bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiotensin AT1 receptor‐mediated excitation of rat carotid body chemoreceptor afferent activity
Authors: Allen A. M.
Published: September 2004

A high density of angiotensin II receptors was observed in the rat carotid body by in vitro autoradiography employing 125I‐[Sar1,Ile8]‐angiotensin II as radioligand. Displacement studies demonstrated that the receptors were of the AT1 subtype.

The binding pattern indicated that the AT1 receptors occurred over clumps of glomus cells, the principal chemoreceptor cell of the carotid body. Selective lesions of the sympathetic or afferent innervation of the carotid body had little effect on the density of receptor binding, demonstrating that the majority of AT1 receptors were intrinsic to the glomus cells.

To determine the direct effect of angiotensin II on chemoreceptor function, without the confounding effects of the vasoconstrictor action of angiotensin II, carotid sinus nerve activity was recorded from the isolated carotid body in vitro. The carotid body was superfused with Tyrode solution saturated with carbogen (95 % O2, 5 % CO2), maintained at 36 °C, and multi‐unit nerve activity recorded with a suction electrode.

Angiotensin II elicited a dose‐dependent excitation of carotid sinus nerve activity (maximum increase of 36 ± 11 % with 10 nM angiotensin II) with a threshold concentration of 1 nM. The response was blocked by the addition of an AT1 receptor antagonist, losartan (1 μM), but not by the addition of an AT2 receptor antagonist, PD123319 (1 μM).

In approximately 50 % of experiments the excitation was preceded by an inhibition of activity (maximum decrease of 24 ± 8 % with 10 nM angiotensin II). This inhibitory response was markedly attenuated by losartan but not affected by PD123319.

These observations demonstrate that angiotensin II, acting through AT1 receptors located on glomus cells in the carotid body, can directly alter carotid chemoreceptor afferent activity. This provides a means whereby humoral information about fluid and electrolyte homeostasis might influence control of cardiorespiratory function.
Low K+‐induced hyperpolarizations trigger transient depolarizations and action potentials in rabbit ventricular myocytes
Authors: Akuzawa‐Tateyama M. , Tateyama M. , Ochi R.
Published: September 2004

The effects of large reductions of [K+]o on membrane potential were studied in isolated rabbit ventricular myocytes using the whole‐cell patch clamp technique.

Decreasing [K+]o from the normal level of 5.4 mm to 0.1 mm increased resting membrane potential (Vrest) from −75.6 ± 0.3 to −140.3 ± 1.9 mV (means ± s.e.m; n= 127), induced irregular, transient depolarizations with mean maximal amplitudes of 19.5 ± 1.5 mV and elicited action potentials in 56.7 % of trials. The action potentials exhibited overshoots of 37.9 ± 1.5 mV (n= 72) and sustained plateaux.

Addition of 0.1 mm La3+ in the presence of 0.1 mm[K+]o significantly increased Vrest but decreased the amplitude of transient depolarizations and suppressed the firing of action potentials.

Replacement of external Na+ or Cl− with N‐methyl‐D‐glucamine or aspartate, respectively, or internal dialysis with 10 mm EGTA or BAPTA had little effect on low [K+]o‐induced membrane potential changes.

Hyperpolarizing voltage clamp pulses to potentials between −110 and −200 mV activated irregular inward currents that increased in amplitude and frequency with increasing hyperpolarization and were depressed by 0.1 mm La3+.

The generation of transient depolarizations by low [K+]o can be explained as being a consequence of decreasing the inward rectifier K+ current (IK1) and the appearance of inward currents reflecting electroporation resulting from strong electric fields across the membrane.
Cytochrome P450: a novel system modulating Ca2+ channels and contraction in mammalian heart cells
Authors: Xiao Yong‐Fu , Huang Lin , Morgan James P.
Published: September 2004

Cytochrome P450 (P450) is a ubiquitous enzyme system that catalyses oxidative reactions of numerous endogenous and exogenous compounds. The modulatory effects of P450 on the L‐type Ca2+ current (ICa), intracellular free Ca2+ signals and cell shortening were assessed in adult rat single ventricular myocytes.

Bath administration of the imidazole antimycotics, clotrimazole, econazole and miconazole, which are potent P450 inhibitors, significantly suppressed cardiac ICa. While the Ca2+ channel antagonist nifedipine blocked ICa within 30 s, clotrimazole‐induced suppression of ICa required 5.1 ± 0.4 min (n= 14) to reach a steady low level. The suppression of ICa was dose dependent and recovered after washout of clotrimazole. Intracellular dialysis with the P450 antibody anti‐rat CYP1A2 also significantly reduced cardiac ICa.

Additional administration of the β‐adrenergic agonist isoprenaline (1 μM) or the membrane‐permeable 8‐bromo‐cAMP (2 mM) completely reversed the suppressant effects of clotrimazole and NaCN on ICa. In addition, intracellular dialysis with 2 mM cAMP abolished the P450 inhibitor‐induced suppression of ICa. Phosphorylation of the channel with hydrolysis‐resistant ATPγS prevented the suppressant effect of clotrimazole on ICa. Furthermore, dephosphorylation of the Ca2+ channel with intracellular dialysis with phosphatase types I and II reduced ICa by 85 ± 3 % and abolished clotrimazole‐induced suppression of ICa.

Extracellular administration of the phospholipase A2 inhibitors mepacrine and 4‐bromophenacyl bromide significantly suppressed ICa.

Clotrimazole, econazole, miconazole and CN− also significantly inhibited intracellular free Ca2+ signals and cell shortening in rat single ventricular myocytes.

Intracellular cAMP content was significantly reduced in isolated ventricular myocytes incubated with clotrimazole or CN−. Extracellular administration of 11, 12‐epoxyeicosatrienoic acid, one of the P450‐mediated metabolites of arachidonic acid, enhanced ICa and intracellular cAMP content. The epoxyeicosatrienoic acid also restored the amplitude of the reduced ICa in P450 antibody‐dialysed myocytes.

