Sympathetic Nerves Inhibit Conducted Vasodilatation Along Feed Arteries during Passive Stretch of Hamster Skeletal Muscle

Ascending vasodilatation is integral to blood flow control in exercising skeletal muscle and is attributable to conduction from intramuscular arterioles into proximal feed arteries. Passive stretch of skeletal muscle can impair muscle blood flow but the mechanism is not well understood. We hypothesized that the conduction of vasodilatation along feed arteries can be modulated by changes in muscle length. In anaesthetized hamsters, acetylcholine (ACh) microiontophoresis triggered conducted vasodilatation along feed arteries (diameter, 50-70 μm) of the retractor muscle secured at 100 % resting length or stretched by 30 %. At 100 % length, ACh evoked local dilatation (> 30 μm) and this response conducted rapidly along the feed artery (14 ± 1 μm dilatation at 1600 μm upstream). During muscle stretch, feed arteries constricted ≈10 μm (   P < 0.05  ) and local vasodilatation to ACh was maintained while conducted vasodilatation was reduced by half (   P < 0.01  ). Resting diameter and conduction recovered upon restoring 100 % length. Sympathetic nerve stimulation (4-8 Hz) produced vasoconstriction and attenuated conduction in the manner observed during muscle stretch, as did noradrenaline or phenylephrine (10 nM). Inhibiting nitric oxide production ( N ^ω-nitro-L-arginine, 50 μM) produced similar vasoconstriction yet had no effect on conduction. Phentolamine, prazosin, or tetrodotoxin (1 μM) during muscle stretch abolished vasoconstriction and restored conduction. Inactivation of sensory nerves with capsaicin had no effect on vasomotor responses. Thus, muscle stretch can attenuate conducted vasodilatation by activating α-adrenoreceptors on feed arteries through noradrenaline released from perivascular sympathetic nerves. This autonomic feedback mechanism can restrict muscle blood flow during passive stretch.     

Angiotensin II-induced modulation of endothelium-dependent relaxation in rabbit mesenteric resistance arteries

The role of local endogenous angiotensin II (Ang II) in endothelial function in resistance arteries was investigated using rabbit mesenteric resistance arteries. First, the presence of immunoreactive Ang II together with Ang II type-1 receptor (AT₁R) and angiotensin converting enzyme (ACE) was confirmed in these arteries. In endothelium-intact strips, the AT₁R-blocker olmesartan (1 μ m ) and the ACE-inhibitor temocaprilat (1 μ m ) each enhanced the ACh (0.03 μ m )-induced relaxation during the contraction induced by noradrenaline (NA, 10 μ m ). Similar effects were obtained using CV-11974 (another AT₁R blocker) and enalaprilat (another ACE inhibitor). The nitric-oxide-synthase inhibitor N ^G-nitro- l -arginine ( l -NNA) abolished the above effect of olmesartan. In endothelium-denuded strips, olmesartan enhanced the relaxation induced by the NO donor NOC-7 (10 n m ). Olmesartan had no effect on cGMP production (1) in endothelium-intact strips (in the absence or presence of ACh) or (2) in endothelium-denuded strips (in the absence or presence of NOC-7). In β-escin-skinned strips, 8-bromoguanosine 3',5' cyclic monophosphate (8-Br-cGMP, 0.01–1 μ m ) concentration dependently inhibited the contractions induced (a) by 0.3 μ m Ca²⁺ in the presence of NA+GTP and (b) by 0.2 μ m Ca²⁺+GTPγS. Olmesartan significantly enhanced, while Ang II (0.1 n m ) significantly inhibited, the 8-Br-cGMP-induced relaxation. We propose the novel hypothesis that in these arteries, Ang II localized within smooth muscle cells activates AT₁Rs and inhibits ACh-induced, endothelium-dependent relaxation at least partly by inhibiting the action of cGMP on these cells.     

