bluuebunny/crossref_metadata_2025 huggingface dataset:
Pituitary adenylate cyclase‐activating polypeptide (PACAP) is an islet substance serving as an intra‐islet amplifier Of glucose‐induced insulin secretion in rats
Authors: Yada Toshihiko , Sakurada Masaya , Ishihara Hisamitsu , Nakata Masanori , Shioda Seiji , Yaekura Kazuro , Hamakawa Noriaki , Yanagida Kazuhiro , Kikuchi Masatoshi , Oka Yoshitomo
Published: September 2004

We examined whether pituitary adenylate cyclase‐activating polypeptide with 38 or 27 residues (PACAP‐38 or PACAP‐27) serves as an intra‐islet regulator of glucose‐induced insulin secretion in rats. PACAP antiserum specific for PACAP‐38 and PACAP‐27 was used to neutralize the effect of endogenous PACAP in islets. PACAP release from islets was bioassayed using the response of cytosolic Ca2+ concentration ([Ca2+]i) in single β‐cells, monitored by dual‐wavelength fura‐2 microfluorometry. Expression of PACAP mRNA was studied by reverse transcription‐polymerase chain reaction (RT‐PCR), while expression of PACAP was studied by metabolic labelling and immunoblotting. Localization of PACAP receptors was studied immunohistochemically.

High glucose‐stimulated insulin release from isolated islets was attenuated by PACAP anti‐serum but not by non‐immune sera.

The islet incubation medium with high glucose (Med) possessed a capacity, which was neutralized by PACAP antiserum, to increase [Ca2+]i in β‐cells. PACAP antiserum also neutralized the [Ca2+]iincreasing action of synthetic PACAP‐38 and PACAP‐27, but not that of vasoactive intestinal polypeptide (VIP) and glucagon.

Both Med and synthetic PACAP increased [Ca2+]i in β‐cells only in the presence of stimulatory, but not basal, glucose concentrations. In contrast, ATP, a substance that is known to be released from β‐cells, increased [Ca2+]i in β‐cells at both basal and stimulatory glucose concentrations.

Expression of PACAP mRNA and biosynthesis of PACAP‐38 were detected in islets and a β‐cell line, MIN6.

Immunoreactivity for PACAP‐selective type‐I receptor was observed in islets.

[Ca2+]i measurements combined with immunocytochemistry with insulin antiserum revealed a substantial population of glucose‐unresponsive β‐cells, many of which were recruited by PACAP‐38 into [Ca2+]i responses.

These results indicate that PACAP‐38 is a novel islet substance that is synthesized and released by islet cells and then, in an autocrine and/or paracrine manner, potentiates and arouses β‐cells responses to glucose, thereby amplifying glucose‐induced insulin secretion in islets.
Receptors couple to L‐type calcium channels via distinct Go proteins in rat neuroendocrine cell lines
Authors: Degtiar Vadim E. , Harhammer Rainer , Nürnberg Bernd
Published: September 2004

The present study examines the hypothesis of G protein subtype selectivity in receptor‐induced inhibition of calcium channel currents (ICa) in the insulin‐secreting RINm5F and pituitary GH3 rat cell lines. Specificity of receptor coupling to G proteins was studied by infusion of purified Ga isoforms into cells via a patch pipette.

In RINm5F cells, the neuropeptide galanin inhibited dihydropyridine (DHP)‐ and ω‐conotoxin‐sensitive components of ICa and slowed down their activation kinetics. In GH3 cells, DHP‐sensitive ICa was inhibited by galanin, as well as by somatostatin and carbachol. Agonist‐induced ICa inhibition was suppressed by pertussis toxin (PTX) pretreatment of the cells. In PTX‐pretreated cells of either cell line, the response to galanin was restored only by the Gα01 subunit. Following PTX treatment of GH3 cells, only the Gα01 subunit restored carbachol‐induced inhibition of ICa, whereas only the Gαo2 subunit restored somatostatin‐induced inhibition of ICa. Gl subtypes had no effect on ICa inhibition.

Both cell lines expressed two distinct immunoreactive Go proteins. Whereas in RINm5F cell membranes G01 was found to be the predominant isoform, we detected more G02 than G01 in GH3 cell membranes. Nevertheless, all agonists stimulated incorporation of the photoreactive GTP analogue [α‐ 32P]GTP azidoanilide into both Go isoforms.

The results indicate that the same Go subtype, i.e. G01, mediates galanin‐induced inhibition of ICa in both cell lines and that the Go subtype specificity of receptor‐G protein coupling is confined to intact cells.
Diadenosine polyphosphates evoke Ca2+ transients in guinea‐pig brain via receptors distinct from those for ATP
Authors: Pintor Jesús , Puche José A. , Gualix Javier , Hoyle Charles H. V. , Miras‐Portugal Maria Teresa
Published: September 2004

The ability of diadenosine polyphosphates, namely P1,P2‐di(adenosine) pyrophosphate (Ap2A), P1,P3‐di(adenosine) triphosphate (Ap3A), P1,P4‐di(adenosine) tetraphosphate (Ap4A), P1,P5‐di(adenosine) pentaphosphate (Ap5A) and P1,P6‐di(adenosine) hexaphosphate (Ap6A) to evoke Ca2+ signals in synaptosomes prepared from three different regions of the guinea‐pig brain was examined.

In synaptosomal preparations from the paleocortex (cortex), diencephalon/brainstem (midbrain) and cerebellum all the dinucleotides evoked Ca2+ signals that were concentration dependent over the range 1‐300 μm. ATP and its synthetic analogues,α,β‐methylene ATP, 2‐methylthio ATP and adenosine 5‘‐O‐(2‐thio)diphosphate (all 100 μm) also evoked Ca2+ signals in these preparations.

