bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the NO‐cGMP pathway in the muscarinic regulation of the L‐type Ca2+ current in human atrial myocytes
Authors: Vandecasteele Grégoire , Eschenhagen Thomas , Fischmeister Rodolphe
Published: September 2004

The whole‐cell patch‐clamp technique was used to examine the participation of nitric oxide synthase (NOS) and soluble guanylyl cyclase in the muscarinic regulation of the L‐type Ca2+ current (ICa) in freshly isolated human atrial myocytes.

Acetylcholine (ACh, 1 μM) decreased basal ICa by 39.1 ± 5.5 % (n= 8) under control conditions, and by 38.0 ± 6.1 % (n= 6) in the presence of 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxaline‐1‐one (ODQ, 10 μM), a potent guanylyl cyclase inhibitor, and NG‐monomethyl‐L‐arginine (L‐NMMA, 1 mM), a competitive NOS inhibitor. L‐NMMA alone had no effect on ICa, whilst ODQ increased ICa in 50 % of the cells.

The accentuated antagonism of ACh on ICa, i.e. its ability to antagonize the stimulatory effect of β‐adrenergic agonists and, by extension, of other cAMP‐elevating agents, was examined after the current was stimulated by either the β‐adrenergic agonist isoprenaline (Iso) or serotonin (5‐HT). ACh (100 nM or 1 μM) completely blocked the stimulatory effects of 10 nM Iso or 10 nM 5‐HT on ICa.

Extracellular application of Methylene Blue (MBlue, 10 μM), a guanylyl cyclase inhibitor, antagonized the inhibitory effect of 1 μM ACh on Iso‐ or 5‐HT‐stimulated ICa. However, this effect was overcome by a 100‐fold higher ACh concentration and was not mimicked by an intracellular application of MBlue.

Inhibition of NOS and soluble guanylyl cyclase activities by addition of ODQ (10 μM) and L‐NMMA (1 mM) to both extracellular and intracellular solutions, or by a 2 h pre‐incubation of the cells with these inhibitors, modified neither the Iso (10 nM) response nor the inhibitory effect of ACh (100 nM or 1 μM) on Iso‐stimulated ICa.

Extracellular application of the NO donor SNAP (S‐nitroso‐N‐acetyl‐D,L‐penicillamine) at 100 nM produced a stimulatory effect on ICa in control conditions. This stimulatory effect was abolished by intracellular MBlue (20 μM) or by intracellular and extracellular application of ODQ (10 μM) in combination with L‐NMMA (1 mM).

We conclude that the NO‐cGMP pathway does not contribute significantly to the muscarinic regulation of ICa in human atrial myocytes.
Two mutations linked to nocturnal frontal lobe epilepsy cause use‐dependent potentiation of the nicotinic ACh response
Authors: Figl Antonio , Viseshakul Nareerat , Shafaee Navid , Forsayeth John , Cohen Bruce N.
Published: September 2004

We constructed rat homologues (S252F and +L264) of two human α4 nicotinic mutations ‐ α4(S248F) and α4(777ins3) ‐ that have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and co‐expressed them with wild‐type rat β2 subunits in Xenopus oocytes.

The S252F and +L264 mutations had three common effects on the ACh response. First, they caused use‐dependent potentiation of the response during a train of brief 100 nm ACh pulses. Second, they delayed the rise times of the 5–15 nm (+L264) and 30 nm (S252F) ACh responses. Third, they reduced extracellular Ca2+‐induced increases in the 30 μm ACh response.

Beside these shared effects, the S252F mutation also reduced the channel burst duration measured from voltage‐jump relaxations, enhanced steady‐state desensitization and reduced the single‐channel conductance. In contrast, the +L264 mutation prolonged the channel burst duration, did not affect desensitization and slightly increased single‐channel conductance. Neither mutation affected the number of surface receptors measured by antibody binding but the S252F mutation reduced the maximum ACh response.

The ACh concentration dependence of use‐dependent potentiation and the delay in the rising phase of the mutant ACh response suggest that these effects are caused by a slow unblocking of the closed mutant receptors. Use‐dependent potentiation of the mutant response during a series of high‐frequency cholinergic inputs to the presynaptic terminal could trigger ADNFLE seizures by suddenly increasing nicotinic‐mediated transmitter release.
Non‐genomic inhibition of human P2X7 purinoceptor by 17β‐oestradiol
Authors: Cario‐Toumaniantz Chrystelle , Loirand Gervaise , Ferrier Laurent , Pacaud Pierre
Published: September 2004

Effects of oestrogen on the current evoked by ATP and benzoylbenzoyl ATP (BzATP) in CV‐1 monkey kidney cells transformed by SV 40 (COS cells) expressing the human P2X7 (hP2X7) purinoceptor were studied using standard patch‐clamp techniques.

17β‐Oestradiol rapidly and reversibly inhibited the whole‐cell hP2X7 receptor cation current. This inhibitory action resulted in a rightward shift of the dose‐response curve to ATP and BzATP in the presence of physiological as well as low divalent cation concentrations.

The inhibitory effect of 17β‐oestradiol on the BzATP‐ or ATP‐induced cation current was concentration dependent. The half‐maximal inhibition was obtained with 3 μM 17β‐oestradiol. Progesterone and 17α‐oestradiol had almost no effect on the hP2X7 receptor cation current.

The inhibition of the hP2X7 receptor cation current by 17β‐oestradiol did not depend on the membrane potential. 17β‐Oestradiol added to the extracellular side of outside‐out patches inhibited BzATP‐activated single‐channel currents.

Activation of the hP2X7 receptor in both COS and U937 (human macrophage) cells did not induce the formation of large non‐specific pores.