The present data suggest that cytochrome P450 modulates cardiac ICa and cell contraction, and the modulation may result from changes in intracellular levels of cAMP by P450‐ mediated metabolites of arachidonic acid.
Mutual occlusion of P2X ATP receptors and nicotinic receptors on sympathetic neurons of the guinea‐pig
Authors: Searl T. J. , Redman R. S. , Silinsky E. M.
Published: September 2004

The interaction of ion channels activated by nicotinic receptor agonists with ion channels gated by extracellular ATP (i.e. P2X receptors) was studied on sympathetic neurons acutely dissociated from coeliac ganglia of the guinea‐pig. Patch clamp methods were used to measure the inward current generated through these non‐selective cationic channels under voltage clamp.

At the whole cell level, the specific nicotinic receptor agonists nicotine (5‐100 μM) or cytisine (50‐75 μM) and the P2X receptor agonists ATP (0.1‐7 μM) or α,β‐methylene ATP (6 μM) were examined separately and in the presence of the other receptor activator. When a nicotinic and P2X receptor agonist were applied together, mutually occlusive effects were generally observed. This occurred even with concentrations of agonists that in themselves generated little to no inward current.

The occlusive effects of nicotinic agonists on ATP‐gated currents were blocked by the nicotinic receptor/ion channel blocker hexamethonium (150 μM). The occlusive effects of ATP analogues on inward currents generated by nicotinic agonists were blocked by the P2X receptor antagonist suramin (100 μM).

Mutual occlusion of the effects of nicotinic agonists and ATP analogues were also observed when currents through single channels were studied in excised (outside‐out) patches.

The results suggest that nicotinic receptors and P2X ATP receptors do not act independently in these sympathetic neurons.
Laminar organization of epileptiform discharges in the rat entorhinal cortex in vitro
Authors: Lopantsev Valeri , Avoli Massimo
Published: September 2004

Interictal and ictal epileptiform discharges induced by 4‐aminopyridine (4AP, 50 μM) were studied in the rat lateral entorhinal cortex with field potential and intracellular recordings in an in vitro slice preparation. Both types of discharge disappeared in layer II, but continued to occur in layers IV‐VI after a knife cut separation was made at approximately 600 μm from the pia (n= 4 slices).

Interictal depolarizations recorded in layer IV‐VI cells (amplitude, 29.4 ± 8.6 mV; duration, 386 ± 177.4 ms, means ± s.d.; n= 17) were capped by action potential bursts, while smaller interictal depolarizations in layer II cells (amplitude, 11.7 ± 5.8 mV; duration, 192.6 ± 47.9 ms; n= 10) were associated with single action potentials and were terminated by a hyperpolarization. Ictal discharges were initiated by an interictal discharge; they were characterized by a depolarization of 31.5 ± 6.2 mV (n= 12) in layer IV‐VI and 11.6 ± 3.5 mV (n= 7) in layer II neurones.

Slow, presumptive Ca2+‐mediated spikes occurred in layer II (n= 4) and IV‐VI (n= 6) cells loaded with the Na+ channel blocker QX‐314 (50 mM). These events were synchronized with population spikes during interictal and ictal discharges, and were abolished by Ni2+ (1 mM, n= 4 cells) along with the 4AP‐induced synchronous activity.

The N‐methyl‐D‐aspartate (NMDA) receptor antagonist 3,3‐(2‐carboxypiperazine‐4‐yl)‐propyl‐1‐phosphonate (CPP, 10 μM) abolished ictal discharges and reduced interictal depolarizations in layer IV‐VI neurones (n= 4). The non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 10 μM) abolished both interictal and ictal activity (n= 4 cells).

These findings provide evidence for a role played by NMDA‐mediated mechanisms in the generation of epileptiform discharges in the entorhinal cortex. Lack of an NMDA‐mediated component along with presence of inhibition in layer II neurones results in attenuation of epileptiform activity at this site. Moreover Ca2+‐mediated spikes may contribute to the appearance of epileptiform discharges in this model.
ADP and inositol trisphosphate evoke oscillations of a monovalent cation conductance in rat megakaryocytes
Authors: Hussain Jamila F. , Mahaut‐Smith Martyn P.
Published: September 2004

A combination of conventional whole‐cell patch clamp recordings and fura‐2 fluorescence photometry was used to study the membrane currents during oscillations of intracellular Ca2+concentration ([Ca2+]i) in single rat megakaryocytes.

At a holding potential of ‐60 mV, in NaCl external saline and KCl internal saline with low levels of Ca2+buffering, 10 μmADP evoked [Ca2+]ioscillations and simultaneous Ca2+‐gated K+currents at a frequency of 3–10 spikes min−1. A smaller inward current was also activated, with a time course that identified this component as the inositol 1,4,5‐trisphosphate (IP3)‐activated monovalent cation current previously demonstrated in rat megakaryocytes.

Cs+replacement of internal K+combined with 100 nM external charybdotoxin (CTX) abolished the outward currents and revealed that an inward current was also transiently activated during each [Ca2+]ispike. This underlying conductance was permeable to Na+and Cs+, but possessed little or no permeability to Cl−or divalent cations.

Intracellular dialysis with IP3(5‐50 μm) activated the monovalent cationic conductance prior to release of Ca2+from intracellular stores. The [Ca2+]iincrease was associated with a second phase of cationic current, implying that both IP3and Ca2+can activate this conductance. Buffering of [Ca2+]iwith BAPTA abolished the second phase of current, leaving monophasic spikes of inward current, often occurring at regular intervals.

