Evidence for ATP as a cotransmitter in dog mesenteric artery

Contractile responses of the dog mesenteric artery were obtained (after removal of endothelium) to transmural stimulation of the perivascular nerves and to exogenous application of ATP, noradrenaline, dopamine, 5-hydroxy-tryptamine and high potassium solution. The alpha-adrenoceptor antagonists prazosin and phentolamine preferentially reduced the response to noradrenaline and the secondary phase of the biphasic contractile response to nerve stimulation, whilst the addition of alpha, beta-methylene-ATP, which selectively desensitizes P2-purinoceptors, reduced only the contractions to ATP and the portion of the nerve-mediated response which was resistant to the alpha-adrenoceptor antagonists. The responses to nerve stimulation were reduced by the selective P1-purinoceptor agonist 2-chloroadenosine, and its effect was reversed by the P1-purinoceptor antagonist 8-phenyltheophylline. These results suggest that in dog mesenteric artery part of the response to sympathetic nerve stimulation is mediated by ATP acting on P1-purinoceptors on the arterial smooth muscle, and that P1-purinoceptors on the sympathetic nerve terminal can inhibit release of the neurotransmitters.     

A comparison between the pattern of dopamine and noradrenaline release from sympathetic neurones of the dog mesenteric artery.

The release of dopamine and noradrenaline (NA), from the main trunk of the mesenteric artery and its proximal branches elicited by electrical nerve stimulation and K+, has been measured by using high pressure liquid chromatography with electrochemical detection. Both stimuli released dopamine and NA. With the main trunk of the mesenteric artery, dopamine represented 8% of the NA tissue content; the dopamine/NA ratio in the catecholamine overflow caused by nerve stimulation or K+-induced depolarization also averaged 8%. For the proximal branches the tissue dopamine/NA ratio was significantly greater than that observed to occur in the overflow caused by nerve stimulation and K+. When the perifusion with a K+-enriched medium was extended to 120 min the amount of NA released from both the main trunk and the proximal branches progressively declined. The same pattern of release was observed for dopamine in the main trunk, whereas for the proximal branches dopamine overflow did not decline throughout the perifusion period. The addition of alpha-methyl-p-tyrosine did not change the pattern of amine overflow. Our interpretation of these results is that both dopamine and NA are derived from the same sympathetic neurone. In the proximal branches of the mesenteric artery dopamine and NA appear to be in two different storage structures, whereas in the main trunk both dopamine and NA are located in only one storage structure.     

Evidence for sympathetic, purinergic transmission in the mesenteric artery of the dog.

Electrical transmural stimulation of isolated mesenteric artery of the dog produced a transient contraction which consisted of adrenergic and non-adrenergic components. In contrast to the adrenergic component, the nonadrenergic component was resistant to prazosin and other adrenoceptor-blocking agents. However, the nonadrenergic component was completely blocked by guanethidine and by desensitization with alpha,beteta-methylene-ATP (alpha,beta-MeATP). Desensitization induced by alpha,beta-MeATP also inhibited the contractile response to ATP but not the adrenergic responses induced by electrical transmural stimulation and exogenous noradrenaline. These results suggest that the nonadrenergic contraction induced by electrical transmural stimulation is a sympathetic, purinergic response.     

Muscarinic inhibition of acetylcholine release from a novel in vitro preparation of the guinea-pig trachea

Summary An isolated preparation of the guinea-pig trachea is described which allows the simultaneous measurement of acetylcholine release and smooth muscle contraction. Incubation of the epithelium-free preparation with [³H]choline resulted in the formation of [³H]acetylcholine. Electrical stimulation caused the release of [³H]acetylcholine and a contractile response. Tetrodotoxin and omission of calcium from the medium abolished both the evoked release and contractions.The muscarinic agonists oxotremorine, carbachol and pilocarpine concentration-dependently inhibited the electrically evoked acetylcholine release and contracted the tracheal smooth muscle. Pre- and postsynaptic EC₅₀ values for a given agonist were not different. Atropine (100 nmol/l) significantly faciliated the evoked acetylcholine release. A concentration of 10 nmol/l atropine did not change the evoked release but antagonized the inhibitory effect of oxotremorine. It is concluded that presynaptic muscarine autoreceptors inhibit the release of acetylcholine from parasympathetic nerves of the guinea-pig trachea.Send offprint requests to G. D'Agostino at the above address     