Since COS cells do not express endogenous nuclear oestrogen receptor, this study shows that, at pharmacological concentrations, 17β‐oestradiol inhibited the hP2X7 receptor cation channel in a non‐genomic manner.
Regulation of Ca2+‐dependent Cl− conductance in a human colonic epithelial cell line (T84): cross‐talk between Ins(3,4,5,6)P4 and protein phosphatases
Authors: Xie Weiwen , Solomons Kevin R. H. , Freeman Sally , Kaetzel Marcia A. , Bruzik Karol S. , Nelson Deborah J. , Shears Stephen B.
Published: September 2004

We have studied the regulation of whole‐cell chloride current in T84 colonic epithelial cells by inositol 3,4,5,6‐tetrakisphosphate (Ins(3,4,5,6)P4). New information was obtained using (a) microcystin and okadaic acid to inhibit serine/threonine protein phosphatases, and (b) a novel functional tetrakisphosphate analogue, 1,2‐bisdeoxy‐1,2‐bisfluoro‐Ins(3,4,5,6)P4 (i.e. F2‐Ins(3,4,5,6)P4).

Calmodulin‐dependent protein kinase II (CaMKII) increased chloride current 20‐fold. This current (ICl,CaMK) continued for 7 ± 1.2 min before its deactivation, or running down, by approximately 60 %. This run‐down was prevented by okadaic acid, whereupon ICl,CaMK remained near its maximum value for ≥ 14.3 ± 0.6 min.

F2‐Ins(3,4,5,6)P4 inhibited ICl,CaMK (IC50= 100 μM) stereo‐specifically, since its enantiomer, F2‐Ins(1,4,5,6)P4 had no effect at <= 500 μM. Dose‐response data (Hill coefficient = 1.3) showed that F2‐Ins(3,4,5,6)P4 imitated only the non‐co‐operative phase of inhibition by Ins(3,4,5,6)P4, and not the co‐operative phase.

Ins(3,4,5,6)P4 was prevented from blocking ICl,CaMK by okadaic acid (IC50= 1.5 nM) and microcystin (IC50= 0.15 nM); these data lead to the novel conclusion that, in situ, protein phosphatase activity is essential for Ins(3,4,5,6)P4 to function. The IC50 values indicate that more than one species of phosphatase was required. One of these may be PP1, since F2‐Ins(3,4,5,6)P4‐dependent current blocking was inhibited by okadaic acid and microcystin with IC50 values of 70 nM and 0.15 nM, respectively.
Intracellular nucleotide‐mediated gating of SUR/Kir6.0 complex potassium channels expressed in a mammalian cell line and its modification by pinacidil
Authors: Satoh Eisaku , Yamada Mitsuhiko , Kondo Chikako , Repunte Vez P. , Horio Yoshiyuki , Iijima Toshihiko , Kurachi Yoshihisa
Published: September 2004

We have examined the properties of intracellular nucleotide‐mediated gating of K+ channel constructs composed of the sulphonylurea receptor 2B and the inwardly rectifying K+ channel subunits Kir6.1 and Kir6.2 (SUR2B/Kir6.1 and SUR2B/Kir6.2 complex K+ channels) heterologously expressed in human embryonic kidney (HEK) 293T cells. In the cell‐attached form, both types of K+ channel were activated by pinacidil.

In inside‐out (IO) patches, the SUR2B/Kir6.2 channels opened spontaneously and were inhibited by intracellular ATP (ATPi). Pinacidil attenuated the ATPi‐mediated channel inhibition in a concentration‐dependent manner. In contrast, the SUR2B/Kir6.1 channels required intracellular nucleoside di‐ or tri‐, but not mono‐, phosphates for opening. The potency of adenine, guanine or uracil nucleotides to activate SUR2B/Kir6.1 channels was enhanced by pinacidil.

In the presence of pinacidil, adenine and guanine, but not uracil, nucleotides exhibited bell‐shaped concentration‐dependent activating effects on SUR2B/Kir6.1 channels. This was due to channel inhibition caused by adenine and guanine nucleotides, which was unaffected by pinacidil.

From power density spectrum analysis of SUR2B/Kir6.1 currents, channel activation could be described by the product of two gates, a nucleotide‐independent fast channel gate and a nucleotide‐dependent slow gate, which controlled the number of functional channels. Pinacidil specifically increased the potency of nucleotide action on the slow gate.

We conclude that Kir6.0 subunits play a crucial role in the nucleotide‐mediated gating of SUR/Kir6.0 complex K+ channels and may determine the molecular mode of pinacidil action.
Four novel myosin heavy chain transcripts define a molecular basis for muscle fibre types in Ranapipiens
Authors: Lutz Gordon J. , Cuizon Denise B. , Ryan Allen F. , Lieber Richard L.
Published: September 2004

Differential expression of myosin heavy chain (MHC) isoforms dramatically affects mechanical and energetic properties of skeletal muscle fibre types. As many as five different fibre types, each with different mechanical properties, have been reported in frog hindlimb muscles. However, only two frog MHC isoforms have previously been detected by SDS‐PAGE and only one adult hindlimb MHC isoform has been cloned.

In the present study, four different fibre types (type 1, type 2, type 3 and tonic) were initially identified in adult Ranapipiens anterior tibialis muscle based on myosin ATPase histochemistry, size and location. Each fibre type exhibited unique reactivity to a panel of MHC monoclonal antibodies. Single fibre analysis using SDS‐PAGE revealed that MHCs from immunohistochemically defined type 1, type 2 and type 3 fibres ran as three distinct isoform bands, while MHC of tonic fibres co‐migrated with type 1 MHC. The combined data from immunohistochemistry and SDS‐PAGE suggests that Rana fibre types are composed of four different MHCs.

Four novel MHC cDNAs were cloned and expression of the corresponding transcripts was measured in single immuno‐identified fibres using specific polymerase chain reaction (PCR) primer pairs. Each of the four transcripts was found to be primarily expressed in a different one of the four fibre types.

Coexpression of MHC isoforms was observed only between types 1/2 and types 2/3 at both the protein and mRNA level.

These data provide a molecular basis for differentiation between frog fibre types and permit future molecular studies of MHC structure/function and gene regulation in this classic physiological system.

