bluuebunny/crossref_metadata_2025 huggingface dataset:
Voltage‐gated and inwardly rectifying potassium channels
Authors: Jan Lily Yeh , Jan Yuh Nung
Published: September 2004

This lecture is dedicated to Max Delbrück and Seymour Benzer. Max Delbrück was our graduate advisor. He introduced us to a variety of biophysical problems, and taught us ways of thinking about these problems by example. Potassium channels was one of the topics included in his journal club in the early seventies; Max also carefully considered the feasibility of purifying potassium channels then. It was in Seymour Benzer's laboratory that we began to look for Drosophila mutants that aÍfect synaptic transmission at the larval neuromuscular junction. Shaker was the first behavioural mutant we tested that gave a robust phenotype, a phenotype that could be mimicked by treating wild‐type preparations with a potassium channel blocker. This mutant fly has led us to our subsequent molecular studies of potassium channels.

Since we settled in the University of California, San Francisco, and began to study neural development as well as potassium channels, we have settled into the pattern of each attending meetings and presenting our studies on one of these two areas so as to avoid both being away from home and our children at the same time. In following this pattern, I will be presenting the studies of potassium channels as part of our long‐term collaboration. In this talk I will first briefly take you through the path that led us to the molecular studies of potassium channels and then discuss the diversity and modulation of these potassium channels at the molecular and physiological level.
Plasticity of Na+ Channels in Afferent Neurones Innervating Rat Urinary Bladder Following Spinal Cord Injury
Authors: Yoshimura Naoki , de Groat William C.
Published: September 2004

Whole‐cell patch‐clamp recordings in combination with axonal tracing techniques were used to investigate the effects of chronic spinal cord injury on the electrical properties of dorsal root ganglion neurones innervating the urinary bladder or colon of the adult rat.

In spinal intact animals, the majority (73–74%) of bladder and colon neurones which were small in size exhibited high‐threshold humped spikes mediated by tetrodotoxin (TTX)‐resistant Na+ channels, whereas large neurones had low‐threshold narrow spikes mediated by TTX‐sensitive Na+ channels.

In chronic spinal transected animals, 60% of bladder afferent neurones exhibited TTX‐sensitive low‐threshold spikes. The average diameter and input capacitance of the cells were significantly larger than those of cells obtained from spinal intact animals.

In bladder afferent neurones from chronic spinal transected rats, the density of TTX‐resistant Na+ currents significantly decreased from 60.5 to 17.9 pA pF−1, whereas that of TTX‐sensitive currents increased from 32.1 to 80.6 pA pF−1.

These changes in action potential and Na+ current characteristics were not detected in colon afferent neurones following spinal cord injury.

The results indicate that spinal cord injury increases bladder afferent neurone excitability by shifting the expression of Na+ channels from a high‐threshold TTX‐resistant type to a low‐threshold TTX‐sensitive type. This change in properties may occur in response to alterations in neurotrophic signals originating in the hypertrophied bladder.
Activation of the Insular Cortex During Dynamic Exercise in Humans
Authors: Williamson J. W. , Nobrega A. C. L. , McColl R. , Mathews D. , Winchester P. , Friberg L. , Mitchell J. H.
Published: September 2004

The insular cortex has been implicated as a region of cortical cardiovascular control, yet its role during exercise remains undefined. The purpose of the present investigation was to determine whether the insular cortex was activated during volitional dynamic exercise and to evaluate further its role as a site for regulation of autonomic activity.

Eight subjects were studied during voluntary active cycling and passively induced cycling. Additionally, four of the subjects underwent passive movement combined with electrical stimulation of the legs.

Increases in regional cerebral blood flow (rCBF) distribution were determined for each individual using single‐photon emission‐computed tomography (SPECT) co‐registered with magnetic resonance (MR) images to define exact anatomical sites of cerebral activation during each condition.

The rCBF significantly increased in the left insula during active, but not passive cycling. There were no significant changes in rCBF for the right insula. Also, the magnitude of rCBF increase for leg primary motor areas was significantly greater for both active cycling and passive cycling combined with electrical stimulation compared with passive cycling alone.

These findings provide the first evidence of insular activation during dynamic exercise in humans, suggesting that the left insular cortex may serve as a site for cortical regulation of cardiac autonomic (parasympathetic) activity. Additionally, findings during passive cycling with electrical stimulation support the role of leg muscle afferent input towards the full activation of leg motor areas.
Interactions between neuropeptide Y and the adenylate cyclase pathway in rat mesenteric small arteries: role of membrane potential
Authors: Prieto Dolores , Buus Carsten , Mulvany Michael J. , Nilsson Holger
Published: September 2004

Simultaneous measurements of membrane potential and tension were performed to investigate the intracellular mechanisms of neuropeptide Y (NPY) in rat mesenteric small arteries.

NPY (0.1 μM)depolarized arterial smooth muscle cells from −55 to −47 mV and increased wall tension by 0.22N m−1, representing 11% of the contraction elicited by a high potassium solution. Isoprenaline (1 μM) and acetylcholine (1 μM) evoked hyper polarizations of 11 and 17 mV, resupectively. NPY inhibited the isoprenaline ‐induced effects on membrane potential without affecting those of acetylcholine.

Forskolin evoked sustained concentration‐dependent hyper polarizations of small mesenteric arteries. NPY (0.1 mM) inhibited the resuponses to 1 μM forskolin, but did not alter the stable hyperpolarization elicited by the supecific activator of protein kinase A (PKA) SP‐5,6‐DCl‐cBIMPS (0.1 mm). Forskolin increased the cyclic AMP (cAMP) content of the arteries 21‐fold, and NPY inhibited the forskolin‐evoked increase in cAMP levels by 91 %.

The hyperpolarization produced by 1 μM forskolin was not affected by either charybdotoxin (0.1 μM) or 4‐aminopyridine (0.5mM), but glibenclamide (5 μM) inhibited the hyperpolarization by 70%. Glibenclamide also inhibited the hyperpolarization evoked by SP‐:5,6‐DCl‐cBIMPSuby59%.

