Application Of Adenosine Triphosphate Research Articles

The actions of adenosine 5′‐triphosphate on guinea‐pig intracardiac neurones in culture

1. The actions of adenosine 5'-triphosphate (ATP) and related nucleotides and nucleosides on the membrane ion conductances of M and AH type intracardiac neurones cultured from ganglia within the atria and interatrial septum of newborn guinea-pig heart were studied with intracellular current- and voltage-clamp techniques. 2. Approximately 74% (120 out of 161) of AH type cells and 41% (5 out of 12) M cells responded to direct application of ATP (500 microM) onto their soma. 3. In 41% of M and 43% of AH type cells, focal application of ATP (500 microM) evoked rapid depolarization with an increase in conductance which frequently elicited action potential discharge. The underlying inward current had a null potential of -11.2 mV and was reduced in solutions containing low extracellular sodium and calcium but unaffected by reduced chloride-containing solutions. 4. In a further 31% of AH type cells, ATP evoked a multi-component response consisting of an initial depolarization followed by a hyperpolarization and a slow prolonged depolarization. The current underlying the initial depolarization resulted from an increase in conductance and had a null potential of -19.1 mV. The current was increased in low chloride-containing solutions and was only slightly reduced in low sodium- and calcium-containing solutions. The subsequent hyperpolarization and outward current resulted from an increase in membrane conductance and had a null potential of -88.5 mV, which was close to the potassium equilibrium potential in these cells. The slow depolarization and inward current was not associated with change in membrane conductance. 5. In less than 2% of AH cells, ATP evoked a second type of slow depolarization. This was associated with a fall in conductance and had a null potential of -90.7 mV. 6. In 40% of AH cells, adenosine (10-100 microM) inhibited the calcium-sensitive potassium current responsible for the after-hyperpolarization. The action of adenosine was antagonized by the P1-purinoceptor antagonist 8-phenyltheophylline (1-10 microM). 7. The potency order of agonists for all of the ATP-evoked responses, except the slow depolarization associated with a fall in conductance was ATP > ADP with AMP and adenosine being ineffective. 8. Responses to ATP were only weakly desensitized by alpha,,Beta-methylene ATP (3 x 10-6 M) and the potency order of analogues was 2-methylthio ATP > ATP > alpha,beta-methylene ATP, indicating the involvement of receptors similar to P2Y purinoceptors.

ATP-activated channels in rat and bullfrog sensory neurons: concentration dependence and kinetics

The concentration dependence and kinetics of ionic currents activated by extracellular adenosine 5'-triphosphate (ATP) were studied in voltage-clamped dorsal root ganglion neurons from rats and bullfrogs. About 40% of neurons of both species responded to ATP with an increase in membrane conductance. The ATP-activated currents were similar in the 2 species, except that currents in rat neurons desensitized faster. In bullfrog neurons, the conductance was half-maximally activated by about 3 microM ATP; at low concentrations, the conductance increased 3- to 7-fold for a doubling in [ATP], suggesting that several ATP molecules must bind in order to activate the current. A steeper concentration-response relationship than expected from 1:1 binding was also seen in rat neurons. The current activated quickly upon application of ATP and decayed quickly when ATP was removed. Activation kinetics were faster at higher [ATP], with time constants decreasing from about 200 msec at 0.3 microM ATP to about 10 msec at 100 microM ATP. Deactivation kinetics (tau approximately 100-200 msec) were independent of the ATP concentration. The rapid activation and deactivation make it seem likely that the ATP-activated current is mediated by direct ligand binding rather than by a second-messenger system. The experimental observations can be mimicked by a simple model in which ATP must bind to 3 identical, noninteracting sites in order to activate a channel. The potency and kinetics of ATP action were voltage-dependent, with hyperpolarization slowing deactivation and increasing ATP's potency. Deactivation kinetics were also sensitive to the concentration of external Ca, becoming faster in higher Ca.

