A comparative study of mammalian tracheal mucous glands

We have compared the distribution, numbers and volume of mucous glands in the tracheas of 11 mammalian species. No glands were present in the rabbit. The mouse only contained glands at the border between the trachea and larynx. In the rat, glands were commonest in the cephalad third of the trachea, but on average were much scarcer than in the larger species. Between species, there was a significant correlation between airway diameter and gland volume per unit surface area, suggesting that the rate of deposition of inhaled particles may increase in large airways. In the ventral portion of the trachea of about half the species, the glands were concentrated between the cartilaginous rings; in others they were evenly distributed over and between the rings. In most species in which the trachealis muscle attached to the internal surface of the cartilaginous rings, the glands were external to the muscle. In all species in which the muscle attached to the external surface of the cartilaginous rings, the glands were internal to the muscle. In the ox, goat, dog and sheep, the volume of glands per unit tracheal surface area was markedly greater in the ventral than the dorsal aspect of the trachea. The reverse was true of the pig. In humans, gland density in the 2 regions was similar. The frequency of gland openings was determined in the ox, goat, pig, dog and sheep tracheas, and ranged from 0.3 per mm² in the dorsal portion of the sheep trachea to 1.5 per mm² in the ventral portion of the ox trachea. For these 5 species, the volume of gland acini per unit luminal surface area varied linearly with the numbers of gland openings, with the volume of individual glands being constant at ∼ 120 nl.     

Mast cell-cholinergic nerve interaction in mouse airways

We addressed the mechanism by which antigen contracts trachea isolated from actively sensitized mice. Trachea were isolated from mice (C57BL/6J) that had been actively sensitized to ovalbumin (OVA). OVA (10 μg ml⁻¹) caused histamine release (∼70% total tissue content), and smooth muscle contraction that was rapid in onset and short-lived ( t _1/2 < 1 min), reaching approximately 25% of the maximum tissue response. OVA contraction was mimicked by 5-HT, and responses to both OVA and 5-HT were sensitive to 10 μ m -ketanserin (5-HT₂ receptor antagonist) and strongly inhibited by atropine (1 μ m ). Epithelial denudation had no effect on the OVA-induced contraction. Histological assessment revealed about five mast cells/tracheal section the vast majority of which contained 5-HT. There were virtually no mast cells in the mast cell-deficient ( sash −/−) mouse trachea. OVA failed to elicit histamine release or contractile responses in trachea isolated from sensitized mast cell-deficient ( sash −/−) mice. Intracellular recordings of the membrane potential of parasympathetic neurons in mouse tracheal ganglia revealed a ketanserin-sensitive 5-HT-induced depolarization and similar depolarization in response to OVA challenge. These data support the hypothesis that antigen-induced contraction of mouse trachea is epithelium-independent, and requires mast cell-derived 5-HT to activate 5-HT₂ receptors on parasympathetic cholinergic neurons. This leads to acetylcholine release from nerve terminals, and airway smooth muscle contraction.     

Action potential initiation and propagation in hippocampal mossy fibre axons

Dentate gyrus granule cells transmit action potentials (APs) along their unmyelinated mossy fibre axons to the CA3 region. Although the initiation and propagation of APs are fundamental steps during neural computation, little is known about the site of AP initiation and the speed of propagation in mossy fibre axons. To address these questions, we performed simultaneous somatic and axonal whole-cell recordings from granule cells in acute hippocampal slices of adult mice at ∼23°C. Injection of short current pulses or synaptic stimulation evoked axonal and somatic APs with similar amplitudes. By contrast, the time course was significantly different, as axonal APs had a higher maximal rate of rise (464 ± 30 V s⁻¹ in the axon versus 297 ± 12 V s⁻¹ in the soma, mean ± s.e.m. ). Furthermore, analysis of latencies between the axonal and somatic signals showed that APs were initiated in the proximal axon at ∼20–30 μm distance from the soma, and propagated orthodromically with a velocity of 0.24 m s⁻¹. Qualitatively similar results were obtained at a recording temperature of ∼34°C. Modelling of AP propagation in detailed cable models of granule cells suggested that a ∼4 times higher Na⁺ channel density (∼1000 pS μm⁻²) in the axon might account for both the higher rate of rise of axonal APs and the robust AP initiation in the proximal mossy fibre axon. This may be of critical importance to separate dendritic integration of thousands of synaptic inputs from the generation and transmission of a common AP output.     

