bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatial Patterns of Fasting and Fed Antropyloric Pressure Waves in Humans
Authors: Sun W. M. , Hebbard G. S. , Malbert C.‐H. , Jones K. L. , Doran S. , Horowitz M. , Dent J.
Published: September 2004

Gastric mechanics were investigated by categorizing the temporal and spatial patterning of pressure waves associated with individual gastric contractions.

In twelve healthy volunteers, intraluminal pressures were monitored from nine side hole recording points spaced at 1.5 cm intervals along the antrum, pylorus and duodenum.

Pressure wave sequences that occurred during phase II fasting contractions (n= 221) and after food (n= 778) were evaluated.

The most common pattern of pressure wave onset along the antrum was a variable combination of antegrade, synchronous and retrograde propagation between side hole pairs. This variable pattern accounted for 42% of sequences after food, and 34% during fasting (P < 0.05). Other common pressure wave sequence patterns were: purely antegrade ‐ 29% after food and 42% during fasting (P < 0.05); purely synchronous ‐ 23% fed and 17% fasting; and purely retrograde ‐ 6% fed and 8% fasting. The length of sequences was shorter after food (P < 0.05). Some sequences ‘skipped’ individual recording points.

The spatial patterning of gastric pressure wave sequences is diverse, and may explain the differing mechanical outcomes among individual gastric contractions.

Better understanding of gastric mechanics may be gained from temporally precise correlations of luminal flows and pressures and gastric wall motion during individual gastric contraction sequences.
Maximum rate of oxygen uptake by human skeletal muscle in relation to maximal activities of enzymes in the Krebs cycle
Authors: Blomstrand Eva , Rådegran Göran , Saltin Bengt
Published: September 2004

Ten subjects performed incremental exercise up to their maximum work rate with the knee extensors of one leg. Measurements of leg blood flow and femoral arteriovenous differences of oxygen were made in order to be able to calculate oxygen uptake of the leg.

The volume of the quadriceps muscle was determined from twenty‐one to twenty‐five computer tomography section images taken from the patella to the anterior inferior iliac spine of each subject.

The maximal activities of three enzymes in the Krebs cycle, citrate synthase, oxoglutarate dehydrogenase and succinate dehydrogenase, were measured in biopsy samples taken from the vastus lateralis muscle.

The average rate of oxygen uptake over the quadriceps muscle at maximal work, 353 ml min−1kg−1, corresponded to a Krebs cycle rate of 4.6 μmol min−1 g−1. This was similar to the maximal activity of oxoglutarate dehydrogenase (5.1 μmol min−1 g−1), whereas the activities of succinate dehydrogenase and citrate synthase averaged 7.2 and 48.0 μmol min−1 g−1, respectively.

It is suggested that of these enzymes, only the maximum activity of oxoglutarate dehydrogenase can provide a quantitative measure of the capacity of oxidative metabolism, and it appears that the enzyme is fully activated during one‐legged knee extension exercise at the maximal work rate.
Reflex cardiac dromotropic responses to stimulation of the carotid and aortic chemoreceptors in the anaesthetized cat
Authors: Jones James F. X. , Daly M. de Burgh
Published: September 2004

The reflex changes in the dromotropic state of the heart (P–R interval or atrioventricular conduction time) in response to selective stimulation of the carotid and aortic bodies by sodium cyanide were studied in the anaesthetized cat. The heart was paced and the arterial blood pressure was kept constant to minimize secondary effects of changes in arterial baroreceptor activity.

Stimulation of the carotid and aortic bodies caused an increase in the atrioventricular conduction time.

Evidence is presented to suggest that this negative dromotropic response was due predominantly to a vagal cholinergic mechanism. There is a small sympathetic component but only in so far as the carotid body reflex is concerned.

The negative dromotropic responses were enhanced during reflex suppression of the central inspiratory neuronal drive combined with minimal activity of the slowly adapting pulmonary stretch afferents indicating that they are respiratory modulated.

The clinical implications of these results are discussed.
Actions of compounds manipulating the nitric Oxide system in the cat primary visual cortex
Authors: Cudeiro Javier , Rivadulla Casto , Rosa Rodriguez  , Grieve Kenneth L. , Martínez‐Conde Susana , Acuña Carlos
Published: September 2004

We iontophoretically applied NG‐nitro‐L‐arginine (l‐NOArg), an inhibitor of nitric oxide synthase (NOS), to cells (n= 77) in area 17 of anaesthetized and paralysed cats while recording single‐unit activity extracellularly. In twenty‐nine out of seventy‐seven cells (38%), compounds altering NO levels affected visual responses.

In twenty‐five out of twenty‐nine cells, l‐NOArg non‐selectively reduced visually elicited responses and spontaneous activity. These effects were reversed by co‐application of l‐arginine (l‐Arg), which was without effect when applied alone. Application of the NO donor diethylamine‐nitric oxide (DEA‐NO) produced excitation in three out of eleven cells, all three cells showing suppression by l‐NOArg. In ten cells the effect of the soluble analogue of cGMP, 8‐bromo‐cGMP, was tested. In three of those in which l‐NOArg application reduced firing, 8‐bromo‐cGMP had an excitatory effect. In six out of fifteen cells tested, L‐NOArg non‐selectively reduced responses to NMDA and α‐amino‐3‐hydroxy‐5‐methylisoxasole‐4‐propionic acid (AMPA). Again, co‐application of l‐Arg reversed this effect, without enhancing activity beyond control values.

In a further subpopulation of ten cells, l‐NOArg decreased responses to ACh in five.

