The effects of beta-adrenoceptor activation on contraction in isolated fast- and slow-twitch skeletal muscle fibres of the rat.

1. The aim of the experiments was to examined the effects of beta-adrenoceptor activation on twitch and tetanic contractions in fast- and slow-twitch mammalian skeletal muscle fibres. Isometric force was recorded from bundles of intact fibres isolated from the normal and denervated slow-twitch soleus and normal fast-twitch sternomastoid muscles of the rat. 2. Terbutaline (10 microM), a beta 2-adrenoceptor agonist, induced an average 15% potentiation of peak twitch and peak tetanic force in normal soleus fibres and abbreviated twitch and tetanic relaxation. In white- and red-sternomastoid fibres, 10 microM terbutaline potentiated peak twitch force by about 7% and slowed twitch relaxation. 3. The potentiation of twitches and tetani by terbutaline was quantitatively similar in normal and denervated soleus fibres. However, in contrast to the normal soleus, terbutaline slowed twitch relaxation and had no effect on tetanic relaxation in denervated soleus fibres. 4. Adrenaline (10 microM) increased peak tetanic force by about 7% in both normal and denervated soleus fibres. 5. Exposure to (+/-)-propranolol (0.1 microM), a general beta-adrenoceptor blocker, completely abolished the tetanus potentiation by terbutaline. 6. Dibutyryl-cyclic AMP (2 mM) mimicked the effects of 10 microM terbutaline on peak tetanic force and tetanic relaxation in normal and denervated soleus fibres. Dibutyryl-cyclic AMP also potentiated peak twitch force in denervated soleus fibres but only after a brief period of twitch depression: the twitch depression might be due to butyrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nebivolol, carvedilol and metoprolol do not influence cardiac Ca(2+) sensitivity.

It has been argued that some beta-adrenoceptor antagonists may directly influence myofibrillar cross-bridge interaction in cardiac skinned fiber preparations of animal models. The present study investigates the effects of nebivolol, metoprolol and carvedilol on tension development of Triton X-100 skinned fibers obtained from human failing myocardium as well as on force of contraction and intracellular Ca(2+) transient in isolated trabeculae. In skinned fiber preparations, none of the beta-adrenoceptor antagonists (10 microM) influenced Ca(2+) sensitivity of tension development or maximal Ca(2+) activated tension (DT(max)): control: EC(50) for Ca(2+): 1.28+/-0.05 microM, DT(max): 14.09+/-0.59 mN/mm(2); nebivolol: 1.36+/-0.1 microM, 14.14+/-0.95 mN/mm(2); carvedilol: 1.32+/-0.11 microM, 13.83+/-0.90 mN/mm(2); metoprolol: 1.34+/-0.14 microM, 13.72+/-0.36 mN/mm(2). Simultaneous measurement of force and Ca(2+) transient in the presence of the beta-adrenoceptor antagonists (3 microM) showed that the decrease in force of contraction was paralleled by a similar decrease in the intracellular Ca(2+) transient. In conclusion, none of the investigated beta-adrenoceptor antagonists influenced Ca(2+) sensitivity of myofibrillar tension development in human failing myocardium.     

Effects of atropine and glycopyrrolate on neuromuscular transmission in the rat phrenic nerve-diaphragm preparation

1. The effects of atropine and glycopyrrolate on neuromuscular transmission and on muscle contraction, were studied, in the rat diaphragm preparation, by analyzing their effects on the indirectly (and directly)-elicited twitch (0.2 Hz), tetanic (50 Hz for 20 sec duration), post-tetanic twitch responses (at 5 sec after the tetanus), and on the phenomenon of post-tetanic twitch potentiation (PTP), which is thought to be of a presynaptic origin, i.e. due to increased transmitter release. 2. Atropine (0.001-10 microM) increased the indirectly-elicited twitch tension by 22 +/- 2.1% (control 0.9 +/- 0.1 g, P less than 0.02), the tetanus by 15 +/- 1.1% (control 3.9 +/- 0.7 g, P less than 0.05), the post-tetanic twitch response by 33 +/- 3.1% (control 1.2 +/- 0.1 g, P less than 0.01) and the PTP value by 36 +/- 1.9% (control 33 +/- 2.3%, P less than 0.01, means +/- SEM = 6). 3. Atropine (0.001-10 microM) had little effect on the directly-elicited twitch tension, but in high concentrations (e.g. 20 microM), it blocked the twitch tension. 4. In contrast, glycopyrrolate (0.1-100 microM) had little effect on the twitch tension (direct or indirect), but it significantly reduced the tetanus (by 38 +/- 3.5%, P less than 0.01), the post-tetanic twitch response (by 17 +/- 1.2%, P less than 0.05) and the PTP values (by 24 +/- 3.1% P less than 0.02). 5. In the presence of hemicholinium (1.3 microM) the responses to atropine and glycopyrrolate were altered (decreased), indicating a possible action on presynaptic mechanism of transmission. 6. It is concluded that atropine and glycopyrrolate produce different (opposite) effects at the rat neuromuscular junction, atropine enhances whereas glycopyrrolate depresses neuromuscular transmission. The effects of these two antimuscarinic drugs may be exerted at the presynaptic nerve terminals, i.e. on presynaptic muscarinic receptors, which are involved in the feedback mechanism of transmitter release.     

