Protective activity of nifedipine and R 58735 upon damage caused by global ischemia in the guinea pig heart-lung preparation.

In the guinea pig heart-lung preparation, the protective effects of nifedipine and R 58735 on cardiovascular alterations following mild (35 min) and severe (60 min) ischemia and reperfusion (30 min) were studied. Nifedipine and R 58735 were equi-protective against the effects of mild ischemia with respect to functional (LVP, dp/dt, and cardiac output) and biochemical (ATP, CrP, and adenylate charge) parameters. A clear difference, however, was observed between nifedipine and R 58735 upon severe ischemia, where R 58735 produced a significantly greater protection of functional, but not of biochemical parameters. Since no significant differences between the two compounds were found with respect to the concentrations of high energy phosphates after 35 and 60 min of ischemia before reperfusion, an energy sparing effect is not likely to be responsible for the difference between nifedipine and R 58735 in severe ischemia. An additional protective effect of R 58735 upon reperfusion in severe ischemia experiments may explain the difference between the two compounds.

A comparison of the cardioprotective effects of calcium antagonists from different classes upon ischaemic damage in the guinea-pig working heart.

The cardioprotective effects of nifedipine, verapamil, diltiazem, bepridil, CERM 11956, lidoflazine, mioflazine and the coronary vasodilator dipyridamole were evaluated in the guinea-pig working heart with respect to cardiac function and high energy phosphate content after 45 min of global ischaemia and 25 min of reperfusion. All drugs, with the exception of dipyridamole, induced a negative inotropic effect, which resulted in a decrease of the aortic pressure (AoP), of its first derivative dAoP/dt and the cardiac output. To compare the anti-ischaemic effect of the calcium antagonists, concentrations were selected that reduced the dAoP/dt by 10% (EC10) and 30% (EC30), respectively. With the exception of nifedipine at the EC10 and bepridil and CERM 11956 at the EC30, perfusion with the calcium antagonists and dipyridamole (3 mumol/l) improved the recovery of contractile function after global ischaemia and reperfusion to a value between 60 and 80% of the controls in normoxic hearts. Pretreatment with nifedipine, verapamil, diltiazem, lidoflazine and mioflazine, but not with bepridil, CERM 11956 and dipyridamole led to slightly increased ATP levels in ischaemic hearts as compared to the control value in ischaemic hearts. After subsequent reperfusion for 25 min, for all drugs, ATP levels were further enhanced to 50% of the level in normoxic hearts; phosphocreatine levels reached normoxic values. In particular at the EC30, the effects of calcium antagonists on cardiac function varied in accordance with their known pharmacological and physiological profile. However, there appeared to exist no direct relationship between their beneficial effects on contractile activity and those on the levels of high energy phosphates after ischaemia and reperfusion.

The anti-ischaemic activity of the novel compound, CERM 11956, compared with that of bepridil and nifedipine in isolated guinea-pig hearts.

A comparison between the protective activity of bepridil, its novel derivative, CERM 11956, and nifedipine in isolated electrically paced guinea-pig hearts after 60 min of global ischaemia followed by 30 min of reperfusion has been made. All three compounds exerted a significant anti-ischaemic effect, as indicated by an improved recovery of functional parameters (left ventricular pressure and coronary perfusion), a delayed onset of the ischaemic contracture, and an enhanced recovery of biochemical (CrP, ATP and adenylate energy charge) parameters. The most pronounced anti-ischaemic activity was shown by the compound CERM 11956 at concentrations that displayed only minor negative inotropic activity. From the results it may be concluded that the new bepridil derivative, CERM 11956, is a promising and potent anti-ischaemic compound, which has little influence on haemodynamic parameters.

Anti-ischaemic activity of various calmodulin antagonists.

The anti-ischaemic activity of the calmodulin antagonists trifluperazine, felodipine, W-7 and calmidazolium has been investigated in electrically paced guinea-pig hearts, perfused according to Langendorff, which were subjected to 60 min of global ischaemia followed by 30 min of reperfusion. At concentrations that induced a comparable reduction in cardiac contractile force, trifluperazine, felodipine and to a lesser extent W-7, were associated with improvement of post-ischaemic functional (LVP and coronary flow) and biochemical parameters (CrP and ATP). Furthermore, felodipine and trifluperazine delayed the onset and suppressed the maximum tension of the ischaemic contracture was observed. In contrast, calmidazolium had no anti-ischaemic effects. This lack of anti-ischaemic activity of the most potent calmodulin antagonist calmidazolium, as well as the significant calcium entry blocking activity of both trifluperazine and felodipine suggest that additional factors besides calmodulin antagonism may contribute to the anti-ischaemic activity of these compounds.