In four out of twenty‐nine cells l‐NOArg produced the opposite effect and increased visual responses. This was reversed by co‐application of l‐Arg. Some cells were also affected by 8‐bromo‐cGMP and DEA‐NO in ways opposite to those described above. It is possible that the variety of effects seen here could also reflect trans‐synaptic activation, or changes in local circuit activity. However, the most parsimonious explanation for our data is that NO differentially affects the activity of two populations of cortical cells, in the main causing a non‐specific excitation.
The effect of tetracaine on supontaneous Ca2+ release and sarcoplasmic reticulum calcium content in rat ventricular myocytes
Authors: Overend C. L. , Eisner D. A. , O'Neill S. C.
Published: September 2004

The effects of tetracaine were studied on voltage‐clamped rat ventricular myocytes, which exhibited Ca2+ overload as identified by supontaneous Ca2+ release from the sarcoplasmic reticulum (SR) as shown by the associated contractions. This Ca2+ release was initially abolished by tetracaine before returning at a lower frequency, but greater amplitude, than the control. On removal of tetracaine, there was a burst of supontaneous Ca2+ release activity. All these effects were dose dependent, from 25 to 200 μm tetracaine.

The supontaneous Ca2+ release activated an inward Na+–Ca2+ exchange current as Ca2+ was pumped out of the cell. The integral of this current (i.e. the Ca2+ efflux) was increased in the presence of tetracaine. The calcium efflux per unit time was unaffected by tetracaine.

The SR Ca2+ content was increased by tetracaine, as shown by the integral of the caffeine‐evoked Na+–Ca2+ exchange current. The increase of SR Ca2+ content was equal to the extra Ca2+ lost from the cell during the burst on removal of tetracaine, and to estimates of the extra calcium gained over the quiescent period following addition of tetracaine.

It is concluded that partial inhibition of calcium‐induced calcium release increases SR Ca2+ content. In the steady state, cell Ca2+ balance is maintained as the lower frequency of supontaneous release (that activates efflux) is compensated for by their greater size.
Orally projecting interneurones in the guinea‐pig small intestine
Authors: Brookes S. J. H. , Meedeniya A. C. B. , Jobling P. , Costa M.
Published: September 2004

Orally projecting, cholinergic interneurones are important in mediating ascending excitatory reflexes in the small intestine. We have shown that there is just one major class of orally projecting interneurone, which we have characterized using retrograde labelling in organ culture, combined with immunohistochemistry, intracellular recording and dye filling.

Orally projecting interneurones, previously shown to be immunoreactive for choline acetyltransferase, tachykinins, enkephalin, calretinin and neurofilament protein triplet, have axons up to 14 mm long and are the only class of cells with orally directed axons more than 8.5 mm long.

They are all small Dogiel type I neurones with short dendrites, usually lamellar in form, and a single axon which sometimes bifurcates. Their axons give rise to short varicose collaterals in myenteric ganglia more than 3 mm oral to their cell bodies.

Orally projecting interneurones receive prominent fast excitatory post synaptic potentials (fast EPSPs). A major source of fast EPSPs is other ascending interneurones located further aborally. They also receive fast EPSPs from circumferential pathways.

In the stretched preparations used in this study, orally projecting interneurones were highly excitable, firing repeatedly to depolarizing current pulses and had negligible long after ‐hyperpolarizations following their action potentials. They did not receive measurable non‐cholinergic slow excitatory synaptic inputs.

Ascending interneurones had a characteristic inflection in their membrane responses to depolarizing current pulses and their first action potential was typically delayed by approximately 30 ms. Under single electrode voltage clamp, ascending interneurones had a transient outward current when depolarized above –70 mV from more hyperpolarized holding potentials. Ascending interneurones also consistently showed marked inward rectification under both current clamp and voltage clamp conditions.

This class of cells has consistent morphological, neurochemical and electrophysiological characteristics and are important in mediating orally directed enteric reflexes.
Characterization of vagal pathways mediating gastric accommodation reflex in rats
Authors: Takahashi Toku , Owyang Chung
Published: September 2004

We investigated the vagal pathways mediating the gastric accommodation reflex in the rat stomach.

Gastric distension (6 ml) evoked an increase of 9.0 ± 1.0 cm H2O of intragastric pressure in vivo. Pretreatment with tetrodotoxin (TTX) caused a significant pressure increase by gastric distension, reaching 17.0 ± 1.7 cm H2O, suggesting mediation by neural pathways.

The pressure increase evoked by gastric distension was significantly enhanced in vivo by acute truncal vagotomy (TV), hexamethonium (C6), and NG‐nitro‐L‐arginine methyl ester (l‐NAME), but not by vasoactive intestinal polypeptide (VIP) antiserum, guanethidine, or splanchnicotomy.

Gastric distension (6 ml) evoked a much larger intragastric pressure in the denervated, vascularly isolated, perfused rat stomach in vitro. Intra‐arterial application of TTX and l‐NAME did not cause further pressure increases evoked by gastric distension.

The pressure increase evoked by gastric distension remained high 2 weeks after TV in vivo. However, the accommodation reflex was fully restored 4 weeks after TV in vivo. This reflex was antagonized by TTX, C6 and l‐NAME, but not by VIP antiserum, guanethidine and splanchnicotomy.

Similar to in vivo studies, gastric distension caused a smaller increase in intragastric pressure in response to gastric distension in the denervated, vascularly isolated, perfused stomach obtained from rats 4 weeks after vagotomies in vitro. The pressure increase evoked by gastric distension was significantly enhanced by l‐NAME, hexamethonium and TTX.

