bluuebunny/crossref_metadata_2025 huggingface dataset:
Non‐specificity of chloride channel blockers in rat cerebral arteries: block of the L‐type calcium channel
Authors: Doughty Joanne M. , Miller Alastair L. , Langton Philip D.
Published: September 2004

The effects of chloride channel blockers on pressure‐induced constriction, K+‐induced force, and whole‐cell calcium channel currents were tested in rat cerebral arteries using isobaric and isometric myography, and patch clamp.

Under isobaric conditions at 75 mmHg, NPPB), a chloride channel blocker, reversibly depressed the myogenic constriction with an IC50 of 32.8 ± 0.52 μm (mean ± s.e.m., n= 5). Blockers of Ca2+‐activated chloride channels, flufenamic acid (100 μm) and 9‐AC; 1 mm), and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel blocker, glibenclamide (100 μm), were without effect in this tissue (n= 3).

Under isobaric conditions at 20 mmHg, 37 °C, raising [K+]o to 45 mm induced a constriction which was unaffected by 100 μm NPPB (n= 4). In contrast, at 75 mmHg and 18‐21 °C, 100 μm NPPB completely and reversibly blocked a 45 mm K+‐induced constriction (n= 3).

Under isometric conditions, NPPB reversibly depressed a 45 mm K+‐induced force with an IC50 of 10.0 ± 0.76 μm (mean ± s.e.m., n= 5). IAA‐94), another chloride channel blocker, depressed the K+‐induced force with an IC50 of 17.0 ± 1.2 μm (mean ± s.e.m., n= 4).

Using whole‐cell patch clamp, 100 μm NPPB or 200 μm IAA‐94 blocked calcium channel currents carried by 10 mm Ba2+ by 79.1 ± 1.7 and 39.8 ± 7.0 %, respectively (mean ± s.e.m., n= 6).

In summary, chloride channel blockers depress calcium channel currents in rat cerebral arteries, which could contribute to a reduction in myogenic contraction.
Involvement of PKC‐α in regulatory volume decrease responses and activation of volume‐sensitive chloride channels in human cervical cancer HT‐3 cells
Authors: Chou Cheng‐Yang , Shen Meng‐Ru , Hsu Kuei‐Sen , Huang Hsueh‐Yin , Lin Hui‐Chuan
Published: September 2004

The present study was carried out to identify the specific protein kinase C (PKC) isoform involved in regulatory volume decrease (RVD) responses, and to investigate the signal transduction pathways underlying the activation of volume‐sensitive chloride channels in human cervical cancer HT‐3 cells. The role of Ca2+ in RVD and in the activation of chloride currents was also studied.

The time course of RVDs was prolonged by microinjection of PKC‐α antibody but not by PKC‐β or PKC‐γ antibody, and also by exposure to Ca2+‐free medium, in particular when combined with microinjection of EDTA. Immunofluorescence staining showed that hypotonic superfusion evoked the translocation of PKC‐α to the cell membrane, whereas PKC‐β or PKC‐γ remained unaffected. The translocation of PKC‐α was observed a few minutes after hypotonic stress, reaching peak intensity at 30 min, and returned to the cytoplasm 60 min after hypotonic exposure. Western blot analyses showed an increased PKC‐α level in terms of intensity and phosphorylation in the cell membrane, while neither PKC‐β nor PKC‐γ was activated upon hyposmotic challenge.

Whole‐cell patch‐clamp studies demonstrated that neomycin and PKC blockers such as staurosporine and H7 inhibited volume‐sensitive chloride currents. The inhibitory effect of neomycin on chloride currents can be reversed by the PKC activator phorbol 12‐myristate, 13‐acetate (PMA). Moreover, the PKC inhibitor and PKC‐α antibody, but not PKC‐β or PKC‐γ antibody, significantly attenuated the chloride currents. The activation of volume‐sensitive chloride currents were insensitive to the changes of intracellular Ca2+ but required the presence of extracellular Ca2+.

Our results suggest the involvement of PKC‐α and extracellular Ca2+ in RVD responses and the activation of volume‐sensitive chloride channels in HT‐3 cells.
Dissociation between electrical and mechanical responses to nitrergic stimulation in the canine gastric fundus
Authors: Bayguinov Orline , Sanders Kenton M.
Published: September 2004

We examined the relationships between membrane potential, intracellular [Ca2+] ([Ca2+]i), and tension in muscles of the canine gastric fundus in response to nitrergic stimulation by NO donors and electrical field stimulation (EFS) of intrinsic enteric inhibitory neurons when adrenergic and cholinergic responses were blocked.

NO donors reduced [Ca2+]i and tension in a concentration‐dependent manner. A close relationship was noted between these parameters.

In terms of the [Ca2+]vs. force relationship, relaxation responses to EFS differed from responses to NO donors. EFS resulted in smaller decreases in [Ca2+]i to produce a given relaxation compared with responses to NO donors. Thus, muscles stimulated with EFS were less sensitive to [Ca2+]i than muscles stimulated with exogenous NO.

When membrane potential, [Ca2+]i and tension were monitored simultaneously in the same muscles, a temporal dissociation was noted between the electrical responses and changes in [Ca2+]i and tension. Brief electrical responses were associated with more sustained changes in [Ca2+]i and tension.

Further dissociation between electrical and mechanical effects was noted. Changes in [Ca2+]i and tension caused by sodium nitroprusside and EFS were blocked by arginine analogues and by oxyhaemoglobin, but electrical responses were unaffected. 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ), an inhibitor of soluble guanylyl cyclase, also blocked the effects of nitrergic stimulation on [Ca2+]i and tension, without affecting hyperpolarization. Thus, in the presence of continued hyperpolarization, the reductions in [Ca2+]i and tension caused by nitrergic stimulation were blocked.

