD (n=12). In addition, patients with a chronic dry cough (n=12) and healthy subjects (n=10) without cough were studied. Cough responses to inhalation of isotonic solutions containing 150, 75, 37.5 and 0 mM Cl- ions and of capsaicin (0.9-500 mM) were measured. PSS patients with ILD reported a significantly higher cough score than PSS patients without ILD (p<0.03). ILD patients coughed more than those without ILD to Cl- of 37.5 and 0 mM (19.1+/-5.0 vs 6.2+/-1.9 coughs x min(-1) (p<0.03), and 29.2+/-5.0 vs 14.1/-4.1 coughs x min(-1) (p<0.04), respectively). The log concentration of capsaicin causing two or five coughs was lower in PSS with ILD compared to PSS without ILD (0.74+/-0.15 mM vs 2.12+/-0.26 mM; p<0.002). Patients with chronic dry cough had a similar degree of response to low-chloride and capsaicin solutions as patients with PSS and ILD, whilst healthy controls had a similar degrees of response to PSS patients. There is an increased cough reflex in patients with interstitial lung disease, which may represent sensitization of airway sensory nerves. This may be the basis for the chronic dry cough in patients with interstitial lung disease.     

Electrical activity in rat tail artery during asynchronous activation of postganglionic nerve terminals by ciguatoxin-1

1. The effects of ciguatoxin-1 (CTX-1) on the membrane potential of smooth muscle cells have been examined in rat proximal tail arteries isolated in vitro. 2. CTX-1 (> or = 10 pM) increased the frequency of spontaneous excitatory junction potentials (s.e.j.ps). At 100-400 pM, there was also a marked and maintained depolarization (19.7 +/- 1.4 mV, n = 14, at 400 pM). 3. In 20-400 pM CTX-1, perivascular stimuli evoked excitatory junction potentials (e.j.ps) which were prolonged in time course relative to control. 4. Although threshold and latency of the e.j.p. were not affected by CTX-1 (< or = 400 pM), propagated impulses were blocked at > or = 100 pM. 5. The spontaneous activity and the depolarization produced by CTX-1 were reduced in the presence of Ca2+ (0.1 mM)/Mg2+ (25 mM), omega-conotoxin (0.1 microM) or Cd2+ (50-100 microM). 6. All effects of CTX-1 were abolished by tetrodotoxin (0.3 microM). 7. Raised Ca2+ (6 mM) reduced the depolarization and spontaneous activity produced by CTX-1. 8. In 400 pM CTX-1, the membrane repolarized (17 +/- 3.2 mV, n = 4) following the addition of phentolamine (1 microM). S.e.j.ps and e.j.ps were selectively abolished by suramin (1 mM), and the membrane repolarized by 1.3 +/- 1.6 mV (n = 4). 9. We conclude that CTX-1 releases noradrenaline and ATP by initiating asynchronous discharge of postganglionic perivascular axons. In 100-400 pM CTX-1, the smooth muscle was depolarized to levels resembling those recorded in this artery during ongoing vasoconstrictor discharge in vivo.     