It is suggested that the vago‐vagal reflex plays an important role in mediating the accommodation reflex. This involves a vagal efferent pathway that uses nitric oxide as a final neurotransmitter mediating gastric relaxation in intact rats. It is also suggested that the adaptive mechanism mediating the accommodation reflex following vagotomy occurs in the gastric myenteric plexus.
Oxygen deprivation inhibits Na+ current in rat hippocampal neurones via protein kinase C
Authors: O'Reilly John P. , Cummins Theodore R. , Haddad Gabriel G.
Published: September 2004

Hippocampal neurones respond to acute oxygen deprivation (hypoxia) with an inhibition of whole‐cell Na+ current (INa), although the mechanism of the inhibition is unknown. Kinases can modulate INa and kinases are activated during hypoxia. We hypothesized that kinase activation may play a role in the hypoxia‐induced inhibition of INa.

Single electrode patch clamp techniques were used in dissociated hippocampal CA1 neurones from the rat. INa was recorded at baseline, during exposure to kinase activators (with and without kinase inhibitors), and during acute hypoxia (with and without kinase inhibitors).

Hypoxia (3min) reduced INa to 38.1 ± 4.5% of initial values, and shifted steady‐state inactivation in the negative direction. Hypoxia produced no effect on activation or fast inactivation.

Protein kinase A (PKA) activation with 2.5 mm adenosine 3′,5′‐cyclic adenosine monophosphate, N6O2‐dibutyryl, sodium salt (db‐cAMP) resulted in reduction of INa to 62.8 ± 5.5% without an effect on activation or steady‐state inactivation. INa was also reduced by activation of protein kinase C (PKC) with 5 nm phorbol 12‐myristate 13‐acetate (PMA; to 40.0 ± 3.7%) or 50 μm 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG; to 46.1 ± 2.8%). In addition, steady‐state inactivation was shifted in the negative direction by PKC activation. Neither the activation curve nor the kinetics of fast inactivation was altered by PKC activation.

The response to PKA activation was blocked by the PKA inhibitor N‐[2‐(p‐bromocinnamyl‐amino) ethyl]‐5‐isoquinolinesulphonamide (H‐89; 30 μm) and by 30 μm of PKA inhibitory peptide PKA5–24 (PKAi). PKC activation was blocked by the kinase inhibitor 1‐(5‐iso‐quinolinesulphonyl)‐2‐methylpiperazine (H‐7; 100 μm), by the PKC inhibitor calphostin C (10 μm), and by 20 μm of the inhibitory peptide PKC19–31 (PKCi).

The hypoxia‐induced inhibition of INa and shift in steady‐state inactivation were greatly attenuated with H‐7, calphostin C, or PKCi, but not with H‐89 or PKAi.

We conclude that hypoxia activates PKC in rat CA1 neurones, and that PKC activation leads to the hypoxia‐induced inhibition of INa.
Thrombin‐dependent calcium signalling in single human erythroleukaemia cells
Authors: Somasundaram Baggi , Mason Michael J. , Mahaut‐Smith Martyn P.
Published: September 2004

A combination of single cell fluorescence and patch clamp techniques were used to study the mechanisms underlying thrombin‐evoked Ca2+ signals in human erythroleukaemia (HEL) cells, a leukaemic cell line of platelet–megakaryocyte lineage.

Thrombin caused a transient increase in intracellular Ca2+ ([Ca2+]j), consisting of both release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Mn2+ quench studies indicated that the thrombin‐evoked divalent cation‐permeable pathway was activated during, but not prior to, release from internal stores.

Thapsigargin (1 μm) irreversibly released internal Ca2+ from the same store as that released by thrombin and continuously activated a Ca2+‐influx mechanism. The amplitude of the thrombin‐ and thapsigargin‐induced Ca2+ influx displayed a marked single cell heterogeneity which showed no correlation with the size of the store Ca2+ transient.

In whole‐cell patch clamp recordings, both thrombin and thapsigargin evoked an inwardly rectifying Ca2+ current which developed with little or no increase in current noise, showed no reversal in the voltage range −110 to +60 mV and was blocked by 1 mm Zn2+. The apparent divalent cation permeability sequence of this pathway was Ca2+≫ Ba2+ > Mn2+, Mg2+. The thapsigargin‐evoked current density at −100 mV varied between 0.42 and 2.1 pA pF−1 in different cells. Thrombin failed to activate additional Ca2+ current if it was added after the thapsigargin‐induced inward current had fully developed.

These studies indicate that thrombin activates Ca2+ influx in HEL cells entirely via a Ca2+‐store‐release‐activated Ca2+ current (ierac) rather than via receptor‐operated or second messenger‐dependent Ca2+ channels. The level of expression of Ierac appears to be a major factor in determining the duration of the thrombin‐evoked [Ca2+]i response and therefore represents a means by which cells can exert control over [Ca2+]i‐dependent events.
Dissociation between lactate and proton exchange in muscle during intense exercise in man
Authors: Bangsbo J. , Juel C. , Hellsten Y. , Saltin B.
Published: September 2004

Transport of lactate, H+ and fluid across muscle sarcolemma was studied in contracting muscles under varying blood acid–base conditions.

Subjects performed two‐legged submaximal knee‐extensor exercise for 29–35 min consisting of warming up for 5 min followed by 10 min of leg exercise (L1), leg and arm exercise for 6–10 min (L2+A) and leg exercise for 10 min (L3). The experimental protocol was performed on two occasions; inspiring air (normoxia, N) or breathing 14% O2 in N2 (hypoxia, H). Leg blood flow was measured and femoral arterial and venous blood was sampled before and during each phase of exercise.