Block of hyperpolarization in response to nitrergic stimulation with tetrapentylammonium chloride (TPEA) had relatively little effect on the [Ca2+]i and tension responses. Thus, hyperpolarization is not required for nitrergic effects on [Ca2+]i and tension.

In summary, reduction in [Ca2+]i and tension in response to nitrergic stimulation of the canine gastric fundus does not depend upon electrical hyperpolarization. Non‐electrical mechanisms such as enhanced uptake of Ca2+ by the sarcoplasmic reticulum or reduction in the Ca2+ sensitivity of the contractile apparatus may be the primary mechanisms mediating nitrergic responses in these muscles.
Identification of an ATP‐sensitive potassium channel current in rat striatal cholinergic interneurones
Authors: Lee K. , Dixon A. K. , Freeman T. C. , Richardson P. J.
Published: September 2004

Whole‐cell patch‐clamp recordings were made from rat striatal cholinergic interneurones in slices of brain tissue in vitro. In the absence of ATP in the electrode solution, these neurones were found to gradually hyperpolarize through the induction of an outward current at −60 mV. This outward current and the resultant hyperpolarization were blocked by the sulphonylureas tolbutamide and glibenclamide and by the photorelease of caged ATP within neurones.

This ATP‐sensitive outward current was not observed when 2 mM ATP was present in the electrode solution. Under these conditions, 500 μM diazoxide was found to induce an outward current that was blocked by tolbutamide.

Using permeabilized patch recordings, neurones were shown to hyperpolarize in response to glucose deprivation or metabolic poisoning with sodium azide (NaN3). The resultant hyperpolarization was blocked by tolbutamide.

In cell‐attached recordings, metabolic inhibition with 1 mM NaN3 revealed the presence of a tolbutamide‐sensitive channel exhibiting a unitary conductance of 44.1 pS.

Reverse transcription followed by the polymerase chain reaction using cytoplasm from single cholinergic interneurones demonstrated the expression of the ATP‐sensitive potassium (KATP) channel subunits Kir6.1 and SUR1 but not Kir6.2 or SUR2.

It is concluded that cholinergic interneurones within the rat striatum exhibit a KATP channel current and that this channel is formed from Kir6.1 and SUR1 subunits.
Intracellular recordings in thalamic neurones during spontaneous spike and wave discharges in rats with absence epilepsy
Authors: Pinault Didier , Leresche Nathalie , Charpier Stéphane , Deniau Jean‐Michel , Marescaux Christian , Vergnes Marguerite , Crunelli Vincenzo
Published: September 2004

In vivo extracellular and intracellular recordings were performed from thalamocortical (TC) neurones in a genetic model of absence epilepsy (genetic absence epilepsy rats from Strasbourg) during spontaneous spike and wave discharges (SWDs).

Extracellularly recorded single units (n= 14) fired either a single action potential or a high frequency burst of up to three action potentials, concomitantly with the spike component of the spike‐wave complex.

Three main events characterized the intracellular activity of twenty‐six out of twenty‐eight TC neurones during SWDs: a small amplitude tonic hyperpolarization that was present throughout the SWD, rhythmic sequences of EPSP/IPSPs occurring concomitantly with the spike‐wave complexes, and a small tonic depolarization at the end of the SWD. The rhythmic IPSPs, but not the tonic hyperpolarization, were mediated by activation of GABAA receptors since they reversed in polarity at ‐68 mV and appeared as depolarizing events when recording with KCl‐filled electrodes.

The intracellular activity of the remaining two TC neurones consisted of rhythmic low threshold Ca2+ potentials, with a few EPSP/IPSP sequences present at the start of the SWD.

These results obtained in a well‐established genetic model of absence epilepsy do not support the hypothesis that the intracellular activity of TC neurones during SWDs involves rhythmic sequences of GABAB IPSPs and low threshold Ca2+ potentials.
Ca2+‐permeable non‐NMDA glutamate receptors in rat magnocellular basal forebrain neurones
Authors: Waters D. Jack , Allen Timothy G. J.
Published: September 2004

Ionotropic glutamate receptor‐mediated responses were recorded from rat magnocellular basal forebrain neurones under voltage clamp from a somatically located patch‐clamp pipette. Currents were recorded from both acutely dissociated neurones and neurones maintained in culture for up to 6 weeks.

Non‐NMDA and NMDA receptor‐mediated events could be distinguished pharmacologically using the selective agonists (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐isoxazolepropionic acid (AMPA), kainate and N‐methyl‐D‐aspartate (NMDA), and antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and D(‐)‐2‐amino‐5‐phosphonopentanoic acid (AP5).

Responses to rapid application of AMPA displayed pronounced and rapid desensitization. Responses to kainate showed no desensitization. Steady‐state EC50 values for AMPA and kainate were 2.7 ± 0.4 μM (n= 5) and 138 ± 25 μM (n= 10), respectively. Cyclothiazide markedly increased current amplitude of responses to both agonists, whereas concanavalin A had no clear effect on either response. The selective AMPA receptor antagonist GYKI 53655 inhibited responses to kainate with an IC50 of 1.2 ± 0.08 μM (n= 5) at ‐70 mV. These data strongly suggest that AMPA receptors are the predominant non‐NMDA receptors expressed by basal forebrain neurones.

At ‐70 mV, approximately 6 % of control current amplitude remained, at a maximally effective concentration of GYKI 53655. This residual response displayed desensitization, was insensitive to cyclothiazide and was potentiated by concanavalin A, suggesting that it was mediated by a kainate receptor.

