bluuebunny/crossref_metadata_2025 huggingface dataset:
K channel activation by nucleotide diphosphates and its inhibition by glibenclamide in vascular smooth muscle cells
Authors: Beech D.J. , Zhang H. , Nakao K. , Bolton T.B.
Published: July 2012

Whole‐cell and inside‐out patch recordings were made from single smooth muscle cells that had been isolated enzymatically and mechanically from the rabbit portal vein.

In whole‐cells the inclusion in the recording pipette solution of nucleotide diphosphates (NDPs), but not tri‐ or monophosphates, induced a K‐current that developed gradually over 5 to 15 min. Intracellular 1 mm guanosine 5′‐diphosphate (GDP) induced a slowly developing outward K‐current at − 37 mV that reached a maximum on average of 72 ± 4 pA (n = 40). Half maximal effect was estimated to occur with about 0.2 mm GDP. Except for ADP, other NDPs had comparable effects. At 0.1 mm, ADP was equivalent to GDP but at higher concentration ADP was less effective. ADP induced its maximum effect at 1 mm but had almost no effect at 10 mm.

In 14% of inside‐out patches exposed to 1 mm GDP at the intracellular surface, characteristic K channel activity was observed which showed long (> 1 s) bursts of openings separated by longer closed periods. The current‐voltage relationship for the channel was linear in a 60 mm:130 mm K‐gradient and the unitary conductance was 24 pS.

Glibenclamide applied via the extracellular solution was found to be a potent inhibitor of GDP‐induced K‐current (IK(GDP)) in the whole‐cell. The Kd was 25 nm and the inhibition was fully reversible on wash‐out.

(IK(GDP)) was not evoked if Mg ions were absent from the pipette solution. In contrast the omission of extracellular Mg ions had no effect on outward or inward (IK(GDP)).

Inclusion of 1 mm ATP in the recording pipette solution reduced (IK(GDP)) and also attenuated its decline during long (25 min) recordings.

When perforated‐patch whole‐cell recording was used, metabolic poisoning with cyanide and 2‐deoxy‐d‐glucose induced a glibenclamide‐sensitive K‐current. This current was not observed when conventional whole‐cell recording was used. Possible reasons for this difference are discussed.

These K channels appear similar to ATP‐sensitive K channels but we refer to them as nucleotide diphosphate‐dependent K channels (KNDP) to emphasise what seems to be a primary role for nucleotide diphosphates in their regulation.
Activation of protein kinase C potentiates postsynaptic acetylcholine response at developing neuromuscular synapses
Authors: Fu Wen‐Mei , Lin Jiann‐Liang
Published: July 2012

Phorbol 12‐myristate 13‐acetate (TPA, 1 μm) and phorbol 12,13‐dibutyrate (PDBu, 2 μm), activators of protein kinase C (PKC), increased the mean amplitude and decay time of the spontaneous synaptic currents of Xenopus nerve‐muscle coculture, whereas, 4α‐phorbol (2 μm) which is an inactive phorbol analogue had no effect.

Staurosporine (0.5 μm) and H‐7 (10 μm), inhibitors of PKC, inhibited the potentiation effects of TPA on the spontaneous synaptic currents.

Effects of TPA on the postsynaptic acetylcholine (ACh) sensitivity were examined by iontophoresis of ACh to the surface of embryonic muscle cells of 1‐day‐old Xenopus cultures. TPA increased both the amplitude and decay time of ACh‐induced whole‐cell currents in isolated myocytes.

TPA concentration‐dependently increased the mean open time of low‐conductance ACh channels but did not affect those of high‐conductance ACh channels. PDBu but not 4α‐phorbol exhibited similar effects to TPA. Staurosporine and H‐7 inhibited the increasing effects of TPA.

These results suggest that activation of PKC might be involved in synaptogenesis at developing neuromuscular synapses by the postsynaptic potentiation of ACh sensitivity.
Repeated administration of desipramine and a GABAB receptor antagonist, CGP 36742, discretely up‐regulates GABAB receptor binding sites in rat frontal cortex
Authors: Pratt Gerard D. , Bowery Norman G.
Published: July 2012

GABAB receptor binding site densities within laminar regions of the rat frontal cortex were examined autoradiographically following repeated administration (21 days) of the antidepressants desipramine, paroxetine and amitriptyline in addition to the GABAB receptor antagonists, CGP 35348 and CGP 36742. β1‐Adrenoceptor autoradiography was studied in parallel with that for GABAB receptor sites.

The effects of these compounds were examined concomitantly on the GABAB receptor‐mediated inhibition of forskolin‐ and enhancement of noradrenaline‐stimulated cyclic AMP production.

GABAB receptor binding was increased by both desipramine (20 mg kg−1, p.o. and 10 mg kg−1, i.p.) and CGP 36742 (100 mg kg−1, i.p.) in the outer laminar region of the frontal cortex by around 50% above control levels. Conversely, no significant changes were mediated by paroxetine, amitriptyline, CGP 35348 or the GABAB receptor agonist, baclofen.

With the exception of paroxetine, all compounds down‐regulated the total β‐adrenoceptor population throughout frontal cortical laminae which was attributable to the β1‐adrenoceptor subtype. In contrast, the reduction in β‐adrenoceptors mediated by CGP 35348 and CGP 36742 did not occur as a consequence of reduced β1‐adrenoceptor numbers.

Protracted treatment with CGP 35348, failed to influence forskolin‐stimulated cyclic AMP production; however, a significant increase in the accumulation of cyclic AMP produced in response to forskolin was seen after treatment with CGP 36742.

Such discretely localized changes in GABAB receptor densities induced by desipramine and CGP 36742 may provide an explanation for the discrepancies reported in membrane binding studies and possibly implicate a role for GABAB receptor antagonists in antidepressant therapy.
Activation of the phospholipase C pathway by ATP is mediated exclusively through nucleotide type P2‐purinoceptors in C2C12 myotubes
Authors: Henning Robert H. , Duin Marry , den Hertog Adriaan , Nelemans Adriaan
Published: July 2012

The presence of a nucleotide receptor and a discrete ATP‐sensitive receptor on C2C12 myotubes has been shown by electrophysiological experiments. In this study, the ATP‐sensitive receptors of C2C12 myotubes were further characterized by measuring the formation of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) and internal Ca2+.