Arterial blood lactate concentration increased progressively during exercise to 5.9 ± 0.8 (N) and 8.2 ± 0.8 mmol l−1 (H) (P≤ 0.05) after 5.5 min of L2+A. Arterial blood pH was higher (P≤ 0.05) in H than in N, whereas arterial blood HCO3‐ concentrations were the same. Leg lactate release was higher in H than in N (3.1 ± 0.7 vs. 2.0 mmol l−1 (P≤ 0.05) during L1. In L2+A a net uptake of lactate was observed in both N and H. The concentration of lactate in the red blood cells increased during exercise to 2.3 ± 0.4 (N) and 4.3 ± 0.7 mmol l−1 (H) (P≤ 0.05) after 5.5 min of L2+A, but no red blood cell femoral arterial–venous lactate difference was observed.

Net proton release, estimated from actual base excess (ABE) adjusted for changes in reduced haemoglobin, was significant (P≤ 0.05) both at rest and during each phase of exercise. Furthermore, the difference between net proton and lactate release was positive throughout exercise and of similar magnitude in N and H.

The present data suggest that (1) H+ exchange in muscle during submaximal exercise can to a large extent occur through mechanisms other than via coupling to lactate; (2) muscle transport of H+ is insensitive to changes in blood pH in the range of 0.02–0.08 pH units; (3) transport of lactate across the membrane of red blood cells appears to be of minor importance for lactate release from active muscles.
Swelling‐ and cAMP‐Activated Cl− Currents in Isolated Rat Carotid Body Type I Cells
Authors: Carpenter E. , Peers C.
Published: September 2004

In the whole‐cell configuration of the patch clamp technique, isolated rat carotid body type I cells exhibited reversible activation of Cl− currents during cell swelling effected by hypotonic extracellular solutions.

Hypotonic solutions evoked outwardly rectifying, non‐inactivating currents which showed time‐independent activation. The reversal potential (Erev) for the hypotonically evoked current was 1.6 ± 0.6 mV (n= 26). Reduction of extracellular Cl− from 133 to 65.5 mm caused a shift in Erev of ± 14.7 ± 0.4 mV (n= 5).

The swelling‐activated Cl− current could not activate when ATP was omitted from the patch pipette or when substituted for the non‐hydrolysable ATP analogues 5′‐adenylylimido‐diphosphate, AMP‐PNP (2 mm) or β,γ–methylene‐adenosine 5′‐triphosphate, AMP‐PCP (2 mm). The current also failed to activate in the absence of free intracellular Ca2+.

The swelling‐activated Cl− current was sensitive to blockade by the Cl− channel blockers niflumic acid (300 μm and 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS; 200 μm), although the blockade by DIDS was voltage dependent.

A similar, non‐inactivating, outwardly rectifying Cl− current was evoked by the inclusion of cAMP (200 μm) in the patch pipette. This current could be inhibited by niflumic acid (300 μm) DIDS (200 μm) and hypertonic solutions, and was virtually abolished in the absence of intracellular ATP.

In conclusion, carotid body type I cells possess Cl− currents activated by cell swelling and rises in intracellular cAMP concentration. These currents may be involved in cell volume regulation, blood volume and osmolarity regulation and the response of the type I cell to chemostimuli.
Activation of small conductance Ca2+‐dependent K+ channels by purinergic agonists in smooth muscle cells of the mouse ileum
Authors: Vogalis Fivos , Goyal Raj K.
Published: September 2004

Whole‐cell and single‐channel K+ currents were recorded at room temperature (22–24 °C), from smooth muscle cells enzymatically dispersed from the mouse ileum, using variations of the patch‐clamp technique.

Net outward K+ currents recorded through amphotericin‐B‐perforated patches in response to step depolarizations positive to −50 mV from a holding potential of −80 mV were decreased by up to 70% by external apamin (0.5/μM). Apamin‐sensitive whole‐cell currents were also recorded from cells perfused internally with 150 nm Ca2+ but not from cells perfused internally with 85nM Ca2+.

Three types of non‐inactivating Ca2+‐sensitive K+ channels were identified in cell‐attached and excised patches under an asymmetrical K+ gradient: (i) large conductance (BKCa; ∼200 pS) channels blocked by 2 mm external TEA; (ii) intermediate conductance (IKCa; ∼39 pS) channels blocked by 2mm external TEA and inhibited by external apamin (0.5 μm); and (iii) small conductance (SKCa; ∼10pS) channels that were not blocked by 5mm external TEA but were sensitive to extracellular apamin (0.5 μm).

The TEA‐resistant SKCa channels were activated by an increase in [Ca2+]i with an EC50 of 1.5 μm and a Hill coefficient of 1.3.

P2 purinoceptor agonists 2‐methylthioATP (2‐MeSATP), 2‐chloroATP and ATP (10–50μm) increased an apamin‐sensitive whole‐cell outward K+ current. Extrapatch application of 2‐MeSATP (20–100μm) stimulated the apamin‐sensitive IKCa and SKCa channels and activated an apamin‐sensitive steady outward current at 0mV.

Smooth muscle cells from the mouse ileum possess two apamin‐sensitive K+ channels (IKCa and SKCa); of these, the IKCa channels are TEA sensitive while the SKCa channels are TEA resistant. These channels, along with an apamin‐sensitive but TEA‐resistant steady outward current, may mediate membrane hyperpolarization elicited by purinergic agonists.
Regulation of the cytosolic Ca2+ concentration by Ca2+ stores in single smooth muscle cells from rat cerebral arteries
Authors: Kamishima T. , McCarron J. G.
Published: September 2004

There is no general agreement on the presence or role of Ca2+‐induced Ca2+ release in smooth muscle. In this paper, Ca2+‐induced Ca2+ release has been investigated in rat resistance‐sized superior cerebral arteries to determine its role in regulating the cytosolic Ca2+ concentration ([Ca2+]i.