Apelin increases contractility in failing cardiac muscle

Apelin, a ligand for apelin-angiotension receptor-like 1 (APJ), has recently been shown to be a potent positive inotropic agent in normal hearts. In humans, levels of apelin have been shown to rise in early-stage heart failure and to fall in late-stage heart failure. In this study, we tested the hypothesis that apelin augments contraction directly in failing rat cardiac muscle. Right ventricular heart failure secondary to pulmonary hypertension was induced by exposing the rats to hypoxia (10% O(2) inhaled air) for 14-16 weeks. Trabeculae were dissected and mounted between a force transducer and a motor arm, superfused with Krebs-Henseleit (K-H) solution (pH 7.4, 22 degrees C), and loaded with fura-2. Both force development and [Ca(2+)](i) transient amplitude increased in a dose-dependent manner in the presence of Apelin-12 (10 approximately 70 nM, [Ca(2+)](o)=0.5 mM) in failing muscles as compared to control (36+/-7% vs. 7.4+/-5% at 70 nM, P<0.05). Also, [Ca(2+)](i) transients increased up to 18.4+/-9.5% as compared to control (4.5+/-1.9%, P<0.05). The increases in contraction in the presence of apelin were also maintained over a range of external Ca(2+) (0.5-2.0 mM). Steady-state force-[Ca(2+)](i) relation of the failing muscles reveals decreased maximal Ca(2+)-activated force (F(max)) (51.45+/-5.3 vs. 98.5+/-11.5 mN/mm(2), P<0.001), with no changes in Ca(2+) required for 50% of maximal activation (Ca(50)) (0.45+/-0.07 vs. 0.30+/-0.04 muM, P>0.05) and Hill coefficient (4.60+/-0.73 vs. 3.17+/-0.92, P>0.05). Apelin (70 nM) had no effect on the steady-state force-[Ca(2+)](i) relation in failing muscles (F(max): 63.03+/-3.5 mN/mm(2); Ca(50): 0.50+/-0.08 microM; Hill coefficient: 4.73+/-0.89). These results indicate that apelin exerts a selective positive inotropic action in failing myocardium. The increased force development is the result of increased [Ca(2+)](i) transients rather than changes in myofilament calcium responsiveness.     

Effects of toxin II from the sea anemone Anemonia sulcata on contractile and electrical responses of frog skeletal muscle fibres

The effects of Anemonia sulcata toxin II (ATX-II) were studied on mechanical and electrical activities of frog muscle fibres isolated from semitendinosus or tibialis anterior muscles of Rana temporaria (2.8-7.7 degrees C). In concentrations ranging between 7.7 and 100 microM, ATX-II greatly potentiated the isometric twitch of single muscle fibres in a time-dependent manner. Increase in twitch amplitude by ATX-II was associated with an increase in time to peak tension and time from peak tension to half relaxation. ATX-II caused no change in maximum force production during fused tetanus, but the tension was maintained for several seconds after the cessation of stimulation. Such long tetanic contractions were also obtained in low-Na Ringer solution, but their duration was somewhat shorter. No specific action of ATX-II was detected on relaxation kinetics during a tetanus. The twitch potentiating effect of ATX-II was markedly increased by 3,4-diaminopyridine. Action potentials recorded from single muscle fibres in the presence of ATX-II showed a delayed onset of repolarization with a reduced rate of fall. In addition, ATX-II caused repetitive spontaneous firing of action potentials after the cessation of tetanic stimulation. ATX-II (3.3 - 10 microM) also increased action potential duration by reducing the rate of repolarization in surface fibres of formamide-treated sartorius or cutaneous pectoris muscles (20 degrees C) stimulated indirectly or directly. The potentiation of twitch force and the prolongation of contractions caused by tetanic stimulation can be attributed to the membrane action of ATX-II, which leads to prolongation of action potentials, to repetitive muscle firing and to the appearance of plateau potentials.     