Purine-induced depression of dorsal horn neurons in the cat spinal cord: Enhancement by tachykinins

The neurokinins, physalaemin, substance P, neurokinin A and bradykinin, were tested on the responses of single spinal neurons to the purines, adenosine 5′-triphosphate (ATP) and adenosine 5′-monophosphate and to GABA. Experiments were done on anaesthetized cats, recording extracellularly from functionally identified sensory neurons in the lumbar dorsal horn. All compounds were administered by iontophoresis. Neurokinins caused a slow, prolonged excitation which outlasted the period of application. Physalaemin was tested on responses to ATP in 24 units. In each case application of ATP caused either depression, excitation or a biphasic response when the application was not pre-conditioned by ejection of physalaemin. For 11 units, with ATP applications subthreshold to alter the on-going firing rate, such applications caused depression when they were preceded by administration of physalaemin. Three units were tested with ATP applications which caused the excitatory response; when the applications of ATP were preceded by ejection of physalaemin, there was then a depressant component in the response. In these 14 cases, the magnitude of the depression or of the depressant component of the response, was measured using currents which failed to produce depression in the absence of physalaemin ejection; the mean magnitude of this depression was 34.7 ± 1.6% (±S.E.M.). With the 10 remaining units, responses to ATP were unaffected by application of physalaemin. The early components of the biphasic and excitatory responses were unaffected by physalaemin and hence it appeared to have a differential effect, enhancing only the depressant effects of ATP. The enhancement of depression was reversible, lasting up to 30 min following a single ejection. Neither control current nor glutamate mimicked the effect of physalaemin in the responses to application of ATP. The depressant response to adenosine 5′-monophosphate was also enhanced by physalaemin: ejections of adenosine 5′-monophosphate subthreshold to affect the on-going firing rate caused depression after physalaemin application in 3 of 8 units (average depression: 35.0 ± 3.3%). On the other hand, depression induced by GABA was unaffected by physalaemin in every case ( n = 8); in 4 of these cases GABA was tested on units for which purine-induced depression was enhanced by physalaemin. Thus, physalaemin preferentially affected depressant responses to ATP and to adenosine 5′-monophosphate. As depressant effects of purines appear to be mediated by P 1-purinergic receptors, physalaemin may specifically enhance depression caused by activation of P 1-purinergic receptors. Substance P enhanced purine-induced depression in 4/7 units tested. Neurokinin A had a similar effect in 5/9 units. Bradykinin failed to affect the depression in any of the 15 units studied. Thus, enhancement of purine-induced depression may be an effect specific to the tachykinins. The results of the present study prompt us to suggest that tachykinins endogenous to the spinal cord may enhance depression induced by activation of P 1-purinergic receptors.

Action of externally applied adenosine triphosphate on single smooth muscle cells dispersed from rabbit ear artery.

1. Adenosine triphosphate (ATP), applied in the bathing solution or ionophoretically, depolarized freshly dispersed single arterial smooth muscle cells obtained by collagenase and elastase treatment of the rabbit ear artery. 2. Ionophoretic application of ATP evoked an inward current with a latency of about 70 ms and a time to peak of about 230 ms in cells held under voltage clamp using whole-cell patch-pipette techniques. 3. Bath application of 10 microM-ATP evoked a transient inward current at negative holding potentials. The amplitude of the ATP-induced current was linearly related to the clamp potential with a reversal potential near 0 mV. Removal of extracellular calcium, buffering intracellular calcium with high EGTA concentration, or depleting calcium stores with caffeine or noradrenaline treatment did not affect the ATP-evoked current. 4. Changing the chloride concentration gradient by decreasing extracellular or intracellular chloride concentration, or using the chloride channel blocker, frusemide, had no effect on the currents. 5. Replacing sodium with Tris shifted the reversal potential to more negative potentials. The reversal potential was not affected by exchanging intracellular potassium for caesium or sodium. Replacing extracellular sodium with 89 mM-barium also had little effect on the reversal potential. 6. These results are consistent with ATP activating a conductance that is cation selective but allows both monovalent and divalent cations to pass across the membrane.