Enhancement of spontaneous synaptic activity in rat Purkinje neurones by ATP during development

The establishment of functional synaptic connections and activity is a pivotal process in the development of neuronal networks. We have studied the synaptic activity in the developing rat cerebellum, and the contribution mediated by purinergic receptors. The mean frequency of the spontaneous postsynaptic currents (sPSCs) recorded with the whole-cell patch-clamp technique from Purkinje neurones in acute brain slices at room temperature, increased fourfold from 4.4 ± 0.8 Hz at postnatal day 9/10 ( n = 23) to 17.8 ± 1.6 Hz at postnatal day 17–20 (p17–p20; n = 113; P < 0.01). ATP, which increased the frequency of sPSCs by up to 100%  (EC₅₀= 18 μ m )  in the third postnatal week, started to modulate the synaptic activity during the second postnatal week, which was determined by three processes: (1) the appearance of functional ATP receptors during p10–p12, (2) the enhancement of the sPSC frequency by endogenous ATP release becoming apparent after inhibition of ecto-ATPases by 6- N , N -diethyl-β,γ-dibromomethylene- d -adenosine-5-triphosphate (ARL67156; 50 μ m ) at p11–p12, and (3) with tonic stimulation of purinoceptors at p14, as revealed by the P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 μ m ). ATP had a similar effect at later stages (p24–p27) and at 35°C. Our results suggest that endogenous release of ATP starts to enhance the synaptic activity in Purkinje neurones by the end of the second postnatal week.     

Prolonged increase in ciliary beat frequency after short-term purinergic stimulation in human airway epithelial cells

Stimulation of ovine airway epithelial cells with 10 μ m ATP for 1 min at 25 °C transiently increased both cytoplasmic calcium (fura-2 epifluorescence microscopy) and ciliary beat frequency (CBF; differential interference contrast microscopy) with a similar time course. Identical purinergic stimulation of human airway epithelial cells at 25 or 35 °C, however, lead to an increase in CBF that outlasted the calcium transient at least 20 min. While a nitric oxide synthase inhibitor had no effect, pre-treatment of human cells with inhibitors of cAMP-dependent kinase (PKA), 10 μ m myristoylated PKA-inhibitory peptide and 1 μ m KT-5720, as well as an inhibitor of adenylyl cyclase, 1 m m SQ22536, blocked the prolonged, but not calcium-coupled CBF increase. Addition of PKA inhibitors after purinergic stimulation only partially reduced CBF from its elevated plateau. Prolonged CBF increases did not depend on adenosine production as 10 μ m UTP had an effect similar to ATP and 8-sulphophenyl-theophylline did not block them. After increasing human CBF in a PKA-dependent manner to a stable plateau with forskolin (10 μ m ), ATP caused only a transient, calcium-coupled CBF increase. Calcium transients were necessary for both short-term and prolonged CBF changes as ATP failed to produce CBF increases after emptying calcium stores with 1 μ m thapsigargin. These data suggest that in human, but not ovine airway epithelial cells, ATP-induced calcium transients activate a signalling cascade including adenylyl cyclase and PKA. The resulting prolonged CBF stimulation does not rely only on PKA activity, suggesting that the decay of CBF is influenced by ciliary phosphatase activity.     

Small- and Intermediate-Conductance Calcium-Activated K+ Channels Provide Different Facets of Endothelium-Dependent Hyperpolarization in Rat Mesenteric Artery

Activation of both small-conductance (SK_Ca) and intermediate-conductance (IK_Ca) Ca²⁺-activated K⁺ channels in endothelial cells leads to vascular smooth muscle hyperpolarization and relaxation in rat mesenteric arteries. The contribution that each endothelial K⁺ channel type makes to the smooth muscle hyperpolarization is unknown. In the presence of a nitric oxide (NO) synthase inhibitor, ACh evoked endothelium and concentration-dependent smooth muscle hyperpolarization, increasing the resting potential (approx. −53 mV) by around 20 mV at 3 μ m . Similar hyperpolarization was evoked with cyclopiazonic acid (10 μ m , an inhibitor of sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA)) while 1-EBIO (300 μ m , an IK_Ca activator) only increased the potential by a few millivolts. Hyperpolarization in response to either ACh or CPA was abolished with apamin (50 n m , an SK_Ca blocker) but was unaltered by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (1 μ m TRAM-34, an IK_Ca blocker). During depolarization and contraction in response to phenylephrine (PE), ACh still increased the membrane potential to around −70 mV, but with apamin present the membrane potential only increased just beyond the original resting potential ( circa −58 mV). TRAM-34 alone did not affect hyperpolarization to ACh but, in combination with apamin, ACh-evoked hyperpolarization was completely abolished. These data suggest that true endothelium-dependent hyperpolarization of smooth muscle cells in response to ACh is attributable to SK_Ca channels, whereas IK_Ca channels play an important role during the ACh-mediated repolarization phase only observed following depolarization.     