Pressurized superior cerebral arteries developed spontaneous oscillations in diameter. These oscillations were abolished by ryanodine (an inhibitor of Ca2+‐induced Ca2+ release) and removal of extracellular Ca2+. This suggests, indirectly, that Ca2+‐induced Ca2+ release may regulate [Ca2+]i in the resistance arteries.

To determine if Ca2+‐induced Ca2+ release could regulate [Ca2+]i, single smooth muscle cells were isolated from the superior cerebral artery, voltage clamped in the whole cell configuration and high temporal resolution [Ca2+]i measurements made. The relationship between the Ca2+ current (ICa) and rise in [Ca2+]i was examined.

Depolarization triggered ICa and increased [Ca2+]i. The time course of the measured increase in [Ca2+]i closely followed the increase in [Ca2+]i expected from the time‐integrated ICa, although about 140‐fold more Ca2+ entered the cytosol than appeared as free Ca2+. When the cells were dialysed with ryanodine (30 μm), the Ca2+ transient evoked by the ICa was substantially reduced indicating that Ca2+ influx triggered Ca2+ release from an internal store.

Voltage pulses to negative membrane potentials were more effective in triggering Ca2+ release than pulses to positive potentials suggesting that the Ca2+‐induced Ca2+ release was voltage dependent. However, the release of Ca2+ from the internal store triggered by caffeine was voltage independent. These results suggest that the voltage dependence of Ca2+ release is indirect and possibly related to the plasmalemma unitary Ca2+ current magnitude.

The results establish that Ca2+‐induced Ca2+ release contributes to depolarization‐evoked increases in [Ca2+]i in rat resistance‐sized superior cerebral arteries over the physiological [Ca2+]i range (100‐200 nm). Compared with more positive membrane potentials the efficacy of Ca2+ in triggering release is high at physiological membrane potentials.
Spatial heterogeneity of intracellular Ca2+ signals in axons of basket cells from rat cerebellar slices
Authors: Llano Isabel , Tan Yusuf P. , Caputo Carlo
Published: September 2004

Using tight‐seal whole‐cell recording and digital fluorescence imaging, we studied intracellular calcium (Cai2+) dynamics in cerebellar basket cells, whose dendrites, axon and presynaptic terminals are coplanar, an optimal configuration for simultaneous optical measurements of all functional domains.

In Cs+‐loaded neurones, depolarizing pulses induced large Cai2+ transients in single axonal varicosities and synaptic terminals, contrasting with much weaker signals between varicosities or in the somato‐dendritic domain.

Axonal branch points consistently displayed [Cai2+] rises of similar magnitude and time course to those in axonal terminals and varicosities.

In biocytin‐filled basket cells, varicosity‐like swellings were present along the axon including its branch points. Thus, axonal enlargements are not due to fluorescence‐induced cell damage.

The spatial heterogeneity of Cai2+ signals was also observed in K+‐loaded cells upon depolarizing trains, suggesting that this behaviour is an intrinsic property of Cai2+ homeostasis in basket cells.

We conclude that depolarization of basket cell axons evokes high local Cai2+ signals in synaptic terminals, en passant varicosities and branch points. While high [Ca2+]1 in presynaptic structures presumably triggers transmitter release, Cai2+ transients at branch points may control signal transmission in the axonal arborization.
Evidence of two mechanisms for the activation of the glucose transporter GLUT1 by anisomycin: p38(MAP kinase) activation and protein synthesis inhibition in mammalian cells
Authors: Barros Luis Felipe , Young Michelle , Saklatvala Jeremy , Baldwin Stephen A.
Published: September 2004

Inhibitors of protein synthesis stimulate sugar transport in mammalian cells through activation of plasma membrane GLUT1, the housekeeping isoform of the glucose transporter. However, it has been reported that some of these compounds, in addition to their effect on protein synthesis, also activate protein kinases.

In the present study we have explored the role of these two effects on GLUT1 activation. In 3T3‐L1 adipocytes and Clone 9 cells, stimulation of sugar transport by puromycin, a translational inhibitor that does not activate kinases, was not detectable until 90 min after exposure. In contrast, stimulation by anisomycin, a potent Jun‐NH2‐terminal kinase (JNK) agonist, exhibited no lag phase. An intermediate response was observed to emetine and cycloheximide, weak activators of JNK.

The potency of anisomycin to stimulate transport acutely (30 min of exposure) was 5‐ to 10‐fold greater than for its chronic stimulation of transport, measured after 4 h of exposure. The stimulation of transport by a low concentration of anisomycin (0.3 μm) was transient, peaked at 30–60 min and it was inhibited (IC50 < 1 μm) by SB203580, which indicates that its mediator is not JNK, but the homologous p38(MAP kinase) (p38(MAPK)). In contrast, the responses to 4 h exposure to 300 μm anisomycin or puromycin were refractory to SB203580.

Exposure to anisomycin resulted in rapid activation of p38(MAPK). Activation of both p38(MAPK) and GLUT1 by 0.3 μm anisomycin was cancelled by puromycin.

We conclude that the activation of GLUT1 in response to anisomycin includes two components: a delayed component involving translational inhibition and a fast, puromycin‐inhibitable component that is secondary to activation of p38(MAPK).
Leptin activates ATP‐sensitive potassium channels in the rat insulin‐secreting cell line, CRI‐G1
Authors: Harvey J. , McKenna F. , Herson P. S. , Spanswick D. , Ashford M. L. J.
Published: September 2004

Whole‐cell current‐clamp recordings demonstrate that leptin (0.3–10 nm) hyperpolarizes CRI‐G1 insulin‐secreting cells. This effect is slow on onset and is not reversed on washout of the leptin.