Current‐voltage relationships for non‐NMDA receptor‐mediated currents were obtained from both nucleated patches pulled from neurones in culture and from acutely dissociated neurones. With 30 μM spermine in the recording pipette, currents frequently displayed double‐rectification characteristic of non‐NMDA receptors with high Ca2+ permeabilities. Ca2+ permeability, relative to Na+ and Cs+, was investigated using constant field theory. The measured Ca2+ to Na+ permeability coefficient ratio was 0.26‐3.6; median, 1.27 (n= 15).

Current flow through non‐NMDA receptors was inhibited by Ca2+, Cd2+ and Co2+ ions. At a holding potential of ‐70 mV, a maximally effective concentration of Cd2+ (> 30 mM) reduced current amplitude by approximately 90 %, with an IC50 of 44 μM. In six out of seven cells tested, block by Cd2+ was voltage sensitive.

Ca2+ permeability of many of the non‐NMDA receptors expressed by magnocellular basal forebrain neurones may underlie the unusual sensitivity of cholinergic basal forebrain neurones to non‐NMDA receptor‐mediated excitotoxicity.
Potassium channels activated in the endothelium‐dependent hyperpolarization in guinea‐pig coronary artery
Authors: Nishiyama Makoto , Hashitani Hikaru , Fukuta Hiroyasu , Yamamoto Yoshimichi , Suzuki Hikaru
Published: September 2004

Properties of the endothelium‐dependent hyperpolarization evoked by acetylcholine (ACh) in smooth muscle of the guinea‐pig coronary artery were investigated using conventional microelectrode techniques.

ACh hyperpolarized the membrane in an endothelium‐dependent manner. The hyperpolarization comprised two components: an initial and a slow hyperpolarization. The former appeared during application of ACh, while the latter occurred after withdrawal of ACh.

Indomethacin and diclofenac, inhibitors of the enzyme cyclo‐oxygenase, blocked only the slow hyperpolarization, indicating that this potential was produced by endothelial prostanoids.

Clotrimazole and SKF 525a, known inhibitors of the enzyme cytochrome P450, inhibited both the initial and the slow hyperpolarizations, suggesting that these chemicals acted as non‐selective inhibitors of arachidonic acid metabolism. Inhibition of the lipoxygenase pathway of arachidonic acid metabolism by nordihydroguaiaretic acid had no effect on either component of the hyperpolarization.

The slow hyperpolarization was inhibited by 4‐aminopyridine (4‐AP; 10−4‐10−3 M) and glibenclamide (10−6 M). The initial hyperpolarization was greatly inhibited by charybdotoxin (CTX; 5 × 10−8 M) and partially inhibited by apamin (10−7 M), but was not inhibited by glibenclamide (10−5 M). Ba2+ (10−4 M) depolarized the membrane and increased the amplitude of both components of the ACh‐induced hyperpolarization.

Hyperpolarizations produced by Y‐26763, a K+ channel opener, were inhibited by glibenclamide, but not by 4‐AP.

The results indicate that the slow hyperpolarization is produced by endothelial prostanoids through activation of 4‐AP‐sensitive K+ channels (possibly delayed rectifier type). The initial hyperpolarization is produced mainly through activation of CTX‐sensitive K+ channels (possibly Ca2+‐sensitive type).
Regulated anion secretion in cultured epithelia from Sertoli cells of immature rats
Authors: Ko W. H. , Chan H. C. , Chew S. B. , Wong P. Y. D.
Published: September 2004

Cultured epithelia of Sertoli cells from prepubertal rats were grown on Matrigel‐coated millipore filters for short‐circuit current (Isc) measurements. Under basal conditions, these epithelia exhibited a ‘zero’ transepithelial potential difference, a ‘zero’ short‐circuit current and a transepithelial resistance of 60 Ω cm2.

Forskolin (100 μm) and 8‐(4‐chlorophenylthio)‐cAMP (cpt‐cAMP) (100 μm) added to the apical side stimulated the Isc (forskolin, peak ΔIsc= 1.32 ± 0.16 μA cm−1; cpt‐cAMP, peak ΔIsc= 0.88 ± 0.16 μA cm−2).

ATP (100 μm) added apically elicited a Isc response (peak ΔIsc= 6.45 ± 0.28 μA cm−2) which was similar in magnitude to that of 1 μm thapsigargin (peak ΔIsc= 6.09 ± 0.44 μA cm−2). The potency of the responses to other nucleotides: UTP ≥ ATP > ADP >> AMP = adenosine indicates the involvement of a mixture of P2Y receptors.

Removal of extracellular Cl− and HCO3− reduced the Isc response to ATP by 70 % and 40 %, respectively. Removal of K+ had no effect, whereas removal of Na+ attenuated the Isc response.

The response to ATP was insensitive to agents known to block anion secretion (except apical diphenylamine‐2‐carboxylate (DPC) and DIDS). The resistance to perturbation by pharmacological agents may be a unique property of the seminiferous epithelium.

Whole‐cell current recordings in cultured rat Sertoli cells demonstrated a DIDS‐sensitive outwardly rectifying Cl− conductance with activating and inactivating characteristics at depolarizing and hyperpolarizing voltages, respectively.

The stimulation of electrogenic ion transport by ATP may be part of a complex mechanism regulating fluid secretion by the testis. Cultured Sertoli cell epithelia are shown to provide a useful model to investigate transepithelial transport in the seminiferous epithelium.
The buffer barrier hypothesis, [Ca2+]i homogeneity, and sarcoplasmic reticulum function in swine carotid artery
Authors: Rembold Christopher M. , Chen Xiao‐Liang
Published: September 2004

The goal of this study was to evaluate the buffer barrier hypothesis in an intact arterial smooth muscle. Specifically, we investigated the interrelationships between intracellular [Ca2+] ([Ca2+]i) homogeneity and sarcoplasmic reticulum function in swine carotid artery.