Somato-dendritic nicotinic receptor responses recorded in vitro from the medial septal diagonal band complex of the rodent

The medial septal diagonal band area (MS/DB), made up of GABAergic and cholinergic neurones, plays an essential role in the generation and modulation of the hippocampal theta rhythm. To understand the part that the cholinergic neurones might play in this activity, we sought to determine whether postsynaptic nicotinic receptor responses can be detected in slices of the rodent MS/DB by puffing on acetylcholine (ACh). Neurones were characterized electrophysiologically into GABAergic and cholinergic neurones according to previous criteria. Responses of the MS/SB neurones to ACh were various combinations of fast depolarizations (1.5–2.5 s), fast hyperpolarizations (3–4 s) and slow depolarizations (20–30 s), the latter two being blocked by atropine. The fast depolarizations were partially or not blocked with cadmium and low calcium, tetrodotoxin, and antagonists of other ionotropic receptors, and were antagonized with 25 μ m mecamylamine. Pharmacological investigation of the responses showed that the α7* nicotinic receptor type is associated with cholinergic neurones and 10% of the GABAergic neurones, and that nonα7* nicotinic receptor subtypes are associated with 50% of the GABAergic neurones. Pharmacological dissection of evoked and spontaneous postsynaptic responses, however, did not provide evidence for synaptic nicotinic receptor transmission in the MS/DB. It was concluded that nicotinic receptors, although prevalent on the somatic and/or dendritic membrane compartments of neurones in the MS/DB, are on extrasynaptic sites where they presumably play a neuromodulatory role. The presence of α7* nicotinic receptors on cholinergic neurones may also render these cells specifically vulnerable to degeneration in Alzheimer's disease.     

Sleep during arousal episodes as a function of prior torpor duration in hibernating European ground squirrels

EEG's were recorded in hibernating European ground squirrels during euthermic arousal episodes at an ambient temperature of 5.5°C. Spontaneous torpor bouts ranged from 6 to 15 days, body temperature during torpor was 7.5°C. The torpor duration prior to EEG measurements was experimentally manipulated: the animals were induced to arouse by gentle handling after torpor of less then 1 day (n=3), 1–2 days (n=6), 3–4 days (n=9) and 5–12 days (n=9). The animals slept 71.5% of euthermic time, of which 61.4% NREM and 10.2% REM sleep. NREM percentage was slightly positively and REM percentage negatively correlated with prior torpor duration (TD). Spectral analysis showed changes in EEG activity during the euthermic phase in the slow wave frequency range (1–4 Hz) and in higher frequencies. Prior TD specifically affected the slow waves. Slow wave activity decreased exponentially during the euthermic phase. The initial slow wave activity showed a systematic increase with prior TD, which could be described by an exponentially saturating function, albeit with a relatively small time constant compared with spontaneous torpor duration. It is concluded that sleep during arousal episodes following torpor at an ambient temperature of 5.5°C is affected both in structure and intensity by prior TD. The results are consistent with the proposition that torpor inhibits the restorative function of sleep.     

Time to fatigue is increased in mouse muscle at 37°C; the role of iron and reactive oxygen species

Studies exploring the rate of fatigue in isolated muscle at 37°C have produced mixed results. In the present study, muscle fibre bundles from the mouse foot were used to study the effect of temperature on the rate of muscle fatigue. Provided iron was excluded from the solutions, time to fatigue at 37°C was increased compared to 22°C (125 ± 8% of 22°C fatigue time). In contrast, when iron was present (∼1 μ m ), fatigue was accelerated (68 ± 10%). Iron can increase reactive oxygen species (ROS), which are believed to accelerate fatigue. The addition of 25–100 μ m H₂O₂ at 22°C reduced time to fatigue to 80–20% of the control, respectively. Iron was added to cultured primary skeletal muscle cells to determine if iron could increase ROS production. Neither iron entry nor ROS production were detected in non-contracting muscle cells. The addition of 8-hydroxyquinoline, which facilitates iron entry, to iron–ascorbic acid solutions caused a rapid rise in intracellular iron and ROS. Our results indicate that time to fatigue in vitro is increased at 37°C relative to 22°C, but the addition of ROS can accelerate fatigue. An increase in muscle iron can accelerate ROS production, which may be important during or following exercise and in haemochromatosis, disuse atrophy and sarcopenia.     