These data demonstrate that a monovalent cation current, which results in Na+influx under normal ionic conditions, oscillates in response to ADP receptor stimulation due to activation by both IP3and [Ca2+]i. This provides a route for long‐term Na+entry in the megakaryocyte following stimulation of receptors coupled to phospholipase C activation and may play a role in cell shape change.
NMDA and GABAAreceptors in the rat Kölliker‐Fuse area control cardiorespiratory responses evoked by trigeminal ethmoidal nerve stimulation
Authors: Dutschmann Mathias , Herbert Horst
Published: September 2004

Electrical stimulation (10 s) of the ethmoidal nerve (EN5) evokes the nasotrigeminal reflex responses, including apnoea, bradycardia and rise in arterial blood pressure. In the present study, we examined the involvement ofN‐methyl‐D‐aspartate (NMDA), AMPA/kainate, (γ‐aminobutyric acidA(GABAA) and glycine receptors in the Kölliker‐Fuse (KF) nucleus in the mediation of the nasotrigeminal reflex responses.

Unilateral injections (n= 6) of 50‐100 nl of the NMDA receptor antagonist AP5 into the KF area led to a significant blockade of the EN5‐evoked respiratory depression and bradycardia. Injections placed into the midlevel of the KF area were most effective (80‐90 % blockade). The rise in arterial blood pressure remained unaffected.

Unilateral injections (n= 6) of the AMPA/kainate receptor antagonist CNQX into the KF area failed to block EN5‐evoked autonomic responses significantly.

Unilateral injections (n= 5) of the GABAAreceptor antagonist bicuculline enhanced the EN5‐evoked respiratory depression and bradycardia. The effect persisted for up to 30 s after stimulation. Bicuculline injections into the midlevel of the KF area were most effective. The increase in arterial blood pressure remained unaffected.

Unilateral injections (n= 5) of the glycine receptor antagonist strychnine into the KF area did not produce any significant effects on EN5‐evoked autonomic responses.

Our results suggest that the KF area represents a mandatory relay for the nasotrigeminally induced apnoea and bradycardia which are predominantly mediated by NMDA receptors in the KF. Furthermore, it appears that KF neurons are under a potent GABAergic inhibitory control. The EN5‐evoked rise in arterial blood pressure was not altered by any of the drugs and, therefore, appears not to be mediated via the KF.
Streptomycin inhibition of myogenic tone, K+‐induced force and block of L‐type calcium current in rat cerebral arteries
Authors: Miller A. L. , Langton P. D.
Published: September 2004

Streptomycin has been demonstrated to inhibit mechanosensitive conductances in a wide variety of cell types, including muscle. The action of streptomycin on rat cerebral arteries that exhibit pressure‐induced myogenic response was investigated.

Pressure‐induced tone, measured using isobaric myography, in isolated pressurized cerebral arteries was reversibly and concentration‐dependently inhibited by streptomycin with an IC50 of 2.6 mM.

Isometric K+‐induced force, measured using isometric myography, is supported by voltage‐gated Ca2+ entry. Streptomycin reversibly and concentration‐dependently inhibited isometric force with an IC50 of 1.71 mM.

Voltage‐gated macroscopic inward Ca2+ channel currents were recorded from freshly isolated rat basilar myocytes. These were reversibly and concentration‐dependently inhibited by streptomycin with an IC50 of 1.79 and 0.47 mM when 10 and 1.8 mM CaCl2, respectively, was used as the charge carrier.

These data suggest that streptomycin inhibits myogenic tone and K+‐induced isometric force largely by blockade of L‐type, dihydropyridine‐sensitive Ca2+ channels. In conclusion, streptomycin is not useful in the investigation of stretch‐activated channels which may underlie the myogenic response of rat small cerebral arteries.
ATP‐activated cation currents in single guinea‐pig hepatocytes
Authors: Capiod Thierry
Published: September 2004

Responses of single guinea‐pig liver cells to the application of external ATP were studied using the whole‐cell voltage clamp technique.

When the cells were loaded with 5 mM EGTA in the absence of K+ and Cl− in both internal and external solutions, application of ATP (0.03‐100 μM) elicited a large cation‐selective inward current at negative holding potentials. The current densities at the peak of the response to 100 μM ATP were 4.5 ± 0.5 pA pF−1 (mean ± s.e.m., n= 18) in the presence of Na+ and Ca2+ in the external medium and 3.3 ± 0.7 pA pF−1 (n= 6) with Ca2+ as the major permeant ion.

Divalent cations, when added during the response to ATP in the presence of Na+ and Ca2+, exerted different effects: CdSO4 (2 mM) totally and NiSO4 (2 mM) partially blocked the inward current whereas MnSO4 (2 mM) did not block it. The ATP‐activated conductance was permeable to all the divalent cations tested in this study, i.e. Ca2+, Cd2+, Ni2+, Mn2+ and Mg2+. No response to ATP was observed in the absence of external cations.

The activation of the inward current was not maintained in the continuous presence of ATP. The effect of Ca2+ ions on the desensitization of the response was studied in different external solutions. The decline in the amplitude of the inward current after the peak was fitted with a single exponential with a time constant of about 2 s for pure Ca2+, Cd2+ or Ni2+ currents, 3 s for Mg2+ or Mn2+ and 4 s in the presence of both Na+ and Ca2+.

Under more physiological conditions, the entry of Ca2+ evoked after the stimulation of P2X purinoceptors was associated with an increase in fluo‐3 fluorescence and a marked reduction in the delay before the mobilization of internal Ca2+ stores following the activation of P2Y purinoceptors.
Fundamental calcium release events revealed by two‐photon excitation photolysis of caged calcium in guinea‐pig cardiac myocytes
Authors: Lipp Peter , Niggli Ernst
Published: September 2004

In cardiac muscle, ‘Ca2+ sparks’ have been proposed to underlie Ca2+‐induced Ca2+ release (CICR), and to result from openings of clusters of Ca2+ channels (ryanodine receptors; RyRs) located in the sarcoplasmic reticulum membrane.