We measured focal changes in [Ca2+]i by exploiting the different characteristics of several [Ca2+]i indicators: (1) aequorin, which can detect focal increases in [Ca2+]i such as those that occur in the subplasmalemmal region ([Ca2+]pm); (2) fura‐2, which is primarily a measure of mean cytoplasmic [Ca2+] ([Ca2+]c); and (3) force, which reflects increases in [Ca2+] near the contractile apparatus. We then estimated the relative degree of [Ca2+]i homogeneity with the aequorin/fura‐2 ratio. Finally, we inhibited sarcoplasmic reticulum Ca2+ pumping with cyclopiazonic acid (CPA), an inhibitor of the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA).

We found that, after Ca2+ depletion, the sarcoplasmic reticulum could be partially reloaded with Ca2+ by manipulations that increased the aequorin signal relatively more than the fura‐2 signal. Complete reloading required large increases in the fura‐2 signal. These data suggest that increases in [Ca2+]pm (as measured with aequorin) can partially reload the sarcoplasmic reticulum, but complete reloading required increases in [Ca2+]c (as measured with fura‐2). Reloading could be partially inhibited by 10 μM CPA, indicating that SERCA function was important for reloading.

In unstimulated arteries, 10 μM CPA increased the fura‐2 signal without altering the aequorin signal, thereby decreasing the aequorin/fura‐2 ratio. Removal of extracellular Ca2+ without CPA also reduced the aequorin/fura‐2 ratio. These data suggest that resting cells have a [Ca2+] gradient with [Ca2+]pm > [Ca2+]c; this gradient is maintained by SERCA function.

CPA slowed the decline in the fura‐2 signal observed when histamine stimulation was removed. This result is consistent with the concept of vectorial Ca2+ efflux in which Ca2+ pumping by SERCA reduces [Ca2+]c after stimulation.

Ca2+ depletion by prior treatment with 100 μM histamine and CPA transiently attenuated subsequent histamine‐induced aequorin and fura‐2 transients. The effect on contraction was smaller: a delay in contraction of approximately 10 s. These data suggest that histamine‐induced Ca2+ release has at least a small role in the initial phase of contraction; however, other contractile mechanisms appear to be able to compensate for loss of Ca2+ release with only modest changes in contraction kinetics.

These data suggest that there is a complex interrelationship between smooth muscle sarcoplasmic reticulum function and [Ca2+] in at least two cytoplasmic compartments. [Ca2+]pm and [Ca2+]c can differentially regulate sarcoplasmic reticulum Ca2+ filling; and sarcoplasmic reticulum function regulates [Ca2+]pm and [Ca2+]c.
The dynamics of synchronized neurotransmitter release determined from compound spontaneous IPSCs in rat dentate granule neurones in vitro
Authors: Williams Stephen R. , Buhl Eberhard H. , Mody Istvan
Published: September 2004

The properties of GABAA receptor‐mediated spontaneous IPSCs generated in hippocampal dentate granule neurones were analysed using whole‐cell voltage‐clamp techniques in order to explore the functional consequences of the low number (6‐12) and close proximity of synaptic contacts made by single GABAergic interneurones.

Spontaneous IPSCs (sIPSCs) occurred with a frequency of 14.0 ± 9.1 Hz (n= 31) and revealed a multi‐modal positively skewed amplitude distribution (39.0 ± 19.8 pA, median values).

The variance of 10–90 % rise times and decay kinetics between IPSCs decreased with increasing peak amplitude. Larger amplitude events had significantly faster rise times, consistent with their site of generation being proximal to the soma. The decay kinetics of sIPSCs did not significantly change with amplitude.

Large amplitude sIPSCs occurred singularly or in discrete bursts, repeated regularly at low frequency. The rising phase of such sIPSCs were multi‐phasic, composed of clear step‐like inflections that were not a product of noise. The variability between the rising phase of individual sIPSCs was quantified by calculating their standard deviation, which produced fast rising (0.22 ± 0.05 ms time to peak, n= 16) functions with half‐widths of 0.38 ± 0.10 ms, which declined to plateaux.

Computer simulations demonstrated that IPSCs with properties similar to those recorded experimentally could be generated by the linear summation of groups of temporally dispersed component events. Standard deviation functions of the rising phase of simulated IPSCs accurately described distributions of the temporal dispersion of unitary components.

The GABA uptake inhibitor (R)‐N‐[4,4‐bis(3‐methyl‐2‐thienyl)but‐3‐enl‐yl] nipecotic acid (tiagabine) (10 μM, n= 12) significantly prolonged the decay of mIPSCs (6.5 ± 0.8 to 8.7 ± 1.0 ms, median values) and sIPSCs (6.2 ± 0.4 to 7.3 ± 1.2 ms, median values), but failed to alter the frequency of occurrence, 10–90 % rise times or peak amplitude of events. The application of flurazepam (30 μM, n= 7; 50 μM, n= 4) prolonged the decay of sIPSCs regardless of their amplitude.

These data indicate that sIPSCs are formed by the summation of unitary components that occur asynchronously and that GABA released from multiple sites has independent postsynaptic actions.
Type I and II models of diabetes produce different modifications of K+ currents in rat heart: role of insulin
Authors: Shimoni Y. , Ewart H. S. , Severson D.
Published: September 2004

Several K+ currents were measured and compared in enzymatically dispersed ventricular myocytes from control and diabetic rats.

Diabetic conditions were established either with a single intravenous injection of streptozotocin (STZ, 100 mg kg−1; 6‐14 days duration) or by feeding with a fructose‐enriched diet for 4‐10 weeks. Both groups became hyperglycaemic, with the former having decreased and the latter having elevated levels of plasma insulin. These conditions therefore mimic type I (insulin‐dependent) and type II (non‐insulin‐dependent) diabetes mellitus, respectively.