In the midbrain and cerebellum preparations, responses to ATP and its analogues were attenuated or abolished by the P2 receptor antagonist suramin (100μm) but responses to the dinucleotides were not. Also, desensitization by a dinucleotide blocked responses to dinucleotides but not mononucleotides, and desensitization by a mononucleotide blocked responses to mononucleotides but not dinucleotides.

In cortical preparations, suramin (100μm) blocked responses to both classes of nucleotides. Furthermore, there was mutual cross‐desensitization between the mono‐ and dinucleotides.

The adenosine A1 receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine, did not affect responses evoked by the dinucleotides, nor did the pyrimidine UTP.

It is concluded that there are specific dinucleotide receptors, activated by diadenosine polyphosphates, but not ATP or UTP, on synaptic terminals in guinea‐pig diencephalon/brainstem and cerebellum. These receptors bear a similarity to the dinucleotide receptor (P4 receptor) in rat brain. In guinea‐pig cerebral cortex synaptosomes, diadenosine polyphosphates appear to act via the same receptor as ATP.
The role of the sarcolemmal Ca2+ ‐ATPase in the pH transients associated with contraction in rat smooth muscle
Authors: Naderali E. K. , Buttell N. , Taggart M. J. , Bullock A. J. , Eisner D. A. , Wray Susan
Published: September 2004

We have investigated the origin of the intracellular acid pH transients that accompany myometrial contraction. Intra‐and extracellular pH were measured with SNARF and intra‐cellular Ca2+ concentration ([Ca2+]i) with indo‐1.

An intracellular acidification accompanied spontaneous contractions and those elicited by KC1 depolarization or the addition of the agonists carbachol or prostaglandin F2x. The size of the acidification increased with the magnitude of the contraction.

The intracellular acidification was accompanied by an extracellular alkalinization, showing that it results from proton movement across the surface membrane. Furthermore, it was decreased either by addition of Cd2+ (20 nm, an inhibitor of the sarcolemmal Ca2+‐ATPase) or by elevating [Ca2+]o.

Extracellular alkalinization increased the magnitude of the rise of [Ca2+]j and force produced by KC1.

An intracellular acidification was also associated with contraction in the portal vein and ureter.

We conclude that the sarcolemmal Ca2+‐ATPase produces a significant intracellular acidification while removing Ca2+. Both the acidification and decrease of [Ca2+]i will promote relaxation. Since Ca2+ and protons have opposite effects on many cellular processes, this dual regulation by these two ions may be of general importance.
Mechanism of Glibenclamide Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator Cl− Channels Expressed in a Murine Cell Line
Authors: Sheppard David N. , Robinson Katherine A.
Published: September 2004

The sulphonylurea drug glibenclamide is a widely used inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR). To investigate how glibenclamide inhibits CFTR, we studied CFTR Cl− channels using excised inside‐out membrane patches from cells expressing wild‐type human CFTR.

Addition of glibenclamide (10–100 μM) to the intracellular solution caused a concentration‐dependent decrease in the open time of CFTR Cl− channels, but closed times did not change. This suggests that glibenclamide is an open‐channel blocker of CFTR.

Glibenclamide is a weak organic acid. Acidification of the intracellular solution relieved glibenclamide inhibition of CFTR, suggesting that the anionic form of glibenclamide inhibits CFTR.

To begin to identify the glibenclamide binding site in CFTR, we investigated whether glibenclamide competes with either MgATP or Cl− ions for a common binding site. Glibenclamide inhibition of CFTR was unaffected by nucleotide‐dependent stimulation of CFTR, suggesting that glibenclamide and intracellular MgATP interact with CFTR at distinct sites.

Glibenclamide inhibition of CFTR was voltage dependent and enhanced when the external Cl− concentration was decreased. The data suggest that glibenclamide and Cl− ions may compete for a common binding site located within a large intracellular vestibule that is part of the CFTR pore.
Variability in frequency and characteristics of Ca2+ sparks at different release sites in rat ventricular myocytes
Authors: Parker Ian , Gil Wier Withrow
Published: September 2004

High spatial resolution confocal imaging was used to investigate spontaneous calcium release events (Ca2+ sparks) in isolated rat cardiac myocytes loaded with the fluorescent calcium indicator fluo‐3.

Frequencies of sparks at different release sites varied widely, with a few sites showing sustained activities as great as 50 times the average. Sites with frequent sparks showed more rapid recovery of activity following Ca2+ waves and locally elevated basal [Ca2+].

In addition to transient sparks, some sites showed bursts of rapid flurries of spark‐like events, or apparently sustained Ca2+ liberation. Bursts remained localized to individual Z‐lines, but adjacent sites on the same Z‐line could be ‘driven’ by a bursting site to generate similar activity.

Individual sites showed long‐term (tens of seconds) changes in ‘modes’ of activity, with abrupt transitions in frequencies of sparking, and between transient sparks and sustained bursts. These transitions were not associated with changes in the amplitude of the sparks.

We conclude that spontaneous sparks are not stereotyped events generated with uniform probability at all sites. Instead, the Ca2+ release event is variable, and some sites have a high probability of spark generation. Both factors show long‐term changes at individual sites, raising the possibility that properties of fundamental Ca2+ release units may be subject to modulation.
Muscarinic Ca2+ responses resistant to muscarinic antagonists at perisynaptic schwann cells of the frog neuromuscular junction
Authors: Robitaille Richard , Jahromi Babak S. , Charlton Milton P.
Published: September 2004

Acetylcholine causes a rise of intracellular Ca2+ in perisynaptic Schwann cells (PSCs) of the frog neuromuscular junction. The signalling pathway was characterized using the fluorescent Ca2+ indicator fluo‐3 and fluorescence microscopy.

Nicotinic antagonists had no effect on Ca2+responses evoked by ACh and no Ca2+ responses were evoked with the nicotinic agonist nicotine. The muscarinic agonists muscarine and oxotremorine‐M induced Ca2+ signals in PSCs.