The nucleotides ATP and UTP caused a concentration‐dependent increase in Ins(1,4,5)P3 content with comparable time courses (EC50: ATP 33 ± 2 μm, UTP 80 ± 4 μm). ADP was less effective in increasing Ins(1,4,5)P3 content of the cells, while selective agonists for P1‐, P2X‐ and P2Y‐purinoceptors, adenosine, α,β‐methylene ATP and 2‐methylthio ATP, appeared to be ineffective.

Under Ca2+‐free conditions, the basal level of Ins(1,4,5)P3 was lower than in the presence of Ca2+, and the ATP‐ and UTP‐induced formation of Ins(1,4,5)P3 was diminished.

The Ins(1,4,5)P3 formation induced by optimal ATP and UTP concentrations was not additive. ATP‐ and UTP‐induced Ins(1,4,5)P3 formation showed cross‐desensitization, whereas cross‐desensitization was absent in responses elicited by one of the nucleotides and bradykinin.

The change in Ins(1,4,5)P3 content induced by effective nucleotides was inhibited by suramin. Schild plots for suramin inhibition of Ins(1,4,5)P3 formation in ATP‐ and UTP‐stimulated myotubes showed slopes greater than unity (1.63 ± 0.09 and 1.37 ± 0.11, respectively). Apparent pA2 values were 4.50 ± 0.48 and 4.41 ± 0.63 for ATP and UTP, respectively.

Stimulation of the cells with ATP or UTP induced a rapid increase in intracellular Ca2+, followed by a slow decline to basal levels. Ca2+ responses reached lower maximal values and did not show the slow phase in the absence of extracellular Ca2+. The ATP and UTP‐evoked increase in intracellular Ca2+ was not additive and showed cross‐desensitization. Cross‐desensitization was absent in myotubes stimulated with one of the nucleotides and bradykinin.

These results show that ATP‐ and UTP‐induced formation of Ins(1,4,5)P3, Ca2+ release from internal stores and Ca2+‐influx from the extracellular space are mediated exclusively via the nucleotide type P2‐purinoceptor in mouse C2C12 myotubes.
Extracellular ATP and UTP activation of phospholipase D is mediated by protein kinase C‐ε in rat renal mesangial cells
Authors: Pfeilschifter Josef , Merriweather Claire
Published: July 2012

We have studied whether a nucleotide receptor mediates the effects of extracellular ATP and UTP on phosphatidylcholine metabolism in rat cultured glomerular mesangial cells.

ATP and UTP stimulated a biphasic 1,2‐diacylglycerol (DAG) formation in [3H]‐arachidonic acid‐labelled mesangial cells. In contrast, in cells labelled with [3H]‐myristic acid, a tracer that preferentially marks phosphatidylcholine, both nucleotides induced a delayed monophasic production of DAG with a concomitant increase in phosphatidic acid and choline formation.

A phospholipase D‐mediated phosphatidylcholine hydrolysis was further suggested by the observation that ATP and UTP stimulate the accumulation of phosphatidylethanol, when ethanol was added to mesangial cells.

The rank order of potency of a series of nucleotide analogues for stimulation of phosphatidylethanol formation was UTP = ATP > ITP > ATPγS > βγ‐imido‐ATP = ADP > 2‐methylthio‐ATP = βγ‐methylene‐ATP = ADPβS, while AMP, adenosine, CTP and GTP were inactive, indicating the presence of a nucleotide receptor.

Elevation of cytosolic free Ca2+ by the calcium ionophore A23187 (1 μm) or the Ca2+‐ATPase inhibitor, thapsigargin (200 nm) slightly increased phosphatidylethanol formation. However, chelation of cytosolic Ca2+ with high concentrations of Quin 2 did not attenuate ATP‐ and UTP‐induced phosphatidylethanol production, thus suggesting that Ca2+ is not crucially involved in agonist‐stimulated phospholipase D activation.

The protein kinase C (PKC) activator, phorbol 12‐myristate 13‐acetate (PMA), but not the biologically inactive 4α‐phorbol 12,13‐didecanoate, increased phospholipase D activity in mesangial cells, suggesting that PKC may mediate nucleotide‐induced phosphatidylcholine hydrolysis.

Down‐regulation of PKC‐α and ‐δ isoenzymes by 8 h PMA treatment still resulted in full phospholipase D activation. In contrast, a 24 h treatment of mesangial cells with PMA, a regimen that also causes depletion of PKC‐ε, markedly attenuated nucleotide‐evoked phosphatidylethanol formation. In addition, the selective PKC inhibitor, calphostin C attenuated ATP‐ and UTP‐induced phosphatidylethanol production.

In summary, these data suggest that extracellular ATP and UTP use a common nucleotide receptor to activate phospholipase D‐mediated phosphatidylcholine hydrolysis. Stimulation of phospholipase D appears to involve the PKC‐ε isoenzyme, activated by DAG derived from phosphoinositide hydrolysis by phospholipase C.
Eicosanoid‐induced Ca2+ release and sustained contraction in Ca2+‐free media are mediated by different signal transduction pathways in rat aorta
Authors: Kurata Ryuichi , Takayanagi Issei , Hisayama Tetsuhiro
Published: July 2012

The effects of 12‐O‐tetradecanoyl 4β‐phorbol 13‐acetate (β‐TPA) on the inositol 1,4,5‐trisphosphate (IP3) production, Ca2+ release from the intracellular Ca2+ stores and sensitization of contractile apparatus, induced by prostaglandin F2α (PGF2α) and U46619, a thromboxane A2‐mimetic, were studied, using fura‐2‐loaded and ‐unloaded rat thoracic aortic strips.

Both eicosanoids had characteristic patterns of responses in Ca2+‐free, 2 mm EGTA‐containing solution (Ca2+‐free solution). They induced transient increases in intracellular Ca2+ concentration ([Ca2+]i) without corresponding transient contraction, but produced delayed, sustained contraction, where [Ca2+]i was returned to the basal level.

Treatment with β‐TPA for 60 min reduced the eicosanoids‐induced IP3 production, suggesting that the treatment inhibits PIP2 breakdown.