Rate, extent and concentration dependence of histamine-evoked Weibel–Palade body exocytosis determined from individual fusion events in human endothelial cells

The rate, concentration dependence and extent of histamine-evoked Weibel–Palade body (WPB) exocytosis were investigated with time-resolved fluorescence microscopy in cultured human umbilical vein endothelial cells expressing WPB-targeted chimeras of enhanced green fluorescent protein (EGFP). Exocytosis of single WPBs was characterized by an increase in EGFP fluorescence, morphological changes and release of WPB contents. The fluorescence increase was due to a rise of intra-WPB pH from resting levels, estimated as pH 5.45 ± 0.26 ( s.d. , n = 144), to pH 7.40. It coincided with uptake of extracellular Alexa-647, indicating the formation of a fusion pore, prior to loss of fluorescent contents. Delays between the increase in intracellular free calcium ion concentration evoked by histamine and the first fusion event were 10.0 ± 4.42 s ( n = 9 cells) at 0.3 μ m histamine and 1.57 ± 0.21 s ( n = 15 cells) at 100 μ m histamine, indicating the existence of a slow process or processes in histamine-evoked WPB exocytosis. The maximum rates of exocytosis were 1.20 ± 0.16 WPB s⁻¹ ( n = 9) at 0.3 μ m and 3.66 ± 0.45 WPB s⁻¹ at 100 μ m histamine ( n = 15). These occurred 2–5 s after histamine addition and declined to lower rates with continued stimulation. The initial delays and maximal rate of exocytosis were unaffected by removal of external Ca²⁺ indicating that the initial burst of secretion is driven by Ca²⁺ release from internal stores, but sustained exocytosis required external Ca²⁺. Data were compared to exocytosis evoked by a maximal concentration of the strong secretagogue ionomycin (1 μ m ), for which there was a delay between calcium elevation and secretion of 1.67 ± 0.24 s ( n = 6), and a peak fusion rate of ∼10 WPB s⁻¹.     

Somatic Exocytosis of Serotonin Mediated by L-Type Calcium Channels in Cultured Leech Neurones

We studied somatic exocytosis of serotonin and its mediation by L-type calcium (Ca²⁺) channels in cultured Retzius neurones of the leech. Exocytosis was induced by trains of impulses at different frequencies or by depolarisation with 40 m m potassium (K⁺), and was quantified by use of the fluorescent dye FM 1–43. Stimulation increased the membrane fluorescence and produced a pattern of FM 1–43 fluorescent spots of 1.28 ± 0.01 μm in diameter, provided that Ca²⁺ was present in the bathing fluid. Individual spots lost their stain during depolarisation with 40 m m K⁺. Electron micrographs showed clusters of dense core vesicles, some of which were in contact with the cell membrane. Presynaptic structures with clear vesicles were absent from the soma. The number of fluorescent spots per soma, but not their diameter or their fluorescence intensity, depended on the frequency of stimulation. Trains at 1 Hz produced 19.5 ± 5 spots per soma, 77.9 ± 13.9 spots per soma were produced at 10 Hz and 91.5 ± 16.9 spots per soma at 20 Hz. Staining patterns were similar for neurones in culture and in situ . In the presence of the L-type Ca²⁺ channel blocker nimodipine (10 μ m ), a 20 Hz train produced only 22.9 ± 6.4 spots per soma, representing a 75 % reduction compared to control cells (   P < 0.05  ). Subsequent incubation with 10 m m caffeine to induce Ca²⁺ release from intracellular stores increased the number of spots to 73.22 ± 12.5. Blockers of N-, P-, Q- or invertebrate Ca²⁺ channels did not affect somatic exocytosis. Our results suggest that somatic exocytosis by neurones shares common mechanisms with excitable endocrine cells.     