To investigate the elementary nature of these Ca2+ signals directly, a diffraction‐limited point source of Ca2+ was created in single cardiac myocytes by two‐photon excitation photolysis of caged Ca2+. Simultaneously, concentration profiles of released Ca2+ were imaged at high temporal and spatial resolution with a laser‐scanning confocal microscope.

This approach enabled us to generate and detect photolytic Ca2+ signals that closely resembled the Ca2+ sparks occurring naturally, not only in amplitude and size, but also in their ability to trigger additional Ca2+ sparks or Ca2+ waves.

Surprisingly, at low photolytic power minuscule events with estimated Ca2+ release fluxes 20‐40 times smaller than those calculated for a typical Ca2+ spark were directly resolved. These events appeared to arise from the opening of a more limited number of RyRs (possibly one) or from RyRs exhibiting a different gating mode and may correspond to the elusive ‘Ca2+ quark’.

The Ca2+ quark represents the fundamental Ca2+ release event of excitable cells implementing hierarchical Ca2+ signalling systems with Ca2+ release events of various but distinct amplitude levels (i.e. Ca2+ quarks, Ca2+ sparks and full cellular Ca2+ transients).

A graded recruitment of nanoscopic Ca2+ release domains (i.e. Ca2+ quarks) exhibiting variable degrees of spatial coherence and coupling may then build up intermediate Ca2+ signalling events (i.e. Ca2+ sparks). This mechanism suggests the existence of Ca2+ sparks caused by gating of a variable fraction of RyRs from within an individual cluster. Additional mobilization of a variable number of these Ca2+ sparks enables cardiac cells to show graded cellular Ca2+ transients. Similar recruitment processes may underlie regulation of Ca2+ signalling on the cellular level in general.
Hypoxia and cyanide induce depolarization and catecholamine release in dispersed guinea‐pig chromaffin cells
Authors: Inoue M. , Fujishiro N. , Imanaga I.
Published: September 2004

The perforated patch method and amperometry were used to determine whether the adrenal medullary cell itself is capable of sensing hypoxia and, if so, how such sensation is transduced to secretion of catecholamines (CA).

Exposure to hypoxia, cyanide (CN), or muscarine facilitated CA secretion from dissociated chromaffin cells. The CN‐induced secretion was not affected by removal of glucose, indicating that the CN release is due to chemical hypoxia.

The secretions induced by CN and muscarine were markedly diminished by removal of Ca2+ ions or by application of Cd2+ or methoxyverapamil (D‐600).

Cyanide and muscarine produced depolarizations with generation of action potentials and increased intracellular Ca2+ concentrations determined using the acetoxymethyl (AM) ester form of fluo‐3 in the presence of external Ca2+ ions, but not in their absence.

Hypoxia and CN produced inward currents at an equilibrium potential for Cl− ions, irrespective of whether or not Na+ ions were present in the cells, and substitution of N‐methyl‐D‐glucamine for 134 mM Na+ ions in the perfusate inhibited the CN current by 71 %. The reversal potential for the CN current was −24 mV in the standard perfusate.

The hypoxia‐, CN‐ and muscarine‐induced currents decreased in parallel with hyperpolarizations, and exposure to CN prevented muscarine, but not nicotine, from inducing a further inward current.

We conclude that hypoxia and CN induce CA secretion through depolarization and the subsequent activation of voltage‐dependent Ca2+ channels and that this depolarization is due to opening of cation channels, which are possibly identical to muscarinic cation channels.
Repolarizing K+ currents in rabbit heart Purkinje cells
Authors: Cordeiro J. M. , Spitzer K. W. , Giles W. R.
Published: September 2004

Electrophysiological experiments on single myocytes obtained from Purkinje fibres and ventricular tissue of adult rabbit hearts were done to compare the contributions of three potassium (K+) currents to the action potentials in these two tissues.

In Purkinje cells reductions in extracellular potassium, [K+]o, from normal (5.4 mM) to 2.0 mM resulted in a large hyperpolarization and marked lengthening of the action potential. In ventricular myocytes, these changes were much less pronounced. Voltage clamp measurements demonstrated that these differences were mainly due to a much smaller inward rectifier K+ current, IK1, in Purkinje cells than in ventricular myocytes.

Application of 4‐aminopyridine (4‐AP, 2 mM) showed that all Purkinje cells exhibited a very substantial Ca2+‐independent transient K+ outward current, It. 4‐AP significantly broadened the early, rapid repolarization phase of the action potential.

Selective inhibitors of the fast component, IK,r (MK‐499, 200 nM) and the slow component IK,s (L‐735821 (propenamide), 20 nM) of the delayed rectifier K+ currents both significantly lengthened the action potential, suggesting that these conductances are present, but very small (< 20 pA) in Purkinje cells. Attempts to identify time‐ and voltage‐dependent delayed rectifier K+ current(s) in Purkinje cells failed, although a slow delayed rectifier was observed in ventricular myocytes.

These results demonstrate significant differences in action potential waveform, and underlying K+ currents in rabbit Purkinje and ventricular myocytes. Purkinje cells express a much smaller IK1, and a larger It than ventricular myocytes. These differences in current densities can explain some of the most important electrophysiological properties of these two tissues.
Activation of L‐arginine transport by protein kinase C in rabbit, rat and mouse alveolar macrophages
Authors: Racké Kurt , Hey Claudia , Mössner Jutta , Hammermann Rainer , Stichnote Christina , Wessler Ignaz
Published: September 2004

The role of protein kinase C in controlling L‐arginine transport in alveolar macrophages was investigated.