Neither depolarization nor contraction caused by NPY were significantly affected by either glibenclamide (5 μM) or nifedipine (1 μM), but they were reduced by gadolinium (10 μM). However, the blocking effect of NPY on forskolin‐elicited hyperpolarization was not affected by gadolinium.

Charybdotoxin (O.1 μM) and 4‐aminopyridine (0.5 mM) strongly enhanced the depolarization and contraction caused by NPY (O.1μM), and nifedipine (1 μM) prevented the enhanced resuponses to NPY in the presence of charybdotoxin.

These findings suggest that NPY acts through at least two different intracellular mechanisms in mesenteric small arteries: a depolarization of arterial smooth muscle which is probably due to activation of non‐selective cation channels, and a marked inhibition of adenylate cyclase activity, which in turn inhibits the hyperpolarization produced by cAMP accumulation in these arteries.
Single channel properties of recombinant GABAA receptors containing γ2 or δ subtypes expressed with α1 and β3 subtypes in mouse L929 Cells
Authors: Fisher Janet L. , Macdonald Robert L.
Published: September 2004

To determine their contributions to GABAA receptor (GABAR) channel properties, rat γ2L and δ subunits were acutely co‐expressed with α1 and β3 subtypes in mouse L929 fibroblasts to produce α1β3, α1β3δ or α1β3γ2L GABAR isoforms.

With whole‐cell recording, the α1β3 isoform had relatively high sensitivity to GABA (EC50 2.1 μm) and low maximum current amplitude. The α1β1δ isoform had similar sensitivity to GABA (EC50 2.8 μm) and low current amplitude. The α1β3γ2L isoform had lower sensitivity to GABA (EC50 11.6 μm) and higher maximum current amplitude.

The single channel conductance of α1β3 channels was low (13 pS) compared with that of α1α3δ and α1β3γ2L channels (27 pS).

The single channel kinetic properties of the channels also differed. The α1β3γ2L channel exhibited three open states, while the α1β3 and α1β3δ channels exhibited only two open states with mean dwell times similar to those of the two shorter open states of the α1β3γ2L channel. All three channels exhibited at least five closed states. Bursts of α1β3δ channels consisted primarily of only one or two openings, while those of α1β3 channels contained multiple openings. α1β3γ2L channels exhibited burst kinetics typical for native GABARs with several long openings per burst.

These results show that the α1β3 heterodimer formed a GABA‐sensitive channel with complex gating kinetics. Addition of a δ subunit to form the α1β3δ heterotrimer altered the single channel conductance and the kinetic properties of the closed components, but did not affect the GABA sensitivity of the receptor nor the open state kinetics. In contrast, addition of a γ subunit (γ2L subtype) to produce the α1β3γ2L heterotrimer affected the GABA sensitivity, channel conductance and kinetic properties of both open and closed states.
Scorpion Toxin Block of the Early K+ Current (IKf) in Rat Dorsal root Ganglion Neurones
Authors: Matteson Donald R. , Blaustein Mordecai P.
Published: September 2004

The ability of three structurally homologous scorpion toxins to block voltage‐dependent K+ currents in rat dorsal root ganglion neurones was examined using the patch‐clamp technique.

Neurones with a diameter > 35 μm had two identifiable components of macroscopic K+ current. The outward current during depolarizations had both inactivating and non‐inactivating components, and the tail currents had both a fast component (IKf) with a time constant of about 2.5 ms and a slow component (IKs) with a time constant of about 10 ms.

The functional properties of IKf and IKs differed in several ways: (i)IKf activated over a more negative voltage range than IKf; (ii)IKf partially inactivated during a depolarization to +70 mV, whereas IKs did not inactivate during a 1 s depolarization to +70 mV; (iii)IKf activated more rapidly than IKs;and (iv)α‐dendrotoxin selectively blocked IKf.

Tityustoxin‐Kα (TsTX‐Kα) selectively blocked IKf, with little or no effect on IKs. The block was concentration dependent, with 50% of the current inhibited at a toxin concentration of about 38nM.

TsTX‐Kα block of IKf was completely reversible, but the washout rate was slow. The time constant of recovery from TsTX‐Kα block was about 11 min.

Charybdotoxin (CTX) also selectively blocked IKf in a reversible manner, but was about 10 times less potent than TsTX‐Kα. The CTX washout rate was over 10 times faster than that of TsTX‐Kα; the time constant of recovery was 0.8 min.

Pandinotoxin‐Kα (PiTX‐Kα) also selectively blocked IKf; the IC50 for block of IKf was about 8.1nM. In contrast to the other two toxins, however, PiTX‐Kα was poorly reversible.

The block of IKf produced by CTX was voltage dependent. In the voltage range from ‐10 to +70 mV, the fraction of blocked IKf fell from 91 to 37%. In contrast, both TsTX‐Kα and PiTX‐Kα blocked IKf in a voltage‐independent manner.

The backbone structure and many of the amino acid side‐chains on the presumed docking surfaces of the toxins are identical or conservatively replaced in all three toxins. Thus, some small differences in a few side‐chains that influence electrostatic, hydrophobic/hydrophilic and/or steric interactions probably account for the marked differences in affinities and dissociation rates.
Cation Permeability of a Cloned Rat Epithelial Amiloride‐Sensitive Na+ Channel
Authors: Ismailov Iskander I. , Shlyonsky Vadim Gh. , Alvarez Osvaldo , Benos Dale J.
Published: September 2004

Conductance of heterotrimeric rat epithelial Na+ channels (α,β,γ‐rENaCs) for Li+ and Na+ in planar lipid bilayers was a non‐linear function of ion concentration, with a maximum of 30.4 ± 2.9 pS and 18.5 ± 1.9 pS at 1 M Li+ and Na+, respectively.