Mexiletine block of wild-type and inactivation-deficient human skeletal muscle hNav1.4 Na+ channels

Mexiletine is a class 1b antiarrhythmic drug used for ventricular arrhythmias but is also found to be effective for paramyotonia congenita, potassium-aggravated myotonia, long QT–3 syndrome, and neuropathic pain. This drug elicits tonic block of Na⁺ channels when cells are stimulated infrequently and produces additional use-dependent block during repetitive pulses. We examined the state-dependent block by mexiletine in human skeletal muscle hNav1.4 wild-type and inactivation-deficient mutant Na⁺ channels (hNav1.4-L443C/A444W) expressed in HEK293t cells with a β1 subunit. The 50% inhibitory concentrations (IC₅₀) for the inactivated-state block and the resting-state block of wild-type Na⁺ channels by mexiletine were measured as 67.8 ± 7.0 μ m and 431.2 ± 9.4 μ m , respectively ( n = 5). In contrast, the IC₅₀ for the block of open inactivation-deficient mutant channels at +30 mV by mexiletine was 3.3 ± 0.1 μ m ( n = 5), which was within the therapeutic plasma concentration range (2.8–11 μ m ). Estimated on- and off-rates for the open-state block by mexiletine at +30 mV were 10.4 μ m ⁻¹ s⁻¹ and 54.4 s⁻¹, respectively. Use-dependent block by mexiletine was greater in inactivation-deficient mutant channels than in wild-type channels during repetitive pulses. Furthermore, the IC₅₀ values for the block of persistent late hNav1.4 currents in chloramine-T-pretreated cells by mexiletine was 7.5 ± 0.8 μ m ( n = 5) at +30 mV. Our results together support the hypothesis that the in vivo efficacy of mexiletine is primarily due to the open-channel block of persistent late Na⁺ currents, which may arise during various pathological conditions.     

Effect of dexamethasone on skeletal muscle Na+,K+ pump subunit specific expression and K+ homeostasis during exercise in humans

The effect of dexamethasone on Na⁺,K⁺ pump subunit expression and muscle exchange of K⁺ during exercise in humans was investigated. Nine healthy male subjects completed a randomized double blind placebo controlled protocol, with ingestion of dexamethasone (Dex: 2 × 2 mg per day) or placebo (Pla) for 5 days. Na⁺,K⁺ pump catalytic α1 and α2 subunit expression was ∼17% higher ( P < 0.05) and the structural β1 and β2 subunit expression was ∼6–8% higher ( P < 0.05) after Dex compared with Pla. During one-legged knee-extension for 10 min at low intensity (LI; 18.6 ± 1.0 W), two moderate intensity (51.7 ± 2.4 W) exercise bouts (MI₁: 5 min; 2 min recovery; MI₂: exhaustive) and two high-intensity (71.7 ± 2.5 W) exercise bouts (HI₁: 1 min 40 s; 2 min recovery; HI₂: exhaustive), femoral venous K⁺ was lower ( P < 0.05) in Dex compared with Pla. Thigh K⁺ release was lower ( P < 0.05) in Dex compared with Pla in LI and MI, but not in HI. Time to exhaustion in MI₂ tended to improve (393 ± 50 s versus 294 ± 41 s; P = 0.07) in Dex compared with Pla, whereas no difference was detected in HI₂ (106 ± 10 s versus 108 ± 9 s). The results indicate that an increased Na⁺,K⁺ pump expression per se is of importance for thigh K⁺ reuptake at the onset of low and moderate intensity exercise, but less important during high intensity exercise.     