Two inotropes with different mechanisms of action: contractile, PDE-inhibitory and direct myofibrillar effects of levosimendan and enoximone

We characterized the Ca2+-sensitizing and phosphodiesterase (PDE)-inhibitory potentials of levosimendan and enoximone to assess their contributions to the positive inotropic effects of these drugs. In guinea pig hearts perfused in the working-heart mode, the maximal increase in cardiac output (55%, P<0.05) was attained at 50 nM levosimendan. The corresponding value for enoximone (36%) was significantly smaller (P<0.05) and was observed at a higher concentration (500 nM). In permeabilized myocyte-sized preparations levosimendan evoked a maximal increase of 55.8+/-8% (mean+/-SEM) in isometric force production via Ca2+ sensitization (pCa 6.2, EC50 8.4 nM). Enoximone up to a concentration of 10 microM failed to influence the isometric force. The PDE-inhibitory effects were probed on the PDE III and PDE IV isoforms. Levosimendan proved to be a 1300-fold more potent and a 90-fold more selective PDE III inhibitor (IC50 for PDE III 1.4 nM, and IC50 for PDE IV 11 microM, selectivity factor approximately 8000) than enoximone (IC50 for PDE III 1.8 microM, and IC50 for PDE IV 160 microM, selectivity factor approximately 90). Hence, our data support the hypothesis that levosimendan exerts positive inotropy via a Ca2+-sensitizing mechanism, whereas enoximone does so via PDE inhibition with a limited PDE III versus PDE IV selectivity.     

Amrinone relaxes potassium-induced contracture of failing right ventricular muscle of cats

The effects of amrinone (5.3 X 10(-4) M) on isometric contraction and K+-induced contracture force of right ventricular muscle isolated from normal cats (n = 6) and cats in right ventricular failure (RVF, n = 6), 3-14 days after partial pulmonary artery ligation, were studied. Peak isometric contractile force (Po) and maximal rate of force development (dP/dt) of RVF muscles (1.38 +/- 0.21 g/mm2; means +/- SEM, and 11 +/- 1 g/s/mm2, respectively) were significantly lower than normal muscles (2.46 +/- 0.41 g/mm2, p less than 0.025, and 24 +/- 3 g/s/mm2, p less than 0.005, respectively). Duration of contraction (DC) was significantly longer in RVF muscles (442 +/- 32 ms) than in normal muscles (361 +/- 11 ms, p less than 0.025). Times to peak twitch force (TTP) and peak K+-induced contracture force (Pc) of RVF and normal muscles were not different. Amrinone increased Po and dP/dt significantly in normal muscles (+78 +/- 24%; means +/- SEM; p less than 0.01, and +53 +/- 17%, p less than 0.025, respectively), but not RVF muscles (+11 +/- 16% and +8 +/- 19%, respectively). TTP and DC of normal muscle were unchanged by amrinone, whereas TTP was unchanged while DC was shortened (-12 +/- 3%, p less than 0.025) in RVF muscle. Amrinone relaxed Pc similarly and significantly in normal (-28 +/- 7%, p less than 0.005) and RVF (-26 +/- 4%, p less than 0.025) muscles. These results suggest that part of amrinone's salutary action in the failing heart occurs through modification of myocardial relaxation.     

Modulation of cross-bridge interaction by 2,3-butanedione monoxime in human ventricular myocardium

OBJECTIVES: 2,3-butanedione monoxime (BDM) has been suggested as an additive to cardioplegic solutions, because it may reduce the energy cost associated with force production in heart muscle. METHODS: The present study investigated the effect of BDM (10 mM) on developed tension (DT), Ca(2+)-dependent myosin ATPase activity (MYO) and tension cost (myosin ATPase activity/ tension ratio), characterizing the cross-bridge detachment rate, of skinned fiber preparations (1% Triton X, 20 h, 4 degrees C) of human left ventricular failing myocardium (dilative cardiomyopathy, heart transplants, n=6) at increasing concentrations of Ca2+ (0.01-32 microM). RESULTS: BDM decreased Ca2+ sensitivity of DT [EC50 Ca2+, control: 1.3+/-0.2 microM, + BDM (10 mM): 4.5+/-0.3 microM] and MYO [EC50 Ca2+, control: 0.9+/-0.2 microM, + BDM (10 mM): 3.1+/-0.3 mM]. In addition, BDM reduced maximal DT [control: 26.0+/-1.9 mN/mm2, + BDM (10 mM): 8.1+/-1.1 mN/mm2] and MYO [control: 124+/-21 RM ADP/s, + BDM (10 mM): 62 9 microM ADP/s]. However, the influence of BDM on maximal DT (-69%) was more pronounced than on maximal MYO (-50%). The myosin ATPase activity/tension relation was significantly higher in the presence of BDM. CONCLUSIONS: BDM exerts negative inotropic activity by reducing the number of force-generating cross-bridges, possibly by increasing the cross-bridge detachment rate as well as by reducing force generation per cross-bridge in human myocardium. As BDM reduces force generation more than ATPase activity, BDM may not necessarily reduce energy demand in human myocardium.     