The α,β,γ‐rENaC conductance measured in symmetrical mixtures of Na+–Li+ (1 M) exhibited an anomalous mole fraction dependence, with a minimum at 4:1 Li+ to Na+ molar ratio.

Permeability ratios PK/PNa and PLi/PNa of the channel calculated from the biionic reversal potentials were dependent on ion concentration: PK/PNa was 0.11 ± 0.01, and PLi/PNa was 1.6 ± 0.3 at 50 mm; PK/PNa was 0.04 ± 0.01 and PLi/PNa was 2.5 ± 0.4 at 3 M, but differed from the ratios of single‐channel conductances in symmetrical Li+, Na+ or K+ solutions. The permeability sequence determined by either method was Li+ > Na+ > K+≫ Rb+ > Cs+.

Predictions of a model featuring two binding sites and three energy barriers (2S3B), and allowing double occupancy, developed on the basis of single ion current–voltage relationships, are in agreement with the observed conductance maximum in single ion experiments, conductance minimum in the mole fraction experiments, non‐linearity of the current–voltage curves in biionic experiments, and the concentration dependence of permeability ratios.

Computer simulations using the 2S3B model recreate the ion concentration dependencies of single‐channel conductance observed for the immunopurified bovine renal amiloride‐sensitive Na+ channel, and short‐circuit current in frog skin, thus supporting the hypothesis that ENaCs form a core conduction unit of epithelial Na+ channels.
Sr2+ supports depolarization‐induced suppression of inhibition and provides new evidence for a presynaptic expression mechanism in rat hippocampal slices
Authors: Morishita W. , Alger B. E.
Published: September 2004

We studied the transient suppression of evoked GABAAergic inhibitory postsynaptic currents (eIPSCs) that follows brief membrane depolarization in rat CA1 hippocampal pyramidal cells, a Prs called depolarization‐induced suppression of inhibition (DSI). We used whole‐cell patch electrodes filled with a CsCl‐based solution to voltage clamp the currents. All experiments were done in the Presence of 50 μm 2‐amino‐5‐phosphonovaleric acid (APV) and 20 μm 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) to block ionotropic glutamate‐induced currents and polysynaptic transmission in the slice Preparation.

Substituting strontium (Sr2+) for extracellular calcium (Ca2+) led to the appearance of numerous ‘asynchronous’ small IPSCs following an eIPSC. These asynchronous IPSCs were indistinguishable from TTX‐insensitive quantal IPSCs.

Although somewhat less effective than Ca2+, Sr2+ was capable of supporting DSI, and both asynchronous and synchronous IPSCs were blocked by the DSI Process.

During DSI, quantal content of eIPSCs, but not quantal size, was significantly reduced.

Sr2+ converted paired‐pulse depression (PPD) of eIPSCs to a paired‐pulse facilitation (PPF), Presumably by altering the Probability of release at inhibitory nerve terminals. DSI had no effect on either PPD or PPF.

The results show that Sr2+ induces asynchronous release of GABA as it does of other neurotransmitters and changes the Probability of release at GABAAergic terminals as well. Most importantly, the results support the hypothesis that, despite being induced postsynaptically, DSI is expressed Presynaptically as a decrease in GABA release, possibly by acting at a site other than the Ca2+‐dependent release step.
Actions of Serum and Plasma Albumin on Intracellular Ca2+ in Human Endothelial Cells
Authors: Fuentes Esther , Nadal Angel , Jacob Ron , McNaughton Peter
Published: September 2004

The effects of serum and plasma albumin on [Ca2+]i in human endothelial cells were examined using single‐cell Ca2+ imaging. Two types of endothelial cell were used: human umbilical vein endothelial cells (HUVEC) in primary culture, and the endothelial‐derived cell line ECV304.

Serum albumin caused a large and transient rise in [Ca2+]i, due to Ca2+ release from an IP3‐sensitive internal store, followed by a maintained elevation in [Ca2+]i attributable to Ca2+ influx from the external medium. A half‐maximal rise in [Ca2+]i was produced by a concentration of serum albumin of about 1 μg ml−1.

The Ca2+‐releasing action of serum albumin is abolished by methanol extraction and is therefore attributable to an attached polar lipid. A possible candidate is lysophosphatidic acid, known to be released from platelets during blood coagulation, which produced similar effects to those of serum albumin.

In HUVEC, plasma albumin caused a sustained decrease in [Ca2+]i from the mean resting level of 114 nm to 58 nm. No effect of plasma albumin was observed in ECV304 cells.

The decrease in [Ca2+]i caused by plasma albumin is due to an uptake into intracellular stores. The store loading substantially potentiates the action of Ca2+‐releasing agonists such as histamine.

The results show that normal plasma albumin, which carries few lipids, lowers [Ca2+]i and potentiates the actions of Ca2+‐releasing agonists by promoting Ca2+ uptake into intracellular stores. When converted to the serum form, by binding lysophosphatidic acid released during blood coagulation, albumin has a potent effect in elevating [Ca2+]i. Blood coagulation may therefore play a role in regulating vascular tone and capillary permeability.
K+ Channels Which Contribute to the Acetylcholine‐Induced Hyperpolarization in Smooth Muscle of the Guinea‐Pig Submucosal Arteriole
Authors: Hashitani H. , Suzuki H.
Published: September 2004

Membrane potentials were recorded from submucosal arterioles (diameter, 30–80 μm) of the guinea‐pig small intestine, using conventional microelectrode techniques. In control solution the resting membrane potential was about −73 mV, and the addition of 0.5 mm Ba2+ depolarized the membrane to about −43 mV.

ACh (10 nm to 10 μM), or substance P (0.1 μM), caused a membrane hyperpolarization in preparations which had been depolarized by Ba2+ but not in control preparations. ACh produced a sustained hyperpolarization, whereas substance P produced a transient hyperpolarization, without being affected by either nitroarginine (0.1 mm) or indomethacin (10 μM).