Electrophysiological properties of BK channels in Xenopus motor nerve terminals

Single channel properties of Ca²⁺-activated K⁺ (BK or Maxi-K) channels have been investigated in presynaptic membranes in Xenopus motoneurone–muscle cell cultures. The occurrence and density of BK channels increased with maturation/synaptogenesis and was not uniform: highest at the release face of bouton-like synaptic varicosities in contact with muscle cells, and lowest in varicosities that did not contact muscle cells. The Ca²⁺ affinity of the channel ( K _d= 7.7 μ m at a membrane potential of +20 mV) was lower than those of BK channels that have been characterized in other terminals. Hill coefficients varied between 1.5 and 2.8 at different potentials and open probability increased e-fold per 16 mV change in membrane potential over a range of [Ca²⁺]_i from 1 μ m to 1 m m . The maximal activation rate of ensembled single BK channel currents was in the submillisecond range at ≥+20 mV. The activation rate increased ∼10-fold in response to a [Ca²⁺]_i increase from 1 to 100 μ m , but increased only ∼2-fold with a voltage change from +20 to +130 mV. The fastest activation kinetics of BK channels in cell-attached patches resembled that in inside-out patches with [Ca²⁺]_i of 100 μ m or more, suggesting that many BK channels are located very close to calcium channels. Given the low Ca²⁺ affinity and rapid Ca²⁺ binding/unbinding properties, we conclude that BK channels in this preparation are adapted to play an important role in regulation of neurotransmitter release, and they are ideal reporters of local [Ca²⁺] at the inner membrane surface.     

Mutation of Walker-A lysine 464 in cystic fibrosis transmembrane conductance regulator reveals functional interaction between its nucleotide-binding domains

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel bears two nucleotide-binding domains (NBD1 and NBD2) that control its ATP-dependent gating. Exactly how these NBDs control gating is controversial. To address this issue, we examined channels with a Walker-A lysine mutation in NBD1 (K464 A) using the patch clamp technique. K464A mutants have an ATP dependence (EC₅₀≈ 60 μ m ) and opening rate at 2.75 m m ATP (∼ 2.1 s⁻¹) similar to wild type (EC₅₀≈ 97 μ m ; ∼ 2.0 s⁻¹). However, K464A's closing rate at 2.75 m m ATP (∼ 3.6 s⁻¹) is faster than that of wild type (∼ 2.1 s⁻¹), suggesting involvement of NBD1 in nucleotide-dependent closing. Delay of closing in wild type by adenylyl imidodiphosphate (AMP-PNP), a non-hydrolysable ATP analogue, is markedly diminished in K464A mutants due to reduction in AMP-PNP's apparent on-rate and acceleration of its apparent off-rate (∼ 2- and ∼ 10-fold, respectively). Since the delay of closing by AMP-PNP is thought to occur via NBD2, K464A's effect on the NBD2 mutant K1250A was examined. In sharp contrast to K464A, K1250A single mutants exhibit reduced opening (∼ 0.055 s⁻¹) and closing (∼ 0.006 s⁻¹) rates at millimolar [ATP], suggesting a role for K1250 in both opening and closing. At millimolar [ATP], K464A-K1250A double mutants close ∼ 5-fold faster (∼ 0.029 s⁻¹) than K1250A but open with a similar rate (∼ 0.059 s⁻¹), indicating an effect of K464A on NBD2 function. In summary, our results reveal that both of CFTR's functionally asymmetric NBDs participate in nucleotide-dependent closing, which provides important constraints for NBD-mediated gating models.     

Differential structural adaptation to haemodynamics along single rat cremaster arterioles

We tested the hypothesis that under physiological conditions, arterioles match their diameter to the level of shear stress. Haemodynamic and anatomical data were obtained in segments of the first-order arteriole of the rat cremaster muscle. Along this segment of ∼10 mm in length, local blood pressure decreased from 68 ± 4 mmHg upstream to 54 ± 3 mmHg downstream ( n = 5). Pulse pressure decreased from 8.2 ± 1.3 mmHg upstream to 4.1 ± 0.6 mmHg downstream. At the same locations, an increase in arteriolar diameter was measured in vivo, from 179 ± 4 μm upstream to 203 ± 4 μm downstream ( n = 10). In vitro pressure-diameter relations of maximally dilated vessels showed that the passive diameter was larger in downstream than upstream segments over a 15–125 mmHg pressure range ( n = 18). The wall stress was similar for the upstream vs. downstream location: 266 ± 16 vs. 260 ± 14 mN mm⁻². However, shear stress decreased from 30 ± 5 to 21 ± 5 dyn cm⁻² (3.0 ± 0.5 to 2.1 ± 0.5 N m⁻²; n = 4) along the artery. In conclusion, these results demonstrate that shear stress is not the only factor in determining vascular calibre. We suggest that arteriolar calibre may rather depend on an interplay between shear stress and the local pressure profile.     