As reported previously, a Ca2+‐independent transient outward K+ current, It, was attenuated in the type I model. This was not observed in the type II model. The two models differed greatly in the changes observed in a quasi‐steady‐state K+ current denoted Iss. In the STZ model Iss was substantially attenuated, whereas in the fructose‐fed model it was augmented. In both models, the background inwardly rectifying current, IK1, was unchanged. Concomitantly, there was a substantial prolongation of the action potential in the STZ model but not in the fructose‐fed model.

Incubation of control myocytes with insulin (100 nM) for 5‐9 h caused a significant augmentation of Iss, with no effect on It or on IK1. Incubation of myocytes from STZ‐diabetic rats with insulin reversed the attenuation of It, but not of Iss.

The effect of insulin was not blocked by wortmannin, an inhibitor of phosphatidylinositol 3‐kinase. However, inhibition of the mitogen‐activated protein kinase pathway with PD98059 prevented restoration of It. Insulin action on It may therefore involve changes in transcription or expression of channel proteins, rather than changes in cellular metabolism.
Sarcolemmal mechanisms for pHi recovery from alkalosis in the guinea‐pig ventricular myocyte
Authors: Leem Chae‐Hun , Vaughan‐Jones Richard D.
Published: September 2004

The mechanism of pHi recovery from an intracellular alkali load (induced by acetate prepulse or by reduction/removal of ambient PCO2) was investigated using intracellular SNARF fluorescence in the guinea‐pig ventricular myocyte.

In Hepes buffer (pHo 7.40), pHi recovery was inhibited by removal of extracellular Cl−, but not by removal of Na+o or elevation of K+o. Recovery was unaffected by the stilbene drug DIDS (4,4‐diisothiocyanatostilbene‐disulphonic acid), but was slowed dose dependently by the stilbene drug DBDS (dibenzamidostilbene‐disulphonic acid).

In 5 % CO2/HCO3− buffer (pHo 7.40), pHi recovery was faster than in Hepes buffer. It consisted of an initial rapid recovery phase followed by a slow phase. Much of the rapid phase has been attributed to CO2‐dependent buffering. The slow phase was inhibited completely by Cl− removal but not by Na+o removal or K+o elevation.

At a test pHi of 7.30 in CO2/HCO3− buffer, the slow phase was inhibited 70 % by DIDS. The mean DIDS‐inhibitable acid influx was equivalent in magnitude to the HCO3−‐stimulated acid influx. Similarly, the DIDS‐insensitive influx was equivalent to that estimated in Hepes buffer.

We conclude that two independent sarcolemmal acid‐loading carriers are stimulated by a rise of pHi and account for the slow phase of recovery from an alkali load. The results are consistent with activation of a DIDS‐sensitive Cl−‐HCO3− anion exchanger (AE) to produce HCO3− efflux, and a DIDS‐insensitive Cl−‐OH− exchanger (CHE) to produce OH− efflux. H+‐Cl− co‐influx as the alternative configuration for CHE is not, however, excluded.

The dual acid‐loading system (AE plus CHE), previously shown to be activated by a fall of extracellular pH, is thus activated by a rise of intracellular pH. Activity of the dual‐loading system is therefore controlled by pH on both sides of the cardiac sarcolemma.
Components of after‐hyperpolarization in magnocellular neurones of the rat supraoptic nucleus in vitro
Authors: Greffrath Wolfgang , Martin Erich , Reuss Stefan , Boehmer Gerd
Published: September 2004

The pharmacological sensitivity of hyperpolarizing components of spike train after‐potentials was examined in sixty‐one magnocellular neurones of the rat supraoptic nucleus using intracellular recording techniques in a brain slice preparation.

In 26 % of all neurones a slow after‐hyperpolarization (AHP) was observed in addition to a fast AHP. In 31 % of all neurones a depolarizing after‐potential (DAP) was observed.

The fast AHP was blocked by apamin whereas the slow AHP was blocked by charybdotoxin (ChTX). The DAP was enhanced by ChTX or a DAP was unmasked if not present during the control period.

Low concentrations of TEA (0.15–1.5 mm) induced effects on the slow AHP and the DAP essentially resembling those of ChTX. The same was true for the effects of CoCl2 (1 mm).

Spike train after‐potentials were not affected by either iberiotoxin (IbTX), a selective high‐conductance potassium (BK) channel antagonist, or margatoxin (MgTX), a Kv1.3 α‐subunit antagonist.

Kv1.3 α‐subunit immunohistochemistry revealed that these units are not expressed in the somato‐dendritic region of supraoptic neurones.

The effects of ChTX, IbTX, MgTX, TEA, CoCl2 and CdCl2 on spike train after‐potentials are interpreted in terms of an induction of the slow AHP by the activation of calcium‐dependent potassium channels of intermediate single channel conductance (IK channels).

The results suggest that at least the majority of supraoptic magnocellular neurones share the capability of generating both a slow AHP and a DAP. The slow AHP may act to control the expression of the DAP, thus regulating the excitability of magnocellular neurones. The interaction of the slow AHP and the DAP may be important for the control of phasic discharge.
Age‐dependent changes in myosin composition correlate with enhanced economy of contraction in guinea‐pig hearts
Authors: van der Velden J. , Moorman A. F. M. , Stienen G. J. M.
Published: September 2004

The composition of myosin heavy chains (MHCs) was investigated in young (1‐ to 8‐week‐old) and mature (9‐ to 26‐week‐old) guinea‐pigs using two monoclonal antibodies directed specifically against α‐MHC and β‐MHC. In addition, maximum force and the rate of ATP consumption during isometric contraction were measured in chemically skinned trabeculae taken from the same hearts.