In the presence of 50 μM BAPTA (acetoxymethyl ester form), the membrane potentials were not altered in the control solution or in the presence of Ba2+, but Ba2+ caused a smooth depolarization of the membrane. Following this procedure, both ACh and substance P caused membrane depolarization instead of hyperpolarization, suggesting that the ACh‐ and substance P‐induced hyperpolarization in arteriolar smooth muscle are intracellular [Ca2+] dependent.

In short segments (200–500 μM) of arteriole, the time constant of electrotonic potentials produced by passing current pulses through the recording electrode was about 75 ms. The addition of Ba2+ increased both the input resistance and the time constant.

The hyperpolarizations produced by ACh or substance P were associated with a reduction in the amplitude and the time constant of electrotonic potential.

The reversal potential for the ACh‐induced hyperpolarization, estimated from the current‐voltage relationship, was about −86 mV, a value close to the equilibrium potential for K+.

In the presence of 50 nM charybdotoxin the hyperpolarization produced by ACh became transient and was reduced in amplitude: the residual response was further reduced by apamin (0.1 μM). The response produced by substance P was also reduced by 50 nM charybdotoxin: again the residual response was sensitive to 0.1 μM apamin. The hyper‐polarizations produced by either ACh or substance P were insensitive to glibenclamide (10 μM) and 4‐aminopyridine (1 mm).

It is suggested that in submucosal arterioles of the guinea‐pig ileum, ACh‐ or substance P‐induced hyperpolarizations of smooth muscle result from activation of both charybdotoxin‐sensitive and apamin‐sensitive K+ channels, with the former being predominant. The results are discussed in relation to the possible involvement of one or more endothelium‐dependent hyperpolarizing factors in ACh‐ and substance P‐induced hyperpolarization.
Evidence Against the Involvement of Cytochrome P450 Metabolites in Endothelium‐Dependent Hyperpolarization of the Rat Main Mesenteric Artery
Authors: Vanheel Bert , Van de Voorde Johan
Published: September 2004

The influence of different inhibitors of cytochrome P450 mono‐oxygenase on the endothelium‐dependent and ‐independent hyperpolarization in the isolated rat main mesenteric artery was investigated.

Application of acetylcholine (ACh; 1 μm) for 10 min evoked an endothelium‐dependent peak hyperpolarization of about 18 mV followed by a partial recovery to a level 7 mV more negative than the resting value (‐50.2 ± 0.5 mV).

Proadifen (30 μm) completely and reversibly inhibited the ACh‐induced hyperpolarization. Conversely, the imidazole antimycotics clotrimazole (30 μm) and miconazole (100 μm) had less effect on the peak endothelium‐dependent hyperpolarization. The suicide substrate inhibitors 17‐octadecynoic acid (17‐ODYA; 5 μm) and 1‐aminobenzotriazole (1‐ABT; 2 mm) did not significantly influence endothelium‐dependent hyperpolarization.

The endothelium‐independent hyperpolarization (16 mV) evoked by levcromakalim (300 nm) was completely inhibited by proadifen as well as by clotrimazole and miconazole but was not affected by 17‐ODYA or 1‐ABT.

These results do not support the view that the ACh‐induced endothelium‐dependent hyperpolarization in the rat mesenteric artery is mediated by cytochrome P450 mono‐oxygenase metabolites. Proadifen and imidazole antimycotics impair the activation of ATP‐regulated K+ channels in mesenteric artery cells, rendering non‐specific inhibition of smooth muscle K+ channel activation an alternative explanation for the inhibitory influence of some (but not all) P450 inhibitors on endothelium‐dependent hyperpolarization in this preparation.
Stretch‐Activated Whole‐Cell Currents in Smooth Muscle Cells from Mesenteric Resistance Artery of Guinea‐Pig
Authors: Setoguchi Motoko , Ohya Yusuke , Abe Isao , Fujishima Masatoshi
Published: September 2004

Stretch‐activated (SA) channels were studied in smooth muscle cells isolated from mesenteric resistance arteries using the whole‐cell patch‐clamp method. Membrane stretch was achieved by cell inflation after application of positive pressure through a patch electrode.

In the voltage‐clamp configuration, cell inflation increased and cell deflation decreased the membrane conductance. Conductance of the evoked current depended on the increase in cross‐sectional area of the cell. The current‐voltage relationship was linear between ‐80 and 0 mV, while further hyperpolarization showed a slight inward rectification.

The reversal potential of the SA current depended on the extracellular Na+ concentration, suggesting that the inward SA current was carried predominantly by Na+. The SA current was also carried by other cations, suggesting that the channel responsible for this current is a non‐selective cation channel. The permeability sequence of cations as assessed by reversal potential was as follows: K+≥ Cs+≥ Na+ > Li+. The channel was also permeable to Ca2+.

Extracellular Ca2+ and Gd3+ inhibited the SA current carried by monovalent cations in a concentration‐dependent manner with IC50 (concentration giving 50% of maximal inhibition) values of 0.9 mm and 14 μm, respectively.

In the current‐clamp configuration, membrane stretch depolarized the cell, and 100 μm Gd3+ inhibited the stretch‐induced depolarization.

The results suggest that SA cation channels exist in arterial smooth muscle cells. Activation of the channels may modify membrane potential and intracellular ionic environment, and promote stretch‐mediated cell responses.
Role of Q‐type Ca2+ Channels in Vasopressin Secretion From Neurohypophysial Terminals of the Rat
Authors: Wang Gang , Dayanithi Govindan , Kim Sinnei , Hom Dennis , Nadasdi Laszlo , Kristipati Ramasharma , Ramachandran J. , Stuenkel Edward L. , Nordmann Jean J. , Newcomb Robert , Lemos José R.
Published: September 2004

The nerve endings of rat neurohypophyses were acutely dissociated and a combination of pharmacological, biophysical and biochemical techniques was used to determine which classes of Ca2+ channels on these central nervous system (CNS) terminals contribute functionally to arginine vasopressin (AVP) and oxytocin (OT) secretion.