Barium decreases endothelium-dependent smooth muscle responses to transient but not to more prolonged acetylcholine applications

The influence of inhibiting the inward rectifier and Na/K pump on endothelium-dependent hyperpolarizations in smooth muscle cells of the mesenteric artery was investigated. Membrane potential was measured with microelectrodes, and the influence of low concentrations of Ba2+ (30 microM) and of high concentrations of ouabain (0.5 mM) on smooth muscle hyperpolarization elicited by prolonged or by transient exposure to acetylcholine (ACh, 3x10(-7) M) was assessed in the continuous presence of NG-nitro-L-arginine (100 microM) and indomethacin (50 microM). Pre-exposure to Ba2+ did not inhibit the magnitude of smooth muscle cell hyperpolarization induced by ACh superfusion, but significantly slowed its onset and time course. The membrane potential response to transient ACh applications, however, was impaired. After combined Ba2+ and ouabain pre-exposure, peak hyperpolarizations to ACh superfusion were somewhat decreased but not abolished. In addition, 4-5 mM increases of the extracellular K+ concentration consistently depolarized smooth muscle cells. These findings argue against the idea that smooth muscle inward rectifier K+ channels and Na/K pumping play a role in the ACh-induced endothelium-dependent hyperpolarization of this preparation. Moreover, the slowing of smooth muscle membrane hyperpolarization by Ba2+ is discussed in terms of the influence of this ion on the release of hyperpolarizing factor.     

Dehydroepiandrosterone Potentiates Native Ionotropic ATP Receptors Containing the P2X2 Subunit in Rat Sensory Neurones

We have studied the modulatory effect of dehydroepiandrosterone (DHEA), the most abundant neurosteroid produced by glial cells and neurones, on membrane currents induced by the activation of ionotropic ATP (P2X) receptors in neonatal rat dorsal root ganglion neurones. ATP (1 μ m ) induced three types of currents/responses termed F (fast and transient), S (slowly desensitizing) and M (mixed, sum of F- and S-type responses). DHEA (10 n m to 100 μ m ) concentration-dependently increased the amplitude of plateau-like currents of S- and M-type responses evoked by submaximal (1 μ m ) but not saturating (100 μ m or 1 mM) concentrations of ATP. αβ-Methylene ATP (αβme-ATP, 5 μ m ) also evoked F-, S- and M-type responses, the plateau phases of which were potentiated by lowering external pH (6.3) and by ivermectin (IVM, 3 μ m ), indicating the presence heteromeric P2X₂-containing receptors and possibly of functional native P2X_4/6 receptors. There was a strict correlation between the potentiating effects of low pH and DHEA on αβme-ATP responses but not between that of IVM and DHEA, suggesting that DHEA selectively modulated P2X₂-containing receptors. DHEA also potentiated putative homomeric P2X₂ receptor responses recorded in the continuous presence of 1 μ m 2'-(or 3')- O -(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP). Our results constitute the first demonstration of a fast potentiation of P2X receptors by a neurosteroid and suggest that DHEA could be an endogenous modulator of P2X₂-containing receptors thereby contributing to the facilitation of the detection and/or the transmission of nociceptive messages, particularly under conditions of inflammatory pain where the P2X receptor signalling pathway appears to be upregulated.     

Role of the medullary raphé in thermoregulatory vasomotor control in rats

To investigate the involvement of the medullary raphé in thermoregulatory vasomotor control, we chemically manipulated raphé neuronal activity while monitoring the tail vasomotor response to preoptic warming. For comparison, neuronal activity in the rostral ventrolateral medulla (RVLM) was manipulated in similar experiments. Injections of d,l -homocysteic acid (DLH; 0.5 m m , 0.3 μl) into a restricted region of the ventral medullary raphé suppressed the tail vasodilatation normally elicited by warming the preoptic area to 42 °C. DLH injection into the RVLM also suppressed the vasodilatation elicited by preoptic warming. Injection of bicuculline (0.5 m m , 0.3 μl) into the same raphé region suppressed the vasodilatation elicited by preoptic warming. Bicuculline injection into the RVLM did not suppress tail vasodilatation. These results suggest that neurones in both the medullary raphé and the RVLM are vasoconstrictor to the tail, but only those in the raphé receive inhibitory input from the preoptic area. That input might be direct and/or indirect (e.g. via the periaqueductal grey matter).     