The treatment also attenuated [Ca2+]i transient induced by the eicosanoids, but not by caffeine (an IP3‐independent releaser of stored Ca2+), in fura‐2‐loaded preparations incubated in Ca2+‐free solution.

In contrast in the presence of β‐TPA, the sustained contractions evoked by the eicosanoids in Ca2+‐free solution were potentiated, suggesting that the sites of actions of β‐TPA and the eicosanoids may differ from each other.

PGF2α and U46619 utilize different and parallel signal transduction pathways to release Ca2+ by IP3 produced by PIP2 breakdown (β‐TPA‐sensitive), and to increase the sensitivity of contractile apparatus, in which protein kinase C may not be involved (β‐TPA‐insensitive).
Pro‐inflammatory activities in elapid snake venoms
Authors: Tambourgi Denise V. , dos Santos Maria Cristina , Furtado Maria de Fátima D. , de Freitas Maria Cristina W. , Dias da Silva Wilmar , Kipnis Thereza L.
Published: July 2012

Snake venoms from the genera Micrurus (M. ibiboboca and M. spixii) and Naja (N. naja, N. melanoleuca and N. nigricollis) were analysed, using biological and immunochemical methods, to detect pro‐inflammatory activities, cobra venom factor (COF), proteolytic enzymes, thrombin‐like substances, haemorrhagic and oedema‐producing substances.

The venoms of the five snake species activate the complement system (C) in normal human serum (NHS) in a dose‐related fashion, at concentrations ranging from 5 μg to 200 μg ml−1 serum. Electrophoretic conversion of C3 was observed with all venoms in NHS containing normal concentrations of Ca2+ and Mg2+, but only by venoms from N. naja and N. melanoleuca when Ca2+ was chelated by adding Mg2+‐EGTA.

Purified human C3 was electrophoretically converted, in the absence of other C components, by the venoms from N. naja, N. nigricollis and M. ibiboboca. However, only the venoms from N. naja and N. melanoleuca contained a 144 kDa protein revealed in Western blot with sera against COF or human C3.

All venoms, at minimum concentrations of 30 ng ml−1, were capable of lysing sheep red blood cells, also in a dose‐related fashion, when incubated with these cells in presence of egg yolk as a source of lecithin. Although the venoms from M. spixii and N. nigricollis showed detectable thrombin‐like activity, these and the other venoms were free of proteolytic activity when fibrin, gelatin and casein, were used as substrates.

When tested on mice skin, all five venoms were capable of inducing an increase in vascular permeability and oedema, but were devoid of haemorrhagic producing substances (haemorrhagins).

These data provide evidence indicating that Elapidae venoms contain various pro‐inflammatory factors which may be important in the spreading of neurotoxins throughout the tissues of the prey or human victim.
Mepacrine‐induced inhibition of the inward current mediated by 5‐HT3 receptors in rat nodose ganglion neurones
Authors: Fan Ping
Published: July 2012

With the whole‐cell patch clamp technique, the effect of the antimalarial drug, mepacrine (quinacrine) on the inward current mediated by 5‐HT3 receptors (5‐hydroxytryptamine (5‐HT)‐induced current) was investigated in isolated nodose ganglion neurones of the rat.

5‐HT and the selective 5‐HT3 receptor agonists, 2‐methyl‐5‐HT and m‐chlorophenylbiguanide elicited an inward current which reversed at around 0 mV and quickly desensitized to a steady state level.

Mepacrine dose‐dependently inhibited the peak current induced by 5‐HT with an IC50 of 2.1 μm and an apparent Hill coefficient of 0.99.

Mepacrine increased the decay rate of the 5‐HT‐induced current.

The effect of mepacrine on the 5‐HT‐induced current was reversible and not dependent on membrane potential. The reversal potential of the 5‐HT‐induced current was not affected.

Intracellular mepacrine had no significant effect on the 5‐HT‐induced current and did not block the extracellular action of mepacrine.

Concentration‐response curves in the presence and absence of mepacrine suggest a non‐competitive inhibition of 5‐HT‐induced current by mepacrine.
A quantitative comparison of functional and anti‐ischaemic effects of the phosphodiesterase‐inhibitors, amrinone, milrinone and levosimendan in rabbit isolated hearts
Authors: Rump A.F.E. , Acar D. , Klaus W.
Published: July 2012

The functional and anti‐ischaemic effects of the phosphodiesterase (PDE)‐inhibitors, amrinone, milrinone and levosimendan, a new agent combining PDE‐inhibitory with calcium‐sensitizing properties, were investigated in rabbit isolated hearts (Langendorff, constant pressure: 70 cmH2O, Tyrode solution, Ca2+ 1.8 mmol l−1, 37°C). Anti‐ischaemic effects were studied in electrically‐driven hearts (200 beats min−1). Acute regional ischaemia was induced by ligature of a branch of the circumflex coronary artery and quantified from epicardial NADH‐fluorescence photography.

Cumulative concentration‐response curves in spontaneously beating hearts in the presence of isoprenaline (10−1 m), showed a higher inotropic and coronary vasodilator potency for levosimendan (EC50: 7 × 10−7 m) compared to milrinone (EC50: 7.7 × 10−6 m) or amrinone (EC50: 2 × 10−5 m). Although the maximal coronary dilator activity was similar for the three agents, the maximal inotropic and chronotropic effects were lower for levosimendan than for amrinone or milrinone (P < 0.05).

In regionally ischaemic hearts, milrinone (10−5 m) or levosimendan (5 × 10−6 m) similarly enhanced the left ventricular pressure (+15–20%) (P < 0.05) and the global coronary flow (+ 40–50%) (P < 0.05). The epicardial NADH‐fluorescence area was significantly diminished by milrinone or levosimendan (– 20–30%) (P < 0.05) and there was no significant difference between the anti‐ischaemic effects of either agent (P > 0.05).