L‐[3H]Arginine uptake in rabbit alveolar macrophages declined by 80 % after 20 h in culture. 4β‐Phorbol 12‐myristate 13‐acetate (PMA), but not 4α‐phorbol 12‐myristate 13‐acetate (α‐PMA), present during 20 h culture, enhanced L‐[3H]arginine uptake more than 10‐fold. Staurosporine and chelerythrine opposed this effect.

L‐[3H]Arginine uptake was saturable and blockable by L‐lysine. After PMA treatment Vmax was increased more than 5‐fold and Km was reduced from 0.65 to 0.32 mM.

Time course experiments showed that PMA increased L‐[3H]arginine uptake almost maximally within 2 h. This short‐term effect was not affected by cycloheximide or actinomycin D.

L‐[3H]Arginine uptake and its stimulation by PMA was also observed in sodium‐free medium.

L‐Leucine (0.1 mM) inhibited L‐[3H]arginine uptake by 50 % in sodium‐containing medium, but not in sodium‐free medium. At 1 mM, L‐leucine caused significant inhibition in sodium‐free medium also. L‐Leucine showed similar effects on PMA‐treated cells.

N‐Ethylmaleimide (200 μm, 10 min) reduced L‐[3H]arginine uptake by 70 % in control cells, but had no effect on PMA‐treated (20 or 2 h) cells.

In alveolar macrophages, multiple transport systems are involved in L‐arginine uptake, which is markedly stimulated by protein kinase C, probably by modulation of the activity of already expressed cationic amino acid transporters.
Metabotropic and ionotropic glutamate receptors regulate calcium channel currents in salamander retinal ganglion cells
Authors: Shen Wen , Slaughter Malcolm M.
Published: September 2004

Glutamate suppressed high‐voltage‐activated barium currents (IBa,HVA) in tiger salamander retinal ganglion cells. Both ionotropic (iGluR) and metabotropic (mGluR) receptors contributed to this calcium channel inhibition.

Trans‐ACPD (1‐aminocyclopentane‐trans‐1S,3R‐dicarboxylic acid), a broad‐spectrum metabotropic glutamate receptor agonist, suppressed a dihydropyridine‐sensitive barium current. Kainate, an ionotropic glutamate receptor agonist, reduced an ω‐conotoxin GVIA‐sensitive current.

The relative effectiveness of selective agonists indicated that the predominant metabotropic receptor was the L‐2‐amino‐4‐phosphonobutyrate (L‐AP4)‐sensitive, group III receptor. This receptor reversed the action of forskolin, but this was not responsible for calcium channel suppression. l‐AP4 raised internal calcium concentration. Antagonists of phospholipase C, inositol trisphosphate (IP3) receptors and ryanodine receptors inhibited the action of metabotropic agonists, indicating that group III receptor transduction was linked to this pathway.

The action of kainate was partially suppressed by BAPTA, by calmodulin antagonists and by blockers of calmodulin‐dependent phosphatase. Suppression by kainate of the calcium channel current was more rapid when calcium was the charge carrier, instead of barium. The results indicate that calcium influx through kainate‐sensitive glutamate receptors can activate calmodulin, which stimulates phosphatases that may directly suppress voltage‐sensitive calcium channels.

Thus, ionotropic and metabotropic glutamate receptors inhibit distinct calcium channels. They could act synergistically, since both increase internal calcium. These pathways provide negative feedback that can reduce calcium influx when ganglion cells are depolarized.
Potentiation of GABAergic synaptic transmission by AMPA receptors in mouse cerebellar stellate cells: changes during development
Authors: Bureau Ingrid , Mulle Christophe
Published: September 2004

The effects of low concentrations of domoate, an agonist at both α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate and kainate receptors (AMPARs and KARs, respectively), were investigated in stellate cells in slices of mouse cerebellum at two developmental stages (postnatal day (PN) 11‐13 and PN21‐25).

Low concentrations of domoate enhanced the frequency of miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin (TTX) at PN11‐13 but not at PN21‐25.

The effects of low concentrations of domoate on synaptic activity were probably mediated by the activation of AMPARs and not KARs, since they were blocked by GYKI 53655 (LY300168), a selective AMPAR antagonist.

Domoate increased mIPSC frequency in part by activation of presynaptic voltage‐dependent Ca2+ channels since potentiation was reduced by 60 % in the presence of Cd2+. AMPARs in stellate cells were found to be permeable to Ca2+. The residual potentiation in the presence of Cd2+ could thus be due to a direct entry of Ca2+ through AMPAR channels.

In the presence of TTX, potentiation of synaptic activity by focal application of domoate was not restricted to the region of the cell body, but was observed within distances of 120 μm. These experiments also revealed a strong spatial correlation between the location of the presynaptic effects of domoate and the activation of postsynaptic AMPARs.

Our data show a developmentally regulated presynaptic potentiation of synaptic transmission between cerebellar interneurones mediated by AMPARs. We discuss the possibility that the developmental switch could be due to a shift in the localization of AMPARs from the axonal to the somato‐dendritic compartment.
Effects of mitochondrial uncouplers on intracellular calcium, pH and membrane potential in rat carotid body type I cells
Authors: Buckler K. J. , Vaughan‐Jones R. D.
Published: September 2004

Mitochondrial uncouplers are potent stimulants of the carotid body. We have therefore investigated their effects upon isolated type I cells. Both 2,4‐dinitrophenol (DNP) and carbonyl cyanide p‐trifluoromethoxyphenyl hydrazone (FCCP) caused an increase in [Ca2+]i which was largely inhibited by removal of extracellular Ca2+ or Na+, or by the addition of 2 mm Ni2+. Methoxyverapamil (D600) also partially inhibited the [Ca2+]i response.

In perforated‐patch recordings, the rise in [Ca2+]i coincided with membrane depolarization and was greatly reduced by voltage clamping the cell to −70 mV. Uncouplers also inhibited a background K+ current and induced a small inward current.