Ca2+ responses remained unchanged when extracellular Ca2+ was removed, indicating that they are due to the release of Ca2+ from internal stores. Incubation with pertussis toxin did not alter the Ca2+ signals induced by muscarine, but did block depression of transmitter release induced by adenosine and prevented Ca2+ responses in PSCs induced by adenosine.

The general muscarinic antagonists atropine, quinuclidinyl benzilate and N‐methyl‐scopolamine failed to block Ca2+ responses to muscarinic agonists. Atropine (at 20000‐fold excess concentration) also failed to reduce the proportion of cells responding to a threshold muscarine concentration sufficient to cause responses in less than 50% of cells. Only the allosteric, non‐specific blocker, gallamine (1–10 μm) was effective in blocking muscarine‐induced Ca2+ responses.

In preparations denervated 7 days prior to experiments, low concentrations of atropine reversibly and completely blocked Ca2+ responses to muscarine.

The lack of blockade by general muscarinic antagonists in innervated, in situ preparations suggests that muscarinic Ca2+ responses at PSCs are not mediated by any of the five known muscarinic receptors or that post‐translational modification prevented antagonist binding.
Role in fast inactivation of the IV/S4–S5 loop of the human muscle Na+ channel probed by cysteine mutagenesis
Authors: Lerche H. , Peter W. , Fleischhauer R. , Pika‐Hartlaub U. , Malina T. , Mitrovic N. , Lehmann‐Horn F.
Published: September 2004

In order to investigate the role in fast inactivation of the cytoplasmic S4‐S5 loop of the fourth domain (IV/S4–S5) within the α‐subunit of the adult human muscle Na+ channel, every single amino acid from R1469 to G1486 was substituted by a cysteine and the mutants were studied by functional expression in human embryonic kidney cells (tsA201) using whole‐cell patch clamping. Effects following intracellular application of the sulfhydryl reagents MTSET and MTSES on the mutants were investigated.

Sixteen of eighteen mutants resulted in the formation of functional channels. For P1480C and N1484C, no Na+ currents could be detected in transfected cells. In the absence of sulfhydryl reagents, F1473C and A1481C slowed fast Na+ channel inactivation by 2‐ and 1 .5‐fold, respectively, and L1482C induced a steady‐state Na+ current (Iss) of 3% of peak current (I≥peak) (1 % for wild‐type).

Upon application of MTSET and MTSES, changes in fast inactivation gating occurred for most of the mutants. The most dramatic destabilizing effects on fast inactivation were observed for M1476C (9‐fold slowing of inactivation; Iss/Ipeak, 3.6%; + 15 mV shift in steady‐state inactivation; 2‐ to 3‐fold acceleration of recovery from inactivation), A1481C (3‐fold; 14%; +20 mV; no change) and F1473C (2.5‐fold; 2.4%; +8 mV; 1.5‐fold). Less pronounced destabilizing effects were observed for M1477C and L1479C. Strongly stabilizing effects on the inactivated state, that is a 20–30 mV hyperpolarizing shift of the inactivation curve associated with a 3‐ to 4‐fold decrease in the rate of recovery from inactivation, occurred for T1470C, L1471C and A1474C. Almost all effects were independent of the membrane potential; however, A1474C only reacted when cells were depolarized. Significant effects on activation were not observed.

We conclude that the IV/S4–S5 loop plays an important role in fast inactivation of the muscle Na+ channel and may contribute to the formation of a receptor for the putative inactivation particle. The effects of sulfhydryl reagents on the various mutations suggest an α‐helical structure of IV/S4–S5 (up to P1480) with destabilizing effects on inactivation for one cluster of amino acids (1473/76/77/79) and a stabilized inactivation at the opposite side of the helix (1470/71/74).
Tetrahydrobiopterin Regulates Cyclic GMP‐Dependent Electrogenic Cl− Secretion in Mouse Ileum In Vitro
Authors: Rolfe V. E. , Brand M. P. , Heales S. J. R. , Lindley K. J. , Milla P. J.
Published: September 2004

Basal electrogenic Cl− secretion, measured as the short‐circuit current (Isc), was variable in ileum removed from tetrahydrobiopterin (BH4)‐deficient hph‐1 mice and wild‐type controls in vitro, although values were not significantly different.

The basal nitrite release and mucosal cyclic guanosine 3′,5′‐monophosphate (cyclic GMP) production were similar in control and BH4‐deficient ileum.

Mucosally added Escherichia coli heat‐stable toxin (STa, 55 ng ml−1) increased the nitrite release, cyclic GMP levels and the Isc in control ileum, but its secretory actions were reduced in BH4‐deficient ileum.

L‐Arginine (1 mM) increased the nitrite release, cyclic GMP production and the Isc in control ileum, but the actions were reduced in BH4‐deficient ileum.

Serosal carbachol (1 mM) stimulated maximum short‐circuit currents of similar magnitude in both control and BH4‐deficient ileum, whilst nitrite release and cyclic GMP production were minimal.

E. coli STa and L‐arginine increased electrogenic Cl− secretion across intact mouse ileum in vitro by releasing nitric oxide and elevating mucosal cyclic GMP. The inhibition of these processes in the hph‐1 mouse ileum suggests that BH4 may be a target for the modulation of electrogenic transport, and highlight the complexity of the interactions between nitric oxide and cyclic GMP in the gut.
Endothelium‐dependent frequency modulation of Ca2+ signalling in individual vascular smooth muscle cells of the rat
Authors: Kasai Yasuyo , Yamazawa Toshiko , Sakurai Takashi , Taketani Yuji , Iino Masamitsu
Published: September 2004

We visualized intracellular Ca2+ concentration ([Ca2+]i) changes, using fluo‐3 as an indicator, of individual vascular smooth muscle cells and endothelial cells within intact rat tail arteries by confocal microscopy.