Evidence for verapamil-induced functional inhibition of noradrenergic neurotransmission in vivo

Summary Contractions of the cat nictitating membrane have been used to explore the effects of calcium channel blockers on neurotransmission in vivo, by comparing the effects of verapamil and nifedipine on contractions of nictitating membrane following either electrical stimulation of the superior cervical ganglion or intravenous injection of the -adrenoceptor agonist phenylephrine. Verapamil (0.3, 0.6 and 1.2 mg/kg, iv) produced a dose related and reversible inhibition of stimulation induced contractions but did not affect phenylephrine responses of nictitating membrane. Intravenous nifedipine (10, 20 and 40 g/kg) produced inconsistent effects on both stimulation- and phenylephrine-induced contractions of the nictitating membrane. Thus only verapamil appears to selectively affect noradrenergic neurotransmission in this model, possibly by altering the neurotransmitter release from the terminals innervating the nictitating membrane in the cat. Send offprint requests to S. Gurtu at the above address     

Evidence that the efficacy (intrinsic activity) of fenoterol is higher than that of salbutamol on beta-adrenoceptors in guinea-pig trachea.

The functional antagonism of carbachol by fenoterol and salbutamol (beta-adrenoceptor agonists) has been used to demonstrate that the efficacy (intrinsic activity) of fenoterol was about twice that of salbutamol on guinea-pig trachea. The mean maximum shifts of the carbachol concentration--response lines by fenoterol and salbutamol were (log units) 1.07 +/- 0.07 (n = 5) and 0.64 +/- 0.07 (n = 5) respectively. This difference in their efficacies could be demonstrated as differences in maximum relaxation on tracheal preparations contracted with carbachol, although this was dependent on the concentration of carbachol used. beta-Adrenoceptor blocking properties of salbutamol (1 mM) but not fenoterol (1 mM) could be demonstrated on trachea in that salbutamol, but not fenoterol, antagonised the shift in the carbachol concentration--response line produced by isoprenaline. The implications of these findings in relation to the use of fenoterol and salbutamol as bronchodilators is discussed.     

Evidence for dopaminergic co-transmission in dog mesenteric arterial vessels.

1. The overflow of dopamine and noradrenaline (NA) from the main trunk of the dog mesenteric artery and its proximal branches during prolonged depolarization (120 min) by K+ (52 mM) was quantified by high performance liquid chromatography with electrochemical detection. 2. K+-induced depolarization resulted in release of both dopamine and NA. The amount of NA released from both blood vessels declined progressively throughout the experiment. In the main trunk the same pattern of release was observed for dopamine, whereas in the proximal branches the overflow of dopamine increased throughout the experiment. 3. The addition of phentolamine (0.2 microM) to the perifusion fluid increased the overflow of both amines. In the presence of sulpiride (1 microM) the overflow of dopamine and NA was found to be increased in the proximal branches, but not in the main trunk. The addition of phentolamine to sulpiride caused a further increase in amine overflow in proximal branches, but not in the main trunk. 4. The addition of alpha-methyl-p-tyrosine (50 microM) to the perifusion fluid caused a decrease in the amounts of dopamine and NA released from both preparations. In alpha-methyl-p-tyrosine-treated preparations phentolamine increased amine overflow to the same extent as in experiments without tyrosine hydroxylase inhibition. The increasing effect of sulpiride on the overflow of dopamine and NA from the proximal branches was completely abolished after alpha-methyl-p-tyrosine. 5. The results presented suggest that in the proximal branches of the dog mesenteric artery, dopamine beta-hydroxylase represents a rate limiting step in the synthesis of NA; dopamine, through activation of prejunctional dopamine receptors acts like a prejunctional co-transmitter in the control of transmitter release, but only newly-synthesized dopamine appears to be responsible for this effect.     