Descending vasa recta pericytes express voltage operated Na+ conductance in the rat

We studied the properties of a voltage-operated Na⁺ conductance in descending vasa recta (DVR) pericytes isolated from the renal outer medulla. Whole-cell patch-clamp recordings revealed a depolarization-induced, rapidly activating and rapidly inactivating inward current that was abolished by removal of Na⁺ but not Ca⁺ from the extracellular buffer. The Na⁺ current ( I _Na) is highly sensitive to tetrodotoxin  (TTX, K _d= 2.2 n m )  . At high concentrations, mibefradil (10 μ m ) and Ni⁺ (1 m m ) blocked I _Na. I _Na was insensitive to nifedipine (10 μ m ). The L-type Ca⁺ channel activator FPL-64176 induced a slowly activating/inactivating inward current that was abolished by nifedipine. Depolarization to membrane potentials between 0 and 30 mV induced inactivation with a time constant of ∼1 ms. Repolarization to membrane potentials between −90 and −120 mV induced recovery from inactivation with a time constant of ∼11 ms. Half-maximal activation and inactivation occurred at −23.9 and −66.1 mV, respectively, with slope factors of 4.8 and 9.5 mV, respectively. The Na⁺ channel activator, veratridine (100 μ m ), reduced peak inward I _Na and prevented inactivation. We conclude that a TTX-sensitive voltage-operated Na⁺ conductance, with properties similar to that in other smooth muscle cells, is expressed by DVR pericytes.     

Tetrahydrobiopterin augments endothelium-dependent dilatation in sedentary but not in habitually exercising older adults

Endothelium-dependent dilatation (EDD) is impaired with ageing in sedentary, but not in regularly exercising adults. We tested the hypotheses that differences in tetrahydrobiopterin (BH₄) bioactivity are key mechanisms explaining the impairment in EDD with sedentary ageing, and the maintenance of EDD with ageing in regularly exercising adults. Brachial artery flow-mediated dilatation (FMD), normalized for local shear stress, was measured after acute oral placebo or BH₄ in young sedentary (YS) ( n = 10; 22 ± 1 years, mean ± s.e.m. ), older sedentary (OS) ( n = 9; 62 ± 2), and older habitually aerobically trained (OT) ( n = 12; 66 ± 1) healthy men. At baseline, FMD was ∼50% lower in OS versus YS (1.12 ± 0.09 versus 0.57 ± 0.09 (Δmm (dyn cm⁻²)) × 10⁻², P < 0.001; 1 dyn = 10⁻⁵ N), but was preserved in OT (0.93 ± 0.08 (Δmm (dyn cm⁻²)) × 10⁻²). BH₄ administration improved FMD by ∼45% in OS (1.00 ± 0.10 (Δmm (dyn cm⁻²)) × 10⁻², P < 0.01 versus baseline), but did not affect FMD in YS or OT. Endothelium-independent dilatation neither differed between groups at baseline nor changed with BH₄ administration. These results suggest that BH₄ bioactivity may be a key mechanism involved in the impairment of conduit artery EDD with sedentary ageing, and the EDD-preserving effect of habitual exercise.     