Using a piezo‐driven objective, we focused on endothelial and smooth muscle cell layers alternately to obtain Ca2+ images of their cells. In the presence of 1 μm acetylcholine (ACh), individual endothelial cells responded with intermittent increases in the [Ca2+]i (Ca2+ oscillations). At the same time, the frequency of Ca2+ oscillations in smooth muscle cells induced by electrical stimulation of the perivascular sympathetic nerve was greatly decreased.

A [Ca2+]i rise during the oscillations in the endothelial cells propagated in the form of a wave along the long axis of the cells.

In the presence of a NO synthase inhibitor, no significant inhibitory effect of ACh on the Ca2+ signalling in the vascular smooth muscle cells was detected, although the Ca2+ oscillations in the endothelial cells persisted.

The inhibitory effect of ACh on the frequency of Ca2+ oscillations in the vascular smooth muscle cells was mimicked by 1 μm sodium nitroprusside, a NO donor.

These results indicate that Ca2+ waves and oscillations in vascular endothelial cells regulate NO production, which modulates vascular tone by decreasing the frequency of Ca2+ oscillations in smooth muscle cells activated by sympathetic agonists.
Frequency‐Dependent Actions of Benzodiazepines on GABAA Receptors in Cultured Murine Cerebellar Granule Cells
Authors: Mellor J. R. , Randall A. D.
Published: September 2004

Miniature IPSCs recorded from cultured murine cerebellar granule cells increased in half‐width and amplitude following application of the benzodiazepine (BDZ) Flunitrazepam (Flu, 1 μm). The increase in the half‐width was much greater than that in the amplitude.

Five‐millisecond applications of 1 mm GABA to nucleated outside‐out patches elicited rapidly rising biexponentially decaying responses that resembled IPSCs. Flu had no effect on the amplitude of such responses, but consistently slowed their deactivation by ∼50%. This effect was reversed by Flu washout or application of the BDZ antagonist Ro 15‐1788. The partial inverse agonist Ro15‐4513 speeded deactivation and depressed peak current amplitude by 23 ± 12%.

The EC50 for GABA was between 45 and 50 μm. At submaximally effective agonist concentrations, Flu increased response amplitude and slowed response deactivation. Both effects were present in all cells taken from young cultures (4–7 days in vitro) but the latter was absent in 55% of the neurones obtained from older cultures (14–27 days in vitro).

With 120 ms applications of 20 μm GABA, responses activated monoexponentially (time constant, 39.8 ± 2.8 ms) and deactivated biexponentially (time constants, 40.4 ± 2.1 and 251 ± 15 ms). Application of Flu slowed both activation and deactivation. The latter effect arose from an increased contribution of the slower component of decay.

Desensitization of responses to 1 mm GABA was biexponential, with time constants of 47 ± 11 and 479 ± 49 ms. Flu speeded desensitization by decreasing both fast and slow time constants. GABAA receptor desensitization consistently slowed subsequent deactivation. No significant relationship between the level of desensitization and the amount of slowing of deactivation produced by Flu was found.

Responses to paired 5 ms applications of 1 mm GABA indicated that the slowing of deactivation and the speeding of desensitization produced by Flu combine to generate a marked frequency dependence in the actions of this BDZ. Thus when compared with control responses, GABA‐induced charge transfer was only enhanced by Flu during the first of two successive agonist applications.
Action potential propagation into the presynaptic dendrites of rat mitral cells
Authors: Bischofberger Josef , Jonas Peter
Published: September 2004

Dendritic patch‐clamp recordings were obtained from mitral cells in rat olfactory bulb slices, up to 350 μm from the soma. Simultaneous dendritic and somatic whole‐cell recordings indicated that action potentials (APs) evoked by somatic or dendritic current injection were initiated near the soma. Both the large amplitude (100.7 ± 1.1 mV) and the short duration (1.38 ± 0.07 ms) of the AP were maintained as the AP propagated back into the primary mitral cell dendrites.

Outside‐out patches isolated from mitral cell dendrites contained voltage‐gated Na+ channels (peak conductance density, 90 pS μm−2 at −10 mV). When an AP was used as a somatic voltage‐clamp command in the presence of 1 μm tetrodotoxin (TTX), the amplitude of the dendritic potential was attenuated to 48 ± 14 mV. This shows that dendritic Na+ channels support the active back‐propagation of APs.

Dendritic patches contained voltage‐gated K+ channels with high density (conductance density, 513 pS μm−2 at 30 mV). Dendritic K+ currents were reduced to 35% by 1 mm external tetraethylammonium chloride (TEACl). When an AP was used as a somatic voltage‐clamp command in the presence of TEACl, the dendritic potential was markedly prolonged. This indicates that dendritic K+ channels mediate the fast repolarization of dendritic APs.

We conclude that voltage‐gated Na+ and K+ channels support dendritic APs with large amplitudes and short durations that may trigger fast transmitter release at dendrodendritic synapses in the olfactory bulb.
Amine precursor uptake and decarboxylation: significance for processing of the rat gastrin precursor
Authors: Voronina Svetlana , Henry Julie , Vaillant Camille , Dockray Graham J. , Varro Andrea
Published: September 2004

Conversion of prohormone precursors to smaller active products occurs in secretory granules, which also have the capacity to concentrate biogenic amines. We have examined how processing of the gastrin precursor, progastrin, in rat antral mucosa is influenced by modulation of the biogenic amine content of secretory granules.

Newly synthesized progastrin‐derived peptides in rat antral mucosa were labelled in vitro with 35SO42− using a pulse–chase protocol and detected after immunoprecipitation by HPLC with on‐line liquid scintillation counting. Secretory granule morphology was examined by electron microscopy. The effects of experimentally manipulating secretory granule pH and amine content were examined.