Purified neurohypophysial plasma membranes not only had a single high‐affinity binding site for the N‐channel‐specific ω‐conopeptide MVIIA, but also a distinct high‐affinity site for another ω‐conopeptide (MVIIC), which affects both N‐ and P/Q‐channels.

Neurohypophysial terminals exhibited, besides L‐ and N‐type currents, another component of the Ca2+ current that was only blocked by low concentrations of MVIIC or by high concentrations of ω‐AgaIVA, a P/Q‐channel‐selective spider toxin.

This Ca2+ current component had pharmacological and biophysical properties similar to those described for the fast‐inactivating form of the P/Q‐channel class, suggesting that in the neurohypophysial terminals this current is mediated by a ‘Q’‐type channel.

Pharmacological additivity studies showed that this Q‐component contributed to rises in intraterminal Ca2+ concentration ([Ca2+]i) in only half of the terminals tested.

Furthermore, the non‐L‐ and non‐N‐component of Ca2+‐dependent AVP release, but not OT release, was effectively abolished by the same blockers of Q‐type current.

Thus Q‐channels are present on a subset of the neurohypophysial terminals where, in combination with N‐ and L‐channels, they control AVP but not OT peptide neurosecretion.
Tetrodotoxin‐blockable calcium currents in rat ventricular myocytes; a third type of cardiac cell sodium current
Authors: Aggarwal R. , Shorofsky S. R. , Goldman L. , Balke C. W.
Published: September 2004

Whole‐cell patch clamp currents from freshly isolated adult rat ventricular cells, recorded in external Ca2+ (Ca2+o) but no external Na+ (Ma4o), displayed two inward current components: a smaller component that activated over more negative potentials and a larger component (L‐type Ca2+ current) that activated at more positive potentials. The smaller component was not generated by Ca2+ channels. It was insensitive to 50 μm M2+ and 10 μm La3+, but suppressed by 10 μm tetrodotoxin (TTX). We refer to this component as ICa(TTx).

The conductance–voltage, g(V), relation in Ca2+o only was well described by a single Boltzmann function (half‐maximum potential, V½, of –44.5; slope factor, k, of –4.49 mV, means of 3 cells). g(V) in Ca2+o plus Na4o was better described as the sum of two Boltzmann functions, one nearly identical to that in Ca2+o only (mean V½ of –45.1 and k of – 3.90 mV), and one clearly distinct (mean V½ of –35.6 and k of –2.31 mV). Mean maximum conductance for ICa(TTX) channels increased 23.7 % on adding 1 mm Na+o to 3 mm Ca2+o. ICa(TTx) channels are permeable to Na+ ions, insensitive to Ni2+ and La3+ and blocked by TTX. They are Na+ channels.

I
Ca(TTx) channels are distinct from classical cardiac Na+ channels. They activate and inactivate over a more negative range of potentials and have a slower time constant of inactivation than the classical Na+ channels. They are also distinct from yet another rat ventricular Na+ current component characterized by a much higher TTX sensitivity and by a persistent, non‐fast‐inactivating fraction. That ICa(TTx) channels activate over a more negative range of potentials than classical cardiac Na+ channels suggests that they may be critical for triggering the ventricular action potential and so of importance for cardiac arrhythmias.
pH‐Dependent Interactions of Cd2+ and a Carboxylate Blocker with the Rat ClC‐1 Chloride Channel and Its R304E Mutant in the Sf‐9 Insect Cell Line
Authors: Rychkov G. Y. , St J. Astill D. , Bennetts B. , Hughes B. P. , Bretag A. H. , Roberts M. L.
Published: September 2004

Gating of the skeletal muscle chloride channel (ClC‐1) is sensitive to extracellular pH. In this study, whole‐cell recording of currents from wild‐type (WT) ClC‐1 and a mutant, R304E, expressed in the Sf‐9 insect cell line was used to investigate further the nature of the pH‐sensitive residues.

Extracellular Cd2+ produced a concentration‐dependent block of WT ClC‐1 with an IC50 of 1.0 ± 0.1 mm and a Hill coefficient of 2.0 ± 0.3. This block was sensitive to external pH, reducing at low pH, with an apparent pKa of 6.8 ± 0.1 and a Hill coefficient for proton binding of 3.0 ± 0.3. Anthracene‐9‐carboxylate (A‐9‐C) block of WT ClC‐1 was also pH sensitive, increasing at low pH, with an apparent pKa of 6.4 ± 0.1 and a Hill coefficient for proton binding of 1.0 ± 0.2.

Compared with WT ClC‐1, R304E had a lower affinity for Cd2+ (IC50, 3.0 ± 0.3 mm) but it had a similar Hill coefficient for transition metal ion binding. The Hill coefficient for proton binding to the Cd2+ binding site was reduced to 1.4 ± 0.3. In contrast, the A‐9‐C binding site in R304E showed the same pH sensitivity and affinity for the blocker as that seen in WT ClC‐1.

ClC‐1 has at least two binding sites for Cd2+, each of which has at least three residues which can be protonated. Binding of A‐9‐C is influenced by protonation of a single residue. Arg 304 is not sufficiently close to the A‐9‐C binding site to affect its characteristics, but it does alter Cd2+ binding, indicating that transition metal ions and aromatic carboxylates interact with distinct sites.