Suppressive effects of R 56865 on triggered propagated contractions and triggered arrhythmias in rat cardiac trabeculae.

Triggered arrhythmias in rat right ventricular trabeculae are induced by triggered propagated contractions (TPCs) that start in damaged regions of the muscle and propagate along the preparation. We analyzed the effects of the Na+/Ca2+ overload inhibiting agents R 56865 on both TPCs and triggered arrhythmias. This compound has been shown to prevent ultrastructural signs of intracellular calcium overload and arrhythmias caused by exposure to toxic concentrations of cardiac glycosides. TPCs were induced by trains of 15 stimuli (2 Hz, 15 s intervals) at 19-21 degrees C and a [Ca2+]o of 1.0-2.5 mM in the superfusate. Force was measured with a silicon strain gauge; length and shortening of sarcomeres were measured at two sites of the muscle using laser diffraction techniques. Exposure to 1.14 x 10(-7) M R 56865 for 30 min decreased the force of the last stimulated twitch (twitch force) to 89.7 +/- 4.7% (mean +/- SEM) of control, the force produced by TPCs to 39.4 +/- 9.8%, and the velocity of propagation of TPCs to 52.8 +/- 6.3%, while TPC latency increased not significantly to 104.7 +/- 2.8% of control. R 56865 suppressed TPC force, for the same small decrease in twitch force (10%), significantly more than 100 nM D-600 did (29.5 +/- 2.0 vs. 12.4 +/- 3.1%). Eventually, TPCs disappeared in 8 of 14 muscles, in 2 of them without any decrease in twitch force. At 5.7 x 10(-7) M, R 56865 abolished TPCs in five additional trabeculae. An increase in [Ca2+]o reintroduced TPCs. During stimulation of 0.5 Hz, 1.14 x 10(-7) M R 56865 increased the stimulus threshold by 21 +/- 4% in 6 of 14 muscles and decreased the twitch force by 26 +/- 3% in 7 of 14 trabeculae. Triggered arrhythmias were induced in six muscles with the use of 0.5 mM caffeine or 5 nM Bay K 8644; R 56865 rapidly terminated these arrhythmias in all muscles. Although the mechanism of the antiarrhythmic effects of R 56865 remains to be determined, we speculate that the drug raises the threshold for both generation of triggered action potentials and calcium-induced calcium release from the sarcoplasmic reticulum.     

Crataegus special extract WS 1442 increases force of contraction in human myocardium cAMP-independently

The mode of action of Crataegus extracts in the treatment of heart failure is still under examination. WS 1442, a standardized special extract from Crataegus leaves with flowers, exerts direct positive inotropic effects. This study was designed to investigate the mode of inotropic action of WS 1442 in human myocardium from patients with congestive heart failure (left ventricular myocardium from explanted hearts; NYHA IV, n = 8) as well as in nonfailing controls (right auricular trabeculae from patients with coronary heart disease, n = 8). WS 1442 effectively displaced specifically bound 3H-ouabain but did not influence the activity of adenylate cyclase [control, + Gpp(NH)p (10(-4) microM) 3,500 pmol cyclic adenosine monophosphate (cAMP)/20 min). In isolated left ventricular papillary muscle strips, WS 1442 significantly increased the force of contraction [basal, 1.8+/-0.2 mN; WS 1442 (50 microg/ml), 2.4+/-0.1 mN (130%)] and improved the frequency-dependent force generation (0.5 vs. 2.5 Hz: control, +0.1+/-0.01 mN; WS 1442, +0.9+/-0.3 mN) even in failing human myocardium. In fura-2-loaded muscle strips (right atrial trabeculae), WS 1442 increased both the Ca2+-transient and force generation. These effects also were observed in the lipophilic ethyl acetate-soluble fraction A, enriched in flavone derivatives. In conclusion, these findings suggest a pharmacologic mechanism of WS 1442 similar to the cAMP-independent positive inotropic action of cardiac glycosides. In addition, WS 1442 improves the force-frequency relation in failing human myocardium.     