Replacement of connexin 43 by connexin 32 in a knock-in mice model attenuates aortic endothelium-derived hyperpolarizing factor-mediated relaxation

Previous studies demonstrated that intercellular communication through endothelial, smooth muscle or myoendothelial connexin channels contributes to the control of vascular tone. At least four connexin types are present in the arterial wall. The aim of the present work was to assess the role played by connexin 43 (Cx43)-formed gap junctions on vessel function. Aortic reactivity to noradrenaline, acetylcholine and sodium nitroprusside, and endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations, were analysed in a Cx43KI32 mouse model in which the coding region of Cx43 was replaced by that of connexin 32 (Cx32). Aortic rings were placed in organ baths containing a Krebs solution oxygenated at 37°C (pH 7.4). Confocal images of aortic rings confirmed connexin substitution in mutant mice. In control conditions, replacement of Cx43 by Cx32 in homozygous mutant mice did not modify endothelium-independent contractile responses to noradrenaline, or relaxations in response to sodium nitroprusside (endothelium independent) or acetylcholine (endothelium dependent). However, residual endothelium-dependent relaxations in response to acetylcholine after nitric oxide synthase and cyclooxygenase inhibition (EDHF type) were significantly reduced in homozygous Cx43KI32 mice (maximal effect values: 4.86 ± 0.37% of noradrenaline precontraction versus 7.06 ± 0.31% in wild-type, n = 8, P < 0.05). This attenuation was mimicked by treatment of rings from wild-type animals with the connexin-mimetic peptide ^37,43Gap27 (5 × 10⁻⁶ m ). In conclusion, replacement of Cx43 by Cx32 attenuates EDHF-mediated relaxations in mice aortic rings, suggesting that they are, at least in part, dependent on Cx43-formed gap junctions. In contrast, aortic responses to tested endothelium-independent agonists were not modified in knock-in animals.     

Attenuation of conducted vasodilatation in rat mesenteric arteries during hypertension: role of inwardly rectifying potassium channels

The present study was designed to elucidate whether the conduction of vasomotor responses mediated by endothelium-derived hyperpolarizing factor (EDHF) in rat mesenteric arteries is altered during hypertension. Iontophoresed acetylcholine (ACh; 500 ms) caused EDHF-mediated hyperpolarization and vasodilatation at the local site and these responses spread through the endothelium to remote sites in 12-week-old Wistar-Kyoto rats (WKY). Conducted responses were significantly attenuated in age-matched spontaneously hypertensive rats (SHR) although the rate of decay with distance did not change. Inhibition of inwardly rectifying potassium (Kir) channels (30 μ m barium) eliminated the difference between WKY and SHR by attenuating conducted responses in WKY but not SHR. At the local site, barium (30 μ m ) significantly reduced the duration but not the amplitude of ACh-induced hyperpolarization in WKY only. Barium had no effect when the iontophoretic stimulus was reduced to 350 ms. After blockade of EDHF in SHR, ACh elicited a depolarization which our indirect data suggest spreads along the vessel in the endothelium. Messenger RNA expression of Kir2.0 genes did not differ between the strains nor did the amplitude of K⁺-induced hyperpolarization, which was abolished by disruption of the endothelium. Immunohistochemistry revealed a decrease in connexin (Cx)37 but not Cx40 or Cx43 protein in endothelial cells of SHR compared to WKY. Results suggest that conduction of EDHF-mediated responses in WKY, but not in SHR, is facilitated by activation of Kir channels at the site of ACh application and not by differences in endothelial connexin expression. Lack of Kir channel involvement in hypertension may result from reduction in the duration of the hyperpolarization due to the development of ACh-mediated depolarization, rather than to any difference in Kir subunit expression or function.     

Voltage is a partial activator of rat thermosensitive TRP channels

TRPV1 and TRPM8 are sensory nerve ion channels activated by heating and cooling, respectively. A variety of physical and chemical stimuli activate these receptors in a synergistic manner but the underlying mechanisms are unclear. Both channels are voltage sensitive, and temperature and ligands modulate this voltage dependence. Thus, a voltage-sensing mechanism has become an attractive model to explain the generalized gating of these and other thermo-sensitive TRP channels. We show here using whole-cell and single channel measurements that voltage produces only a partial activation of TRPV1 and TRPM8. At room temperature (20–25°C) membrane depolarization evokes responses that saturate at ∼50–60% of the maximum open probability. Furthermore, high concentrations of capsaicin (10 μ m ), resiniferatoxin (5 μ m ) and menthol (6 m m ) reveal voltage-independent gating. Similarly, other modes of TRPV1 regulation including heat, protein kinase C-dependent phosphorylation, and protons enhance both the efficacy and sensitivity of voltage activation. In contrast, the TRPV1 antagonist capsazepine produces the opposite effects. These data can be explained by an allosteric model in which voltage, temperature, agonists and inverse agonists are independently coupled, either positively or negatively, to channel gating. Thus, voltage acts separately but in concert with other stimuli to regulate channel activation, and, therefore, a voltage-sensitive mechanism is unlikely to represent a final, gating mechanism for these channels.     