The block of ClC‐1 by transition metal ions and the apparent pKa of this block, together with the apparent pKa for A‐9‐C block and gating are all compatible with the involvement of His residues in the pore and gate of ClC‐1.
Activation of NMDA receptors is necessary for the induction of associative long‐term potentiation in area CA1 of the rat hippocampal slice
Authors: Murphy K. P. S. J. , Reid G. P. , Trentham D. R. , Bliss T. V. P.
Published: September 2004

It is commonly assumed that the role of the strongly activated heterosynaptic input during the induction of associative long‐term potentiation (LTP) is to relieve the magnesium blockade of NMDA receptors located at the weakly stimulated synapses and thereby allow the weak input to undergo potentiation. We tested this assumption by using a caged form of the NMDA receptor antagonist, d‐(–)‐2‐amino‐5‐phosphonopentanoic acid (d‐AP5) to block the activation of NMDA receptors at the weak input in a conditioning protocol for the induction of associative LTP in area CA1 of the rat hippocampal slice.

The effect of releasing d‐AP5 by flash photolysis of 100 μm caged d‐AP5 (N‐[l‐(2‐nitro‐phenyl)ethoxycarbonyl]‐d‐AP5) on pharmacologically isolated NMDA receptor‐mediated field EPSPs was examined in area CA1. The slope of the EPSP was reduced by 71% within 50 ms of the initiation of the photolytic reaction when the concentration of released d‐AP5 had reached 2.0‐2.5 μm and was reduced by 95% within 1 min (10 μmd‐AP5 released).

Associative LTP was induced by pairing a strong tetanus to one input with a weak tetanus (subthreshold for homosynaptic LTP) to a second input. The strong tetanus preceded the weak by 50 ms. Rapid application of d‐AP5, by flash photolysis of caged d‐AP5, coincident with the last shock of the strong tetanus, resulted in the blockade of NMDA receptor activation during the period of the weak tetanus. Associative LTP was blocked by photolysis of caged d‐AP5 but was normally expressed in experiments using caged l‐AP5.

We conclude that activation of NMDA receptors at the weakly activated input is an essential requirement for synaptically induced associative LTP.
Sarcoplasmic reticulum Ca2+ content, L‐type Ca2+ current and the Ca2+ transient in rat myocytes during β‐adrenergic Stimulation
Authors: Hussain M. , Orchard C. H.
Published: September 2004

The effect of β‐adrenergic stimulation on the relationship between the intracellular Ca2+ transient and the amplitude of the L‐type Ca2+ current (ICa) has been investigated in ventricular myocytes isolated from rat hearts. Intracellular [Ca2+] was monitored using fura‐2 during field stimulation and while membrane potential was controlled using voltage clamp techniques.

The increase in the amplitude, and the rate of decline, of the Ca2+ transient produced by isoprenaline (1.0 μmol l−1) was not significantly different in myocytes generating action potentials and in those voltage clamped with pulses of constant duration and amplitude.

Under control conditions, the current‐voltage (I‐ V) relationship for ICa was bell shaped. The amplitude of the Ca2+ transient also showed a bell‐shaped voltage dependence. In the presence of isoprenaline, the amplitude of both ICa and the Ca2+ transient was greater at all test potentials and the I–V relationship maintained its bell‐shaped voltage dependence. However, the size of the Ca2+ transient was no longer graded with changes in the amplitude of ICa: a small ICa could now elicit a maximal Ca2+ transient.

Rapid application of caffeine (10 mmol l−1) was used to elicit Ca2+ release from the sarcoplasmic reticulum (SR). Isoprenaline increased the integral of the subsequent rise in cytoplasmic [Ca2+] to 175 ± 13% of control.

Abbreviation of conditioning pulse duration in the presence of isoprenaline was used to reduce the amplitude of the Ca2+ transient to control levels. Under these conditions, the amplitude of the Ca2+ transient was again graded with the amplitude of ICa in the same way as under control conditions.

Nifedipine (2 μmol l−1) was also used to decrease Ca2+ transient amplitude in the presence of isoprenaline. In the presence of isoprenaline and nifedipine, the amplitude of the Ca2+ transient again showed a bell‐shaped voltage dependence.

The SR Ca2+ ‐ATPase inhibitor thapsigargin (2.5 μmol 1−1) reduced the effect of isoprenaline on the amplitude of the Ca2+ transient. In the presence of thapsigargin, the size of the Ca2+ transient increased as ICa increased in response to isoprenaline.

These data suggest that the increase in the amplitude of the Ca2+ transient produced by β‐adrenergic stimulation in cardiac muscle is due to an increase in the gain of the SR Ca2+ release process, due principally to an increase in the Ca2+ content of the SR.
Multiple Modulatory Effects of the Neuroactive Steroid Pregnanolone on GABAA Receptor in Frog Pituitary Melanotrophs
Authors: Le Foll Frank , Castel Hélène , Louiset Estelle , Vaudry Hubert , Cazin Lionel
Published: September 2004

The effects of the neuroactive steroid pregnanolone (5β‐pregnan‐3α‐ol‐20‐one) on the electrical response to GABA were investigated in cultured frog pituitary melanotrophs using the patch‐clamp technique.

Low concentrations of pregnanolone (0.01–1 μm) in the extracellular solution enhanced the current evoked by submaximal concentrations of GABAA receptor agonists and prolonged the GABA‐induced inhibition of the spontaneous action potentials in a dose‐dependent manner.

Pregnanolone augmented the opening probability of the single GABA‐activated channels but did not modify the conductance levels.

Pregnanolone (1 μm) shifted the GABA dose–response curve towards the low GABA concentrations, reducing the EC50 from 4.2 to 1.8 μm.

Internal cell dialysis with pregnanolone (1 or 10 μm) did not alter the GABA‐evoked current.

Pregnanolone accelerated the desensitization of both the current and conductance increases caused by GABA.

High concentrations of pregnanolone (30 μm) markedly and reversibly diminished the current evoked by 10 μm GABA.

At high concentrations (10–30 μm), pregnanolone induced an outward current which reversed at the chloride equilibrium potential.