It is concluded that amrinone and milrinone possess similar functional profiles in rabbit isolated hearts and a higher inotropic and chronotropic efficacy than levosimendan. At functionally equieffective concentrations, milrinone and levosimendan show similar anti‐ischaemic effects, related to an improvement of myocardial perfusion. The calcium‐sensitizing properties seem not to be relevant for cardioprotection by levosimendan at the concentration used.
The effects of SB 204070, a highly potent and selective 5‐HT4 receptor antagonist, on guinea‐pig distal colon
Authors: Wardle K.A. , Ellis E.S. , Baxter G.S. , Kennett G.A. , Gaster L.M. , Sanger G.J.
Published: July 2012

The pharmacology of a novel 5‐HT4 receptor antagonist, SB 204070 has been evaluated in the guinea‐pig isolated distal colon longitudinal muscle‐myenteric plexus (LMMP).

SB 204070 is a highly potent antagonist of 5‐HT‐evoked cholinergically‐mediated contractions in the guinea‐pig distal colon. Low concentrations (10–100 pm) produced a shift to the right of the curve (apparent pA2 10.8 ± 0.1) with no significant effect on the maximum response. With higher concentrations of SB 204070 (300 pm and above), the maximum response to 5‐HT was reduced.

When tested against the partial 5‐HT4 receptor agonist, BIMU 1, SB 204070 was active at similar low concentrations (10 pm and above) but produced a reduction in maximum, with no prior shift to the right of the curve, at all concentrations tested (10–300 pm).

The antagonism seen with SB 204070 is unlikely to be due to a non‐selective effect since high concentrations (10 nm and 1 μm) of the compound had no effect on cholinergically‐mediated contractions evoked by the nicotinic receptor agonist, DMPP, in the same preparation. SB 204070 is unlikely to be an irreversible antagonist since the effects of the compound could be reversed upon washing of the tissue.

Radioligand binding studies show that SB 204070 has a greater that 5000 fold selectivity for the 5‐HT4 receptor over 5‐HT1A, 5‐HT1D, 5‐HT1E, 5‐HT2A, 5‐HT2C, 5‐HT3, GABAA, BDZ, TBPS, A1 adenosine receptors, α1, α2, β1, β2 adrenoceptors and D1, D2 and D3 dopamine receptors.

SB 204070 is a highly potent, highly selective 5‐HT4 receptor antagonist and as such is an important new tool in evaluating the functional role of the 5‐HT4 receptor.
Comparison of antinociception induced by supraspinally administered l‐arginine and kyotorphin
Authors: Kawabata Atsufumi , Manabe Sachiko , Takagi Hiroshi
Published: July 2012

Intracerebroventricular (i.c.v.) or intracisternal (i.cist.) administration of kyotorphin (KTP), an endogenous Met‐enkephalin releaser, at 5 μg per mouse, and l‐arginine (l‐Arg), a possible KTP precursor, at 30 μg per mouse, elicited antinociception in mice to a similar extent, as assessed by the tail‐flick test.

Intracisternal preadministration of anti‐KTP serum abolished the effects of i.cist. KTP and i.c.v. or i.cist. l‐Arg, but not of i.c.v. KTP.

The antinociceptive effects of i.cist. KTP and of i.c.v. or i.cist. l‐Arg disappeared in reserpinized mice, whereas the effect of i.c.v. KTP was unaffected by treatment of mice with reserpine.

Intrathecal (i.t.) phentolamine markedly reduced the antinocieption induced by i.cist. KTP and by i.c.v. or i.cist. l‐Arg, but not by i.c.v. KTP.

Intrathecal methysergide attenuated the antinociceptive effects of i.cist. KTP, but not of i.c.v. KTP and i.c.v. or i.cist. l‐Arg.

These results suggest that the antinociception produced by i.cist. KTP, but not by i.c.v. KTP, is mediated by the brainstem‐spinal noradrenergic and 5‐hydroxytryptaminergic systems, and that l‐Arg given i.c.v. or i.cist. increases KTP formation in the lower brain, possibly the brainstem, resulting in antinociception mediated by the descending noradrenergic system. Therefore, the regional distribution of KTP receptors and KTP synthetase in the brain does not appear to be common.
The effect of 5‐HT1A receptor agonists on locomotor activity in the guinea‐pig
Authors: Evenden J.L.
Published: July 2012

The present study examined the effects of 8‐hydroxy‐2‐(din‐propylamino)tetralin (8‐OH‐DPAT), flesinoxan, ipsapirone and buspirone, all agonists at the 5‐HT1A receptor, on the locomotor activity of guinea‐pigs. The effects of these drugs were contrasted with those of the non‐selective 5‐HT agonist, 5‐methoxy‐N,N‐dimethyl tryptamine (5‐MeO‐DMT) and the dopamine D2 antagonist, raclopride.

8‐OH‐DPAT, flesinoxan and 5‐MeO‐DMT markedly increased the locomotor activity of naive, unhabituated guinea‐pigs in a dose‐dependent manner. Buspirone also did so, although to a lesser extent and for a shorter time. The doses at which this effect was seen were higher than those normally employed in rats. Ipsapirone and raclopride had no significant effects on locomotor activity.

The locomotor activity increasing effect of 1.0 mg kg−1 8‐OH‐DPAT was blocked by the selective 5‐HT1A antagonist (S)‐UH‐301 (3.0 and 10.0 mg kg−1), but not by (−)‐alprenolol (15.0 mg kg−1). Ipsapirone (30.0 mg kg−1) and raclopride (3.0 mg kg−1) antagonized 8‐OH‐DPAT‐induced locomotor activity but only to a small extent. The 5‐HT reuptake inhibitor, zimelidine (10.0 mg kg−1) had no effect.

The effect of the 5‐HT1A agonists in the guinea‐pig contrasts with the effects of 8‐OH‐DPAT on the locomotor activity of unhabituated rats and mice tested in the same apparatus, but are similar to the effects of 8‐OH‐DPAT on habituated rats, which show a low baseline of activity.

These results support the suggestion that 5‐HT1A agonists may have an intrinsic activating effect which may be masked by other effects of the drug (e.g. hypothermia, 5‐HT syndrome). The rank ordering of the 5‐HT1A agonists also suggests that the degree to which the drugs increase locomotor activity is related to their agonist efficacy at the postsynaptic 5‐HT1A receptor.
All‐or‐nothing responses to carbachol in single intestinal smooth muscle cells of rat
Authors: Ohta Toshio , Ito Shigeo , Nakazato Yoshikazu
Published: July 2012

Concentration‐response relationships for carbachol (CCh)‐induced increases in the cytosolic calcium concentration ([Ca2+]i) and membrane currents were studied by use of fura‐2 microfluorimetry and nystatin‐perforated whole‐cell recording in single smooth muscle cells isolated from rat intestine.