Acetylcholine stores in rat diaphragm are increased by higher concentrations of dexamethasone.

In indirectly stimulated hemidiaphragms, dexamethasone (Dex, 2 microM) causes a significant increase in tissue acetylcholine (ACh) content. No increase in tissue ACh is found with 0.2, 0.6 or 6 microM Dex. Physostigmine (Physo, 15 microM) also causes an increase in tissue ACh, which is even greater in the presence of Dex (6-25 microM). There is no increase in ACh due to Dex, with 50 microM Dex plus Physo. The order of addition is important, as the Dex-induced increase in ACh is only found when Dex is added before Physo. No increase in twitch tension is found with any of these treatments. No Dex-induced increase in ACh is found with unstimulated hemidiaphragms. Similar increases in ACh are also found with other glucocorticoids (plus Physo), namely prednisolone (6-9 microM), corticosterone (2 microM) and hydrocortisone (2 microM). The mineralocorticoid aldosterone (2 microM), and other types of steroids cause no increase in tissue ACh. The increases in hemidiaphragm ACh are not found in a Na+-depleted medium, or in a medium containing 20 mM Mg2+ extra. The increase in ACh due to Physo is Ca2+-dependent, even though an increase in ACh due to Dex plus Physo is found in the absence of Ca2+. No increase in ACh due to either Physo, or Dex plus Physo are found in the presence of the nicotinic antagonists (+)-tubocurarine (5 microM) or alpha-cobrotoxin (5 micrograms/ml), while the muscarinic ligands atropine or oxotremorine (10 microM) abolish the extra increase in ACh due to Dex.(ABSTRACT TRUNCATED AT 250 WORDS)

Dexamethasone reduces the high-affinity binding of hemicholinium-3 to rat phrenic nerve endings in vitro: an autoradiographic study.

Receptor autoradiography has revealed high-affinity binding sites for hemicholinium-3 (HC-3) in the phrenic nerve endings of rat diaphragm. It has been demonstrated that 200 nM dexamethasone (Dex) in vitro reduces the binding of HC-3 to these high-affinity sites. It seems likely that the decrease in inhibitory effects of HC-3 on choline uptake and acetylcholine synthesis in rat diaphragm caused by Dex, should be the result of an interference of Dex with the binding of HC-3 to its high affinity sites.

Presynaptic, high-affinity glutamate uptake in excitatory neuromuscular junctions of the retractor unguis muscle of the locust: determination of the Km value using high resolution autoradiography.

Using high resolution autoradiography the Km of the presynaptic, Na+-dependent and aspartate inhibited, high affinity glutamate uptake in insect neuromuscular junctions was determined. The values found (approx. 15 microM) did not differ for the glia and terminal axons. This Km value agrees with those values determined by classical biochemical techniques for presynaptic high affinity uptake systems in insect neuromuscular junctions and glutamatergic central nervous systems.

Dexamethasone affects radioactive choline uptake in rat diaphragm nerve endings and not in muscle fibres.

The initial rate of radioactive choline (Ch) uptake in the endplate-rich area (EPA) of both stimulated and unstimulated hemidiaphragms is significantly increased by 0.2 microM dexamethasone (Dex) in the presence of 10 microM Ch. In autoradiographs, the mean grain densities above the muscle fibres are not altered by Dex. The mean grain densities above the nerve endings are significantly increased in the presence of Dex in stimulated tissue, and slightly but not significantly increased in unstimulated tissue. There is a positive correlation between the initial rate of Ch uptake in the EPA and the amount of isotope in the nerve terminals, in the absence and presence of Dex. Without correcting for the large amount of diffusion which occurs, the ratio of the grain densities above the nerve terminals to that above the muscle fibres in the presence of Dex is 2.12 in stimulated tissue, and 1.40 in unstimulated tissue. The ratio in the stimulated tissue is significantly greater than the control ratio in the absence of Dex (1.66). Therefore, Dex affects radioactive Ch uptake in nerve endings and not in muscle fibres in the rat diaphragm. The stimulation-induced increase in the uptake of isotope into the nerve endings is abolished in a Na+-depleted medium, and in the absence of Ca2+. Dex has no effect on this abolition. We conclude that relatively low concentrations of Dex affect Ch transport in rat diaphragm nerve endings by a mechanism as yet to be defined.