Comparison of sequence homology among amphibian, avian and mammalian MHC families supports the concept of independent evolution of fast MHC genes within vertebrate classes subsequent to the amphibian/avian/mammalian radiation.
Termination of Ca2+ release during Ca2+ sparks in rat ventricular myocytes
Authors: Lukyanenko Valeriy , Wiesner Theodore F. , Györke Sandor
Published: September 2004

Confocal Ca2+ imaging was used to measure spontaneous release events (Ca2+ sparks) in fluo‐3‐loaded isolated rat ventricular myocytes.

The microscopic Ca2+ release flux underlying Ca2+ sparks was derived by adapting the methods used previously to describe macroscopic Ca2+ release from cell‐averaged Ca2+ transients.

The magnitude of the local release fluxes varied from 2 to 5 μM ms−1, depending on SR Ca2+ loading conditions. Following spontaneous activation, the release flux rapidly decayed (τ= 6–12 ms). The rate of termination of release flux was found to be directly related to the magnitude of the flux (r2= 0.88).

The rate of termination of local release flux was slowed in the presence of FK506, a compound that is known to reduce inactivation of SR Ca2+ channels in vitro.

These results suggest that termination of release flux during sparks is not due to a spontaneous stochastic decay process or local depletion of Ca2+ from the SR, but rather involves an active extinguishing mechanism such as Ca2+‐dependent inactivation or adaptation.
Functional interactions between nicotinic and P2X channels in short‐term cultures of guinea‐pig submucosal neurons
Authors: Barajas‐López Carlos , Espinosa‐Luna Rosa , Zhu Yaohui
Published: September 2004

Functional interactions between nicotinic and P2X receptors in submucosal neurons were investigated. Whole‐cell currents induced by ACh (IACh) and ATP (IATP) were blocked by hexamethonium and PPADS), respectively. Currents induced by simultaneous application of the two transmitters (IACh+ATP) were only as large as the current induced by the most effective of these substances. This current occlusion indicates that activation of nicotinic and P2X channels is not independent.

Kinetic parameters of IACh+ATP indicate that they are carried through channels activated by either substance. In agreement with this interpretation, both IACh and IATP amplitudes were decreased when ATP and ACh were applied simultaneously, whereas no cross‐desensitization was observed when nicotinic and P2X receptors were desensitized individually.

Current occlusion was observed at membrane potentials of −60 and +10 mV, when IACh and IATP were inward. However, when these currents were outward (at +40 mV), current occlusion was not observed. Current occlusion was still observed at +40 mV in experiments in which the reversal potential of these currents had been adjusted to more positive values.

Current occlusion occurred as soon as currents were detected (< 5 ms), was still present in the absence of Ca2+, Na+ or Mg2+, and after adding staurosporine, genistein, K‐252a, or N‐ethylmaleimide to the pipette solution. Similar observations were noted after substituting α,β‐methylene ATP for ATP, or GTP for GTP‐γ‐S in the pipette and in experiments carried out at 36, 23 and 9 °C.

We propose that nicotinic and P2X channels are in functional clusters of at least two, and that the influx of ions through one activates (through allosteric interactions) a mechanism that inhibits the other channel.
Neutralization of aspartate residues in the murine inwardly rectifying K+ channel IRK1 affects the substate behaviour in Mg2+ block
Authors: Oishi Keiko , Omori Koichiro , Ohyama Hideo , Shingu Koh , Matsuda Hiroko
Published: September 2004

To investigate the molecular basis of the sublevels induced in the outward current during block by intracellular Mg2+, single‐channel currents through inwardly rectifying K+ (IRK1) channels were studied.

cDNA encoding a functional murine IRK1 channel was transfected into COS‐1 cells (a Green Monkey kidney cell line) using the liposome method, and voltage clamp experiments were done after 48‐72 h.

Intracellular Mg2+ at micromolar concentrations induced sublevels in the outward current at one‐third and two‐thirds of the unitary amplitude seen in wild‐type channels. Replacing Asp 172 with Asn (D172N) and Gln (D172Q) abolished these sublevels, i.e. the channel showed only the fully open and fully blocked states.

Both mutations reduced the Mg2+ sensitivity of the channel at 2 μM Mg2+. However, the Mg2+ sensitivity did not differ significantly at higher concentrations (10 μM) and voltages (+70 mV).

Channels expressed from D172E showed the sublevels, indicating that a negative charge is indispensable to the substate behaviour.

Channels from tandem tetramers of IRK1 with one and two D172N mutant subunits mainly showed sublevels with two‐thirds amplitude, while those from tetramers with three D172N mutant subunits showed no sublevels.

These findings suggest that differences in Mg2+ binding patterns lead to different conductive states in a single‐barrelled channel.
Direct measurement of SR release flux by tracking ‘Ca2+ spikes’ in rat cardiac myocytes
Authors: Song Long‐Sheng , Sham James S. K. , Stern Michael D. , Lakatta Edward G. , Cheng Heping
Published: September 2004

Ca2+ release flux across the sarcoplasmic reticulum (SR) during cardiac excitation‐contraction coupling was investigated using a novel fluorescence method. Under whole‐cell voltage‐clamp conditions, rat ventricular myocytes were dialysed with a high concentration of EGTA (4.0 mm, 150 nm free Ca2+), to minimize the residence time of released Ca2+ in the cytoplasm, and a low‐affinity, fast Ca2+ indicator, Oregon Green 488 BAPTA‐5N (OG‐5N; 1.0 mm, Kd≈ 31 μm), to optimize the detection of localized high [Ca2+] in release site microdomains. Confocal microscopy was employed to resolve intracellular [Ca2+] at high spatial and temporal resolution.

Analytical and numerical analyses indicated that, under conditions of high EGTA concentration, the free [Ca2+] change is the sum of two terms: one major term proportional to the SR release flux/Ca2+ influx, and the other reflecting the running integral of the released Ca2+.