CCh produced an initial peak rise in [Ca2+]i followed by a small sustained rise. In individual cells, the peak rise in [Ca2+]i did not increase in amplitude even with increasing concentrations of CCh, though the threshold concentration varied in different cells. The initial peak rise in [Ca2+]i, but not the sustained rise, was due to the release of stored Ca2+, because it was unchanged after removal of external Ca2+ and the addition of nifedipine (1 μm) or La3+ (1 mm).

CCh elicited an outward and inward current in a cell dialyzed with a pipette solution containing KCl at a holding potential of – 30 mV and with one containing NaCl at – 60 mV, respectively. In individual cells, the amplitude of each current was similar in cells stimulated at over the threshold concentration of CCh, but the threshold was different among cells.

The percentage of cells showing Ca2+‐transient responses to CCh at given concentrations was similar to those showing current responses and contractile responses.

In thin muscle bundles, a concentration‐dependent contraction was evoked by CCh in the absence of external Ca2+. Its threshold was similar to those of Ca2+‐transient and current responses in single cells.

These results suggest that CCh‐induced release of stored Ca2+ takes place in an all‐or‐nothing fashion in individual cells of the rat intestinal smooth muscle.
Diosmin‐induced increase in sensitivity to Ca2+ of the smooth muscle contractile apparatus in the rat isolated femoral vein
Authors: Savineau Jean‐Pierre , Marthan Roger
Published: July 2012

The effect of diosmin, a flavone derivative, on the Ca2+ sensitivity of the venous contractile apparatus was investigated in chemically (β‐escin) skinned strips from the rat isolated femoral vein. Diosmin (0.5–10 μm) shifted to the left the concentration‐response curve to Ca2+ (0.05–5 μm). The maximal effect was observed in the presence of 1 μm diosmin which increased the contractile response evoked by 0.15 μm Ca2+ from 26.3% to 78.9% of the maximal Ca2+‐induced response. This work demonstrates that the venotonic action of diosmin involves an increase in the Ca2+ sensitivity of the contractile apparatus. Such a mechanism of action could represent a new and important means of therapeutic control of vasomotor activity.
Regional haemodynamic effects of dopamine and its prodrugs l‐dopa and gludopa in the rat and in the glycerol‐treated rat as a model for acute renal failure
Authors: Drieman J.C. , van Kan F.J.P.M. , Thijssen H.H.W. , van Essen H. , Smits J.F.M. , Boudier H.A.J. Struijker
Published: July 2012

In this study the renal selectivity of dopamine and its prodrugs l‐dopa and gludopa, with respect to their effects on regional blood flow, vascular resistance and central haemodynamics was investigated in normal rats and in rats with glycerol‐induced acute renal failure (ARF).

In normal, anaesthetized rats, dopamine as well as its prodrugs caused a dose‐dependent reduction of vascular resistance in the kidney (RR), mesentery (MR) and hindquarters (HQR) (dose range: dopamine: 0.1–5 μmol kg−1h−1; l‐dopa and gludopa: 1–200 μmol kg−1h−1). Blood pressure and heart rate were affected at the highest dose only.

Administration of glycerol induced a preferential renal vasoconstriction; renal blood flow (– 60%) and vascular resistance (+ 190%) were significantly more affected than MR (+ 40%) and HQR (+ 60%). This was only ameliorated by a low rate (10 μmol kg−1h−1) infusion of gludopa: the glycerol‐induced reduction of renal flow and increase in RR were significantly attenuated. A high dose of gludopa (100 μmol kg−1h−1) or any dose of l‐dopa or dopamine did not induce this beneficial effect. The glycerol‐induced increase in MR and HQR was not attenuated by any of the treatments used.

The results indicate that gludopa is not renally selective at a pharmacodynamic level in normal, anaesthetized rats. Contrary to this, a low dose of gludopa does cause a renal selective vasodilatation and reduction of RR in rats with glycerol‐induced ARF. This difference could be explained by a difference in renal vascular tone between normal rats and glycerol‐induced ARF rats. A high dose of gludopa does not cause these renal‐selective effects: renal resistance and renal flow are at the same level as following glycerol and saline. This is probably due to the systemic effects of the released dopamine.
Effects of Cl channel blockers on Ca‐activated chloride and potassium currents in smooth muscle cells from rabbit portal vein
Authors: Hogg R.C. , Wang Q. , Large W.A.
Published: July 2012

The effects of some chloride channel antagonists were studied on the calcium‐activated chloride current (ICl(ca)) in smooth muscle cells from the rabbit portal vein with the perforated patch technique.

4‐Acetamido‐4′‐isothiocyanatostilbene‐2,2′‐disulphonic acid (SITS) and 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulphonic acid (DIDS) reduced the amplitude of spontaneous transient inward currents (STICs, calcium‐activated chloride currents) in a concentration‐dependent manner. The concentrations required to reduce the amplitude by 50% (IC50) of STICs were 2.1 × 10−4m and 6.4 × 10−4m for DIDS and SITS, respectively. This effect was not voltage‐dependent.

The time constant of decay of STICs (τ), which is voltage‐dependent, was increased by about 30% by SITS and decreased by about 20% by DIDS. The effect of DIDS and SITS on τ was similar at holding potentials of − 50 and + 50 mV.

These compounds did not modify the characteristics of spontaneous transient outward currents (STOCs, calcium‐activated potassium currents).

DIDS and SITS decreased the amplitude of ICl(Ca) evoked by noradrenaline and caffeine less potently than STICs with IC50 values of 7.5 × 10−4m and 1.8 × 10−3m, respectively.

DIDS and SITS increased the calcium‐activated potassium current (IK(Ca)) evoked by noradrenaline and caffeine by 3–6 fold.

Anthracene‐9‐carboxylic acid (A‐9‐C) inhibited STICs in a voltage‐dependent fashion and was about 3 fold more active at + 50 mV than at − 50 mV. A‐9‐C increased STIC τ and this effect was enhanced by depolarization.