Depletion of total acetylcholine by hemicholinium-3 in isolated rat diaphragm is less in the presence of dexamethasone.

Low concentrations of dexamethasone (Dex) stimulate the initial rate of radioactive choline (Ch) accumulation in the endplate-rich area (EPA) of indirectly stimulated hemidiaphragms, while higher concentrations (greater than 0.6 microM) inhibit. This biphasic concentration-effect curve is found even in the presence of 26 microM hemicholinium-3 (HC-3), an inhibitor of Ch accumulation. In incubations (3 min) where the total hemidiaphragm acetylcholine (ACh) content is not altered by 26 microM HC-3, the inhibition by HC-3 of both the Ch accumulation rate and the incorporation of radioactive Ch into ACh in the EPA of stimulated tissues is less in the presence of 0.2 microM Dex. In 120 min incubations with 15 microM HC-3 and without added Ch, the tissue ACh content is depleted in both stimulated and unstimulated hemidiaphragms. In both cases the depletion of ACh is significantly less in the presence of 0.2 microM Dex. In stimulated tissues a comparable depletion of ACh due to 15 microM HC-3 is also found with 1 and 10 microM Ch added to the medium. It is significantly less when 0.2 microM Dex and 1 microM Ch are added to the medium. In 120 min incubations with stimulated tissue, the amount of "bound' ACh is increased by addition of 30 microM Ch to the medium, decreased in the presence of 0.2 microM Dex, and greatly decreased in the presence of 15 microM HC-3. In the presence of Dex plus HC-3, the decrease in the amount of "bound' ACh due to either Dex or HC-3 alone, is abolished provided that 30 microM Ch is also present.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioactive choline uptake in the isolated rat phrenic nerve-hemidiaphragm preparation. A biochemical and autoradiographic study.

When hemidiaphragms are stimulated via the phrenic nerve in the presence of 10 microM radioactive choline (Ch), the rate of radioactive Ch uptake in the endplate-rich area (EPA) is greater than that in the endplate-poor muscle (M). Ch uptake in the EPA is temperature-dependent, with a Q10 of 2.9 and an activation energy of 19.5 kcal/mol. It is inhibited in a Na+-depleted medium, in the absence of Ca2+, and by 10-20 microM hemicholinium-3 (HC-3) and it is not inhibited by alpha-bungarotoxin even when the muscle is completely paralyzed. In the absence of stimulation the rate of uptake in the EPA is slightly, but not significantly, greater than in M. Using autoradiography, we find an enhanced amount of isotope in the nerve terminals and their immediate vicinities compared with the muscle fibres, in both stimulated and unstimulated hemidiaphragms. There is no enhanced uptake of isotope into the nerve terminals in stimulated tissues in the presence of 26 microM HC-3. The uptake of isotope into the muscle is not altered by any of these treatments. There is a positive correlation between the initial rate of radioactive Ch uptake in the EPA and the amount of isotope in the nerve terminals (the mean corrected grain density above the nerve terminals). Without correcting for the large amount of diffusion that occurs, the ratio of the grain density above the synapses to that above the muscle fibres is 1.66 in tissue stimulated at 1 Hz, 1.04 in stimulated tissues in the presence of 26 microM HC-3, and 1.31 in unstimulated tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Presynaptic, facilitatory effects of the corticosteroid dexamethasone in rat diaphragm: modulation by beta-bungarotoxin.

Low concentrations of dexamethasone (up to 200 nM) increase the accumulation of choline (Ch) and its incorporation into acetylcholine (ACh) in the endplate rich area (EPA) of stimulated and unstimulated diaphragms in the presence of 10 microM Ch. Tissue ACh is not significantly altered, even after 140 min incubation. The specific radioactivity of the ACh in the EPA is thus increased by dexamethasone (Dex). The corticosteroid has no effects on acetylcholinesterase or choline acetyltransferase in diaphragm extracts. In the same medium, the amplitudes of the MEPPs, MEPCs and EPCs are also increased by Dex. Neither the quantal content of the EPCs nor the MEPP frequency, nor the half decay time of the MEPCs are altered. Therefore Dex (200 nM) increases both the resting and evoked output, and turnover of ACh in rat diaphragm. Beta-bungarotoxin (beta-BuTx) antagonizes the Dex-induced increase in Ch accumulation and its incorporation into ACh, and abolishes the increases in MEPC- and EPC-amplitudes, providing further argument for a presynaptic effect of Dex. In continuously-stimulated diaphragms, beta-BuTx causes an accumulation of ACh which is much greater than in unstimulated tissue. This accumulation of ACh is less in the presence of Dex, provided that Dex is added before beta-BuTx. The interaction of Dex and beta-BuTx is discussed in terms of their possible presynaptic sites of action.