Evidence for an intrastriatal GABA-ergic influence on dopamine neurones of the cat

In the gallamine-immobilized cat, dopamine (DA) and acetylcholine (ACh) liberated within the caudate nucleus were collected by means of a push-pull cannula and measured radioenzymatically.The release of DA was markedly enhanced during perfusion with Ringer solution containing bicuculline or picrotoxin. In contrast, the ACh liberation was only slightly increased by bicuculline and remained unchanged after picrotoxin. In homogenates of the caudate nucleus perfused with Ringer plus bicuculline, the acetylcholine-esterase activity was slightly reduced whereas it was unaffected after picrotoxin. Addition of GABA to the Ringer solution reduced the bicuculline- and the picrotoxininduced enhancement in DA release. GABA alone diminished the spontaneous DA output.These data provide evidence for the presence of GABA-ergic terminals within the neostriatum and their possible influence on the DA neurones; this influence is probably not mediated by changes of an intrastriatal cholinergic activity.     

Modulation by noradrenaline and yohimbine of noradrenergic transmission in the guinea-pig mesenteric artery

In the guinea-pig mesenteric artery, transmitter release modulated by noradrenaline (NA) or yohimbine was estimated from changes in amplitude of the excitatory junction potential (e.j.p.) recorded from smooth muscle cells. NA decreased the amplitude of the e.j.p. with no change in the facilitation. Yohimbine antagonized the effect of NA on the e.j.p. amplitude and enhanced the facilitation of e.j.p.; the latter action was not antagonized by NA. TTX-resistant e.j.p.s evoked by stronger intensity of stimuli were not affected by NA or yohimbine. It is concluded that NA inhibits and yohimbine enhances the release of transmitter, and that the latter event involves prejunctional alpha-adrenoceptor-dependent and -independent processes.     

Modulation of noradrenergic transmission in the rabbit ear artery by dopamine.

1. The effects of dopamine on vasoconstrictor responses to field stimulation of sympathetic nerves and to exogenous noradrenaline were studied in the isolated ear artery of the rabbit. Responses to noradrenaline were unchanged at the start of the dopamine infusions but were enhanced as the infusions continued and also after cessation of the infustion. 2 Dopamine (0.5 muM) reduced the stimulation-induced efflux of tritium from segments of ear artery labelled with [3H]-noradrenaline. The reduction persisted during 65 min of dopamine infusion, after which time the vasoconstrictor responses had generally recovered to 93% of control level. On ceasing the infusion, the stimulation-induced efflux and the vasoconstrictor responses were enhanced. 3 Metoclopramide, haloperidol and ergometrine, each in a concentration of 0.2 muM, prevented the inhibitory effect of 0.5 muM dopamine on the stimulation-induced tritium release, but not the inhibitory effect of 0.5 muM noradrenaline. Phenoxybenzamine (0.2 and 1 muM) and phentolamine (1 muM) prevented the inhibitory effects of both noradrenaline and dopamine on the stimulation-induced efflux, and phentolamine (0.2 muM) prevented the inhibition of the stimulation-induced release by noradrenaline but only partially prevented the inhibitory effect of dopamine on the stimulation-induced efflux. 4 A possible role for dopamine in the modulation of noradrenergic transmission is suggested.     

Effects of dopamine on neuromuscular transmission in the guinea-pig mesenteric artery

The effects of dopamine on neuromuscular transmission in the guinea-pig mesenteric artery were investigated using a microelectrode method. Dopamine did not modify the membrane potential or the membrane resistance of the smooth muscle, but did reduce the amplitude of excitatory junction potentials (e.j.p.) and enhance the facilitation of e.j.p. produced by repetitive stimulation. Phentolamine (10(-7) M) enhanced the amplitude and the facilitation of the e.j.p., and with the addition of dopamine (10(-6) M) there was a reduction in the amplitude of e.j.p. but not in the facilitation. Haloperidol and sulpiride (greater than 10(-6) M) increased the amplitude of e.j.p. without altering the postjunctional membrane properties. Haloperidol and sulpiride did not increase the facilitation of e.j.p. produced by repetitive stimulation. In the presence of haloperidol or sulpiride (10(-5) M), dopamine (10(-6) M) did not suppress the amplitude of the e.j.p. These results indicate that in the guinea-pig mesenteric artery, dopamine inhibits the release of transmitter at the presynaptic membrane.     