The dopamine precursor l‐β‐3,4‐dihydroxyphenylalanine (l‐DOPA) inhibited cleavage of 35S‐labelled thirty‐four amino acid amidated gastrin, i.e. [35S]G34, and of [35S]G34 with COOH‐terminal glycine, i.e. [35S]G34–Gly, at a pair of lysine residues, but did not influence cleavage of progastrin at pairs of arginine residues. The effect of l‐DOPA was reversed by reserpine, which inhibits the amine–proton exchangers VMAT1 and VMAT2, and by carbidopa, which inhibits aromatic L‐amino acid decarboxylase.

Treatments that raise intragranular pH, e.g. the weak base chloroquine, the ionophore monensin and the vacuolar proton pump inhibitor bafilomycin A1, had similar effects to l‐DOPA.

Electron microscopical studies showed that the electron‐dense aggregrates in gastrin cell secretory granules were lost after inhibition of the vacuolar proton pump. Treatment with l‐DOPA produced reserpine‐sensitive dissipation of the electron‐dense aggregates, compatible with the idea that increased amine delivery raised intragranular pH.

The data suggest that the processes of amine precursor uptake, decarboxylation and sequestration in secretory granules are associated with selective modulation of progastrin cleavage, possibly by raising intragranular pH and thereby inhibiting pH‐sensitive prohormone convertases.
Calcium release from intracellular stores evoked by extracellular ATP in a Xenopus renal epithelial cell line
Authors: Mori Masahiro , Hosomi Hidenobu , Nishizaki Tomoyuki , Kawahara Katsumasa , Okada Yasuhiro
Published: September 2004

The signal transduction mechanism mediating extracellular adenosine 5′‐triphosphate (ATP)‐induced calcium release in a renal epithelial cell line (A6) was investigated using the whole‐cell voltage‐clamp technique and fura‐2 fluorescence measurement.

ATP (10 μM) activated calcium‐dependent non‐selective cation channels in cells held under voltage clamp.

Guanosine 5′‐O‐(2‐thiodiphosphate) (GDPβS; 0.1–1.0 mM) in the pipette inhibited the ATP‐activated calcium‐dependent currents. With guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS; 0.1–1.0 mM) in the pipette, the currents were spontaneously elicited without application of ATP. Pretreatment with pertussis toxin (PTX) affected neither the ATP‐activated currents nor the increase in intracellular free calcium concentration ([Ca 2+])i evoked by ATP.

Intracellular application of neomycin or heparin inhibited the ATP‐activated currents. Inositol 1,4,5‐trisphosphate (IP3; 0.1–100μM) in the internal solution produced currents similar to those due to ATP activation.

These results suggest that a PTX ‐insensitive guanosine 5′‐triphosphate (GTP)‐binding regulatory protein (G protein) is involved in extracellular ATP‐induced phosphoinositide turnover and subsequent calcium release from IP3 ‐sensitive stores, which subsequently activates the calcium ‐dependent channels in A6 cells.
Mechanism underlying corticotropin‐releasing hormone (CRH) triggered cytosolic Ca2+ rise in identified rat corticotrophs
Authors: Lee Andy K. , Tse Amy
Published: September 2004

The patch‐clamp technique was used in conjunction with the fluorescent Ca2+ indicator indo‐1 to measure simultaneously cytosolic Ca2+ concentration ([Ca2+]i) and membrane potential in single rat corticotrophs identified with the reverse haemolytic plaque assay.

Application of the adrenocorticotropin (ACTH) secretagogue, corticotropin‐releasing hormone (CRH), triggered a sustained [Ca2+]i elevation and membrane depolarization.

The CRH action was mediated via the cAMP‐dependent protein kinase cascade. Both the CRH‐induced depolarization and [Ca2+]i elevation could be mimicked by extracellular application of the adenylate cyclase activator forskolin or the membrane‐permeable cAMP analogue, 8‐(4‐chlorophenylthio)‐adenosine‐3’,5’‐cyclic monophosphate (8‐CPT‐cAMP). Intracellular adenosine cyclic 3’,5’‐(Rp)‐phosphothioate (Rp‐cAMPS), a protein kinase A inhibitor, abolished the CRH effects.

Voltage‐clamp studies suggest that the CRH‐triggered depolarization was due to the reduction of background K+ conductances. The CRH‐sensitive current was Ca2+ independent and was insensitive to the K+ channel blockers tetraethylammonium (TEA) or 4‐amino‐pyridine (4‐AP), but could be partially inhibited by Ba2+.

The CRH‐triggered steady‐state depolarization stimulated extracellular Ca2+ entry via voltage‐gated Ca2+ channels and raised [Ca2+]i. CRH failed to stimulate [Ca2+]i rise in cells that were voltage clamped at their resting potential. Removal of extracellular Ca2+ or inhibition of Ca2+ channels by Ni2+ abolished the [Ca2+]i rise.

Voltage‐clamp studies of voltage‐gated Ca2+ channels using Ba2+ as charge carrier show that ∼90% of the channels were available for activation at the resting potential. CRH did not enhance the voltage‐gated Ca2+ channels.
Intramembrane charge movement and sarcoplasmic calcium release in enzymatically isolated mammalian skeletal muscle fibres
Authors: Szentesi Peter , Jacquemond Vincent , Kovács László , Csernoch László
Published: September 2004

Single muscle fibres were dissociated enzymatically from the extensor digitorum longus and communis muscles of rats and guinea‐pigs. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation–contraction coupling were studied under voltage clamp conditions.

The voltage dependence of intramembrane charge movement followed a two‐state Boltzmann distribution with maximal available charge of 26.1 ± 1.5 and 26.1 ± 1.3 nC μF−1, mid‐point voltage of –35.1 ± 5.0 and –42.2 ± 1.2 mV and steepness of 16.7 ± 2.2 and 17.0 ± 1.9 mV (means ±s.e.m., n= 7 and 4) in rats and guinea‐pigs, respectively.