It is concluded that, in frog pituitary melanotrophs, pregnanolone exerts a dual inverse modulation and a direct activation of the GABAA receptor–channel depending on the concentrations of both GABA and steroid. Pregnanolone acts on an extracellular site on the GABAA receptor inducing conformational changes of the receptor–channel complex, resulting in a desensitized less‐conducting state.
Differential Na+–K+‐ATPase activity in rat lemniscal and non ‐ lemniscal auditory Thalami
Authors: Senatorov V. V. , Hu Bin
Published: September 2004

Using whole‐cell recording and confocal immunofluorescent microscopy, we have investigated the differential electrogenic activity, subunit expression and subcellular distribution of the Na+–K+‐ATPase in the lemniscal (ventral) and non‐lemniscal (dorsal) pathways of the rat medial geniculate body (MGB)in vitro.

Bath application of Na+–K+‐ATPase inhibitors strophanthidin or dihydroouabain produced a transient, dose‐dependent inward current or membrane depolarization which were significantly larger in dorsal MGB neurones than in ventral cells (45.9 ± 6.45vs. 24.3 ± 4.l pA;P < 0.05). Electrophysiological and morphometric measurements showed that the dorsal MGB neurones had a significantly lower input conductance and a smaller somata than their ventral counterparts. The level of the resting membrane potential also differed by about 6 mV between the two cell populations, with the dorsal cells being more hyperpolarized (−74.2 ± 0.6vs. −67.7 ± 1.3 mV;P< 0.001).

Incubation of enzymatically dissociated MGB neurones with fluorescent monoclonal antibodies against α1‐α3 isoforms of Na+–K+‐ATPase showed that both dorsal and ventral cells expressed primarily α3 subunits. Confocal laser scanning revealed, however, that the mean pixel density of α3 fluorescent antibodies in the plasma membrane domain, but not in the cytoplasmic compartment, was about 40 % higher in dorsal neurones than in the ventral cells (29.7 ± 4.7vs. 16.9 ± 2.3 grey shadow per pixel;P< 0.05).

The above results suggest that the electrogenic activity of the Na+–K+‐ATPase is differentially regulated between lemniscal and non‐lemniscal auditory thalami through a mechanism that probably involves differential pump densities in the cell membrane.
Regulation by gastric acid of the processing of progastrin‐derived peptides in rat antral mucosa
Authors: Macro J. A. , Bate G. W. , Varro A. , Vaillant C. , Seidah N. G. , Dimaline R. , Dockray G. J.
Published: September 2004

Inhibition of gastric acid secretion by proton pump inhibitors like omeprazole increases the synthesis and secretion of the pyloric antral hormone gastrin. We report here how omeprazole influences the conversion of the gastrin precursor to its final products, and the abundance of mRNAs encoding proteins associated with progastrin processing in rat antral mucosa.

Progastrin processing was studied using a pulse–chase protocol in antral mucosa, incubated in vitro, from rats treated with omeprazole for up to 5 days. Labelled peptides were detected by on–line scintillation counting after immunoprecipitation and HPLC. The mRNAs encoding prohormone‐processing enzymes were identified by Northern blot, polymerase chain reaction or ribonuclease protection assay, and their cellular origins identified by immunocytochemistry.

Cleavage of [3H]‐ and [35S]‐labelled progastrins at Arg‐94–95 or Arg‐57–58, and amidation at Phe‐92 were not influenced by pretreatment with omeprazole. In contrast, cleavage of G34 (the thirty‐four amino acid amidated gastrin) at Lys‐74–75 to give G17 (the seventeen amino acid amidated gastrin), and of G34–Gly to Gl7–Gly (G34 and G17 with COOH‐terminal glycine), was increased 3‐fold after treatment with omeprazole for either 1 or 5 days.

Approximately 20% of newly synthesized amidated and Gly‐extended gastrins were secreted within 240 min of the labelling period in omeprazole‐treated samples, but secretion of labelled gastrins from control tissue was undetectable over a comparable period.

The amidating enzyme, peptidylglycine α‐amidating mono‐oxygenase (PAM), the prohormone convertases PC1/3, PC2, PC5 and the PC2 chaperone 7B2 were localized to rat antral gastrin cells by immunocytochemistry. The relative abundance of mRNA species encoding 7B2, PC5 and PAM were unchanged after treatment with omeprazole for 5 days, whereas gastrin, PC1/3 and PC2 mRNAs are known to increase at this time.

The main consequence of increased cleavage at Lys‐74–75 is the production of G17 and G17–Gly at the expense of G34 and G34–Gly, respectively. The latter have longer plasma half‐lives, and so their increased cleavage may serve to limit the rise in plasma gastrin concentration after inhibition of acid secretion. Changes in the abundance of mRNAs encoding prohormone‐processing enzymes cannot account for the rapidity of the changes in cleavage of progastrin at Lys residues after omeprazole.
Effect of Cell Shrinkage on Permeability of Cultured Bovine Aortic Endothelia and Frog Mesenteric Capillaries
Authors: Kajimura M. , O'Donnell M. E. , Curry F. E.
Published: September 2004

We have tested the hypothesis that a reduction in endothelial cell volume increases microvessel permeability and that the degree of endothelial cell attachment to their basement membranes determines the magnitude of permeability changes caused by a reduction in endothelial cell volume.

A decrease in endothelial cell volume was imposed on both intact microvessels and cultured endothelial monolayers by raising osmolarity by 100 mosmol l−1

We found that hypertonic solutions did not increase the hydraulic permeability (Lp) of individually perfused venular microvessels in frog mesentery when the perfusate contained albumin. Hypertonic solutions did increase Lp, however, after we perfused the microvessels with the peptide Gly‐Arg‐Gly‐Asp‐Thr‐Pro (GEGDTP; 0.3 mmol l−1), to disrupt integrin‐dependent endothelial cell (EC) attachment to the extracellular matrix (ECM).

After albumin was removed from the perfusate, hypertonic solutions increased Lp of microvessels and the permeability of endothelial monolayers to α‐lactalbumin.