Effects of Perfusion Rate on Permeability of Frog and Rat Mesenteric Microvessels to Sodium Fluorescein

The permeability, P _S, to sodium fluorescein (Stokes-Einstein radius = 0.45 nm) has been measured in single mesenteric capillaries of pithed frogs and anaesthetised rats as perfusion velocity, U , was varied over a range from 400 up to 2000–10 000 μm s⁻¹. P _S increased linearly with U . In 20 frog capillaries, mean (± S.E.M.) P _S (in μm s⁻¹) = 9.35 (± 1.55) U × 10⁻⁵+ 0.244 (± 0.0291). Similarly, in nine rat venules, mean P _S= 1.62 (± 0.385) U × 10⁻⁴+ 0.375 (± 0.025). The flow-dependent component of permeability could be reversibly abolished in frog capillaries by superfusing with 100 μM noradrenaline and by superfusing rat venules with the nitric oxide synthase inhibitor, N ^G-nitro-L-arginine (20 μM). It was shown that changes in microvascular pressure accompanying changes in U during free perfusion could account for only 15 % of the changes in P _S, i.e. 85 % of the changes in P _S were changes in the permeability coefficient itself. A comparison between the changes in P _S with U and the previously described changes in microvascular permeability to K⁺ with U , suggest that if the flow-dependent component of permeability is modelled as a population of pores of constant size, these have radii of 0.8 nm. Such a pathway would limit flow-dependent permeability to small hydrophilic molecules and have minimal effect on net fluid exchange.     

Cerebral ammonia uptake and accumulation during prolonged exercise in humans

We evaluated whether peripheral ammonia production during prolonged exercise enhances the uptake and subsequent accumulation of ammonia within the brain. Two studies determined the cerebral uptake of ammonia (arterial and jugular venous blood sampling combined with Kety–Schmidt-determined cerebral blood flow; n = 5) and the ammonia concentration in the cerebrospinal fluid (CSF; n = 8) at rest and immediately following prolonged exercise either with or without glucose supplementation. There was a net balance of ammonia across the brain at rest and at 30 min of exercise, whereas 3 h of exercise elicited an uptake of 3.7 ± 1.3 μmol min⁻¹ (mean ± s.e.m. ) in the placebo trial and 2.5 ± 1.0 μmol min⁻¹ in the glucose trial ( P < 0.05 compared to rest, not different across trials). At rest, CSF ammonia was below the detection limit of 2 μ m in all subjects, but it increased to 5.3 ± 1.1 μ m following exercise with glucose, and further to 16.1 ± 3.3 μ m after the placebo trial ( P < 0.05). Correlations were established between both the cerebral uptake  ( r ²= 0.87; P < 0.05)  and the CSF concentration  ( r ²= 0.72; P < 0.05)  and the arterial ammonia level and, in addition, a weaker correlation  ( r ²= 0.37; P < 0.05)  was established between perceived exertion and CSF ammonia at the end of exercise. The results let us suggest that during prolonged exercise the cerebral uptake and accumulation of ammonia may provoke fatigue, e.g. by affecting neurotransmitter metabolism.     

Mechanical load plays little role in contraction-mediated glucose transport in mouse skeletal muscle

The factors responsible for control of glucose transport during exercise are not fully understood. We investigated the role of mechanical load in contraction-mediated glucose transport in an isolated muscle preparation. Mouse extensor digitorum longus muscles were stimulated with repeated contractions for 10 min with or without N -benzyl-p-toluene sulphonamide (BTS, an inhibitor of myosin II ATPase) to block crossbridge activity. BTS inhibited force production during repeated contraction to ∼5% of control. In contrast, BTS had little effect on glucose transport in the basal state (control = 0.55 ± 0.04; BTS = 0.47 ± 0.09 μmol (20 min)⁻¹ ml⁻¹) or after contraction (control = 2.27 ± 0.15; BTS = 2.10 ± 0.16 μmol (20 min)⁻¹ ml⁻¹). BTS did not significantly alter the contraction-mediated changes in high-energy phosphates, glutathione status (a measure of oxidant status) or AMP-activated protein kinase activity. In conclusion, these data show that mechanical load plays little role in contraction-mediated glucose transport. Instead, it is likely that the increased glucose transport during contraction is a consequence of the increase in myoplasmic Ca²⁺ and the subsequent alterations in metabolism, e.g. increased energy turnover and production of reactive oxygen species.     