Pharmacological comparison between the actions of methamphetamine and 1-aminoindan stereoisomers on sympathetic nervous function in rat vas deferens

The selective monoamine oxidase-B inhibitor selegiline (deprenyl) causes sympathomimetic effects and is metabolised to R(-)-methamphetamine and R(-)-amphetamine. The new monoamine oxidase-B inhibitor rasagiline is devoid of sympathomimetic effects and is metabolised to R(+)-1-aminoindan. Sympathomimetic effects of methamphetamine and 1-aminoindan enantiomers were compared in the rat vas deferens. R(-)-methamphetamine and S(+)-methamphetamine caused initial potentiation and subsequent inhibition of the field stimulation-induced twitch response of isolated rat vas deferens (0.1 Hz). EC(50) values for inhibition of twitch in prazosin-treated vas deferens were 0.36+/-0.13 and 1.64+/-0.10 microM (mean+/-S.E.M.) for S(+)- and R(-)-methamphetamine, respectively. There was no difference between S(+)-methamphetamine and R(-)-methamphetamine in potentiation of postsynaptic contractile response to noradrenaline. R(+)- and S(-)-1-aminoindan increased twitch response only at concentrations above 30 microM. R(-)-methamphetamine has similar potency to S(+)-methamphetamine in potentiation of noradrenaline-mediated responses and can therefore play a role in the sympathomimetic effects of selegiline.     

Inhibition of neuromuscular transmission in isolated mouse phrenic nerve-diaphragm by the enterotoxin of Clostridium perfringens type A.

The enterotoxin of Clostridium perfringens type A, a channel forming protein toxin, inhibited neuromuscular transmission under conditions of low calcium. Twitch tension of isolated phrenic nerve-diaphragm preparations elicited by electrical stimulations to the phrenic nerve was recorded isometrically, and the preparations were exposed to the purified enterotoxin. In Krebs solution containing 0.5 mM calcium, the enterotoxin (20 micrograms/ml) reduced within 10 min the amplitude of the twitch tension to 34 +/- 7% (mean +/- S.D., n = 11) of that recorded before the treatment. The effects of the enterotoxin on the twitch tension were irreversible and proceeded independently of stimulation. The reduction of the twitch tension by the enterotoxin was apparent in Krebs solution containing less than 0.6 mM calcium and the degree of reduction was inversely related to the concentration of calcium. The reduction of the twitch tension by the enterotoxin was also dependent on temperature and concentration of the toxin. At temperatures below 20 degrees C, no obvious reduction of twitch tension was observed with 20 micrograms/ml of the enterotoxin. Enterotoxin at a concentration of 0.4 micrograms/ml caused 16 +/- 2% (mean +/- S.D., n = 4) reduction of twitch tension, and the degree of the reduction in twitch tension increased with toxin concentration, reaching a plateau of 65 +/- 4% (mean +/- S.D., n = 7) at 6.5 micrograms/ml of the enterotoxin. The effects of the enterotoxin were antagonized by 2 microM physostigmine. Unlike curare, pretreatment of the preparation with enterotoxin did not antagonize the neuromuscular block by decamethonium. Neither the tension of muscular twitch elicited by direct electrical stimulation to the muscle nor the resting membrane potentials of muscle fibers recorded intracellularly were affected by the enterotoxin. The enterotoxin (2.2 micrograms/ml) reduced the frequency, but not mean amplitude or amplitude distribution, of miniature end-plate potentials, from 0.91 +/- 0.07/sec to 0.72 +/- 0.07 (mean +/- S.E., n = 5). The results suggest that the enterotoxin will provide a novel tool for the studies on the mechanism of the neuromuscular transmission because of the unique characteristics of the inhibition and of the known mechanism of its action on the cell membrane.     

Blocking effect of diltiazem in the isolated rat phrenic hemidiaphragm

The effects of diltiazem on indirectly and directly elicited twitch were studied in the isolated rat phrenic hemidiaphragm preparation. Diltiazem (30-500 microM) blocked the indirectly elicited twitch response and this effect was not affected by reducing the extracellular calcium from 2.5 to 1.25 mM. An effect on the directly elicited twitch was also observed (100-300 microM). Diltiazem (30-300 microM) blocked the peak tetanic tension and tetanic fade was present. The results were consistent with an action of diltiazem on the nicotinic receptor-ion channel complex.     