Evidence for M1 muscarinic cholinoceptors mediating facilitation of noradrenaline release in guinea-pig carotid artery

Summary The muscarinic agonists acetylcholine (150 mol/l), carbachol (1–10 mol/l) and McN-A-343 (1–50 mol/l, selective for M₁ receptors) increased, in a concentration-dependent manner, the electrically-evoked tritium overflow from guinea-pig carotid arteries preincubated with [³H]-noradrenaline. The increase caused by acetylcholine was not modified by hexamethonium (300 mol/l) but was reduced by the muscarinic receptor antagonists methylatropinium (0.5 and 1 nmol/l, nonselective), pirenzepine (1 and 5 mol/l, M₁-selective), methoctramine (1 and 5 mol/l, M₂-selective) and pfluoro-hexahydro-sila-difenidol (0.1–1 mol/l, M₃-selective). The order of potencies (expressed as negative logarithms of concentrations that reduced by 50% the facilitatory effect of acetylcholine) was: methylatropinium (9.93) > pirenzepine (8.83) > p-fluoro-hexahydro-siladifenidol (6.81) methoctramine (6.20). These results demonstrate the existence of facilitatory M₁ receptors modulating noradrenaline release in blood vessels. Correspondence to M. Salaices at the above address     

Studies on the action and mechanism of action of oxyfedrine on isolated tracheal chains

Oxyfedrine, a β-sympathomimetic drug, did not affect isolated rat and rabbit trachea in concentration from 2.86 × 10^?8 to 2.86 × 10^?4 M, but on the guinea-pig trachea, it caused a dose dependent relaxation of natural tone in lower concentrations (1.79 × 10^?7 to 2.86 × 10^?6 M. In higher concentrations (1.14 × 10^?5 to 2.86 × 10^?4 M), however, a contraction was observed, which was also dose dependent. This contraction was not affected by atropine, lysergic acid diethylamide or by pretreatment with reserpine but was blocked by antihistaminics (isothipendyl and clemastine). Adrenaline, noradrenaline, phenylephrine and isoprenaline did not contract the guinea-pig trachea, whereas contractions were observed after high concentrations of norephedrine, amphetamine, ephedrine and tyramine. These contractions were also unaffected by reserpine pretreatment.It is concluded that the contraction of the guinea-pig trachea by oxyfedrine is related to its structural relationship to the phenylethylamines and might be due to histamine release, an action on histamine receptors or a histamine-like action.     

Pharmacological evidence for a stimulation of dopamine neurons by noradrenaline neurons in the brain.

The effects of yohimbine (3 mg/kg i.p.), phenoxybenzamine (20 mg/kg i.p.) and clonidine (0.1 mg/kg i.p.) on the synthesis and the utilization of dopamine and noradrenaline in the central nervous system of rats were investigated. Dopa accumulation following decarboxylase inhibition and the alpha-methyltyrosine-induced disappearance of the amines were used as the measure of these effects. The synthesis and the utilization of dopamine and noradrenaline were accelerated by yohimbine. Clonidine plus phenoxybenzamine inhibited the synthesis and utilization of dopamine and the combination also partly antagonized the effects of yohimbine on the trunover of dopamine. The effects of the three drugs on the synthesis and utilization of dopamine might be secondary to their actions on alpha-adrenoreceptors of noradrenaline synapses since, at the doses used, yohimbine increases the release of noradrenaline, phenoxybenzamine blocks postsynapptic noradrenaline receptors and clonidine reduces the release of noradrenaline. This hypothesis is supported by the findings that yohimbine and phenoxybenzamine did not change the increased synthesis of dopamine in reserpine-treated rats and that clonidine did not inhibit the increased synthesis of dopamine after axotomy or treatment with reserpine.     

Comparison of the effects of caffeine and procaine on noradrenergic transmission in the guinea-pig mesenteric artery.