A‐9‐C also inhibited caffeine‐evoked ICl(ca) but less potently than STICs and also increased the evoked IK(ca) without altering spontaneous IK(ca).

The results from the present work are compared with the pharmacology of other chloride conductances and the mechanism of action of the chloride channel antagonists in vascular smooth muscle is discussed.
Phorbol ester‐induced M‐current suppression in bull‐frog sympathetic ganglion cells: insensitivity to kinase inhibitors
Authors: Chen Hsinyo , Jassar Balvinder S. , Kurenny Dmitry E. , Smith Peter A.
Published: July 2012

The effects of 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG), phorbol 12‐myristate 13‐acetate (PMA), 4‐α‐phorbol and muscarine on B‐neurones from bull‐frog sympathetic ganglion were studied by means of whole‐cell patch‐clamp recording. With the exception of 4‐α‐phorbol, all of these agonists reduced the steady‐state outward current recorded at −30 mV as a result of suppression of a voltage‐dependent, non‐inactivating K+‐current, the M‐current, (IM).

Of the cells tested, 34% displayed bona fide responses to OAG (20 μm). The chance of recording a response was not decreased when the protein kinase inhibitor, 1‐(5‐isoquinolinylsulphonyl)‐2‐methyl‐piperazine (H‐7; 50 or 75 μm) was included simultaneously in the extracellular solution and in the pipette solution.

The presence of 50 μm H‐7 on both sides of the membrane or 500 nm staurosporine in the pipette solution did not prevent responses to brief (1–2 min) or prolonged (>20 min) applications of PMA.

Brief (1–2 min) extracellular application of H‐7 (300 μm) suppressed IM by about 29%.

The most likely explanation of these data is that PMA and OAG modulate IM via a mechanism that is independent of protein kinase C (PKC). The availability of such a mechanism poses new questions as to the mechanism of muscarine‐induced IM suppression.
Role of intrathecal tachykinins for micturition in unanaesthetized rats with and without bladder outlet obstruction
Authors: Ishizuka Osamu , Igawa Yasuhiko , Lecci Alessandro , Maggi Carlo Alberto , Mattiasson Anders , Andersson Karl‐Erik
Published: July 2012

The effects on micturition of RP 67,580, a selective NK1 receptor antagonist, and SR 48,968, a highly potent antagonist at NK2 receptor sites, given intrathecally (i.t.) or intra‐arterially (i.a.) near the bladder, were investigated in unanaesthetized rats with and without bladder outlet obstruction.

In normal rats, RP 67,580, given i.t. in doses of 2 and 20 nmol per rat, decreased micturition pressure, but did not change other cystometric parameters. After 20 nmol of RP 67,580, dribbling incontinence due to retention was observed in 1 out of 7 animals. This effect was reversible. I.t. RP 67,580 in a dose of 2 nmol, had no effect on hyperactivity induced by intravesically instilled capsaicin.

In animals with bladder hypertrophy secondary to outflow obstruction, RP 67,580, given i.t. in a dose of 2 nmol per rat, decreased the micturiton pressure, but had no effect on other cystometric parameters. After 20 nmol, dribbling incontinence due to retention was observed in 5 out of 7 animals.

RP 67,580, given i.a. in a dose of 4 nmol, had little effect on the cystometric parameters investigated, both in normal animals and rats with bladder hypertrophy.

SR 48,968, given i.t. in doses of 2 and 20 nmol per rat, had no clear‐cut effects on the micturition pattern in normal rats, or rats with bladder hypertrophy. However, the drug reduced capsaicin‐induced bladder hyperactivity. When given i.a. in a dose of 4 nmol, SR 48,968 had no effect on cystometric parameters in normal rats or rats with bladder hypertrophy.

The effects of both RP 67,580 and SR 48,968 were stereoselective, their enantiomers (RP 68,651 and SR 48,965) being inactive.

These results thus suggest that at the spinal level there is a tachykinin involvement (via NK1 receptors) in the micturition reflex induced by bladder filling, both in normal rats, and, more clearly, in animals with bladder hypertrophy secondary to outflow obstruction. The bladder response to filling was not influenced by blockade of vesical NK1 and NK2 receptors. On the other hand, the bladder hyperactivity evoked by intravesical capsaicin seems to involve NK2 receptors both at the bladder and spinal levels.
Infection by HIV‐1 blocked by binding of dextrin 2‐sulphate to the cell surface of activated human peripheral blood mononuclear cells and cultured T‐cells
Authors: Shaunak Sunil , Gooderham Nigel J. , Edwards Robert J. , Payvandi Nassrin , Javan Caroline M. , Baggett Neil , MacDermot John , Weber Jonathan N. , Davies Donald S.
Published: July 2012

Structural analogues of a sulphated polysaccharide, dextrin sulphate, were synthesized and tested for their ability to block infection by HIV‐1. Using the T‐cell lines, C8166 and HPB‐ALL, and the laboratory adapted strains of HIV‐1.MN, HIV‐1.IIIb and HIV‐1.RF, dextrin 2‐sulphate (D2S) combined the best combination of high anti‐HIV‐1 activity (95% inhibitory concentration (IC95) = 230 nM) and low anticoagulant activity. It also blocked infection of activated peripheral blood mononuclear (PBMN) cells by five primary viral isolates at an IC95 of 230–3700 nM depending upon the primary viral isolate tested.

In saturation binding studies, [3H]‐D2S bound to a cell surface protein on HPB‐ALL cells in a specific and saturable manner with a Kd of 82 ± 14 nM and a Bmax of 4.8 ± 0.3 pmol/106 cells. It bound to other human T‐cell lines in a similar manner.

There was very little binding of [3H]‐D2S to freshly isolated PBMN cells (Bmax 0.18 ± 0.03 pmol/106 cells) and these cells could not be infected by HIV‐1. Culture of PBMN cells in lymphocyte growth medium (LGM) containing IL‐2 did not significantly change the Bmax of [3H]‐D2S. In contrast, PBMN cells which had been cultured with phytohaemagglutinin (PHA; 5 μg ml−1) for 72 h had a Bmax of [3H]‐D2S binding of 7.2 ± 0.1 pmol/106 cells and these cells could be infected by HIV‐1. Removal of the PHA and further culture of the PBMN cells in LGM containing IL‐2 resulted in a fall in the Bmax to 2.0 ± 0.1 pmol/106 cells. The Kd of binding did not change significantly during the course of these experiments.