Indeed, the OG‐5N transients in EGTA‐containing cells consisted of a prominent spike followed by a small pedestal. The OG‐5N spike closely resembled the first derivative (d[Ca2+]/dt) of the conventional Ca2+ transient (with no EGTA), and mimicked the model‐derived SR Ca2+ release function reported previously. In SR Ca2+‐depleted cells, the OG‐5N transient also closely followed the waveform of L‐type Ca2+ current (ICa). Using ICa as a known source of Ca2+ influx, SR flux can be calibrated in vivo by a linear extrapolation of the ICa‐elicited OG‐5N signal.

The OG‐5N image signal was localized to discrete release sites at the Z‐line level of sarcomeres, indicating that the local OG‐5N spike arises from ‘Ca2+ spikes’ at transverse (T) tubule‐SR junctions (due to the imbalance between calcium ions entering the cytosol and the buffer molecules).

Both peak SR release flux and total amount of released Ca2+ exhibited a bell‐shaped voltage dependence. The temporal pattern of SR release also varied with membrane voltage: Ca2+ release was most synchronized and produced maximal peak release flux (4.2 mm s−1) at 0 mV; in contrast, maximal total release occurred at −20 mV (71 versus 61 μm at 0 mV), but the localized release signals were partially asynchronous. Since the maximal conventional [Ca2+] transient and contraction were elicited at 0 mV, it appears that not only the amount of Ca2+ released, but also the synchronization among release sites affects the whole‐cell Ca2+ transient and the Ca2+‐myofilament interaction.
A neuronal C5a receptor and an associated apoptotic signal transduction pathway
Authors: Farkas I. , Baranyi L. , Takahashi M. , Fukuda A. , Liposits Zs. , Yamamoto T. , Okada H.
Published: September 2004

We report the first experimental evidence of a neuronal C5a receptor (nC5aR) in human cells of neuronal origin. Expression of nC5aR mRNA was demonstrated by the reverse transcriptase‐polymerase chain reaction (RT‐PCR) in TGW human neuroblastoma cells.

Expression of a functional C5aR was supported by the finding that C5a evoked a transient increase in the intracellular calcium level as measured by flow cytometry (FACS).

To analyse the function of the nC5aR, an antisense peptide fragment of the C5aR was used. Previous data showed that a C5aR fragment (a peptide termed PR226) has C5aR agonist and antagonist effects in U‐937 cells depending on the concentration of the peptide. We found that a multiple antigenic peptide (MAP) form of the same peptide (termed PR226‐MAP) induced rapid elevation of nuclear c‐fos immunoreactivity and resulted in DNA fragmentation, a characteristic sign of apoptosis, in TGW cells.

Early electrophysiological events characteristic of apoptosis were also detected: intermittent calcium current pulses were recorded within 1–2 min of peptide administration. C5a pretreatment delayed the onset of this calcium influx.

We also demonstrated that the apoptotic pathway is linked to nC5aR via pertussis toxin‐sensitive G‐proteins.

Although the function of C5a and its receptor on neurons is unknown, these results suggest that an abnormal activation of this signal transduction pathway can result in apoptosis and, subsequently, in neurodegeneration.
Protein kinases modulate two glycine currents in salamander retinal ganglion cells
Authors: Han Yi , Slaughter Malcolm M.
Published: September 2004

Protein kinase modulation of glycine‐activated currents was examined in acutely dissociated ganglion cells from tiger salamander retina using whole‐cell voltage‐clamp techniques.

Glycine (100 μM) induced an outward chloride current in cells clamped at 0 mV. Co‐application of 50 μM forskolin made the glycine‐induced current more transient. The combination of forskolin and glycine reduced the later portion of current response without changing the initial peak amplitude.

3‐Isobutyl‐1‐methylxanthine (IBMX) or 8‐bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP) produced effects similar to those of forskolin. H‐89, a protein kinase A (PKA) inhibitor, blocked the effect of forskolin.

A protein kinase C (PKC) activator, OAG (1‐oleoyl‐2‐acetyl‐sn‐glycerol), also made the glycine response more transient. Unlike PKA analogues, OAG enhanced the glycine peak response without changing the glycine late response. OAG effects were blocked by 1 μM GF‐109203X, a PKC inhibitor.

Nanomolar concentrations of strychnine selectively blocked the fast phase of the glycine current and reversed the effect of OAG, but not that of forskolin. Conversely, forskolin occluded the effect of 5,7‐dichlorokynurenic acid, which selectively suppresses the late phase of the glycine current. The action of OAG was not blocked by 5,7‐dichlorokynurenic acid.

Thus, through a differential modulation, both protein kinase A and C shorten the decay time of the glycine current. PKA suppresses the slow component, while PKC potentiates the fast component.
Spontaneous activity regulates calcium‐dependent K+ current expression in developing ascidian muscle
Authors: Dallman Julia E. , Davis Anna K. , Moody William J.
Published: September 2004

In embryonic ascidian muscle, outward K+ currents develop in two stages: the initial expression of a slowly activating, voltage‐gated K+ current (IKv) near the time of neurulation is followed about 6 h later by a rapidly activating calcium‐activated K+ current (IK(Ca)). During this 6 h interval, inward Ca2+ currents (ICa) appear and the inward rectifier (IK(IR)), the sole resting conductance, is transiently downregulated. These events predict a period of spontaneous activity. The following experiments were designed to test this prediction and to examine the relevance of spontaneous activity for muscle cell development.

By recording activity in cell‐attached patches, we have found that muscle cells generate spontaneous action potentials during this 6 h window of time when IK(IR) is downregulated and outward K+ currents are slow. Action potentials occur at a mean frequency of 13.9 min−1.

When activity is blocked by the transient application of the Ca2+ channel blocker Cd2+, IK(Ca) fails to develop. This disruption is specific for IK(Ca): IK(IR) and ICa develop normally in activity‐blocked cells. Application of Cd2+ either before or after the window of activity has no effect.

The reappearance of IK(IR) and the development of IK(Ca) and the mature form of ICa are all prevented by transcription blockers, with a sensitive period corresponding to the period of activity.