Effects of mefloquine on cardiac contractility and electrical activity in vivo, in isolated cardiac preparations, and in single ventricular myocytes

1. To examine the possible cardiotoxicity of the antimalarial drug mefloquine, increasing doses (0.3 - 30 mg kg(-1)) were given i.v. to anaesthetized guinea-pigs. Mefloquine did not alter ECG intervals significantly but gradually increased systolic blood pressure (at 3 mg kg(-1)) then had a depressor effect (at 10 mg kg(-1)). Death due to profound hypotension, probably resulting from cardiac contractile failure or AV block, occurred after either 10 mg kg(-1) (2/6) or 30 mg kg(-1) (4/6) mefloquine. 2. In isolated cardiac preparations mefloquine (3 - 100 microM) did not alter the effective refractory period but at the higher concentrations resting tension increased. Developed tension was reduced by 100 microM mefloquine in left atria (from 5.8+/-1.7 to 2.2+/-0.4 mN) whereas in papillary muscles although 30 microM mefloquine reduced developed tension (from 2. 6+/-0.5 to 1.1+/-0.1 mN) subsequent addition of 100 microM caused a marked, but not sustained, positive inotropic effect (from 1.2+/-0.1 to 3.8+/-0.8 mN). 3. In single ventricular myocytes, mefloquine (10 microM) shortened action potential duration (e.g. APD(90) from 285+/-29 to 141+/-12 ms) and reduced the amplitude of the systolic Ca(2+) transient. 4. These effects were accompanied by a decrease in the L-type Ca(2+) current. These results indicate that the main adverse effect of mefloquine on the heart is a negative inotropic action. This action can be explained by blockade of L-type Ca(2+) channels.     

The role of sensory neuropeptides in motor innervation of the hamster isolated urinary bladder

In this study we have characterized the role of sensory fibers and of the sensory peptides, neurokinin A (NKA) and calcitonin gene-related peptide (CGRP), on the contractile responses evoked by single pulse electrical field stimulation (EFS) in the hamster urinary bladder. EFS of the hamster isolated urinary bladder produced twitch contractions which were unaffected by atropine but abolished by tetrodotoxin. The P2 purinoreceptor antagonist PPADS (30 microM) inhibited twitches by 66+/-4% on its own and by 78+/-3% in the presence of atropine. The selective tachykinin NK2 receptor antagonist nepadutant produced a slight but consistent reduction of twitch amplitude (-21+/-3%) at 1 microM. Addition of nepadutant to atropine and PPADS did not further increase their inhibitory effect. The application of hCGRP (10-300 nM) produced a concentration-dependent inhibition of twitches (Emax -38+/-3%, EC50=12 nM) and a small reduction of tone (0.5+/-0.09 mN). Similar effects were obtained with capsaicin (0.1-10 microM) which inhibited EFS-evoked contractions with an EC50 of 100.0 nM and a maximal effect of 34+/-4% inhibition at 1 microM. Under submaximal parameters of stimulation NKA (10 nM) increased the amplitude of twitches by 45+/-6% and produced a concentration-dependent tonic contraction (EC50=55.9 nM). The CGRP1 receptor subtype antagonist, hCGRP(8-37), increased by 29+/-8% the EFS-evoked contractions and significantly reduced the response to 0.1 microM CGRP. Capsaicin (10 microM) increased both CGRP-LI and NKA-LI release from superfused slices of hamster urinary bladder by about sixfold and by about 70%, over baseline, respectively. A second application of capsaicin was ineffective, indicating a complete desensitization of sensory nerve efferent function. In the hamster urinary bladder the sensory neuropeptides NKA and CGRP are co-released by sensory fibers after stimulation either by EFS or capsaicin. However, the role of CGRP appears functionally predominant.     

Effects of selective phosphodiesterase 3 inhibition in the perfused liver of the rat after endotoxin treatment.