Uncouplers reduced pHi by 0.1 unit. Alkaline media diminished this acidification but had no effect on the [Ca2+]i response.

FCCP and DNP also depolarized type I cell mitochondria. The onset of mitochondrial depolarization preceded changes in cell membrane conductance by 3–4 s.

We conclude that uncouplers excite the carotid body by inhibiting a background K+ conductance and inducing a small inward current, both of which lead to membrane depolarization and voltage‐gated Ca2+ entry. These effects are unlikely to be caused by cell acidification. The inhibition of background K+ current may be related to the uncoupling of oxidative phosphorylation.
Cl− transport by cystic fibrosis transmembrane conductance regulator (CFTR) contributes to the inhibition of epithelial Na+ channels (ENaCs) in Xenopus oocytes co‐expressing CFTR and ENaC
Authors: Briel M. , Greger R. , Kunzelmann K.
Published: September 2004

Epithelial Na+ channels (ENaCs) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is activated by protein kinase A. Since cAMP‐dependent activation of CFTR Cl− conductance is defective in cystic fibrosis (CF), ENaC currents are not inhibited by CFTR. This could explain the enhanced Na+ conductance found in CF. In the present study, we examined possible mechanisms of interaction between CFTR and ENaC co‐expressed in Xenopus oocytes.

The magnitude of CFTR Cl− currents activated by 3‐isobutyl‐1‐methylxanthine (IBMX) in oocytes co‐expressing either wild‐type or mutant CFTR and ENaC determined the degree of downregulation of ENaC currents.

The ability of CFTR to inhibit ENaC currents was significantly reduced either when extracellular Cl− was replaced by poorly conductive anions, e.g. SCN− or gluconate, or when CFTR was inhibited by diphenylamine‐carboxylate (DPC, 1 mmol l−1).

Downregulation of ENaC was more pronounced at positive when compared with negative clamp voltages. This suggests that outward currents, i.e. influx of Cl− through activated CFTR most effectively downregulated ENaC.

Activation of endogenous Ca2+‐activated Cl− currents by 1 μmol l−1 ionomycin did not inhibit ENaC current. This suggests that inhibition of ENaC mediated by Cl− currents may be specific to CFTR.

The present findings indicate that downregulation of ENaC by CFTR is correlated to the ability of CFTR to conduct Cl−. The data have implications for how epithelia switch from NaCl absorption to NaCl secretion when CFTR is activated by secretagogues.
Interruption of central noradrenergic pathways and morphine withdrawal excitation of oxytocin neurones in the rat
Authors: Brown C. H. , Murphy N. P. , Munro G. , Ludwig M. , Bull P. M. , Leng G. , Russell J. A.
Published: September 2004

We have tested the hypothesis that morphine withdrawal excitation of oxytocin neurones that follows from administration of naloxone to morphine‐dependent rats is a consequence of excitation of noradrenergic neurones.

Female rats were made morphine dependent by intracerebroventricular (i.c.v.) infusion of the opioid at increasing doses over 5 days. On the sixth day, the rats were anaesthetized with urethane or pentobarbitone and prepared for blood sampling to determine plasma oxytocin by radioimmunoassay or for in vivo extracellular recording of the firing rate of identified oxytocin neurones from the supraoptic nucleus. Morphine withdrawal was induced by intravenous (i.v.) injection of the opioid antagonist naloxone (5 mg kg−1).

In one group of rats the noradrenergic projections to the hypothalamus were lesioned by i.c.v. injection of 6‐hydroxydopamine immediately prior to the induction of morphine dependence. In these rats the oxytocin secretion induced by i.v. cholecystokinin was reduced to 9 % of that seen in sham‐lesioned rats but in contrast, no attenuation of morphine withdrawal‐induced oxytocin secretion was observed.

i.c.v. infusion of the α1‐adrenoreceptor antagonist benoxathian, at up to 5.3 μg min−1, dose‐ dependently inhibited the withdrawal excitation of oxytocin neurones in morphine‐dependent rats under urethane anaesthesia, and benoxathian reduced withdrawal‐induced oxytocin secretion to 37 % of that of vehicle‐infused rats. i.c.v. benoxathian also inhibited the activity of oxytocin neurones in morphine‐naïve rats. Similarly, microdialysis administration of 2 mM benoxathian directly onto the surface of the supraoptic nucleus reduced the activity of oxytocin neurones by 53 %.

Thus noradrenergic systems are not essential for the expression of morphine withdrawal excitation, since chronic neurotoxic destruction of the noradrenergic inputs to the hypothalamus did not affect the magnitude of withdrawal‐induced oxytocin secretion. However, tonically active noradrenergic inputs influence the excitability of oxytocin neurones, and acute antagonism of this noradrenergic tone can powerfully impair the ability of oxytocin neurones to exhibit morphine withdrawal excitation.
Kainate receptor‐mediated inhibition of presynaptic Ca2+ influx and EPSP in area CA1 of the rat hippocampus
Authors: Kamiya Haruyuki , Ozawa Seiji
Published: September 2004

The effect of a low concentration (1 μM) of kainate (kainic acid; KA) on presynaptic calcium (Ca2+) influx at the Schaffer collateral‐commissural (SCC) synapse was examined in rat hippocampal slices.

Following selective loading of the presynaptic terminals with the fluorescent Ca2+ indicator rhod‐2 AM, transient increases in the presynaptic Ca2+ concentration (pre[Ca2+]t) and field excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of the SCC pathway were recorded simultaneously.

Bath application of 1 μM KA reversibly suppressed field EPSPs and pre[Ca2+]t to 37.7 ± 4.0 % and 72.9 ± 2.4 % of control, respectively. Excitatory postsynaptic currents (EPSCs) recorded with the use of the whole‐cell patch‐clamp technique were also suppressed by 1 μM KA to 42.6 ± 6.3 % of control. A quantitative analysis of the decreases in pre[Ca2+]t and the amplitude of field EPSP during KA application suggests that KA inhibits transmission primarily by reducing the pre[Ca2+]t.