Intracellular calcium concentration ([Ca2+]i) was monitored using the calcium‐sensitive dyes antipyrylazo III, fura‐2 and mag‐fura‐5. Resting [Ca2+]i was similar in rats and guinea‐pigs with 125 ± 18 and 115 ± 8 nm (n= 10 and 9), respectively, while the maximal increase for a 100 ms depolarization to 0 mV was larger in rats (6.3 ± 1.0 μm; n= 7), than in guinea‐pigs (2.8 ± 0.3; n= 4).

The rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) displayed an early peak followed by a fast and a slow decline to a quasi maintained steady level. After normalizing Rrel to the estimated SR calcium content (1.2 ± 0.1 and 0.9 ± 0.1 mm in rats and guinea‐pigs, respectively) and correcting for depletion of calcium in the SR the peak and steady levels at 0 mV, respectively, were found to be 2.50 ± 0.08 and 0.81 ± 0.06% ms−1 in rats and 2.43 ± 0.25 and 0.88 ± 0.01 % ms−1 in guinea‐pigs. The voltage dependence was essentially the same in both species, but different from that in amphibians.

These experiments show that enzymatic isolation yields functionally intact mammalian skeletal muscle fibres for Vaseline gap experiments. The data also suggest a close connection in the regulation of the different kinetic components of SR calcium release in mammalian skeletal muscle.
Functional Distribution of Three Types of Na+ Channel on Soma and Processes of Dorsal Horn Neurones of Rat Spinal Cord
Authors: Safronov Boris V. , Wolff Matthias , Vogel Werner
Published: September 2004

Voltage‐gated Na+ channels and their distribution were studied by the patch‐clamp technique in intact dorsal horn neurones in slices of newborn rat spinal cord and in neurones isolated from the slice by slow withdrawal of the recording pipette. This new method of neurone isolation was further used to study the roles of soma and axon in generation of action potentials.

Tetrodotoxin (TTX)‐sensitive Na+ currents in intact neurones consisted of three components. A fast component with an inactivation time constant (τf) of 0.6–2.0 ms formed the major part (80–90%) of the total Na+ current. The remaining parts consisted of a slowly inactivating component (τs of 5–20 ms) and a steady‐state component.

Single fast and slow inactivating Na+ channels with conductances of 11.6 and 15.5 pS, respectively, were identified in the soma of intact neurones in the slice. Steady‐state Na+ channels were not found in the soma, suggesting an axonal or dendritic localization of these channels.

In the whole‐cell recording mode, the entire soma of a dorsal horn neurone could be isolated from the slice by slow withdrawal of the recording pipette, leaving all or nearly all of its processes in the slice. The isolated structure was classified as: (1) ‘soma’ if it lost all of its processes, (2) ‘soma+axon’ complex if it preserved one process and at least 85% of its original peak Na+ current or (3) ‘soma+dendrite’ complex if it preserved one process but the remaining Na+ current did not exceed those observed in the isolated ‘somata’.

The spatial distribution of Na+ channels in the neurone was studied by comparing Na+ currents recorded before and after isolation. The isolated ‘soma’ contained 13.8 ± 1.3% of inactivating Na+ current but no steady‐state Na+ current. ‘Soma+axon’ complexes contained 93.6 ± 1.4% of inactivating and 46% of steady‐state Na+ current.

In current‐clamp experiments, the intact neurones and isolated ‘soma+axon’ complexes responded with ‘all‐or‐nothing’ action potentials to current injections. In contrast, isolated ‘somata’ showed only passive or local responses and were unable to generate action potentials.

It is concluded that dorsal horn neurones of the spinal cord possess three types of TTX‐sensitive voltage‐gated Na+ channels. The method of entire soma isolation described here shows that the majority of inactivating Na+ channels are localized in the axon hillock and only a small proportion (ca 1/7) are distributed in the soma. Steady‐state Na+ channels are most probably expressed in the axonal and dendritic membranes. The soma itself is not able to generate action potentials. The axon or its initial segment plays a crucial role in the generation of action potentials.
The electrogenic effects of Na+–K+‐ATPase in rat auditory thalamus
Authors: Senatorov V. V. , Mooney D. , Hu B.
Published: September 2004

The electrogenic effects of the Na+–K+‐ATPase in thalamic neurones were investigated by means of intracellular and whole‐cell patch‐clamp recording techniques in rat medial geniculate body (MGB) maintained in vitro.

In twenty‐six out of thirty‐one neurones recorded intracellularly, application of the Na+–K+ pump inhibitor strophanthidin induced two different types of membrane depolarization: a small, reversible depolarization with a peak amplitude of 4 ± 2.6 mV or a prolonged depolarization of large amplitude (48.6 ± 9.0 mV) with or without a decrease in apparent membrane resistance. Blockade of glutamate receptors with kynurenic acid or 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione and (±)‐2‐amino‐5‐phosphonopentanoic acid did not prevent either type of pump response, but the large depolarization was not seen when the medium contained the sodium channel blocker TTX.

Whole‐cell patch‐clamp recording showed that the small membrane depolarization is mediated by an inward membrane current (39.00 ± 5.70 pA) that exhibited a weak voltage dependence. An inward current of similar amplitude was also induced in MGB cells when the pipette solution contained nominally zero Na+ or when K+ was temporarily omitted from the extracellular medium. The large membrane depolarization or the corresponding membrane current was not observed in whole‐cell conditions.

Transient inhibition of the electrogenic Na+–K+‐ATPase consistently led to a change in the mode of synaptic transmission in MGB cells, during which the synaptically evoked burst response was either blocked or converted into a single spike discharge.

Taken together, these data suggest that blockade of the electrogenic pump produces a dual membrane effect in mammalian thalamic neurones: a small electrogenic membrane depolarization and a large depolarization response that can be prevented by artificially maintaining the transmembrane ionic gradients. The electrogenic activity of the Na+–K+‐ATPase may play an important role in setting the mode of synaptic transmission in sensory thalamus.
Contribution of Selectins to Leucocyte Sequestration in Pulmonary Microvessels by Intravital Microscopy in Rabbits
Authors: Kuebler W. M. , Kuhnle G. E. H. , Groh J. , Goetz A. E.
Published: September 2004

Sequestration of leucocytes in the lung is the net result of leucocyte rolling and sticking in pulmonary arterioles and venules and their retention in alveolar capillaries.