Voltage‐clamp recordings indicate that leptin activates a potassium conductance in the presence of intracellular ATP (5 mm), but has no effect in its absence. Following activation of ATP‐sensitive K+ (KATP) current by diazoxide (0.2 mm), addition of leptin did not alter cell membrane potential or potassium current further.

The leptin‐induced hyperpolarization and increased potassium conductance are completely inhibited by the application of the sulphonylureas tolbutamide (100 μm) and glibenclamide (0.5 μm).

Cell‐attached and inside‐out single‐channel recordings indicate that leptin activates tolbutamide‐sensitive KATP channels in CRI‐G1 insulin‐secreting cells.
Effects of acetylcholine on the Na+–K+ pump current in guinea‐pig ventricular myocytes
Authors: Gao J. , Mathias R. T. , Cohen I. S. , Baldo G. J.
Published: September 2004

The whole‐cell patch clamp technique was used to study the effects of acetylcholine (ACh) on Na+‐K+ pump current (Ip) in acutely isolated guinea‐pig ventricular myocytes. Studies were performed in the absence and presence of the β‐agonist isoprenaline (Iso).

ACh had no effect on Ip at low or high [Ca2+]i at any voltage in the absence of Iso. Iso alone inhibited Ip at low [Ca2+]i and shifted the Ip–V relationship at high [Ca2+]i in a negative direction. Addition of 1 μm ACh reversed these effects of Iso. K0.5 for the effects of ACh was about 16 nm, regardless of [Ca2+]i.

The actions of ACh on the heart are usually mediated via muscarinic receptors. Atropine, a muscarinic antagonist, blocked the effects of ACh on Ip in the presence of Iso, suggesting that these effects are also mediated by muscarinic receptors.

Muscarinic receptors are usually coupled to a Gi protein, leading to inhibition of adenylyl cyclase and a reduction of cAMP levels. We have shown previously that basal levels of cAMP are very low in guinea‐pig ventricular myocytes, and that a membrane‐permeant cAMP analogue, chlorophenylthio‐cAMP (CPTcAMP), mimics the effects of Iso. ACh did not reverse the effects of CPTcAMP, supporting the hypothesis that the effects of ACh on Ip are also mediated via inhibition of adenylyl cyclase.

The present results suggest that a high level of parasympathetic tone alone does not affect the activity of ventricular Na+–K+ pumps. However, if sympathetic tone is high, then muscarinic stimulation can reciprocally modulate Na+–K+ pump activity.
How the thyroid controls metabolism in the rat: different roles for triiodothyronine and diiodothyronines
Authors: Moreno Maria , Lanni Antonia , Lombardi Assunta , Goglia Fernando
Published: September 2004

Although the first evidence of a relationship between the thyroid and metabolism was reported in 1895, the mechanism by which thyroid hormones influence resting metabolic rate in whole animals is still poorly understood. This paper reports an attempt to test whether diiodothyronines (T2s) and triiodothyronine (T3) have different roles in the control of resting metabolism (RM).

Changes in resting metabolic rate were measured in hypothyroid rats treated acutely (25μg (100 g body weight)−1) either with one of the T2s or with T3. Injection of T3 induced an increase of about 35% in RM that started 25–30 h after the injection and lasted until 5–6 days after the injection, the maximal value being observed at 50–75 h. The injection of T2s evoked a temporally different pattern of response. The increases in RM started 6–12 h after the injection, had almost disappeared after 48 h, and the maximal stimulation was observed at 28–30 h.

When actinomycin D (an inhibitor of protein synthesis) and T3 were given together, the stimulation of RM was almost completely abolished. The simultaneous injection of actinomycin D and either of the T2s, on the other hand, did not cause any attenuation of the stimulation seen with the T2s alone.

Following chronic treatment (3 weeks) with either T3 or T2s there was a stimulation of organ growth only after the administration of T3.

Chronic administration of either T2s or T3 to hypothyroid rats significantly enhanced the oxidative capacity of each of the tissues considered. In the case of T2s the stimulation was almost the same whether it was expressed as an increase in specific activity or total tissue activity. In the case of T3 the increases were, in the main, secondary to the hypertrophic or hyper plastic effect.

These results indicate that T2s and T3 exert different effects on RM. The effects of T2s are rapid and possibly mediated by their direct interaction with mitochondria. Those of T3 are slower and more prolonged, and at least partly attributable to a modulation of the cellularity of tissues that are metabolically very active.
Histamine H1 receptor activation blocks two classes of potassium current, IK(rest) and IAHP to excite ferret vagal afferents
Authors: Jafri M. Samir , Moore Kimberly A. , Taylor Glen E. , Weinreich Daniel
Published: September 2004

Intracellular recordings were made in intact and acutely dissociated vagal afferent neurones (nodose ganglion cells) of the ferret to investigate the membrane effects of histamine.

In current‐clamp or voltage‐clamp recordings, histamine (10 μm) depolarized the membrane potential (10 ± 0.8 mV; mean ±s.e.m.; n= 27) or produced an inward current of 1.6 ± 0.35 nA (n= 27) in ∼80% of the neurones.

Histamine (10 μm) also blocked the post‐spike slow after‐hyperpolarization (AHPslow) present in 80% of these neurones (95 ± 3.2%; n= 5). All neurones possessing AHPslow in ferret nodose were C fibre neurones; all AHPslow neurones had conduction velocities ≤ 1 m s−1(n= 7).