Consistent with a presynaptic site for these effects, paired‐pulse facilitation (PPF) was enhanced by 1 μM KA.

A substantial KA‐induced suppression of NMDA receptor‐mediated EPSPs was detected when AMPA receptors were blocked by the AMPA receptor‐selective antagonist GYKI 52466 (100 μM).

The suppressive effect of KA on field EPSPs and pre[Ca2+]t was antagonized by the KA antagonist NS‐102 (10 μM).

These results suggest that the presynaptic inhibitory action of KA at the hippocampal CA1 synapse is primarily due to the inhibition of Ca2+ influx into the presynaptic terminals.
Chloride‐sensitive nature of the histamine‐induced Ca2+ entry in cultured human aortic endothelial cells
Authors: Ono Kyoichi , Nakao Miki , Iijima Toshihiko
Published: September 2004

Whole‐cell currents and intracellular Ca2+ concentration ([Ca2+]i) were recorded in cultured human aortic endothelial cells (HAECs) to study the mechanisms underlying Cl−‐sensitive Ca2+ entry.

In the absence of histamine the membrane potential ranged between ‐90 and +5 mV and showed bimodal distribution with peaks at ‐17.8 and ‐67.5 mV.

Histamine (1‐100 μm) activated an outward current, followed by a sustained inward current at ‐50 mV. The reversal potential (Vrev) was more negative than ‐60 mV for the initial outward current, and approximately ‐30 mV for the sustained inward current with normal Tyrode solution and internal solution containing 30 mM Cl−.

V
rev of the sustained inward current was hardly affected by varying the external concentrations of K+, Na+ and Ca2+, but was greatly changed by varying the external Cl− concentration ([Cl−]o). The relationship between Vrev and log[Cl−]o showed a slope of ‐44.8 mV per tenfold increase of [Cl−]o.

The Cl− channel blockers 9‐anthracene carboxylic acid (1 mM), N‐phenylanthranilic acid (0.1 mM) and niflumic acid (0.1 mM) all depressed the histamine‐induced inward current. The non‐selective cation channel blocker Gd3+ (10 μm) was without effect on the current.

In the absence of histamine, [Ca2+]i was not affected by varying the membrane potential. During the continuous presence of histamine, however, hyperpolarization increased and depolarization decreased [Ca2+]i, indicating that Ca2+ entry through the plasma membrane was activated by histamine.

V
rev of the histamine‐induced Cl− current, measured by the gramicidin‐perforated patch clamp method, was ‐28.4 ± 6.6 mV (n= 8), which gave an intracellular Cl− concentration of approximately 34 mM. Under the current clamp condition, the membrane potential varied from cell to cell in the control, but application of histamine induced either depolarization or hyperpolarization, depending on the membrane potential before histamine application, and the membrane potential became stable near the equilibrium potential for Cl−.

We conclude that the histamine‐induced inward current is carried mainly by Cl−. Although Ca2+ entry was also activated, we consider that its amplitude was too small to be resolved by the patch clamp method. The Cl− current may play a functional role in the sustained [Ca2+]i elevation by providing a constant driving force for Ca2+ entry in the presence of histamine.
Presynaptic adrenergic facilitation of parasympathetic neurotransmission in sympathectomized rat smooth muscle
Authors: Krizsan‐Agbas Dora , Zhang Renjie , Marzban Farshid , Smith Peter G.
Published: September 2004

Parasympathetic innervation of rat eyelid tarsal smooth muscle normally inhibits sympathetic neurotransmission prejunctionally without significant direct postjunctional effects. Following surgical sympathectomy, parasympathetic stimulation elicits smooth muscle contraction. This study examined the relative contributions of cholinergic and adrenergic mechanisms mediating these contractions.

Electrical stimulation of the superior salivatory nucleus, which activates tarsal muscle parasympathetic nerves, elicited large contractions at 2 days postsympathectomy, which were abolished by atropine and were decreased by 65 % by α1‐adrenoceptor blockade or spinal cord transection.

Contractions in response to direct cholinergic stimulation by bethanechol at 2 days postsympathectomy were increased following spinal cord transection (C2) and suppressed by the α1‐adrenoceptor agonist phenylephrine, indicating that adrenoceptors on smooth muscle attenuate cholinergic contractions. However, phenylephrine infusion enhanced contractile responses to parasympathetic stimulation.

Reverse transcription‐polymerase chain reaction revealed α1D‐adrenoceptor mRNA within pterygopalatine ganglia.

At 5 weeks and 14 months postsympathectomy, adrenergic facilitation was significantly less than at 2 days, whereas prazosin‐insensitive muscarinic contraction was increased.

We conclude that degeneration of sympathetic innervation is followed rapidly by adrenoceptor‐mediated prejunctional enhancement of parasympathetic nerve‐smooth muscle neurotransmission, which occurs prior to neuroeffector junction formation as determined previously by electron microscopy. Subsequently, noradrenergic enhancement is diminished as cholinergic neurotransmission becomes established.
Activation of P2X receptors for adenosine triphosphate evokes cardiorespiratory reflexes in anaesthetized rats
Authors: McQueen D. S. , Bond S. M. , Moores C. , Chessell I. , Humphrey P. P. A. , Dowd E.
Published: September 2004

We tested the hypothesis that activation of P2X receptors associated with vagal afferent nerves can evoke a Bezold‐Jarisch (B‐J) depressor reflex in anaesthetized rats.