1. This study was designed to investigate the role of rat phosphodiesterase 3 (RPDE3) in regulation of liver metabolism in sepsis. We studied the effects of the phosphodiesterase 3 inhibitor (PDI), enoximone, alone and in combination with regulating factors of hepatic carbohydrate metabolism and bile secretion in the perfused liver of rats treated 4 h earlier with endotoxin. In addition, cyclic AMP and cyclic GMP levels were determined in the effluate and bile by radio immunoassay methods. 2. After endotoxin treatment, infusion of enoximone at three concentrations (1 microM, 10 microM) resulted in an increased glucose output from -1.4 +/- 0.9 to 7.8 +/- 2.5 mumol l-1 20 min-1. Bile acid-independent bile flow increased also, in a dose-dependent manner. 3. In untreated livers, cyclic AMP release increased in the effluate from 1000 +/- 73 fmol g-1 min-1 to 1710 +/- 143 fmol g-1 min-1 when enoximone (10 microM) was administered. In bile from untreated livers, the level of cyclic AMP was also significantly increased by enoximone. After endotoxin treatment, the enoximone (10 microM) effect on cyclic AMP levels in effluate and bile was greatly reduced. Levels of cyclic GMP in the effluate and bile appeared unchanged in the presence of enoximone. 4. During co-infusion of glucagon (1 nM) and enoximone (10 microM), cyclic nucleotide levels in the effluate and bile of livers after endotoxin treatment were determined. In the effluate, cyclic AMP release increased from 827 +/- 144 fmol g-1 min-1 to 17802 +/- 2821 fmol g-1 min-1 when glucagon was administered. The presence of enoximone enhanced cyclic AMP further to 41696 +/- 920 fmol g-1 min-1. The same changes in cyclic AMP release were found in bile. Levels of cyclic GMP in the effluate and bile were not significantly affected by the administration of glucagon and the PDI. 5. Glucose release was determined during glucagon, sympathetic nerves stimulation and phenylephrine administration in the presence and absence of enoximone. The addition of enoximone to glucagon increased glucose release by 8.2 +/- 2.8 mumol g-1 20 min-1, without alteration of lactate balance. The PDI enhanced the glycogenolytic effects of nerve stimulation and of phenylephrine, accompanied by a reduction in lactate production. 6. Enoximone significantly enhanced the bile acid independent bile flow after glucagon, nerves stimulation and after administration of phenylephrine. Bile acid secretion was unaffected by the PDI. The vasoconstrictor effect of nerve stimulation was reduced by the PDI. 7. We conclude that endotoxin treatment reduces the ability of the PDI, enoximone, to increase cyclic AMP release in the perfused liver. The significant increase in cyclic AMP release after stimulation with glucagon and enoximone favours the view that RPDE3 is involved in the degradation of cyclic AMP in the liver after exposure to endotoxin. Additionally, the inhibition of the RPDE3 results in glucose release, vasodilatation and choleresis in endotoxin pretreated livers.     

Photolysis of the novel inotropes EMD 57033 and EMD 57439: evidence that Ca2+ sensitization and phosphodiesterase inhibition depend upon the same enantiomeric site.

1. We studied the effects of flash photolysis on the novel enantiomeric cardiac inotropes EMD 57033 (a calcium sensitizer) and EMD 57439 (a phosphodiesterase III inhibitor) in rat isolated ventricular trabeculae. 2. In skinned trabeculae, EMD 57439 had no effect on force production, consistent with lack of an active cyclic AMP system in this preparation. In contrast, EMD 57033 potentiated force at partial and maximal activation. A single flash of near u.v. light given at partial activation (30-70%) reduced force potentiation by 52.4 +/- 5.2%. No effect was produced by flashes in the presence of EMD 57439 or in the absence of either drug. 3. The time course of relaxation induced by EMD 57033 photolysis was indistinguishable from that obtained on deactivating the muscle by rapidly lowering Ca2+ using photolysis of the caged chelator of calcium, diazo-2. 4. In intact, twitching trabeculae, EMD 57033 increased diastolic and peak force and slowed relaxation. These effects were simultaneously reduced by a light flash. In these muscles EMD 57439 reduced force, without affecting the twitch time course. These effects were also reduced by a light flash. 5. The u.v. absorbance spectra of EMD 57033 and EMD 57439 were identical. After photolysis optical density decreased substantially and the peak shifted from 320 nm to 280 nm. 6. The proton n.m.r. spectra of these compounds were identical. The main change post-photolysis was a decrease in the proton signal associated with the enantiomeric carbon atom. 7. This novel manipulation of the molecular structure of EMD 57033 and EMD 57439 within an experiment thus provides direct evidence linking calcium sensitization to a particular molecular structure. The three main effects of the sensitizer on the twitch were simultaneously abolished and may be mechanistically linked. Flash photolysis may be a useful tool for further investigations of the actions of these compounds. In particular, flash photolysis of the sensitizer represents a novel method of rapidly deactivating cardiac muscle.     

ET(A) receptors are the primary mediators of myofilament calcium sensitization induced by ET-1 in rat pulmonary artery smooth muscle: a tyrosine kinase independent pathway.