An age‐dependent shift in the MHC composition was found. The α‐MHC fraction decreased from 0.17 ± 0.02 (mean ± s.e.m.; n= 24) in young to 0.04 ± 0.01 (n= 43) in mature hearts. This shift was correlated with a decrease in tension cost (i.e. ATP consumption per second per trabecula volume/force per cross‐sectional area) from 4.1 ± 0.2 mmol kN−1 m−1 s−1 (n= 23) in young to 2.5 ± 0.1 mmol kN−1 m−1 s−1 (n= 57) in mature hearts.

From the results it follows that the slow β‐MHC isoform, which predominates in hearts of mature guinea‐pigs, is about 5 times more economical than the fast α‐MHC isoform. Calcium sensitivity of force and ATP consumption decreased with age, but stabilized within a few weeks after birth. The pronounced dependence of cardiac energetics on MHC composition should be taken into account in long‐term studies of cardiac overload.
Intracellular calcium clearance in Purkinje cell somata from rat cerebellar slices
Authors: Fierro Leonardo , DiPolo Reinaldo , Llano Isabel
Published: September 2004

The mechanisms governing the return of intracellular calcium (Ca2+i) to baseline levels following depolarization‐evoked [Ca2+]i rises were investigated in Purkinje cell somata using tight‐seal whole‐cell recordings and fura‐2 microfluorometry, for peak [Ca2+]i ranging from 50 nM to 2 μM.

Ca2+i decay was well fitted by a double exponential with time constants of 0.6 and 3 s. Both time constants were independent of peak [Ca2+]i but the contribution of the faster component increased with [Ca2+]i.

Thapsigargin (10 μM) and cyclopiazonic acid (50 μM) prolonged Ca2+i decay indicating that sarco‐endoplasmic reticulum Ca2+ (SERCA) pumps contribute to Purkinje cell Ca2+i clearance.

A modest participation in clearance was found for the plasma membrane Ca2+ (PMCA) pumps using 5,6‐succinimidyl carboxyeosin (40 μM).

The Na+‐Ca2+ exchanger also contributed to the clearance process, since replacement of extracellular Na+ by Li+ slowed Ca2+i decay.

Carbonyl cyanide m‐chlorophenylhydrazone (CCCP, 2 μM) and rotenone (10 μM) increased [Ca2+]i and elicited large inward currents at −60 mV. Both effects were also obtained with CCCP in the absence of external Ca2+, suggesting that mitochondrial Ca2+ uptake uncouplers release Ca2+ from intracellular stores and may alter the membrane permeability to Ca2+. These effects were irreversible and impeded tests on the role of mitochondria in Ca2+i clearance.

The relative contribution of the clearance systems characterized in this study varied as a function of [Ca2+]i. At 0.5 μM Ca2+i, SERCA pumps, PMCA pumps and the Na+‐Ca2+ exchanger contribute equally to removal and account for 78 % of the process. Only 45 % of the removal at 2 μM Ca2+i can be explained by these systems. In this high [Ca2+]i range the major contribution is that of SERCA pumps (21 %) and of the Na+‐Ca2+ exchanger (18 %), whereas the contribution of PMCA pumps is only 6 %.
Changes in the maximum speed of shortening of frog muscle fibres early in a tetanic contraction and during relaxation
Authors: Josephson R. K. , Edman K. A. P.
Published: September 2004

Isotonic shortening velocities at very light loads were examined in single fibres of the anterior tibialis muscle of the frog, Rana temporaria, using load‐clamp recording and slack tests (temperature, 1‐3 °C; initial sarcomere length, 2.25 μm).

Shortening velocities at very light loads (force‐clamp recording) were found to be higher early in the rise of a tetanic contraction than during the plateau of the contraction. The upper limit of the load at which there was elevated shortening velocity early in the contraction was 1.5‐5.4 % of the maximum tetanic tension (Fo) depending on the particular fibre.

The maximum shortening velocity determined using the slack test method (Vo) was as much as 30 % greater early in a contraction than at the tetanic plateau. Vo was elevated above the plateau level up to about 30 ms after the end of the latent period, which is equivalent to the time required for the force in an isometric contraction to rise to about 30 % of Fo. Vo is depressed below the plateau value during relaxation at the cessation of stimulation.

Simulation studies show that the cross‐bridge model of Huxley (1957) predicts the maximum shortening velocity to be greater early in a contraction, when new actin binding sites are becoming activated and new cross‐bridge connections are being formed rapidly, than during steady‐state contraction. The elevated shortening velocity in the model is a consequence of new cross‐bridges being formed in the pulling configuration, and there being a delay before the newly added bridges are dragged beyond their equilibrium position so they begin to retard shortening. The model also predicts that maximum shortening velocity should be depressed below the plateau level during early relaxation as cross‐bridge binding sites are rapidly removed from the active population.
A leu‐enkephalin depresses transmission from muscle and skin non‐nociceptors to first‐order feline spinal neurones
Authors: Jankowska E. , Schomburg E. D.
Published: September 2004

The effects of an opioid (D‐Ser‐Leu‐enkephalin‐Thr; DSLET) were tested on synaptic actions of non‐nociceptive afferents: group I and II muscle afferents and low‐threshold skin afferents. They were tested on population EPSPs (field potentials) evoked in the dorsal horn and the intermediate zone of mid‐lumbar segments, and on monosynaptically evoked responses of single interneurones at the same locations. DSLET was applied locally (ionophoretically) at locations at which the field potentials were maximal and close to the selected neurones.

DSLET potently depressed transmission from group II muscle afferents and from low‐threshold skin afferents. Transmission to neurones located in the dorsal horn or in the intermediate zone was depressed to a similar extent. The depression was readily antagonized by naloxone. Transmission from group I a or I b muscle afferents to neurones located in the intermediate zone was not affected, or was facilitated by DSLET.