Acetylcholine and an unidentified, muscle-contracting factor in the venom of the bumblebee, Bombus terrestris L.

Extracts of venom reservoirs as well as dilutions of venom droplets collected from the tip of the sting of Bombus terrestris were tested for biological activity on the isolated guinea-pig ileum, the frog rectus abdominis, and the rat phrenic nerve-hemidiaphragm preparation. The venom contains a cholinergic factor, apparently identical to acetylcholine, at a concentration of about 30 micrograms per venom reservoir. This was confirmed using radioimmunoassay of acetylcholine. The venom also contains a component causing a slow contraction of guinea-pig ileum and rat diaphragm. This component is heat stable, non dialysable, and is slowly destroyed in the venom preparation at about 0 degrees C.

Smooth muscle contracting compounds in venoms of sphecid wasps (Hym: Sphecidae).

1. The presence of smooth muscle contracting substances in venom preparations made from six species of sphecid wasps was investigated using a number of standard pharmacological techniques. 2. In addition, acetylcholine was identified by enzymatic degradation as well as by mass fragmentography, while histamine was determined using a radio-enzymatic method. 3. The venom reservoir of P. triangulum contains about 400 ng acetylcholine, but no histamine-, 5-HT- or bradykinin-like activity. 4. The venoms of the five other species contain histamine in amounts varying from 20 to 2000 ng per venom reservoir, but no acetylcholine-, 5-HT- or bradykinin-like activity.

Effects of corticosteroids on neuromuscular blocking actions of d-tubocurarine.

Dexamethasone (50 microgram/kg) significantly increased the LD50 of d-tubocurarine (d-TC) when administered i.p. simultaneously with d-TC. Choline (50 and 100 mg/kg) gave some protection against the lethal effects of d-TC and the cholinesterase inhibitors neostigmine (250 microgram/kg) and physostigmine (1000 microgram/kg) provided full protection against doses of d-TC twice the LD50. The blocking effect of d-TC (75 microgram/kg) on the sciatic nerve-tibialis anterior muscle preparation was antagonized by dexamethasone. Prednisolone delayed the occurrence of a complete neuromuscular block caused by d-TC in the phrenic nerve-diaphragm preparation, and antagonized the effect of d-TC on short tetanic contractions. d-TC (5 mumol/l) inhibited the [14C]choline uptake in the endplate-rich region of the rat diaphragm during stimulation. This inhibition was antagonized by dexamethasone as well as by physostigmine. The incorporation of radioactive choline into acetylcholine was inhibited in the presence of d-TC (15 mumol/l), and both dexamethasone and physostigmine counteracted this inhibition. It is concluded from these experiments that d-TC very probably has an effect on the choline carrier system. These experimental results support the hypothesis that glucocorticoids may improve reduced muscle performance by direct presynaptic effects at the neuromuscular junction.

Cooperative effects of hemicholinium-3 on high-affinity choline uptake by rat diaphragm.

Choline uptake in the end-plate rich area of rat diaphragm is saturable between 0.7 and 16 micron. This choline uptake obeys hyperbolic kinetics and indicates the presence of high-affinity carrier with Km values of 11.8 and 13.2 micron and Vmax values of 37 and 31 nmol choline/h/g tissue respectively. In the presence of hemicholinium-3 the carrier shows sigmoidal kinetics, and hemicholinium-3 is apparently a cooperative effector of choline uptake. The carrier is more calcium than sodium dependent.

The effect of corticosteroids and hemicholinium-3 on choline uptake and incorporation into acetylcholine in rat diaphragm.

The glucocorticoids prednisolone and dexamethasone antagonize the inhibition by hemicholinium-3 of both the rate of choline uptake and the incorporation of choline into acetylcholine in the rat diaphragm. Aldosterone has no such effects. It is concluded that the beneficial effect of glucocorticoids in the treatment of myasthenia gravis may be due not only to immunosuppression, but also to some direct effect on presynaptic events perhaps via a choline carrier or an enzyme of choline metabolism.