A micromechanical model of lung tissue rheology

We evaluated the acute effects of ibuprofen and salicylic acid on cAMP-mediated Cl- secretion (Isc) in both colonic and airway epithelia. In T84 cells, ibuprofen inhibited the forskolin-dependent Isc in a concentration-dependent manner, having an apparent Ki of 142 microM. Salicylic acid inhibited Isc with an apparent Ki of 646 microM. We determined whether ibuprofen would also inhibit the forskolin-stimulated Isc in primary cultures of mouse trachea epithelia (MTE) and human bronchial epithelia (HBE). Similar to our results in T84 cells, ibuprofen (500 microM) inhibited the forskolin-induced Isc in MTEs and HBEs by 59+/-4% (n = 11) and 39+/-6% (n = 8), respectively. Nystatin was employed to selectively permeabilize the basolateral or apical membrane to determine the effect of ibuprofen on apical Cl- (ICl) and basolateral K+ (IK) currents after stimulation by forskolin. After forskolin stimulation, ibuprofen (500 microM) reduced both the ICl and IK; reducing ICl and IK by 60 and 15%, respectively. To determine whether this inhibition of ICl was due to the inhibition of CFTR, the effects of ibuprofen and salicylic acid on CFTR Cl- channels in excised, inside-out patches from L-cells were evaluated. Ibuprofen (300 microM) reduced CFTR Cl- current by 60+/-16% and this was explained by a short-lived block (approximately 1.2 ms) which causes an apparent reduction in single channel amplitude from 1.07+/-0.04 pA to 0.59+/-0.04 pA (n = 3). Similarly, salicylic acid (3 mM) reduced CFTR Cl- current by 50+/-8% with an apparent reduction in single channel amplitude from 1.08+/-0.03 pA to 0.48+/-0.06 pA (n = 4). Based on these results, we conclude that the NSAIDs ibuprofen and salicylic acid inhibit cAMP-mediated Cl- secretion in human colonic and airway epithelia via a direct inhibition of CFTR Cl- channels as well as basolateral membrane K+ channels. This may reduce their efficacy in conjunction with other therapeutic strategies designed to increase CFTR expression and/or function in secretory epithelia.     

Role of cyclic GMP-inhibitable phosphodiesterase and nitric oxide in the beta adrenoceptor control of renin secretion

We have reported that inhibition of nitric oxide synthesis with N(G)-nitro-L-arginine methyl ester (L-NAME) attenuates the renin secretory response to beta adrenoceptor stimulation. We proposed that the attenuation results from disinhibition of the cyclic GMP-inhibitable isoform of phosphodiesterase (PDE III) with a resultant increase in cyclic AMP hydrolysis in the juxtaglomerular cells. In our investigation, experiments were performed in conscious rabbits to test the effects of the specific PDE III inhibitor milrinone on resting renin secretion and on the renin responses to isoproterenol and L-NAME. In the first series of experiments, infusion of milrinone increased plasma renin activity from 5.4 +/- 0.6 to 10.2 +/- 1.4 ng/ml/2 hr (P < .01). Heart rate increased markedly, but arterial pressure did not change. In the second series, infusion of isoproterenol increased plasma renin activity from 6.3 +/- 1.1 to 15.0 +/- 1.0 ng/ml/2 hr (P < .01). The renin response to isoproterenol was increased (P < .01) in the presence of milrinone (15.3 +/- 3.7 to 38.4 +/- 6.2 ng/ml/2 hr, P < .01). In the third series, L-NAME alone decreased plasma renin activity from 5.0 +/- 1.0 to 3.3 +/- 1.0 ng/ml/2 hr (P < .01). Milrinone again increased plasma renin activity and prevented the suppression of plasma renin activity by L-NAME. By contrast, milrinone did not alter the suppression of plasma renin activity produced by infusion of phenylephrine. In addition, a PDE IV inhibitor failed to prevent the suppression of PRA by L-NAME. Finally, administration of milrinone completely reversed the L-NAME-induced suppression of the renin response to isoproterenol. These results provide evidence that PDE III participates in the regulation of renin secretion, and support the proposal that the L-NAME-induced reductions in renin secretion and in the renin response to beta adrenoceptor stimulation result from disinhibition of PDE III and increased hydrolysis of cyclic AMP in the juxtaglomerular cells.     