Ca2+ release-dependent hyperpolarizations modulate the firing pattern of juvenile GABA neurons in mouse substantia nigra pars reticulata in vitro

A phasic activation of small-conductance Ca²⁺-dependent K⁺ channels (SK channels) underlies spike-afterhyperpolarizations and spike-independent, transient hyperpolarizations in juvenile substantia nigra neurons. Outward current pulses that cause the spike-independent hyperpolarizations result from ryanodine receptor-mediated Ca²⁺ release from intracellular stores. To study the modulation of excitability by the outward current pulses, we recorded from GABAergic pars reticulata neurons of mice at postnatal days 12–16. We induced a prolongation of SK channel open states by 1-ethyl-2-benzimidazolinone (1-EBIO). In addition to a prolongation of spike-afterhyperpolarizations, 1-EBIO (200 μ m ) potentiated outward current pulses by increasing their duration. Neurons were manipulated by current injection to display continuous or discontinuous discharge. Despite the prolongation of the outward current pulses by 1-EBIO, continuous action potential discharge became more regular, although its frequency declined. Durations of silent periods (periods of >2× average interspike interval) increased. Caffeine (1 m m ) further increased the duration of such silent periods. Caffeine, however, had no effect at short interspike intervals (<600 ms). Cyclopiazonic acid (10 μ m ) silenced discharge in 1-EBIO, but discharge reappeared with the depletion of Ca²⁺ stores. We conclude that the modulation of excitability by an activation of SK channels through ryanodine receptor-mediated release of Ca²⁺ critically depends on the frequency of discharge. Outward current pulses occur only if interspike intervals exceed the duration of spike-afterhyperpolarizations. In this instance, the phasic, spike-independent activation of SK channels supports pauses to interrupt autonomous discharge in juvenile GABAergic pars reticulata neurons.     

Activation of bronchopulmonary vagal afferent nerves with bradykinin, acid and vanilloid receptor agonists in wild-type and TRPV1–/– mice

The vanilloid receptor TRPV1 (formerly VR1) has been implicated in the activation of nociceptive sensory nerves by capsaicin, noxious heat, protons, bradykinin, cannabinoids such as anandamide, and certain metabolites of arachidonic acid. Using TRPV1 knockout mouse (TRPV1–/–) we address the question of whether TRPV1 is obligatory for action potential discharge in vagal C-fibre terminals evoked by capsaicin, anandamide, acid and bradykinin. The response of a defined subtype of the vagal afferent bronchopulmonary C-fibres (conduction velocity < 0.7 ms⁻¹) to the putative TRPV1 activators was studied in vitro in the mouse isolated/perfused lung–nerve preparation. Capsaicin (1 μ m ) evoked action potential discharge of ∼90% (28/31) of C-fibres in the TRPV1+/+ mice, but failed to activate bronchopulmonary C-fibres in TRPV1–/– animals  ( n = 10)  . Anandamide (3–100 μ m ) induced concentration-dependent activation of capsaicin-sensitive TRPV1+/+ C-fibres with a threshold of 3–10 μ m , but failed to evoke substantive discharge in TRPV1–/– C-fibres. In the TRPV1+/+ mice, the B₂ receptor-mediated activation by bradykinin (1 μ m ) was restricted to the capsaicin-sensitive C-fibres. Bradykinin was effective in evoking B₂ receptor-mediated action potential discharge in TRPV1–/– C-fibres, but the response was significantly ( P < 0.05) less persistent than in TRPV1+/+ C-fibres. Exposing the tissue to acid (pH = 5) excited both TRPV1+/+ and TRPV1–/– C-fibres. We conclude that TRPV1 is obligatory for vagal C-fibre activation by capsaicin and anandamide. By contrast, whereas TRPV1 may have a modulatory role in bradykinin and acid-induced activation of bronchopulmonary C-fibres, it is not required for action potential discharge evoked by these stimuli.     