These data show that, although the expression of three channel types depends on transcription during the period of spontaneous activity, only the development of IK(Ca) depends on activity.
Presynaptic 5‐HT3 receptors evoke an excitatory response in dorsal vagal preganglionic neurones in anaesthetized rats
Authors: Wang Yun , Ramage Andrew G. , Jordan David
Published: September 2004

Recordings were made from a total of sixty‐four vagal preganglionic neurones in the dorsal vagal motor nucleus (DVMN) of pentobarbitone sodium anaesthetized rats. The effects of ionophoretic administration of Mg2+ and Cd2+, inhibitors of neurotransmitter release, and the selective NMDA and non‐NMDA receptor antagonists (±)‐2‐amino‐5‐phosphono‐pentanoic acid (AP5) and 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX) on the excitatory actions of the 5‐HT3 receptor agonist 1‐phenylbiguanide (PBG) were studied.

In extracellular recording experiments, PBG (0‐40 nA) increased the firing rate of thirty‐five of the thirty‐nine neurones tested. The PBG‐evoked excitation was attenuated by application of Mg2+ (1‐10 nA) in sixteen of seventeen neurones or Cd2+ (2‐10 nA) in seven of eight neurones tested. At these low ejection currents neither Mg2+ nor Cd2+ altered baseline firing rates and Mg2+ had no effect on the excitations evoked by DL‐homocysteic acid (n= 4), NMDA (n= 4) or (AMPA; n= 2).

Ionophoresis of AP5 (2‐10 nA), at currents which selectively inhibited NMDA‐evoked excitations, attenuated PBG‐evoked excitations in all eight neurones tested. DNQX (5‐20 nA), at currents which selectively inhibited AMPA‐evoked excitations, also attenuated PBG‐evoked excitations (n= 3).

Intracellular activity was recorded in nine DVMN neurones. In six neurones ionophoretic application of PBG (10‐200 nA) depolarized the membrane and increased firing rate whilst in the other three neurones, PBG had no effect on membrane potential though it increased synaptic noise (n= 3) and firing rate (n= 2). In all six neurones tested, ionophoresis of Mg2+ (10‐120 nA) attenuated the PBG‐evoked increases in synaptic noise and firing rate.

In conclusion, the data are consistent with the hypothesis that 5‐HT3 receptor agonists activate DVMN neurones partly by acting on receptors located at sites presynaptic to the neurones. Activation of these receptors appears to facilitate release of glutamate, which, in turn, acts on postsynaptic NMDA and non‐NMDA receptors to activate the neurones.
Human sodium channel gating defects caused by missense mutations in S6 segments associated with myotonia: S804F and V1293I
Authors: Green Donnella S. , George Alfred L. , Cannon Stephen C.
Published: September 2004

Missense mutations in the α‐subunit of the human skeletal muscle sodium channel (hSkM1) have been detected in some heritable forms of myotonia. By recording Na+ currents from cells transfected with cDNA encoding either wild‐type or mutant hSkM1, we characterized the functional consequences of two myotonia‐associated mutations that lie at the cytoplasmic end of the sixth transmembrane segment in domain II (S804F) or domain III (V1293I).

Both mutations caused modest, but unequivocal, alterations in the voltage‐dependent gating behaviour of hSkM1. For S804F, the abnormalities were limited to fast inactivation: the persistent Na+ current at the end of a 50 ms depolarization was increased 3‐fold, the rate of inactivation from the open state was slowed 2‐fold, and the voltage dependence of fast inactivation (h∞) was shifted by +3 mV. V1293I also disrupted fast inactivation, as evidenced by a 3‐fold faster rate of recovery at hyperpolarized potentials (<= ‐70 mV). Activation was altered as well for V1293I: the voltage dependence was shifted by ‐6 mV (hyperpolarized).

Slow inactivation was not altered by S804F or V1293I.

We conclude that S804F and V1293I are not benign polymorphisms. Either mutation causes detectable alterations in channel gating and, in model simulations, the magnitude of the defects is sufficient to produce runs of myotonic discharges.
Calcitonin gene‐related peptide acts presynaptically to increase quantal size and output at frog neuromuscular junctions
Authors: Van der Kloot William , Benjamin William B. , Balezina Olga P.
Published: September 2004

Calcitonin gene‐related peptide (CGRP) is found in dense‐cored vesicles in the motor nerve terminal.

Exogenous CGRP increased the size of the quanta. The increase in size reached a maximum after about 40 min. The lowest effective concentration of human CGRP (hCGRP) was 0.8 nM. The action of hCGRP was antagonized by (−)‐vesamicol, a drug that blocks active acetylcholine (ACh) uptake into synaptic vesicles, so it appears that hCGRP increases size by adding more ACh to the quanta. The action of hCGRP was antagonized by drugs that block the activation of protein kinase A (PKA). (In other preparations CGRP also activates PKA.)

The hCGRP effect was not blocked by fragment 8–37, an antagonist of one class of CGRP receptor.

hCGRP increases evoked quantal output and miniature endplate potential (MEPP) frequency, again by activating PKA.

CGRP release was measured by radioimmunoassay. Release was increased by depolarization with elevated K+, but the amounts released appear to be below those needed to affect quantal size or output. Moreover, although elevated K+ can increase quantal size it acts by a pathway that does not involve PKA. We suggest that the most likely target of endogenously released CGRP is the regulation of circulation of the muscle.
NH4+ as a substrate for apical and basolateral Na+‐H+ exchangers of thick ascending limbs of rat kidney: evidence from isolated membranes
Authors: Blanchard Anne , Eladari Dominique , Leviel Françoise , Tsimaratos Michel , Paillard Michel , Podevin René‐Alexandre
Published: September 2004

We have used highly purified right‐side‐out luminal and basolateral membrane vesicles (LMVs and BLMVs) isolated from rat medullary thick ascending limb (MTAL) to study directly the possible roles of the LMV and BLMV Na+‐H+ exchangers in the transport of NH4+.

Extravesicular NH4+ ((NH4+)o) inhibited outward H+ gradient‐stimulated 22Na+ uptake in both types of vesicles. This inhibition could not be accounted for by alteration of intravesicular pH (pHi).