In order to investigate whether adhesion molecules of the selectin family contribute to these phenomena the effects of fucoidin (an inhibitor of L‐ and P‐selectin) on micro‐haemodynamics and leucocyte kinetics were studied in pulmonary arterioles, capillaries and venules by means of intravital fluorescence microscopy in a rabbit model.

Fucoidin reduced leucocyte rolling in pulmonary arterioles and venules by 75 and 83%, respectively, without affecting leucocyte sticking. In alveolar capillaries, fucoidin reduced leucocyte retention and accelerated leucocyte passage, thus reducing the alveolar transit time of leucocytes by 62%.

It is concluded that rolling of leucocytes in pulmonary microvessels is mediated by selectins, whereas sticking relies on selectin‐independent mechanisms.

Leucocyte retention in alveolar capillaries is not due solely to mechanical hindrance of leucocyte passage through narrow vessel segments, as previously hypothesized, but also depends on interaction of leucocytes with the capillary endothelium.
Emptying of Intracellular Ca2+ Stores Stimulates Ca2+ Entry in Mouse Pancreatic β‐Cells by Both Direct and Indirect Mechanisms
Authors: Miura Yoshikazu , Henquin Jean‐Claude , Gilon Patrick
Published: September 2004

In non‐excitable cells, the depletion of intracellular Ca2+ stores triggers Ca2+ influx by a process called capacitative Ca2+ entry. In the present study, we have investigated how the emptying of these stores by thapsigargin (1 μM) influences Ca2+ influx in electrically excitable pancreatic β‐cells. The cytoplasmic Ca2+ concentration ([Ca2+]i) was monitored in clusters of mouse β‐cells or in whole islets loaded with fura‐2.

The membrane was first held hyperpolarized by diazoxide, an opener of ATP‐sensitive K+(KATP) channels, in the presence of 4.8 mM K+. Alternating between Ca2+‐free medium and medium containing 2.5 mM Ca2+ caused a minor rise in [Ca2+]i (∼14 nM) in clusters of β‐cells. A larger rise (∼65 nM), resistant to the blockade of voltage‐dependent Ca2+ channels by D600, occurred when extracellular Ca2+ was readmitted after emptying intracellular Ca2+ stores with thapsigargin or acetylcholine. Thus there exists a small capacitative Ca2+ entry in β‐cells.

When the membrane potential was clamped at depolarized levels with 10, 20 or 45 mM K+ in the presence of diazoxide, [Ca2+]i increased to different plateau levels ranging between 100 and 900 nM. Thapsigargin consistently caused a further transient rise in [Ca2+]i, but had little (at 10 mM K+) or no effect on the plateau level. This confirms that the capacitative Ca2+ entry is small.

In clusters of cells whose membrane potential was not clamped with diazoxide, 15 mM glucose (in 4.8 mM K+) induced [Ca2+]i oscillations by promoting Ca2+ influx through voltage‐dependent Ca2+ channels. The application of thapsigargin accelerated these oscillations and increased their amplitude, sometimes causing a sustained elevation of [Ca2+]i. Similar results were obtained from whole islets perifused with a medium containing ≥ 6 mM glucose. The effect of thapsigargin was always much larger than expected from the capacitative Ca2+ entry, probably because of a potentiation of Ca2+ influx through voltage‐dependent Ca2+ channels.

This potentiating effect of thapsigargin did not result from an acceleration of cell metabolism since the drug did not affect glucose‐induced changes in NAD(P)H fluorescence. It is also unlikely to involve the inhibition of KATP channels because thapsigargin steadily elevated [Ca2+]i in cells in which [Ca2+]i oscillations persisted in the presence of a maximally effective concentration of tolbutamide.

In conclusion, the emptying of intracellular Ca2+ stores in β‐cells induces a small capacitative Ca2+ entry and activates a depolarizing current which potentiates glucose‐induced Ca2+ influx through voltage‐dependent Ca2+ channels.
Hyperpolarization of isolated capillaries from guinea‐pig heart induced by K+ channel openers and glucose deprivation
Authors: Langheinrich Ulrike , Daut Jürgen
Published: September 2004

The present study was designed to test if microvascular coronary endothelial cells express ATP–sensitive K+ channels (KATP channels). We performed microfluorometric measurements of the membrane potential of freshly isolated guinea‐pig coronary capillaries equilibrated with the voltage–sensitive dye bis‐oxonol (bis‐[1,3–dibutylbarbituric acid] trimethineoxonol, [DiBAC4(3)]).

The resting membrane potential of capillaries in physiological salt solution was −46±4.2 mV (n= 8) at room temperature (22 °C) as determined after calibration of the fluorescence using the Na+–K+ ionophore gramicidin in the presence of different K+ concentrations. Spontaneous membrane potential fluctuations of 10–20 mV amplitude were often observed.

A reversible, sustained hyperpolarization to a new membrane potential close to the K+ equilibrium potential (EK) could be induced by application of the K+ channel openers HOE 234 (100 nm to 1 μM), diazoxide (10 pm to 100 nM) or pinacidil (100 nM). Subsequent addition of glibenclamide (200 nm to 2 μM) reversed this hyperpolarization.

A glibenclamide–sensitive hyperpolarization of coronary capillaries to values near EK was also observed upon omission of D‐glucose (10 mM) from the superfusing solution or by substituting L‐glucose for D‐glucose. Maximum hyperpolarization was reached in less than 10 min.