The results show that DSLET has similar depressive actions on spinal neurones to monoamines, but its actions are more widespread. Like monoamines it affects transmission from nociceptors and group II muscle afferents, but in addition it gates transmission from low‐threshold cutaneous afferents. Furthermore its effects do not appear to be restricted to interneurones at particular locations since it depressed responses of dorsal horn interneurones (gated by serotonin) as well as intermediate zone interneurones (gated by noradrenaline).
Brain‐derived neurotrophic factor is required for normal development of the central respiratory rhythm in mice
Authors: Balkowiec Agnieszka , Katz David M.
Published: September 2004

Molecular mechanisms underlying maturation of the central respiratory rhythm are largely unknown. Previously, we found that brain‐derived neurotrophic factor (BDNF) is required for expression of normal breathing behaviour in newborn mice, raising the possibility that maturation of central respiratory output is dependent on BDNF.

Respiratory activity was recorded in vitro from cervical ventral roots (C1 or C4) using the isolated brainstem‐spinal cord preparation from postnatal day (P) 0.5–2.0 and P4.5 wild‐type mice and mice lacking functional bdnf alleles.

Loss of one or both bdnf alleles resulted in an approximately 50 % depression of central respiratory frequency compared with wild‐type controls. In addition, respiratory cycle length variability was 214 % higher in bdnf null (bdnf−/−) animals compared with controls at P4.5. In contrast, respiratory burst duration was unaffected by bdnf gene mutation.

These derangements of central respiratory rhythm paralleled the ventilatory depression and irregular breathing characteristic of bdnf mutants in vivo, indicating that central deficits can largely account for the abnormalities in resting ventilation produced by genetic loss of BDNF. BDNF is thus the first growth factor identified that is required for normal development of the central respiratory rhythm, including the stabilization of central respiratory output that occurs after birth.
The C fibre reflex of the cat urinary bladder
Authors: Mazières L. , Jiang C.‐H. , Lindström S.
Published: September 2004

Reflexes evoked in bladder parasympathetic neurones by electrical stimulation of bladder C afferent fibres were studied in cats anaesthetized with α‐chloralose. The responses were compared with the ordinary micturition reflex evoked by low‐threshold Aδ afferents from bladder mechanoreceptors and mediated by a spino‐ponto‐spinal reflex pathway.

The bladder was catheterized for fluid instillations and pressure recordings. Efferent reflex discharges were recorded from the cut central end of a small distal bladder branch of the pelvic nerve. The remaining bladder pelvic nerve branches were stimulated electrically close to the bladder.

Stimulation at C afferent intensity evoked a late reflex discharge in bladder pelvic efferents in all animals. The response was centrally mediated, had a latency of 150−250 ms, and was much weaker after stimulation on the contralateral nerve.

The bladder C fibre reflex differed in several functional aspects from the ordinary Aδ micturition reflex. It could be evoked at a low rate of stimulation, with an empty bladder and no background activity from bladder mechanoreceptors. In this situation, the normal Aδ micturition reflex is not elicited. The C fibre reflex also survived an acute spinalization at a low thoracic level.

The C fibre reflex was strongly inhibited by dorsal clitoris or dorsal penis nerve stimulation, an effect that was maintained after spinalization. It was facilitated by bladder or urethra exposure to cold and menthol, stimuli that activate specific cold‐sensitive receptors associated with unmyelinated C afferents.

It is concluded that the central pathway of the C fibre reflex is spinal and partly separate from that of the ordinary micturition reflex. These observations are in keeping with the clinical finding that a bladder cooling reflex can be elicited in patients with disturbed descending control of the bladder.
Arachidonic acid increases cerebral microvascular permeability by free radicals in single pial microvessels of the anaesthetized rat
Authors: Easton A. S. , Fraser P. A.
Published: September 2004

We have investigated the permeability‐increasing effect of arachidonic acid on pial venular capillaries in vivo using the single microvessel occlusion technique.

Permeability to Lucifer Yellow dye (476 Da) increased dose dependently when arachidonic acid was applied focally to the abluminal surface of the vessels. This was completely reversible at all but the highest dose. The permeability increase was 1.65 × 10−6± 0.247 × 10−6 cm s−1 (mean ± s.e.m.) at 0.25 mm, 3.53 × 10−6± 0.872 × 10−6 cm s−1 at 0.5 mm, 12.61 × 10−6± 3.44 × 10−6 cm s−1 at 1 mm and 18.46 × 10−6± 5.90 × 10−6 cm s−1 at 2 mm arachidonic acid. There was a similar reversible dose‐dependent permeability increase to eicosapentaenoic acid.

These permeability increases could be prevented by co‐application of a mixture of the anti‐oxidants superoxide dismutase and catalase (30 and 100 U ml−1), or by the iron chelator desferrioxamine (100 μm).

The permeability increases were also prevented by the cyclooxygenase and 5‐lipoxygenase blockers indomethacin (100 μm) and nordihydroguariaretic acid (100 μm), respectively, when applied together, but not singly.

It was concluded that the permeability response to arachidonic acid depends on the formation of free radicals and subsequent lipid peroxidation.
Synaptic potentials in respiratory neurones during evoked phase switching after NMDA receptor blockade in the cat
Authors: Pierrefiche O. , Haji A. , Foutz A. S. , Takeda R. , Champagna J. , Denavit‐Saubié M.
Published: September 2004

Blockade of NMDA receptors by dizocilpine impairs the inspiratory off‐switch (IOS) of central origin but not the IOS evoked by stimulation of sensory afferents. To investigate whether this difference was due to the effects of different patterns of synaptic interactions on respiratory neurones, we stimulated electrically the superior laryngeal nerve (SLN) or vagus nerve in decerebrate cats before and after i.v. administration of dizocilpine, whilst recording intracellularly.