[3H]‐D2S did not bind to freshly isolated erythrocytes or to erythrocytes which had been cultured in PHA for 72 h.

These results suggest that there is a relationship between the expression of the [3H]‐D2S binding protein on the plasma membrane of PBMN cells and the susceptibility of these cells to infection by HIV‐1.
Natriuretic peptide‐induced cyclic GMP accumulation in adult guinea‐pig cerebellar slices
Authors: Hernández Félix , Alexander Stephen P.H. , Kendall David A.
Published: July 2012

Second messenger responses to natriuretic peptides were studied in guinea‐pig cerebellar slices by use of radioactive precursors.

The rank order of potency of the different natriuretic peptides in generating [3H]‐guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) was atrial natriuretic peptide (ANP) > brain natriuretic peptide (BNP) > C‐type natriuretic peptide (CNP) with EC50 values of 19.5 ± 8.8 nM for ANP and 169 ± 41 nM for BNP. CNP induced [3H]‐cyclic GMP accumulation only at concentrations greater than 1 μm.

An additive response to ANP (1 μm) was observed in the presence of the adenosine receptor agonist, 5′‐N‐ethylcarboxamidoadenosine (NECA, 10 μm) or the soluble guanylyl cyclase activator, sodium nitroprusside (SNP, 100 μm) for [3H]‐cyclic GMP accumulation.

ANP, BNP and CNP (all at 1 μm) failed to alter significantly either basal‐, forskolin‐ (10 μm), isoprenaline‐ (100 μm), or NECA‐ (10 μm) induced [3H]‐cyclic AMP generation. Natriuretic peptides also did not change the [3H]‐cyclic AMP steady‐state reached after 10 min of treatment with 10 μm forskolin.

Natriuretic peptides failed to elicit significant accumulation of [3H]‐inositol phosphates at concentrations up to 10 μm.

These data are consistent with the presence of ANPA, rather than ANPB or clearance receptors (C‐receptors), linked to second messenger cascades in guinea‐pig cerebellar slices.
Antagonism by riluzole of entry of calcium evoked by NMDA and veratridine in rat cultured granule cells: evidence for a dual mechanism of action
Authors: Hubert J.P. , Delumeau J.C. , Glowinski J. , Prémont J. , Doble A.
Published: July 2012

Intracellular calcium levels were measured in cultured cerebellar granule cells of the rat by use of the fluorescent dye, indo‐1/AM.

Intracellular calcium levels were increased by depolarizing stimuli such as N‐methyl‐d‐aspartate (NMDA) (100 μm), glutamic acid (20 μm), and veratridine (10 μm). This increase was essentially due to entry of external calcium.

Riluzole (10 μm) blocked responses to all the depolarizing agents.

Riluzole could still block the increase in intracellular calcium evoked by NMDA or glutamic acid when sodium channels were blocked by tetrodotoxin, suggesting that this effect is not mediated by a direct action of riluzole on the voltage‐dependent sodium channel.

Pretreatment of the cells with pertussis toxin (0.1 μg ml−1) did not modify the increases in intracellular calcium evoked by NMDA, glutamic acid or veratridine.

In pertussis toxin‐treated cells, riluzole could no longer block responses to excitatory amino acids, but still blocked responses to veratridine.

It is concluded that riluzole has a dual action on cerebellar granule cells, both blocking voltage‐dependent sodium channels and interfering with NMDA receptor‐mediated responses via a pertussis toxin‐sensitive mechanism. Furthermore, these two processes have been shown to be independent.
Pharmacological characteristics of liriodenine, isolated from Fissistigma glaucescens, a novel muscarinic receptor antagonist in guinea‐pigs
Authors: Lin Chien‐Huang , Chang Gwo‐Jyh , Su Ming‐Jai , Wu Yang‐Chang , Teng Che‐Ming , Ko Feng‐Nien
Published: July 2012

The pharmacological activities of liriodenine, isolated from Fissistigma glaucescens, were determined in isolated trachea, ileum and cardiac tissues of guinea‐pigs.

Liriodenine was found to be a muscarinic receptor antagonist in guinea‐pig trachea as revealed by its competitive antagonism of carbachol (pA2 = 6.22 ± 0.08)‐induced smooth muscle contraction. It was slightly more potent than methoctramine (pA2 = 5.92 ± 0.05), but was less potent than atropine (pA2 = 8.93 ± 0.07), pirenzepine (pA2 = 7.02 ± 0.09) and 4‐diphenylacetoxy‐N‐methylpiperidine (4‐DAMP, pA2 = 8.72 ± 0.07).

Liriodenine was also a muscarinic antagonist in guinea‐pig ileum (pA2 = 6.36 ± 0.10) with a pA2 value that closely resembled that obtained in the trachea.

Liriodenine was 10 fold less potent in atrial preparations (left atria, pA2 = 5.24 ± 0.04; right atria, pA2 = 5.35 ± 0.09 and 5.28 ± 0.07 for inotropic and chronotropic effects, respectively) than in smooth muscle preparations.

High concentration of liriodenine (300 μm) partially depressed the contractions induced by U‐46619, histamine, prostaglandin F2a, neurokinin A, leukotriene C4 and high K+ in the guinea‐pig trachea. The inhibitions were characterized by a rightward shift in the concentration‐response curves with suppression of their maximal contraction.

High concentration of liriodenine (300 μm) did not affect U‐46619‐ or neurokinin A‐induced tracheal contraction in the presence of nifedipine (1 μm) or in Ca2+‐free (containing 0.2 mm EGTA) medium.

Neither cyclic AMP nor cyclic GMP content of guinea‐pig trachealis was changed by liriodenine (30–300 μm).