A quantitative analysis of the acetylcholine-activated potassium current in single cells from frog atrium

The K⁺ current activated by acetylcholine (ACh) in single cells from the frog atrium was analyzed using the whole-cell patch clamp technique. The ACh current was analyzed quantitatively by subtracting the currents elicited in response to voltage steps in the presence and absence of a steady bath-application of 1 M ACh. The net ACh currents were voltage- and time-dependent. With depolarizing jumps, the ACh-activated current declined from an instantaneous peak to a new steady level. With hyperpolarizations, the instantaneous current change was followed by a time-dependent increase in current. The current relaxations were well fitted by the sum of two exponentials with time constants of 20 ms and 300 ms at –120 mV. Both time constants decreased with depolarization. The current-voltage relationship inwardly-rectified. This inward rectification was due both to a decrease in the single channel conductance and a decrease in the number of open channels with depolarization. The ACh-activated K⁺ current differs from the background K⁺ current in several respects. The kinetics of the background K⁺ current are much more rapid and the background K⁺ channel passes much less current in the outward direction than the ACh channel.     

Presynaptic short-term depression is maintained during regulation of transmitter release at a GABAergic synapse in rat hippocampus

To examine possible interactions between fast depression and modulation of inhibitory synaptic transmission in the hippocampus, we recorded from pairs of synaptically connected basket cells (BCs) and granule cells (GCs) in the dentate gyrus of rat brain slices at 34 °C. Multiple-pulse depression (MPD) was examined in trains of 5 or 10 inhibitory postsynaptic currents (IPSCs) evoked at frequencies of 10–00 Hz under several conditions that inhibit transmitter release: block of voltage-dependent Ca²⁺ channels by Cd²⁺ (10 μ m ), activation of γ-amino-butyric acid type B receptors (GABA_BRs) by baclofen (10 μ m ) and activation of muscarinic acetylcholine receptors (mAchRs) by carbachol (2 μ m ). All manipulations led to a substantial inhibition of synaptic transmission, reducing the amplitude of the first IPSC in the train (IPSC₁) by 72 %, 61 % and 29 %, respectively. However, MPD was largely preserved under these conditions (0.34 in control versus 0.31, 0.50 and 0.47 in the respective conditions at 50 Hz). Similarly, a theta burst stimulation (TBS) protocol reduced IPSC₁ by 54 %, but left MPD unchanged (0.40 in control and 0.39 during TBS). Analysis of both fractions of transmission failures and coefficients of variation (CV) of IPSC peak amplitudes suggested that MPD had a presynaptic expression site, independent of release probability. In conclusion, different types of presynaptic modulation of inhibitory synaptic transmission converge on a reduction of synaptic strength, while short-term dynamics are largely unchanged.     

Development of pressurized retinal resistance-sized arteriolar preparation with special reference to acetylcholine-induced nitric-oxide-mediated vasodilatation

We examined the responses of pressurized bovine retinal functional arterioles (97-185 microm in diameter and approximately 3 mm long) to vasoactive substances and the mode of action of acetylcholine (ACh) on the pressurized arterioles. The retinal arterioles were cannulated at both ends with glass micropipettes and perfused at a constant pressure of 60 mmHg. Vasoconstrictions of the retinal arterioles were induced by prostaglandin F(2 alpha) (PG F(2 alpha)), U46,619, noradrenaline (NA), and 5-hydroxytryptamine (5-HT) in a dose-dependent manner. The decreasing order of potency (pD(2) value) in the constrictive responses was as follows: 5-HT = U46,619 > NA > PG F(2 alpha). On the other hand, sodium nitroprusside (SNP), isocarbacyclin (a stable prostaglandin I(2) analog), ACh, and isoproterenol (ISP) caused dose-dependent vasodilatation in the pressurized retinal arterioles preconstricted with high-potassium solution (40 mM K+). The decreasing order of potency in the vasodilative responses was as follows: isocarbacyclin > SNP > ACh. The ACh-induced vasodilatation was suppressed significantly by pretreatment with N omega-nitro-L-arginine methyl ester (L-NAME) (3 x 10(-5) M). A treatment with L-arginine (10(-3) M) in the presence of 3 x 10(-5) M L-NAME reversed completely the L-NAME-induced reduction of the vasodilatation. These results suggest that ACh causes the production and release of endogenous nitric oxide or its related compounds, which results in vasodilatation of the pressurized bovine retinal functional arterioles.