Phrenic nerve responses to ipsilateral SLN or vagal stimulation were: at mid‐inspiration, a transient inhibition often followed by a brief burst of activity; at late inspiration, an IOS; and at mid‐expiration, a late burst of activity.

In all neurones (n= 16), SLN stimulation at mid‐inspiration evoked an early EPSP during phase 1 (latency to the arrest of phrenic nerve activity), followed by an IPSP in inspiratory (I) neurones (n= 8) and by a wave of EPSPs in post‐inspiratory (PI) neurones (n= 8) during phase 2 (inhibition of phrenic activity). An EPSP in I neurones and an IPSP in PI neurones occurred during phase 3 (brief phrenic burst) following phase 2.

Evoked IOS was associated with a fast (phase 1) activation of PI neurones, whereas during spontaneous IOS, a progressive (30‐50 ms) depolarization of PI neurones preceded the arrest of phrenic activity.

Phase 3 PSPs were similar to those occurring during the burst of activity seen at the start of spontaneous inspiration.

Dizocilpine did not suppress the evoked phrenic inhibition and the late burst of activity. The shapes and timing of the evoked PSPs and the changes in membrane potential in I and PI neurones during the phase transition were not altered.

We hypothesize that afferent sensory pathways not requiring NMDA receptors (1) terminate inspiration through a premature activation of PI neurones, and (2) evoke a late burst of phrenic activity which might be the first stage of the inspiratory on‐switch.
Modification of activity‐dependent increases of cerebral blood flow by excitatory synaptic activity and spikes in rat cerebellar cortex
Authors: Mathiesen Claus , Caesar Kirsten , Akgören Nuran , Lauritzen Martin
Published: September 2004

Mechanisms of activity‐dependent increases in cerebral blood flow (CBF) were examined in rat cerebellar cortex using the laser Doppler flow technique and extracellular recordings of single unit activity and field potentials.

Stimulation of the monosynaptic climbing fibre system evoked long‐lasting complex spikes in Purkinje cells, and extracellular field potentials with a characteristic profile that indicated contributions from both passive and active membrane mechanisms. The concomitant CBF increases were reproducible at fairly short intervals, and suggest that both synaptic activity and spikes may contribute to increased CBF.

Stimulation of the disynaptic parallel fibre system inhibited the spiking activity in Purkinje cells, while the postsynaptic activity increased as indicated by the simultaneously recorded field potential. Nevertheless, CBF always increased. The inhibition of spike firing activity was partly dependent on GABAergic transmission, but may also relate to the intrinsic membrane properties of Purkinje cells.

The CBF increases evoked by parallel or climbing fibre stimulation were highly correlated to the sum of neural activities, i.e. the negativity of field potentials multiplied by the stimulus frequency. This suggests a robust link between extracellular current flow and activity‐dependent increases in CBF.

AMPA receptor blockade attenuated CBF increases and field potential amplitudes, while NMDA receptor antagonism did not. This is consistent with the idea that the CBF responses are of neuronal origin.

This study has shown that activity‐dependent CBF increases evoked by stimulation of cerebellar parallel fibres are dependent on synaptic excitation, including excitation of inhibitory interneurones, whereas the net activity of Purkinje cells, the principal neurones of the cerebellar cortex, is unimportant for the vascular response. For the climbing fibre system, not only synaptic activity but also the generation of complex spikes from Purkinje cells contribute to the increases in CBF. The strong correlation between CBF and field potential amplitudes suggests that extracellular ion fluxes contribute to the coupling of brain activity to blood flow.
Two neural mechanisms for respiration‐induced cutaneous vasodilatation in humans?
Authors: Wallin B. Gunnar , Båtelsson Karin , Kienbaum Peter , Karlsson Tomas , Gazelius Bertil , Elam Mikael
Published: September 2004

In humans, a deep breath is known to induce cutaneous vasoconstriction in the warm state, and vasodilatation in the cold state. To investigate whether vasodilatation in the cold state is related to reduction of sympathetic vasoconstrictor nerve traffic, we studied the effect of a deep breath on vascular resistance in a skin area on the dorsum of the hand, in which release of noradrenaline from sympathetic nerves was blocked by iontophoretic pretreatment with bretylium tosylate. Simultaneous measurements were made in two control areas. In eight healthy subjects, data were obtained from deep breaths taken before bretylium in the warm state, after general cooling to a finger skin temperature below 25 °C and after rewarming to above 32 °C.

In the warm state before bretylium pretreatment, the deep breath evoked short‐lasting vasoconstrictions at all sites. In the cold state there was no change of vascular resistance in the bretylium‐pretreated area, whereas in the control areas an initial tendency towards vasoconstriction was followed by a significant transient vasodilatation. After rewarming, transient vasoconstrictions reappeared at the control sites, whereas only a transient vasodilatation occurred at the bretylium‐pretreated site.

In six healthy subjects we also monitored the effects of a deep breath on skin sympathetic nerve activity (recorded by microneurography in the peroneal nerve), and skin vascular resistance within the innervation zone of the impaled nerve fascicle in the foot. Data from thirty deep breaths per subject were averaged.

In the cold state, the deep breath induced a strong increase in neural discharge, followed by a transient reduction of nerve traffic lasting approximately 15 s and associated with a subsequent reduction of vascular resistance.

We conclude that the deep breath‐induced vasodilatation in the cold state is due to reduction of sympathetic vasoconstrictor nerve traffic. The vasodilatation after bretylium treatment in the warm state raises the possibility that a deep breath induces two simultaneous neural reactions, a vasoconstrictor and an active vasodilator component, the latter being weaker and normally masked by the strong vasoconstrictor component.