8 It is concluded that liriodenine is a selective muscarinic receptor antagonist in isolated trachea, ileum and cardiac tissues of guinea‐pigs. It is more potent in smooth muscle than in cardiac preparations. It also acts as a blocker of voltage‐dependent Ca2+ channels at a high concentration (300 μm).
Effects of caffeine on intracellular sodium activity in cardiac Purkinje fibres: relation to force
Authors: Iacono Giovanni , Vassalle Mario
Published: July 2012

An increase in cytoplasmic calcium by caffeine would lead to Ca extrusion via the Na/Ca exchange. The hypotheses were investigated that, as a consequence, caffeine might increase intracellular sodium activity (aiNa) and that the relation between aiNa and force might be conditioned by the Ca load.

Action potential, aiNa and contractile force were recorded in sheep Purkinje fibres during exposure to caffeine under conditions that decrease or increase the Ca load by different mechanisms.

In Tyrode solution, caffeine (8 mm) increased aiNa from 8.05 ± 0.20 to 10.52 ± 0.40 mm (+ 30.5%) and had a triphasic effect on force: an initial transient increase (+ 93.6%), a subsequent decrease (‐37.1%) (negative inotropy) and slow partial recovery (+8.9%).

Decreasing the Ca load by means of manganese (1 mm) decreased aiNa and force. Adding caffeine re‐increased aiNa and no longer caused a negative inotropic action. Cadmium (0.2 mm) also decreased aiNa, and caffeine reincreased it although far less than in Tyrode solution.

High [K]o (10 mm) and tetrodotoxin (5 μm) decreased aiNa as well as force. In their presence, caffeine re‐increased aiNa and no longer had a negative inotropic action.

Increasing the Ca load by means of high [Ca]o (8.1 mm) increased force (+ 195%) and decreased aiNa, (‐20.3%). Adding caffeine re‐increased aiNa (+ 28.1%), but immediately decreased force (‐32.3%).

Addition of pyruvate (10 mm) to caffeine increased force, as it does in the presence of Ca overload.

Noradrenaline (0.1–1 μm) decreased aiNa and increased contractile force. In its presence, caffeine decreased aiNa further and increased force.

It is concluded that caffeine increases aiNa, even during the negative inotropic effect. The decrease in force appears to depend on Ca load. Thus, caffeine no longer decreases force under conditions that decrease Ca load (Mn, high [K]o, TTX) and immediately decreases force when the Ca load is increased (high [Ca]o). However, in the presence of noradrenaline, caffeine decreases aiNa and markedly increases force, as the Ca load is increased, but Ca can be removed from the cytoplasm into the SR.
Involvement of enzymatic degradation in the inactivation of tachykinin neurotransmitters in neonatal rat spinal cord
Authors: Suzuki H. , Yoshioka K. , Yanagisawa M. , Urayama O. , Kurihara T. , Hosoki R. , Saito K. , Otsuka M.
Published: July 2012

The possible involvement of enzymatic degradation in the inactivation of tachykinin neurotransmitters was examined in the spinal cord of the neonatal rat.

The magnitude of substance P (SP)‐ or neurokinin A (NKA)‐evoked depolarization of a lumbar ventral root in the isolated spinal cord preparation was increased by a mixture of peptidase inhibitors, consisting of actinonin (6 μm), arphamenine B (6 μm), bestatin (10 μm), captopril (10 μm) and thiorphan (0.3 μm). The mixture augmented the response to NKA more markedly than that to SP.

In the isolated spinal cord‐cutaneous nerve preparation, the saphenous nerve‐evoked slow depolarization of the L3 ventral root was augmented by the mixture of peptidase inhibitors in the presence of naloxone (0.5 μm) but not in the presence of both naloxone and a tachykinin receptor antagonist, GR71251 (5 μm).

Application of capsaicin (0.5 μm) for 6 min to the spinal cord evoked an increase in the release of SP from the spinal cord. The amount of SP released was significantly augmented by the mixture of peptidase inhibitors.

Synaptic membrane fractions were prepared from neonatal rat spinal cords. These fractions showed degrading activities for SP and NKA and the activities were inhibited by the mixture of peptidase inhibitors. The degrading activity for NKA was higher than that for SP and the inhibitory effect of the mixture for NKA was more marked than that for SP. Although some other fractions obtained from homogenates of spinal cords showed higher degrading activities for SP, these activities were insensitive to the mixture of peptidase inhibitors.

Effects of individual peptidase inhibitors on the enzymatic degradation of SP and NKA by synaptic membrane fractions were examined. Thiorphan, actinonin and captopril inhibited SP degradation, while thiorphan and actinonin, but not captopril, inhibited NKA degradation. The potency of the inhibition of each peptidase inhibitor was lower than that of the mixture.

The present results suggest that enzymatic degradation is involved in the inactivation of tachykinin neurotransmitters in the spinal cord of the neonatal rat.
Synergistic inhibition by BQ‐123 and BQ‐788 of endothelin‐1‐induced contractions of the rabbit pulmonary artery
Authors: Fukuroda Takahiro , Ozaki Satoshi , Ihara Masaki , Ishikawa Kiyofumi , Yano Mitsuo , Nishikibe Masaru
Published: July 2012

In the rabbit isolated pulmonary artery, neither the ETA receptor antagonist, BQ‐123 (10 μm), nor the ETB receptor antagonist, BQ‐788 (10 μm), inhibited the contractions induced by 1 nm endothelin‐1 (ET‐1). However, the combination of BQ‐123 and BQ‐788 completely inhibited the ET‐1‐induced contraction. In contrast, the ETB‐selective agonist, sarafotoxin S6c (1 nm)‐induced contraction was completely inhibited by BQ‐788 but not by BQ‐123. In receptor binding assays, [125I]‐ET‐1 specific binding to pulmonary arterial membranes was inhibited by BQ‐123 (1 μm) by approximately 20% and additive treatment with BQ‐788 (1 μm) completely inhibited the BQ‐123‐resistant component of [125I]‐ET‐1 specific binding. The present study demonstrates synergistic inhibition by BQ‐123 and BQ‐788 of ET‐1‐induced contraction of the rabbit pulmonary artery and the coexistence of ETA and ETB receptors, suggesting that the activation of either only ETA or only ETB receptors may be sufficient to cause complete vasoconstriction. Therefore, blockade of both receptor subtypes would be necessary for the inhibition of some ETA/ETB composite types of responses.