The effects of caffeine and procaine on noradrenergic transmission in the guinea-pig mesenteric artery were investigated by recording electrical responses of smooth muscle cells and by measuring the outflow of noradrenaline (NA) and 3,4-dihydroxyphenylglycol (DOPEG) induced by perivascular nerve stimulation. Caffeine possessed dual actions on the membrane, i.e., at low concentrations (2.5 X 10(-4)-5 X 10(-4)M), it hyperpolarized the membrane and decreased the membrane resistance and at high concentrations (over 2.5 X 10(-3)M) it depolarized the membrane and increased the membrane resistance. Procaine (over 10(-4)M) consistently depolarized the membrane and increased the membrane resistance. The amplitude of the excitatory junction potential (e.j.p.) produced by perivascular nerve stimulation was increased by low concentrations of procaine (2.5 X 10(-5)-10(-4)M) or high concentrations (10(-3)-5 X 10(-3)M) of caffeine and was decreased by low concentrations of caffeine (2.5 X 10(-5)-10(-4)M) or high concentrations of procaine (5 X 10(-4)-10(-3)M). Higher concentrations of caffeine (over 5 X 10(-3)M) induced a spike potential on the e.j.p., while higher concentrations of procaine (over 2.5 X 10(-3)M) inhibited the generation of e.j.ps. Facilitation of e.j.ps produced by repetitive stimulation of perivascular nerves remained unchanged by caffeine, while it was enhanced by procaine at any given concentration (caffeine 2.5 X 10(-4)-10(-3)M; procaine 10(-4)-10(-3)M). The membrane depolarization produced by exogenously applied NA (10(-5)M) was not blocked by pretreatment with procaine. Conduction velocity of perivascular nerve excitation remained unchanged by application of caffeine (up to 5 X 10(-3)M), and was reduced by application of procaine (over 2.5 X 10(-4)M). Outflow of NA during perivascular nerve stimulation remained unchanged by caffeine (10(-4)-3 X 10(-3)M), while it was enhanced by procaine (over 2.5 X 10(-4)M). The outflow of DOPEG was slightly reduced by caffeine (10(-3)-5 X 10(-3)M) and by lower concentrations of procaine (10(-4)-2.5 X 10(-4)M) but was not altered by higher concentrations of procaine (10(-3)-5 X 10(-3)M). It is concluded that in the guinea-pig mesenteric artery, high concentrations of caffeine (over 10(-3)M) increased the e.j.p. amplitude which might be due to an increase in membrane resistance of the smooth muscle cells. No marked effect of caffeine was observed on transmitter release from the nerve terminals. Procaine (over 2.5 X 10(-4)M) increased transmitter release from perivascular nerves and blocked the re-uptake mechanism of released NA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of low temperature and pharmacological interventions on the responses of the isolated guinea-pig trachea

Summary Cooling the guinea-pig isolated trachea from 37°C to 20°C virtually abolished the response to CaCl₂ (in K⁺-depolarized tissues) and depressed that to histamine (about 75% reduction), KCl and 5-hydroxytryptamine (around 50% inhibition) while the response to acetylcholine remained unaffected. A further cooling to 10°C was necessary to inhibit acetylcholine-induced contractions. Hyporesponsiveness to spasmogens by cooling was not associated with subsensitivity (increased EC₅₀) except for 5-hydroxytryptamine. Contractile responses to KCl (50 mmol/l), histamine (1 mmol/l) and 5-hydroxytryptamine (0.1 mmol/l) in a Ca²⁺-free EGTA (0.1 mmol/l)-containing solution were inhibited by cooling to 20°C but responses to acetylcholine (1 mmol/l) in the same experimental conditions were not affected. Cooling to 20°C after treatment with an antagonist (ouabain 10 µmol/l, amiloride 0.1 mmol/l or vanadate 0.1 mmol/l) or after incubation in K⁺-free medium or low Na⁺ (25 mmol/l) solution produced the same or greater inhibitions of tracheal responses to spasmogens than cooling alone. The guinea-pig trachea treated with phorbol 12,13-diacetate (PDA; 1 µmol/l) and cooled to 20°C responded to spasmogens similarly to a trachea untreated with PDA at 37°C. In contrast, PDA (1 µmol/l) did not counteract the depressed responsiveness to histamine of ouabain (10 µmol/l)- or amiloride (0.1 mmol/l)- treated tracheal strips at 37°C. PDA (1 mol/l) enhanced tracheal contractions caused by KCl (50 mmol/l) in Ca²⁺-free medium at 20°C but failed to augment those to histamine in the same conditions. PDA (0.1 or 1 µmol/l) did not change the concentration-response curve for Ca²⁺ in skinned trachea. In conclusion, low temperature inhibits the responses to spasmogens in the guinea-pig trachea. Probably, reduced temperature interferes with extracellular and intracellular sources of Ca²⁺ which are differently affected by various spasmogens. Alteration by cooling of the electrogenic Na⁺ pump, Na⁺/Ca²⁺ exchange system, and Ca²⁺-ATPase may participate in the decrease of tracheal responsiveness to agonists. Reversion by PDA of the inhibitory effect of low temperature suggests a role for protein kinase C in the cooling-induced changes of tracheal responses. Correspondence to J. Cortijo at the above address     