Conversely, in both plasma membrane preparations, the imposition of outward NH4+ gradients stimulated 22Na+ uptake at the acidic pHi (6.60) of MTAL cells, under conditions in which possible alterations in pHi were prevented. All NH4+ gradient‐stimulated Na+ uptake was sensitive to 0.5 mM 5‐(N,N‐dimethyl)‐amiloride.

The BLMV and LMV Na+‐H+ exchangers had a similar apparent affinity for internal H+ (H+i), with pK (‐log of dissociation constant) values of 6.58 and 6.52, respectively.

These findings indicate that NH4+ interacts with the external and internal transport sites of the LMV and BLMV Na+‐H+ antiporters, and that both of these exchangers can mediate the exchange of internal NH4+ ((NH4+)i) for external Na+ (Na+o) at the prevailing pHi of MTAL cells.

We conclude that operation of the BLMV Na+‐H+ exchanger on the NH4+‐Na+ mode may represent an important pathway for mediating the final step of NH4+ absorption, whereas transport of NH4+ on the apical antiporter may provide negative feedback regulation of NH4+ absorption.
ATP and glutamate are released from separate neurones in the rat medial habenula nucleus: frequency dependence and adenosine‐mediated inhibition of release
Authors: Robertson Susan J. , Edwards Frances A.
Published: September 2004

ATP and glutamatergic synaptic currents were compared in slices of rat medial habenula nucleus using whole‐cell patch‐clamp techniques.

In most cells low voltage stimulation resulted in glutamatergic responses and not purinergic responses. In five cells where ATP currents could be stimulated with low voltages, wash out of glutamate antagonists did not reveal evoked glutamate currents. Spontaneous glutamate currents confirmed washout of antagonist.

Modulation of release probability of glutamate and ATP, assessed by changes in failure rate of synaptic currents, was compared under conditions of different stimulation frequencies and in the presence of adenosine agonists and antagonists.

ATP release, but not glutamate release, was shown to be modulated by increased stimulation frequency which resulted in inhibition of ATP release via A2‐like adenosine receptors. A1 receptors caused inhibition of both ATP and glutamate release.

Endogenous adenosine inhibited glutamate release via A1 receptors but only inhibited ATP release via A2‐like receptors.

Attempts to inhibit the degradation of ATP to adenosine did not alter the frequency dependence of the failure rate.

We conclude, from the direct demonstration and from the differences in pharmacology and frequency dependence of the modulation of release, that ATP and glutamate responses are due to release from separate neurones.
Insulin occludes leptin activation of ATP‐sensitive K+ channels in rat CRI‐G1 insulin secreting cells
Authors: Harvey J. , Ashford M. L. J.
Published: September 2004

Using whole‐cell and cell‐attached recording configurations, the effects of insulin on leptin activation of ATP‐sensitive K+ (KATP) channels were examined in the CRI‐G1 insulinoma cell line.

Whole‐cell recordings demonstrated that the leptin‐induced hyperpolarization and increased potassium conductance are completely occluded by prior exposure to insulin (1‐50 nM). In cell‐attached recordings, insulin prevented leptin activation of tolbutamide‐sensitive KATP channels. Furthermore, insulin (50 nM) slowly and completely reversed the effects of leptin (10 nM), an action not attributable to direct inhibition of KATP channels per se.

Low concentrations of insulin‐like growth factor‐1 (IGF‐1; 10–100 nM) failed to prevent leptin activation of KATP channels, although higher concentrations (1 μm) did inhibit leptin actions.

The action of insulin was specific for leptin, as the hyperglycaemic agent diazoxide activated KATP channels following prior exposure to insulin.

Wortmannin (1‐10 nM) and LY 294002 (10 μm) prevented leptin activation of KATP channels, indicating an involvement of phosphoinositide 3‐kinase (PI 3‐kinase).

In conclusion, leptin activation of KATP channels is counter‐regulated by insulin in the CRI‐G1 insulinoma cell line. This feedback mechanism may be important in the local integration of hormonal signals which regulate insulin secretion and in alterations of metabolic homeostasis associated with obesity and non‐insulin dependent diabetes mellitus (NIDDM).
Substrate upregulation of the human small intestinal peptide transporter, hPepT1
Authors: Walker Dianne , Thwaites David T. , Simmons Nicholas L. , Gilbert Harry J. , Hirst Barry H.
Published: September 2004

Molecular mechanisms underlying physiological adaptation to increased levels of dietary peptides have been elucidated by studying the response to the substrate glycyl‐L‐glutamine (Gly‐Gln) of the proton‐coupled peptide transporter, hPepT1, in the Caco‐2 human intestinal cell line. Vmax for apical uptake of [14C]glycyl‐[14C]sarcosine was increased 1.64 (± 0.34)‐fold after incubation of Caco‐2 cells for 3 days in a peptide‐rich medium (4 mM Gly‐Gln replacing 4 mM Gln).

A full‐length Caco‐2 hPepT1 cDNA clone was identical to human small intestinal hPepT1 with the exception of a single amino acid substitution Ile‐662 to Val. Transcript sizes, on Northern blots of Caco‐2 poly(A)+ RNA probed with a 630 bp 5′ hPepT1 cDNA probe, correspond to the reported band pattern seen with human small intestinal RNA. The dipeptide‐induced increase in substrate transport was accompanied by a parallel increase of 1.92 (± 0.30)‐fold (n= 9) in hPepT1 mRNA. This was in part due to an increase in hPepT1 mRNA half‐life from 8.9 ± 1.1 to 12.5 ± 1.6 h (n= 3), but the increase in half‐life does not account fully for the observed increase in mRNA levels, suggesting that there was also a dipeptide‐mediated increase in hPepT1 transcription.

Anti‐hPepT1‐specific antipeptide antibodies localized hPepT1 exclusively to the apical membrane of human small intestinal enterocytes and Caco‐2 cells. Gly‐Gln supplementation of media resulted in a 1.72 (± 0.26)‐fold (n= 5) increase in staining intensity of Caco‐2 cells.