Cyclic movement stimulates hyaluronan secretion into the synovial cavity of rabbit joints

The novel hypothesis that the secretion of the joint lubricant hyaluronan (HA) is coupled to movement has implications for normal function and osteoarthritis, and was tested in the knee joints of anaesthetized rabbits. After washing out the endogenous synovial fluid HA (miscibility coefficient 0.4), secretion into the joint cavity was measured over 5 h in static joints and in passively cycled joints. The net static secretion rate (11.2 ± 0.7 μg h⁻¹, mean ± s.e.m. , n = 90) correlated with the variable endogenous HA mass (mean 367 ± 8 μg), with a normalized value of 3.4 ± 0.2 μg h⁻¹ (100 μg)⁻¹     . Cyclic joint movement approximately doubled the net HA secretion rate to 22.6 ± 1.2 μg h⁻¹ ( n = 77) and raised the normalized percentage     to 5.9 ± 0.3 μg h⁻¹ (100 μg)⁻¹. Secretion was inhibited by 2-deoxyglucose and iodoacetate, confirming active secretion. The net accumulation rate underestimated true secretion rate due to some trans-synovial loss. HA turnover time (endogenous mass/secretion rate) was 17–30 h (static) to 8–15 h (moved) The results demonstrate for the first time that the active secretion of HA is coupled to joint usage. Movement–secretion coupling may protect joints against the damaging effects of repetitive joint use, replace HA lost during periods of immobility (overnight), and contribute to the clinical benefit of exercise therapy in moderate osteoarthritis.     

The density of AMPA receptors activated by a transmitter quantum at the climbing fibre-Purkinje cell synapse in immature rats

We aimed to estimate the number of AMPA receptors (AMPARs) bound by the quantal transmitter packet, their single-channel conductance and their density in the postsynaptic membrane at cerebellar Purkinje cell synapses. The synaptic and extrasynaptic AMPARs were examined in Purkinje cells in 2- to 4-day-old rats, when they receive synaptic inputs solely from climbing fibres (CFs). Evoked CF EPSCs and whole-cell AMPA currents displayed roughly linear current-voltage relationships, consistent with the presence of GluR2 subunits in synaptic and extrasynaptic AMPARs. The mean quantal size, estimated from the miniature EPSCs (MEPSCs), was ∼300 pS. Peak-scaled non-stationary fluctuation analysis of spontaneous EPSCs and MEPSCs gave a weighted-mean synaptic channel conductance of ∼5 pS (∼7 pS when corrected for filtering). By applying non-stationary fluctuation analysis to extrasynaptic currents activated by brief glutamate pulses (5 m m ), we also obtained a small single-channel conductance estimate for extrasynaptic AMPARs (∼11 pS). This approach allowed us to obtain a maximum open probability ( P _o,max) value for the extrasynaptic receptors ( P _o,max= 0.72). Directly resolved extrasynaptic channel openings in the continued presence of glutamate exhibited clear multiple-conductance levels. The mean area of the postsynaptic density (PSD) of these synapses was 0.074 μm², measured by reconstructing electron-microscopic (EM) serial sections. Postembedding immunogold labelling by anti-GluR2/3 antibody revealed that AMPARs are localised in PSDs. From these data and by simulating error factors, we estimate that at least 66 AMPARs are bound by a quantal transmitter packet at CF-Purkinje cell synapses, and the receptors are packed at a minimum density of ∼900 μm⁻² in the postsynaptic membrane.     

A-Type potassium currents active at subthreshold potentials in mouse cerebellar purkinje cells