Injection of αβ‐methylene ATP (αβ‐MeATP; 0.6‐600 nmol i.v.) evoked a dose‐dependent B‐J reflex comprising bradycardia, hypotension and apnoea in rats anaesthetized with pentobarbitone. Apnoea was commonly preceded by hyperventilation. Bilateral vagotomy significantly reduced the bradycardia and most of the apnoeic response without affecting hyperventilation, and unmasked a vasopressor response. Hypotension and apnoea were subject to desensitization, and ATP was about 100 times less potent than αβ‐MeATP in evoking the B‐J reflex.

ED50 values for responses to αβ‐MeATP were: bradycardia 14.6 ± 3.8 nmol; apnoea 47.1 ± 8.5 nmol; hyperventilation 23.3 ± 6.0 nmol, n= 14. The ED50 for apnoea was significantly greater than that for bradycardia or hyperventilation (P < 0.05). Atropine (2.8 μmol (kg body wt)−1 i.v.) antagonized the reflex bradycardia and hypotension.

The P2 antagonists suramin (14 μmol (kg body wt)−1 i.v.) and PPADS (17 μmol (kg body wt)−1 i.v.) antagonized the bradycardic and apnoeic components of the reflex response to αβ‐MeATP, without reducing the vasopressor or hyperventilatory responses to the agonist.

Recordings from vagal afferents showed that pulmonary inflation receptors were activated by αβ‐MeATP in 62 % of units recorded (ED50 22 ± 5 nmol) and this was blocked by PPADS (17 μmol (kg body wt)−1 i.v.); unidentified vagal afferents were also activated.

αβ‐MeATP activated carotid chemoreceptor afferents (ED50 23 ± 9 nmol), an action that was unaffected by PPADS or suramin.

The results support the hypothesis that P2X receptor subtypes for ATP are associated with specific sensory nerves that form part of the homeostatic mechanism for cardiovascular and respiratory regulation and these receptors therefore have physiological, pathological and therapeutic significance.
Co‐operation between neural and myogenic mechanisms in the control of distension‐induced peristalsis in the mouse small intestine
Authors: Huizinga Jan D. , Ambrous Krista , Der‐Silaphet Tara
Published: September 2004

Myogenic and neural control of intestinal transit were investigated in a model of distension‐induced peristalsis. A comparison was made between the electrical and mechanical activities and outflow of contents observed in control mice and in W/W v mice, which lack the interstitial cells of Cajal associated with Auerbach's plexus.

Distension caused a periodic appearance of increased motor activity due to stimulation of enteric nerves in both control and W/W v mice. Excitation was primarily delivered by cholinergic nerves, whereas periodic inhibition was mediated by neuronal nitric oxide.

In control mice, outflow was driven by propagating slow‐wave activity and was only in the aboral direction. Outflow only occurred when slow waves carried sufficient action potentials to cause phasic intraluminal pressure increases of ≥ 1 cmH2O through direct stimulation of the musculature or by distension‐induced neurally mediated activation.

In W/W v mice, outflow was associated with propagating action potentials that occurred due to either neural stimulation or direct muscle stimulation. Action potential propagation and outflow occurred in both oral and aboral directions.

In summary, in both control and W/W v mice, distension induced periodic motor activity through stimulation of the enteric nervous system. Intraluminal contents were not moved in front of such motor activity. Rather, within such periods of activity that occurred concurrently throughout an entire segment, pulsatile outflow was directed by individual propagating slow waves with superimposed action potentials in control tissue, and by propagating action potentials in W/W v mice, which lack interstitial cells of Cajal.
Submillisecond kinetics and low efficacy of parallel fibre‐Golgi cell synaptic currents in the rat cerebellum
Authors: Dieudonné Stéphane
Published: September 2004

The whole‐cell configuration of the patch clamp technique was used to record from Golgi cells in thin slices of the rat cerebellum (P12‐P25). Their active membrane properties and the input that they receive from the parallel fibres were characterized.

Most cells were filled with biocytin and morphologically identified by the presence of a large axonal arbor restricted to the granular layer. The morphological parameters of eighteen of the best‐preserved cells were quantified.

A slow capacitive current transient, characteristic of the Golgi cell axon, was used to identify Golgi cells whenever their morphology could not be preserved.

Golgi cells fire action potentials spontaneously at 3 ± 1.7 Hz (n= 17). Their firing frequency increases linearly with the amplitude of depolarizing current pulses and displays marked adaptation.

When hyperpolarized Golgi cells display an anomalous rectification which is blocked by 2 mM CsCl, indicating the presence of an Ih‐like current.

Golgi cells receive a spontaneous excitatory input from parallel fibres. This input is composed of small amplitude, mostly monoquantal, EPSCs. Chemical stimulation of granule cells by locally applied kainate evokes tetrodotoxin (TTX)‐dependent events with similar properties.

The parallel fibre‐Golgi cell EPSCs have both AMPA and NMDA components. The NMDA component is blocked by 1 mM external magnesium at ‐60 mV and decays with time constants of 31 ± 9 ms and 170 ± 15 ms (at +61 mV in the presence of magnesium).

In the presence of 10 μM internal spermine, the AMPA component of the spontaneous EPSCs is markedly slowed (0.96 ± 0.25 ms to 1.86 ± 0.47 ms; n= 4) and reduced in amplitude (49 ± 7 %; n= 4) when depolarizing the cell from ‐70 mV to +61 mV.

The decay kinetics of individual AMPA EPSCs were found to be variable, in part because of dendritic filtering. A more detailed analysis reveals that the synaptic AMPA conductances are regulated during development and close faster at days P19‐P25 than at days P13‐P16.

These data suggest that the efficacy of the parallel fibre‐Golgi cell input is rather low. This places strong constraints on the conditions in which the inhibitory feedback exerted by the Golgi cell can be operational.

The possibility is considered that the Golgi cell‐granule cell circuit shows an oscillatory behaviour. This hypothesis is discussed in relation to the models of Albus and Marr.