We conclude that Caco‐2 cells provide an appropriate model for the study of adaptation of intestinal hPepT1, at the molecular level, to increased levels of dietary peptide. The magnitude of functional increase in apical peptide transport activity in response to Gly‐Gln can be fully accounted for by the increased levels of hPepT1 protein and mRNA, the latter mediated by both enhanced hPepT1 mRNA stability and increased transcription. The signalling pathway between increased dietary peptide and hPepT1 upregulation, therefore, involves direct action on the enterocyte, independent of hormonal and/or neural control.
Extracellular ATP directly gates a cation‐selective channel in rabbit airway ciliated epithelial cells
Authors: Korngreen Alon , Ma Weiyuan , Priel Zvi , Silberberg Shai D.
Published: September 2004

A membrane conductance activated by extracellular ATP was identified and characterized in freshly dissociated rabbit airway ciliated cells using the whole‐cell and outside‐out patch configurations of the patch‐clamp technique.

In solutions designed to maximize currents through voltage‐gated calcium channels, there were no indications of voltage‐gated Ba2+ currents.

Extracellular ATP (but not UTP or ADP) activated a membrane conductance which remained activated for several minutes in the presence of ATP. The conductance was permeable to monovalent and divalent cations with approximate relative permeabilities (P) for PBa:PCs:PTEA of 4:1:0.1. Permeability to Cl− was negligible.

Including GDP‐β‐S in the intracellular solution did not inhibit the effects of ATP, nor did GTP‐γ‐S irreversibly activate the conductance.

In outside‐out membrane patches, with GDP‐β‐S in the pipette solution, ATP activated ion channels which had a chord conductance of approximately 6 pS in symmetrical 150 mM CsCl solutions at ‐120 mV.

Suramin (100 μM) inhibited the whole‐cell currents activated by ATP (200 μM) by 93 ± 3 %. Similar effects of suramin were observed on ATP‐activated channels in outside‐out membrane patches.

Extracellular ATP had a priming action on the response to subsequent exposure to ATP. At ‐40 mV, the time to half‐maximal current activation (t½) was 46 ± 9 s during the first exposure to 200 μM ATP and decreased to 5 ± 3 s during a second exposure to the same concentration of ATP. The priming action of ATP was not inhibited by including GDP‐β‐S in the intracellular solution.

The initial rate of activation increased with the concentration of ATP, and was voltage sensitive. During the first exposure to 200 μM ATP, t½ at +40 mV was 4‐fold longer than t½ at ‐40 mV.

Half‐maximal activation of the conductance shifted from 210 ± 30 to 14 ± 4 μM added ATP when CaCl2 in the extracellular solution was reduced from 1.58 to 0.01 mM. The Hill coefficient for ATP was 1.2 in both solutions.

These observations suggest that a form of ATP uncomplexed with divalent cations directly gates an ion channel (P2X receptor) in rabbit airway ciliated cells, which serves as a pathway for Ca2+ influx. This purinoceptor may contribute to sustained ciliary activation during prolonged exposures to ATP.
Ca2+ images and K+ current during depolarization in smooth muscle cells of the guinea‐pig vas deferens and urinary bladder
Authors: Imaizumi Yuji , Torii Yuichi , Ohi Yoshiaki , Nagano Norihiro , Atsuki Kaoru , Yamamura Hisao , Muraki Katsuhiko , Watanabe Minoru , Bolton Thomas B.
Published: September 2004

Electrical events and intracellular calcium concentration ([Ca2+]) imaged using fluo‐3 and laser scanning confocal microscopy were simultaneously monitored in single smooth muscle cells freshly isolated from guinea‐pig vas deferens or urinary bladder.

Images obtained every 8 ms, during stepping from ‐60 to 0 or +10 mV for 50 ms under voltage clamp, showed that a rise in [Ca2+] could be detected within 20 ms of depolarization in five to twenty small (< 2 μm diameter) ‘hot spots’, over 95 % of which were located within 1.5 μm of the cell membrane. Depolarization at 30 s intervals activated hot spots at the same places.

Cd2+ or verapamil abolished both hot spots and Ca2+‐activated K+ current (IK,Ca). Caffeine almost abolished hot spots and markedly reduced IK,Ca. Cyclopiazonic acid, which raised basal global [Ca2+], decreased the rise in hot spot [Ca2+] and IK,Ca amplitude during depolarization. These results suggest that Ca2+ entry caused Ca2+‐induced Ca2+ release (CICR).

Under voltage clamp, hot spot [Ca2+] closely paralleled the rise in IK,Ca and reached a peak within 20 ms of the start of depolarization, but the rise in global [Ca2+] over the whole cell area was much slower. Step depolarization to potentials positive to ‐20 mV caused hot spots to grow in size and coalesce, leading to a rise in global [Ca2+] and contraction. Ca2+ hot spots also occurred during the up‐stroke of an evoked action potential under current clamp.

It is concluded that the entry of Ca2+ in the early stages of an action potential evokes CICR from discrete subplasmalemma Ca2+ storage sites and generates hot spots that spread to initiate a contraction. The activation of Ca2+‐dependent K+ channels in the plasmalemma over hot spots initiates IK,Ca and action potential repolarization.
The influence of caffeine on intramembrane charge movements in intact frog striated muscle
Authors: L.‐H. Huang Christopher
Published: September 2004

The influence of caffeine, applied over a 25‐fold range of concentrations, on intramembrane charge movements was examined in intact voltage‐clamped amphibian muscle fibres studied in the hypertonic gluconate‐containing solutions that were hitherto reported to emphasize the features of qγ at the expense of those of qβ charge.

The total charge, Qmax, the transition voltage, V*, and the steepness factor, k, of the steady‐state charge‐voltage relationships, Q(V), were all conserved to values expected with significant contributions from the steeply voltage‐dependent qγ species (Qmax≈ 20 nC μF−1, V*≈−50 mV, k≈ 8 mV) through all the applications of caffeine concentrations between 0.2 and 5.0 mm. This differs from recent reports from studies in cut as opposed to intact fibres.