Both the histamine‐induced inward current and the block of AHPslow were concentration dependent and each had an estimated EC50 value of 2 μm. These histamine‐induced effects were mimicked by the histamine H1 receptor agonist 2‐(2‐aminoethyl)thiazole dihydrochloride (10 μm) and blocked by the H1 antagonists pyrilamine (100 nm) or diphenhydramine (100 nm). Schild plot analysis of the effect of pyrilamine on the histamine‐induced inward current revealed a pA2 value of 9.7, consistent with that expected for an H1 receptor. Neither impromidine (10 μm) nor R(−)‐α‐methylhistamine (10 μm), selective H2 or H3 agonists, respectively, significantly affected the membrane potential, input resistance or AHPslow.

The reversal potential (Vrev) for the histamine‐induced inward current was −84 ± 2.1 mV (n= 4). The Vrev for the histamine response shifted in a Nernstian manner with changes in the extracellular potassium concentration. Alterations in the extracellular chloride concentration had no significant effect on the Vrev of the histamine response (n= 3). The Vrev for the AHPslow was –85 ± 1.7 mV (n= 4).

These results indicate that histamine increases the excitability of ferret vagal afferent somata by interfering with two classes of potassium current: the resting or ‘leak’ potassium current (IK(rest)) and the potassium current underlying a post‐spike slow after‐hyper‐polarization (IAHP). Both these effects can occur in the same neurone and involve activation of the same histamine receptor subtype, the histamine H1 receptor.
Epoxyeicosatrienoic acids activate a high‐conductance, Ca2+‐dependent K+ channel on pig coronary artery endothelial cells
Authors: Baron A. , Frieden M. , Bény J.‐L.
Published: September 2004

Epoxyeicosatrienoic acids (EETs) have been described as endothelium‐derived hyper‐polarizing factors (EDHFs), based on their stimulatory effects on smooth muscle K+channels. In order to reveal a putative autocrine effect of EETs on endothelial channels, we have studied the effects of the four EET regioisomers (5,6‐EET, 8,9‐EET, 11,12‐EET and 14,15‐EET) on the high‐conductance, Ca2+‐dependent K+ (BKCa) channel recorded in inside‐out patches of primary cultured pig coronary artery endothelial cells. Currents were recorded in the presence of either 500 nm or 1 μm free Ca2+ on the cytosolic side of the membrane.

In 81% of experiments, EETs at < 156 nm, applied on the cytosolic side of the membrane, transiently increased BKCa channel open state probability (P0) without affecting its unitary conductance, thus providing evidence for direct action of EETs, without involvement of a cytosolic transduction pathway.

The four EET regioisomers appeared to be equally active, multiplying the BKCa channel Po by a mean factor of 4.3 ± 0.6 (n= 15), and involving an increase in the number and duration of openings.

The EET‐induced increase in BKCa channel activity was more pronounced with low initial Po. When the BKCa channel was activated by 500 nm Ca2+, application of EETs increased the initial Po value of below 0.1 by a factor of 5. When the channel was activated by 1 μm Ca2+, application of EETs increased the initial Po value by a factor of 3.

Our results show that EETs potentiate endothelial BKCa channel activation by Ca2+. The autocrine action of EETs on endothelial cells, which occurs in the same concentration range as their action on muscle cells, should therefore fully participate in the vasoactive effects of EETs, and thus be taken into account when considering their putative EDHF function.
Amino terminal‐dependent gating of the potassium channel rat eag is compensated by a mutation in the S4 segment
Authors: Terlau Heinrich , Heinemann Stefan H. , Stühmer Walter , Pongs Olaf , Ludwig Jost
Published: September 2004

Rat eag potassium channels (r‐eag) were expressed in Xenopus oocytes. They gave rise to delayed rectifying K+ currents with a strong Cole‐Moore effect.

Deletions in the N‐terminal structure of r‐eag either shifted the activation threshold to more negative potentials and slowed the activation kinetics (Δ2–190, Δ2–12 and Δ7–12) or resulted in a shift to more positive potentials and faster activation kinetics (Δ150–162).

The impact of the deletion Δ7–12 was investigated in more detail: it almost abolished the Cole‐Moore effect and markedly slowed down channel deactivation.

Unlike wild‐type channels, the deletion mutants Δ7–12 exhibited a rapid inactivation which, in combination with the slow deactivation, resulted in current characteristics which were similar to those of the related potassium channel HERG.

Both the slowing of deactivation and the inactivation induced by the deletion Δ7–12 were compensated by a single histidine‐to‐arginine change in the S4 segment, while this mutation (H343R) only had minor effects on the gating kinetics of the full‐length r‐eag channel.

These results demonstrate a functional role of the N‐terminus in the voltage‐dependent gating of potassium channels which is presumably mediated by an interaction of the N‐terminal protein structure with the S4 motif during the gating process.
Characterization of apical potassium channels induced in rat distal colon during potassium adaptation
Authors: Butterfield I. , Warhurst G. , Jones M. N. , Sandle G. I.
Published: September 2004

Chronic dietary K+ loading stimulates an active K+ secretory process in rat distal colon, which involves an increase in the macroscopic apical K+ conductance of surface epithelial cells. In the present study, the abundance and characteristics of K+ channels constituting this enhanced apical K+ conductance were evaluated using patch clamp recording techniques.2. In isolated non‐polarized surface cells, K+ channels were seen in 9 of 90 (10%) cell‐attached patches in cells from control animals, and in 247 of 437 (57%) cell‐attached patches in cells from K+‐loaded animals, with a significant (P < 0.001) shift in distribution density. Similarly, recordings from cell‐attached patches of the apical membrane of surface cells surrounding the openings of distal colonic crypts revealed identical K+ channels in 1 of 11 (9%) patches in control animals, and in 9 of 13 (69%) patches in K+‐loaded animals.