Our findings indicate that endothelial cell integrin‐ECM binding plays a role in transducing changes in cell volume and/or shape into changes in permeability. We hypothesize that removal of albumin from the vascular perfusate may compromise EC–ECM interactions via an integrin‐dependent mechanism.
Mechanical study of rat soleus muscle using caged ATP and X‐ray diffraction: high ADP affinity of slow cross‐bridges
Authors: Horiuti K. , Yagi N. , Takemori S.
Published: September 2004

The cross‐bridges in slow‐ and fast‐twitch fibres (taken, respectively, from soleus and psoas muscles of rats) were examined in mechanical experiments using caged ATP and X‐ray diffraction, to compare their binding of ATP and ADP.

Caged ATP was photolysed in rigor fibres. When ADP was removed from pre‐photolysis fibres, the initial relaxation (±Ca2+) in soleus was as fast as that in psoas fibres, whereas the subsequent contraction (+Ca2+) was slower in soleus than in psoas. The ATPase rate during the steady‐state contraction was also slower in soleus fibres.

When ADP was added to pre‐photolysis fibres (±Ca2+), tension developed even in the initial phase, the overall tension development being biphasic. Both initial and late components of the Ca2+‐free contraction were enhanced when ADP was added before photolysis, although pre‐photolysis ADP was not a prerequisite for the late component. The effect of ADP was greater in soleus than in psoas fibres. Static experiments on rigor fibres revealed a higher ADP affinity in soleus fibres.

The intensity of the actin layer‐line from ADP rigor soleus fibres decreased rapidly on photorelease of ATP. We conclude that, despite the tight ADP binding of the soleus cross‐bridge, its isometric reaction is not rate limited by the ‘off’ rate of ADP.
Role of Joint Receptors in Modulation of Inspiratory Intercostals Activity by Rib Motion in Dogs
Authors: De Troyer André
Published: September 2004

Inspiratory activity in the canine external intercostal muscles is exquisitely sensitive to the direction and amplitude of the inspiratory displacement of the ribs. This study was designed to investigate the role of muscle receptors, in particular the muscle spindles, in mediating this phenomenon.

External intercostal inspiratory activity showed a reflex increase when the normal cranial motion of the ribs and the normal shortening of the muscles was reduced, and showed a reflex decrease when the cranial motion of the ribs and the shortening of the muscles was augmented. However, clamping the two ribs making up the interspace and maintaining muscle length constant only moderately attenuated these responses.

These persistent responses remained unchanged after section of the levator costae muscles.

The responses were attenuated but still present after section of the external intercostals in the contiguous segments and denervation of the internal intercostals.

These reflex responses are therefore mediated in part by non‐muscular receptors, which most likely lie within the costovertebral joints. These joint receptors might be a primary determinant of the load‐compensating reflex.
Chemo‐mechanical energy transduction in relation to myosin isoform composition in skeletal muscle fibres of the rat
Authors: Reggiani C. , Potma E. J. , Bottinelli R. , Canepari M. , Pellegrino M. A. , Stienen G. J. M.
Published: September 2004

ATP consumption and force development were determined in single skinned muscle fibres of the rat at 12 °C. Myofibrillar ATPase consumption was measured photometrically from NADH oxidation which was coupled to ATP hydrolysis. Myosin heavy chain (MHC) and light chain (MLC) isoforms were identified by gel electrophoresis.

Slow fibres (n= 14) containing MHCI and fast fibres (n= 18) containing MHCIIB were compared. Maximum shortening velocity was 1.02 ± 0.63 and 3.05 ± 0.23 lengths s−1, maximum power was 1.47 ± 0.22 and 9.59 ± 0.84 W l−1, and isometric ATPase activity was 0.034 ± 0.003 and 0.25 ± 0.01 mM s−1 in slow and in fast fibres, respectively.

In fast as well as in slow fibres ATP consumption during shortening increased above isometric ATP consumption. The increase was much greater in fast fibres than in slow fibres, but became similar when expressed relative to the isometric ATPase rate.

Efficiency was calculated from mechanical power and free energy change associated with ATP hydrolysis. Maximum efficiency was larger in slow than in fast fibres (0.38 ± 0.04 versus 0.28 ± 0.03) and was reached at a lower shortening velocity.

Within the group of fast fibres efficiency was lower in fibres which contained more MLC3f. We conclude that both MHC and essential MLC isoforms contribute to determine efficiency of chemo‐mechanical transduction.
Is energy expenditure in the hamster primarily under homeostatic or circadian control?
Authors: Refinetti Roberto , Menaker Michael
Published: September 2004

In order to discriminate between homeostatic and circadian control of energy expenditure, this paper considers whether a shorter circadian cycle will produce a proportional reduction in energy expenditure (so that expenditure per unit time is conserved) or alternatively whether energy expenditure will be compressed into the shorter cycle (so that energy expenditure per cycle is conserved). To answer this question, we measured energy expenditure in tau mutant hamsters (whose free‐running circadian period has been reduced to about 20 h by a single gene mutation) and wild‐type hamsters (whose free‐running circadian period is about 24 h).

In one experiment, the circadian rhythm of running‐wheel activity of tau mutant hamsters was compared with that of wild‐type hamsters. The rate of running was not affected by the mutation and, consequently, the total amount of activity per cycle was significantly less in mutants than in wild‐type hamsters, whereas the total amount of activity per unit time was nearly the same.

In a second experiment, we measured energy expenditure by indirect calorimetry. Metabolic rate was not affected by the mutation and, consequently, the total amount of energy expended per cycle was significantly less in mutants than in wild‐type hamsters but equivalent per unit time.

Because the amount of energy expenditure and locomotor activity was found to be proportional to the circadian cycle, we conclude that expenditure per unit time – rather than expenditure per circadian cycle – is conserved in the mutant animals. Therefore, we infer that energy expenditure in hamsters is primarily under homeostatic, not circadian, control. Further research is necessary to determine whether this inference can be applied to other species.