The delayed transients that have been attributed to transitions within the qγ charge persisted at low (0.2 mm) and intermediate (1.0 mm) caffeine concentrations.

In contrast, the time courses of such qγ currents became more rapid and their waveforms consequently merged with the earlier qβ decays at higher (5.0 mm) reagent concentrations. The charging records became single monotonic decays from which individual contributions could not be distinguished. This suggests that caffeine modified the kinetic properties of the qγ system but preserved its steady‐state properties. These findings thus differ from earlier reports that high caffeine concentrations enhanced the prominence of delayed transient components in cut fibres.

Caffeine (5.0 mm) and ryanodine (0.1 mm) exerted antagonistic actions upon qγ charge movements. The addition of caffeine restored the delayed time courses that were lost in ryanodine‐containing solutions, reversed the shift these produced in the steady‐state charge‐voltage relationship but preserved both the maximum charge, Qmax, and the steepness, k, of the steady‐state Q(V) relationships.

Caffeine also antagonized the actions of tetracaine on the total available qγ charge, but did so only at the low and not at the high applied concentrations. Thus, 0.2 mm caffeine restored the steady‐state qγ charge, the steepness of the overall Q(V) function and the appearance of delayed qγ charge movements that had been previously abolished by the addition of 2.0 mm tetracaine.

In contrast, the higher applied (1.0 and 5.0 mm) caffeine concentrations paradoxically did not modify these actions of tetracaine. The total charge and voltage dependence of the Q(V) curves, and the amplitude and time course of charge movements remained at the reduced values expected for the tetracaine‐resistant qβ charge.

These results permit a scheme in which caffeine acts directly upon ryanodine receptor (RyR)‐Ca2+ release channels whose consequent activation then dissociates them from the tubular dihydropyridine receptor (DHPR) voltage sensors that produce qγ charge movement, with which they normally are coupled in reciprocal allosteric contact.
Variability in spontaneous subcellular calcium release in guinea‐pig ileum smooth muscle cells
Authors: Gordienko D. V. , Bolton T. B. , Cannell M. B.
Published: September 2004

Spontaneous, localized transient increases in [Ca2+]i (‘Ca2+ sparks’) were observed in about 40 % of fluo‐3‐loaded myocytes examined using laser scanning confocal microscopy. Ca2+ sparks persisted after application of Cd2+ (200 μM), but were abolished by ryanodine (30 μM) or thapsigargin (0.1 μM), suggesting that they arise from the spontaneous activation of ryanodine receptors (RyR) in the sarcoplasmic reticulum (SR).

Ca2+ sparks occurred much more frequently at certain sites (or ‘frequent discharge sites’, FDSs) within any confocal plane of the cell and line‐scan imaging revealed a wide variation in their spatial size, amplitude and time course. Some spontaneous local transients were very similar to ‘Ca2+ sparks’ observed in heart, i.e. lasting ≈200 ms with a peak fluorescence ratio of 1.75 ± 0.23 (mean ± s.d., n= 33). Other events were faster and smaller, lasting only ≈40 ms with a peak normalized fluorescence of 1.36 ± 0.09 (mean ± s.d., n= 28).

Spontaneous Ca2+ waves with a wide range of propagation velocities (between 30 and 260 μm s−1) were also observed. In about 60 % of records (n= 33), Ca2+ sparks could be detected at the sites of wave initiation. Waves of elevated [Ca2+]i propagated with non‐constant velocity and in some cases terminated. These observations could be explained by heterogeneity in the distribution of subcellular release sites as well as variability in the contribution of each release site to the wave.

Spontaneous [Ca2+]i transients in single dispersed visceral smooth muscle cells have a wide spectrum of behaviour that is likely to be the result of spatio‐temporal recruitment of smaller local events, probably via a calcium‐induced calcium release (CICR) mechanism. The spatial non‐uniformity of SR and RyR distribution within the cell may account for the existence of ‘frequent discharge sites’ firing the majority of the smooth muscle Ca2+ sparks and the wide variation in the Ca2+ wave propagation velocities observed.
Ca2+ sparks activate K+ and Cl− channels, resulting in spontaneous transient currents in guinea‐pig tracheal myocytes
Authors: ZhuGe Ronghua , Sims Stephen M. , Tuft Richard A. , Fogarty Kevin E. , Walsh John V.
Published: September 2004

Local changes in cytosolic [Ca2+] were imaged with a wide‐field, high‐speed, digital imaging system while membrane currents were simultaneously recorded using whole‐cell, perforated patch recording in freshly dissociated guinea‐pig tracheal myocytes.

Depending on membrane potential, Ca2+ sparks triggered ‘spontaneous’ transient inward currents (STICs), ‘spontaneous’ transient outward currents (STOCs) and biphasic currents in which the outward phase always preceded the inward (STOICs). The outward currents resulted from the opening of large‐conductance Ca2+‐activated K+ (BK) channels and the inward currents from Ca2+‐activated Cl− (ClCa) channels.

A single Ca2+ spark elicited both phases of a STOIC, and sparks originating from the same site triggered STOCs, STICs and STOICs, depending on membrane potential.

STOCs had a shorter time to peak (TTP) than Ca2+ sparks and a much shorter half‐time of decay. In contrast, STICs had a somewhat longer TTP than sparks but the same half‐time of decay. Thus, the STIC, not the STOC, more closely reflected the time course of cytosolic Ca2+ elevation during a Ca2+ spark.

These findings suggest that ClCa channels and BK channels may be organized spatially in quite different ways in relation to points of Ca2+ release from intracellular Ca2+ stores. The results also suggest that Ca2+ sparks may have functions in smooth muscle not previously suggested, such as a stabilizing effect on membrane potential and hence on the contractile state of the cell, or as activators of voltage‐gated Ca2+ channels due to depolarization mediated by STICs.