In isolated surface cells and surface cells in situ, K+ channels had mean slope conductances of 209 ± 6 and 233 ± 14 pS, respectively, when inside‐out patches were bathed symmetrically in K2SO4 solution. The channels were sensitive to ‘cytosolic’ Ca2+ concentration, were voltage sensitive at ‘cytosolic’ Ca2+ concentrations encountered in colonic epithelial cells, and were inhibited by 1 mm quinidine, 20 mm TEA or 5 mm Ba2+ ions.

The data show that dietary K+ loading increases the abundance of Ca2+‐ and voltage‐sensitive large‐conductance K+ channels in the apical membrane of surface cells in rat distal colon. These channels constitute the enhanced macroscopic apical K+ conductance previously identified in these cells, and are likely to play a critical role in the active K+ secretory process that typifies this model of colonic K+ adaptation.
Contraction of the human diaphragm during rapid postural adjustments
Authors: Hodges P. W. , Butler J. E. , McKenzie D. K. , Gandevia S. C.
Published: September 2004

The response of the diaphragm to the postural perturbation produced by rapid flexion of the shoulder to a visual stimulus was evaluated in standing subjects. Gastric, oesophageal and transdiaphragmatic pressures were measured together with intramuscular and oesophageal recordings of electromyographic activity (EMG) in the diaphragm. To assess the mechanics of contraction of the diaphragm, dynamic changes in the length of the diaphragm were measured with ultrasonography.

With rapid flexion of the shoulder in response to a visual stimulus, EMG activity in the costal and crural diaphragm occurred about 20 ms prior to the onset of deltoid EMG. This anticipatory contraction occurred irrespective of the phase of respiration in which arm movement began. The onset of diaphragm EMG coincided with that of transverses abdominis.

Gastric and transdiaphragmatic pressures increased in association with the rapid arm flexion by 13.8 ± 1.9 (mean ±s.e.m.) and 13.5 ± 1.8 cmH2O, respectively. The increases occurred 49 ± 4 ms after the onset of diaphragm EMG, but preceded the onset of movement of the limb by 63 ± 7 ms.

Ultrasonographic measurements revealed that the costal diaphragm shortened and then lengthened progressively during the increase in transdiaphragmatic pressure.

This study provides definitive evidence that the human diaphragm is involved in the control of postural stability during sudden voluntary movement of the limbs.
Molecular constituents of maxi KCa channels in human coronary smooth muscle: predominant α+β subunit complexes
Authors: Tanaka Yoshio , Meera Pratap , Song Min , Knaus Hans‐Günther , Toro Ligia
Published: September 2004

Human large‐conductance voltage‐ and calcium‐sensitive K+ (maxi KCa) channels are composed of at least two subunits: the pore‐forming subunit, α, and a modulatory subunit, β. Expression of the β subunit induces dramatic changes in α subunit function. It increases the apparent Ca2+ sensitivity and it allows dehydrosoyasaponin I (DHS‐I) to upregulate the channel.

The functional coupling of maxi KCa channel α and β subunits in freshly dissociated human coronary smooth muscle cells was assessed. To distinguish maxi KCa currents modulated by the β subunit, we examined (a) their apparent Ca2+ sensitivity, as judged from the voltage necessary to half‐activate the channel (V1/2), and (b) their activation by DHS‐I.

In patches with unitary currents, the majority of channels were half‐activated near –85 mV at 18 μm Ca2+, a value similar to that obtained when the human KCa channel α (HSLO) and β (HKVCaβ) subunits are co‐expressed. A small number of channels half‐activated around 0 mV, suggesting the activity of the α subunit alone.

The properties of macroscopic currents were consistent with the view that most pore‐forming α subunits were coupled to β subunits, since the majority of currents had values for V1/2 near to –90 mV, and currents were potentiated by DHS‐I.

We conclude that in human coronary artery smooth muscle cells, most maxi KCa channels are composed of α and β subunits. The higher Ca2+ sensitivity of maxi KCa channels, resulting from their coupling to β subunits, suggests an important role of this channel in regulating coronary tone. Their massive activation by micromolar Ca2+ concentrations may lead to a large hyperpolarization causing profound changes in coronary blood flow and cardiac function.
Activation of f‐channels by cAMP analogues in macropatches from rabbit sino‐atrial node myocytes
Authors: Bois P. , Renaudon B. , Baruscotti M. , Lenfant J. , DiFrancesco D.
Published: September 2004

The action of the two diastereometric phosphorothioate derivatives of cAMP, Rp‐cAMPs and Sp‐cAMPs, was investigated on hyperpolarization‐activated ‘pacemaker’ current (if) recorded in inside‐out macropatches from rabbit sino‐atrial (SA) node myocytes.

When superfused on the intracellular side of f‐channels at the concentration of 10 μm, both cAMP derivatives accelerated if activation; their action was moderately less pronounced than that due to the same concentration of cAMP.

The measurement of the if conductance‐voltage relation by voltage ramp protocols indicated that both cAMP analogues shift the activation curve of if to more positive voltages with no change in maximal (fully activated) conductance.

Dose–response relationships of the shift of the if activation curve showed that both Rp‐cAMPs and Sp‐cAMPs act as agonists in the cAMP‐dependent direct f‐channel activation. Fitting data to the Hill equation resulted in maximal shifts of 9.6 and 9.5 mV, apparent dissociation constants of 0.82 and 5.4 μm, and Hill coefficients of 0.82 and 1.12 for Sp‐cAMPs and Rp‐cAMPs, respectively.

The activating action of Rp‐cAMPs, a known antagonist of cAMP in the activation of cAMP‐dependent protein kinase, confirms previously established evidence that f‐channel activation does not involve phosphorylation. These results also suggest that the cAMP binding site of f‐channels may be structurally similar to the cyclic nucleotide binding site of olfactory receptor channels.