Voltage-dependent and calcium-independent K⁺ currents were whole-cell recorded from cerebellar Purkinje cells in slices. Tetraethylammonium (TEA, 4 m m ) application isolated an A-type K⁺ current ( I _{k ( a )}) with a peak amplitude, at +20 mV, of about one third of the total voltage-dependent and calcium-independent K⁺ current. The I _{k ( a )} activated at about −60 mV, had a V _0.5 of activation of −24.9 mV and a V _0.5 of inactivation of −69.2 mV. The deactivation time constant at −70 mV was 3.4 ± 0.4 ms, while the activation time constant at +20 mV was 0.9 ± 0.2 ms. The inactivation kinetics was weakly voltage dependent, with two time constants; those at +20 mV were 19.3 ± 3.1 and 97.6 ± 9.8 ms. The recovery from inactivation had two time constants of 60.8 ms (78.4%) and 962.3 ms (21.6%). The I _{k ( a )} was blocked by 4-aminopyridine with an IC₅₀ of 67.6 μM. Agitoxin-2 (2 n m ) blocked 17.4 ± 2.1% of the I _{k ( a )}. Flecainide completely blocked the I _{k ( a )} with a biphasic effect with IC₅₀ values of 4.4 and 183.2 μM. In current-clamp recordings the duration of evoked action potentials was affected neither by agitoxin-2 (2 n m ) nor by flecainide (3 μM), but action potentials that were already broadened by TEA were further prolonged by 4-aminopyridine (100 μM). The amplitude of the hyperpolarisation at the end of depolarising steps was reduced by all these blockers.     

Quantitative assessment of intestinal eosinophils and mast cells in inflammatory bowel disease

Previous studies on the frequency of intestinal mast cells and eosinophils in patients with inflammatory bowel disease yielded conflicting results. In the present morphometric study, we quantified mast cells and eosinophils in the lamina propria by histological and immunohistochemical methods in 64 patients suffering from Crohn's disease (33 cases) or ulcerative colitis (31 cases), and in 29 controls. Histological data from 206 biopsies were related to the presence of mucosal inflammation and clinical parameters. The number of eosinophils was increased in patients with inflammatory bowel conditions (mean ±  SE : 331 ± 44/mm²) as compared to controls (258 ± 27/mm²), and was dependent on disease activity and drug treatment. Mean mast cell numbers did not differ between patients and controls. However, a reduced mast cell number was found in toluidine blue-stained sections of actively inflamed tissue areas (143 ± 16/mm², versus 206 ± 18/mm² in non-inflamed tissue). Immunohistochemical studies using antibodies against the granule proteins tryptase and chymase suggest that this decrease in mast cell numbers is due to mast cell degranulation. The present data show that the number of intestinal mast cells and eosinophils is altered in patients with inflammatory bowel diseases, suggesting that both cell types are involved in the pathogenesis of chronic intestinal inflammation.     

Differential effect of angiotensin II on blood circulation in the renal medulla and cortex of anaesthetised rats

The renal medulla is sensitive to hypoxia, and a depression of medullary circulation, e.g. in response to angiotensin II (Ang II), could endanger the function of this zone. Earlier data on Ang II effects on medullary vasculature were contradictory. The effects of Ang II on total renal blood flow (RBF), and cortical and medullary blood flow (CBF and MBF: by laser-Doppler flux) were studied in anaesthetised rats. Ang II infusion (30 ng kg⁻¹ min⁻¹ i.v. ) decreased RBF 27 ± 2 % (mean ± s.e.m. ), whereas MBF increased 12 ± 2 % (both P < 0.001). Non-selective blockade of Ang II receptors with saralasin (3 μg kg⁻¹ min⁻¹ i.v. ) increased RBF 12 ± 2 % and decreased MBF 8 ± 2 % ( P < 0.001). Blockade of AT₁ receptors with losartan (10 mg kg⁻¹) increased CBF 10 ± 2 % ( P < 0.002) and did not change MBF. Losartan given during Ang II infusion significantly increased RBF (53 ± 7 %) and decreased MBF (27 ± 7 %). Blockade of AT₂ receptors with PD 123319 (50 μg kg⁻¹ min⁻¹ i.v. ) did not change CBF or MBF. Intramedullary infusion of PD 123319 (10 μg min⁻¹) superimposed on intravenous Ang II infusion did not change RBF, but slightly decreased MBF (4 ± 2 %, P < 0.05). We conclude that in anaesthetised surgically prepared rats, exogenous or endogenous Ang II may not depress medullary circulation. In contrast to the usual vasoconstriction in the cortex, vasodilatation was observed, possibly related to secondary activation of vasodilator paracrine agents rather than to a direct action via AT₂ receptors.