ERCC1 mutations in UV-sensitive Chinese hamster ovary (CHO) cell lines

Bronchodilatory substances such as the phosphodisterase inhibitor (PDE-I) theophylline stimulate mucociliary activity. With the introduction of selective PDE-Is it has become possible to study the functional importance of each phosphodiesterase enzyme (PDE) concerning the regulation of the ciliary beat. The effects of rolipram (inhibiting a cAMP specific PDE (PDE4), milrinone (inhibiting a cGMP inhibited PDE (PDE3)) and zaprinast (inhibiting a cGMP specific PDE (PDE5)) were investigated in in vitro preparations from the rabbit maxillary sinus and trachea. Ciliary beat frequency (CBF) was measured with a photoelectrical method. In sinus mucosa all three compounds accelerated CBF. Milrinone (10(-5) M) by 22.6 +/- 5.3% (n = 6; P < 0.01), rolipram (10(-5) M) by 29.7 +/- 5.7% (n = 7; P < 0.01), and zaprinast (10(-5) M) by 19.4 +/- 6.3% (n = 6; P < 0.05). In the tracheal specimens at a concentration of 10(-5) M, milrinone accelerated CBF by 27.5 +/- 9.0% (n = 7; < 0.05), rolipram by 11.6 +/- 2.8% (n = 6; P < 0.05) and zaprinast by 24.3 +/- 5.3% (n = 7; P < 0.01). Comparison of the effects in the upper and lower airways showed that at concentrations of 10(-5) and 10(-4) M rolipram was more effective in the upper than in the lower airways. The reverse was true of milrinone which concentrations of 10(-7) and 10(-6) M had a significant effect in tracheal specimens but not in sinus specimens. Zaprinast was equally effective in both the upper and lower airways. It is concluded that in both the upper and lower airways selective PDE-Is have an accelerating effect on the CBF that may be beneficial in the treatment of airway diseases.     

Activation of the cloned human NK3 receptor in Chinese Hamster Ovary cells characterized by the cellular acidification response using the Cytosensor microphysiometer

1. The aim of the present study was to validate the Cytosensor microphysiometer, a novel system that measures the extracellular acidification rate as a reliable index of the integrated functional response to receptor activation, as a method for studying NK3 receptor pharmacology, and then to use this system to assess the functional activity of novel compounds at this receptor. 2. The selective NK3 agonist senktide caused reproducible, concentration-related increases in acidification ratein CHO-NK3 cells, with a pEC50 value of 8.72+/-0.11 (n=15). [Beta-Ala8]NKA(4-10), the selective NK2 agonist, elicited a much weaker response (pEC50=6.68+/-0.08, n=4), while the NK1-selective agonist substance P methylester only caused a very weak response at concentrations > or =3 microM (n=2). The rank order of potency for the endogenous tachykinins NKB>NKA>substance P (n=3) confirmed the response was mediated by the NK3 receptor. Moreover, the actual potencies obtained were consistent with affinities measured in radioligand binding studies. 3. The novel compounds PD156319-121 (0.3-1 microM), PD161182 (10-300 nM), PD168001 (10-100 nM) and PD168073 (10-100 nM) all acted as surmountable antagonists of the senktide-induced acidification response, with pA2 values of 7.49, 8.67, 9.17 and 9.25 respectively (n=3-5). In comparison the known NK3 antagonist SR142801 (10-100 nM) had a pA2 value of 8.83 (n=8) for the interaction with senktide. Again, these values are consistent with the radioligand binding data. 4. Amiloride (1 mM) inhibited the senktide-induced acidification response by 68.3+/-3.3 (n=4), indicating that the Na+/H+ antiporter plays an important role in this response, and this is consistent with the importance of this antiporter in other acidification responses. 5. Inhibition of protein kinase C with staurosporine (0.1 microM), or depletion of the intracellular Ca2+ stores with thapsigargin (1 microM), both resulted in a reduction in the maximum response to senktide (63.3+/-1.7 and 68.9+/-3.2% respectively, n=3-5), and co-application of these inhibitors abolished the response (n=3). This strongly suggested that the NK3 receptor was coupling via phospholipase C (PLC), as would be expected, although this could not be confirmed by the use of the putative PLC/PLA2 inhibitor U73122. 6. In conclusion, we have demonstrated the utility of the Cytosensor in the characterization of functional responses to agonists, and assessment of the affinities of antagonists in CHO cells expressing the human NK3, and have shown that our series of novel compounds are non-peptide NK3 antagonists of high affinity, as exemplified by PD168073.