Our results suggest that microvascular coronary endothelial cells express KATP channels which may be activated during hypoglycaemia.
Rapid ATP‐Dependent Priming of Secretory Granules Precedes Ca2+ ‐Induced Exocytosis in Mouse Pancreatic B‐Cells
Authors: Eliasson Lena , Renström Erik , Ding Wei‐Guang , Proks Peter , Rorsman Patrik
Published: September 2004

The glucose and ATP dependence of exocytosis were investigated in single mouse pancreatic B‐cells by monitoring changes in cell capacitance evoked by voltage‐clamp depolarizations, infusion of high‐[Ca2+]i buffers or photorelease of caged Ca2+ or ATP.

In intact B‐cells, using the perforated patch whole‐cell technique, glucose (5 mM) increased exocytotic responses evoked by membrane depolarization 5–fold over that observed in the absence of the sugar. Increasing the glucose concentration to 20 mM produced a further doubling of exocytosis. The stimulatory action of glucose was attributable to glucose metabolism and abolished by mannoheptulose, an inhibitor of glucose phosphorylation.

Exocytosis triggered by infusion of high [Ca2+]i and ATP was reduced by 80 % when ATP was replaced by its non‐hydrolysable analogue adenosine 5′‐[β,γ‐methylene]triphosphate (AMP‐PCP) in standard whole‐cell experiments. Exocytosis elicited by GTPγS was similarly affected by replacement of ATP with the stable analogue.

Photoreleasing ATP in the presence of 170 nm [Ca2+]i following the complete wash‐out of endogenous ATP, produced a prompt (latency, < 400 ms) and biphasic stimulation of exocytosis.

Elevation of [Ca2+]i to exocytotic levels by photorelease from Ca2+‐nitrophenyl EGTA preloaded into the cell evoked a biphasic stimulation in the presence of Mg‐ATP. The response consisted of an initial rapid (completed in < 200 ms) phase followed by a slower (lasting ≥ 10 s) sustained component. Replacement of ATP with AMP‐PCP abolished the late component but did not affect the initial phase. The latency between elevation of [Ca2+]i and exocytosis was determined as < 45 ms. Inclusion of N‐ethylmaleimide (NEM; 0.5 mM for 3 min) in the intracellular solution exerted effects similar to those obtained by substituting AMP‐PCP for ATP.

We conclude that the B‐cell contains a small pool (40 granules) of primed granules which are immediately available for release and which are capable of undergoing exocytosis in an ATP‐independent fashion. We propose that this pool of granules is preferentially released during first phase glucose‐stimulated insulin secretion. The short latency between the application of ATP and the onset of exocytosis finally suggests that priming takes place with sufficient speed to participate in the rapid adjustment of the secretory capacity of the B‐cell.
Apparent Ca2+ dissociation constant of Ca2+ chelators incorporated non‐disruptively into intact human red cells
Authors: Tiffert Teresa , Lew Virgilio L.
Published: September 2004

A recently developed method of measuring cytoplasmic Ca2+ buffering in intact red cells was applied to re‐evaluate the intracellular Ca2+ binding properties of the Ca2+ chelators benz2 and BAPTA. Incorporation of the free chelators was accomplished by incubating the cells with the acetoxymethyl ester forms (benz2 AM or BAPTA AM). The divalent cation ionophore A23187 was used to induce equilibrium distribution of Ca2+ between cells and medium. 45Ca2+ was added stepwise to cell suspensions in the presence and absence of external BAPTA. To induce full Ca2+ equilibration, the plasma membrane Ca2+ pump was inhibited either by depleting the cells of ATP or by adding vanadate to the cell suspension.

The properties of the incorporated chelators were assessed from the difference in cytoplasmic Ca2+ buffering between chelator‐free and chelator‐loaded cells, over a wide range of intracellular ionized calcium concentrations ([Ca2+]i), from nanomolar to millimolar.

Under the experimental conditions applied, incorporation of benz2 and BAPTA into the red cells increased their Ca2+ buffering capacity by 300–600 μmol (340 g Hb)−1. The intracellular apparent Ca2+ dissociation constants (KDi were about 500 nm for benz2 and 800 nm for BAPTA, values much higher than those reported for standard salt solutions (KD) of about 40 and 130 nm, respectively. These results suggest that, contrary to earlier observations, the intracellular red cell environment may cause large shifts in the apparent Ca2+ binding behaviour of incorporated chelators.

The possibility that the observed KD shifts are due to reversible binding of the chelators to haemoglobin is considered, and the implications of the present results for early estimates of physiological [Ca2+]i levels is discussed.
Spatial and temporal frequency selectivity of cells in area 21a of the cat.
Authors: Morley J. W. , Vickery R. M.
Published: September 2004

The spatial and temporal response properties of single cells in area 21a of the anaesthetized cat were assessed using drifting sinusoidal gratings presented at the optimum orientation for each cell.

Responses to sinusoidal gratings were dominated by an elevation of the mean discharge, with a relatively small modulated component at the temporal frequency of grating drift. The relative modulation ratio for the majority of cells was less than 1, similar to complex cells in the striate cortex.

Of those cells responsive to stimulation with sinusoidal gratings, 94% displayed spatial bandpass characteristics. Values derived from spatial frequency tuning curves were: mean optimum spatial frequency, 0.26 cycles deg−1; mean spatial resolution, 0.86 cycles deg−1; mean spatial bandwidth, 1.8 octaves; and mean normalized bandwidth, 1.3. Two cells (6%) displayed spatial low‐pass characteristics.

Approximately half our sample of cells (44%) displayed temporal low‐pass tuning, while 35% displayed temporal bandpass characteristics. The mean optimum temporal frequency of bandpass cells was 3.3 Hz and the mean temporal bandwidth 1.9 octaves. The remaining cells were classified as temporal broadband (17%) and temporal high‐pass (4%).

We conclude that the dominant functional input to cells with relatively high spatial frequency selectivity and/or temporal low‐pass response properties most probably arises from area 17. The responses of the remaining cells may be explained by input from area 17 or 18.