Evidence for elevated levels of dopamine in the rabbit pulmonary and carotid artery

Catecholamine levels in six arteries, two aortae, and atria of rabbits were determined by high-performance liquid chromatography (HPLC) coupled with electrochemical detection. An appreciable concentration of dopamine (DA) was found in the pulmonary and carotid arteries. The content in these two arteries was greater than 200 ng/g, although the mesenteric, celiac, renal, and femoral arteries had less than 55 ng/g. The ratio of DA to norepinephrine (NE) in the pulmonary artery was 32.2 +/- 6.3%, and that in the carotid artery was 9.0 +/- 3.8%. These values are equivalent to the ratio in the canine paw pad and kidney, which are considered to be innervated by a dopaminergic system among peripheral tissues. The presence of DA in the pulmonary artery was confirmed by gas chromatography and mass spectrometry. In addition, uptake and distribution of [3H]DA and significant enhancement of [3H]DA release by transmural stimulation in the pulmonary artery were observed. These results suggested the possibility of dopaminergic innervation in the rabbit pulmonary and, possibly, carotid arteries.     

An analysis of the B-adrenergic action of oxyfedrine

1-3-methoxy-ω-(1-hydroxy-1-phenylisopropylamino)-propiophenone hydrochloride (oxyfedrine) (10^?8–10^?6 g/ml) produces a positive inotropic effect on electrically driven guinea-pig left atria which is prevented by propranolol; higher concentrations depress contractile force. Oxyfedrine (10^?8–10^?6 g/ml) effectively antagonizes the action of isoprenaline but not that of theophylline ethylenediamine. Oxyfedrine (10^?5 g/ml)-induced relaxation of the guinea-pig tracheal chain is not reversed by repeated washing but is antagonized by propranolol (10^?7 g/ml). The relaxed tracheal chain contracts when an additional dose of oxyfedrine (10^?5 g/ml) or quinidine (10^?5 g/ml) was administered. The oxyfedrine-induced contraction is relaxed by papaverine (5 × 10^?6 g/ml), theophylline ethylene-diamine (10^?5 g/ml) or adenosine (5 × 10^?5 g/ml) but not by isoprenaline (2 × 10^?8 g/ml) or adrenaline (10^?7 g/ml) nor is it prevented by dibenamine (10^?5 g/ml), methysergide (10^?6 g/ml), atropine (10^?5 g/ml) or tripelennamine (10^?7 g/ml). These observations suggest that oxyfedrine interacts with the ß-adrenergic receptor, inducing at adequate concentrations, stimulation and blockade to catecholamines simultaneously; in this interaction, higher concentrations of oxyfedrine produces a direct depression (quinidine-like) of cardiac muscle and a contraction of tracheal muscle.     

Contribution of RhoA kinase and protein kinase C to weak relaxant effect of pinacidil on carbachol-induced contractions in sensitized guinea-pig trachealis

The exact mechanisms underlying the weak bronchodilator effect of K_ATP channel openers on cholinergic stimulations is unknown. The present study was designed to examine the relaxant efect of pinacidil in guinea-pig trachea stimulated with carbachol by the presence of calcium sensitizer inhibitors; HA 1077, a rhoA kinase inhibitor, and chelerythrine, a protein kinase C inhibitor. Adenosine (10 μM) was used as other contractile agent for comparison. Tracheal tissues were isolated from ovalbumin sensitized guineapigs and changes in tension were recorded isometrically. Pinacidil (1–100 μM, cumulatively) and HA 1077 (0.01–30 μM, cumulatively) produced concentration-dependent relaxations in unstimulated tisues. The relaxant response to pinacidil decreased in carbachol contracted tissues, but increased in adenosine-stimulated tissues. Pretreatment of the tissues with HA 1077 (0.1 μM) and chelerythrine (10 μM) increased the pinacidil-induced relaxations by ∼%100 and %40, respectively. Glibenclamide, a KATP channel blocker, partially antagonized the pinacidil response in contracted tissues. Glibenclamide also inhibited the carbachol and adenosine induced contractions. These results suggest that diminish effect of pinacidil may have related to the enhanced calcium sensitization by cholinergic stimulation. Rho kinase inhibitors appear more effective than PKC inhibitors to achieve of this failure. Two authors made an equal contribution to this study.