We have investigated the possibility that ET-1 can induce an increase in myofilament calcium sensitivity in pulmonary artery smooth muscle. Arterial rings were permeabilized using alpha-toxin (120 microg ml(-1)), in the presence of A23187 (10 microM) to 'knock out' Ca2+ stores, and pre-constricted with pCa 6.8 (buffered with 10 mM EGTA). In the presence of this fixed Ca2+ concentration, 1 microM ET-1 induced a sustained, reversible constriction of 0.15 mN. Pulmonary arterial rings were freeze-clamped at the peak of the induced constriction (time matched). Subsequent densitometric analysis revealed that ET-1 (1 microM) increased the level of phosphorylated myosin light chains by 34% compared to an 11% increase in the presence of pCa 6.8 alone. In contrast to ET-1, the selective ET(B) receptor agonist Sarafotoxin S6C (100 nM) failed to induce a significant constriction. The constriction induced by 1 microM ET-1 was reversibly inhibited when the preparation was preincubated (15 min) with the ETA receptor antagonist BQ 123 (100 microM). The constriction measured 0.13 mN in the absence and 0.07 mN in the presence of 100 microM BQ 123. In contrast, the constriction induced by 1 microM ET-1 measured 0.19 mN in the absence and 0.175 mN following a 15 min pre-incubation with the ET(B) antagonist BQ 788 (100 microM). The constriction induced by 1 microM ET-1 measured 0.14 mN in the presence and 0.13 mN following pre-incubation with the tyrosine kinase inhibitor Tyrphostin A23 (100 microM). We conclude that ET-1 induced an increase in myofilament calcium sensitivity in rat pulmonary arteries via the activation of ET(A) receptors and by a mechanism(s) independent of tyrosine kinase.     

Effects of 3,4-diaminopyridine on mechanical and electrical responses of frog single muscle fibres

The effects of 3,4-diaminopyridine (3,4-DAP) were studied on isolated muscle fibres of the frog in concentrations ranging between 0.025 and 5.0 mM. Isometric twitch and tetanus responses were recorded at temperatures between 2.5 and 3.9 degrees. 3,4-DAP caused a concentration-dependent increase in twitch amplitude, maximum effects being obtained at a concentration of 3 mM with a mean increase in tension of 70 +/- 12% of control (n = 7). 3,4-DAP in 3 mM concentration had only a slight increase in initial rate of rise of twitch tension (mean increase 8 +/- 4%) but increased the time to half peak tension by 59 +/- 9% and the time from peak tension to half relaxation by 78 +/- 10%. No significant effect of 3,4-DAP was observed on the initial rate of rise and total amplitude of the isometric tetanus. The twitch potentiating effect of 3,4-DAP developed gradually with the number of times the fibre was stimulated and reached a maximum level after 40-50 stimulations. A gradual increase in the duration of the action potential was also observed. It is suggested that 3,4-DAP, like 4-aminopyridine, potentiates the twitch by means of prolonging the duration of the action potential.     

Levosimendan improves diastolic and systolic function in failing human myocardium

Ca(2+)-sensitizers increase myocardial contractility, but may worsen diastolic dysfunction. Levosimendan, through its unique troponin-C interaction, may preserve diastolic function. We investigated the effects of levosimendan (10(-7)-10(-5) M) on diastolic and systolic function in multicellular cardiac muscle preparations from end-stage failing human hearts (1 and 2.5 Hz, 37 degrees C, 1.25 mM [Ca(2+)], pH 7.4). Levosimendan improved systolic function: at 1 Hz, developed force (F(dev)) increased from 13.84+/-3.27 to 16.40+/-3.57 (10(-7) M, P<0.05), while diastolic force (F(dia)) decreased from 5.32+/-0.67 to 4.94+/-0.61 mN/mm(2) (P<0.05). Under control conditions, the increase in stimulation frequency from 1 to 2.5 Hz resulted in a decrease in F(dev) of -0.51+/-1.80 mN/mm(2) (negative force-frequency relationship). Levosimendan improved this relationship: at 10(-7) M, this change became positive (+1.81+/-2.06 mN/mm(2), P<0.05). Diastolic function was markedly improved in the presence of levosimendan; the increase in F(dia) of 1.56+/-0.42 mN/mm(2) (control) was attenuated to 0.70+/-0.19 mN/mm(2) (P<0.05). To allow for a more detailed analysis, preparations were sometimes divided into two groups, based on their force-frequency behavior. Twitch timing parameters were accelerated by levosimendan in preparations with a negative force-frequency relationship. Levosimendan improves both systolic and diastolic function in failing human myocardium. Effects are even more pronounced at higher heart rates and under prevailing diastolic dysfunction.