Inhibition of excitatory junction potentials in guinea-pig vas deferens by α, β-methylene-ATP: Further evidence for ATP and noradrenaline as cotransmitters

Excitatory junction potentials (e.j.p.s) and spontaneous excitatory junction potentials (s.e.j.p.s) recorded from guinea-pig vas deferens were greatly reduced or abolished by α,β-methylene-ATP, the stable analogue of adenosine 5′-triphosphate (ATP). Brief (20–50 ms) local application of ATP by pressure ejection from a micropipette produced a transient depolarisation comparable to the e.j.p. Noradrenaline (NA) applied in a similar manner produced no such response. The depolarisation produced by the local application of ATP was also inhibited by α,β-methylene-ATP. Superfusion of the tissue with ATP or NA produced depolarisation of muscle cells; in the presence of α,β-methylene-ATP the depolarisation produced by ATP was almost abolished, whereas that produced by NA was not reduced. These results are consistent with the hypothesis that the e.j.p.s in the guinea-pig vas deferens are mediated by ATP, acting as a cotransmitter with NA in the sympathetic nerves supplying this organ.

The effect of adenosine triphosphate, magnesium chloride and phospholipids on crystal formation in the demineralized shell-repair membrane of the snail, Helix pomatia L. An in vitro study.

The effect of adenosine triphosphate (ATP), magnesium chloride (MgCl2) and phospholipids on the calcium-binding activity and crystal formation within the decalcified shell-repair membrane of the snail, Helix pomatia, was studied in vitro. The application of ATP produced a characteristic dual effect on calcification: (1) It strongly inhibited the formation of inorganic calcium carbonate (CaCO3) crystals. (2) It stimulated the development of organic crystalline bodies and induced deposition of amorphous calcium carbonate. The demineralized shell-repair membranes became white and rigid after incubation for 7 days in the medium containing 1.0 mM ATP. The inhibitory effect of Mg2+ on CaCO3 crystal formation was diminished by reduction of the concentration of MgCl2 in the incubation solution. Thus, after incubation for only 24h, 1.0 mM MgCl2 promoted the formation of birefringent CaCO3 crystals within the repair membranes. The principal effect of phospholipids on the demineralized shell-repair membrane was stimulatory, but after application of phospholipids to the medium, the formation of crystals proceeded slowly. The very large, composite crystals that were formed within the repair membranes showed strong birefringence. In all cases the development of the crystals and the organic crystalline bodies occurred in close vicinity to the amoebocytes. The role of ATP, MgCl2 and phospholipids in the recalcification of shell-repair membrane is discussed.

Effect of adenosine triphosphate and some derivatives on cerebral blood flow and metabolism.

1. Responses of cerebral blood vessels to peri- and intravascular doses of ATP (adenosine triphosphate) and some derivatives were studied in cat and baboon. 2. Perivascular application of ATP to cat pial arterioles gave a threshold dilatory effect at a concentration of 10(-11) M. This figure is comparable to the amount of ATP calculated to be released from electrically stimulated brain slices. 3. It is concluded that adenine nucleotides have a major role to play in the local control of cerebral blood flow. 4. Intracarotid injection of ATP showed a calculated threshold effect at 4 x 10(8) M in the cat and 4 x 10(-9) M in the baboon. 5. The threshold response of the vasculature to intracarotid adenosine lay between 4 x 10(-7) M and 4 x 10(-6) M in the baboon. Little effect was produced with AMP, pyrophosphate and inorganic phosphate. 6. Intracarotid ATP increased the oxygen consumption of the baboon brain parenchyma. This effect was attributed in part to an elevation of the cellular cyclic AMP levels. 7. Osmotic disruption of the blood-brain barrier in baboon did not affect the vasodilatory or metabolic effect of intracarotid ATP. 8. It is postulated that circulating purine compounds mediate a form of metabolic communication inthe body. Also, release of purine compounds from active local nerves might influence cerebral blood flow.

Reversal of Hydroxyproline-Induced Inhibition of Elongation of Avena Coleoptiles

Hydroxy-l-proline-induced inhibition of elongation of Avena coleoptile segments was measured in water and in indole-3-acetic acid. This inhibition was completely reversed by l-proline.Time-sequence experiments revealed that some time had to elapse, or some elongation had to occur, prior to the onset of hydroxyproline inhibition. The presence of sucrose supported the rate of auxin-induced elongation throughout a 24-hour period, and enhanced the effectiveness of hydroxyproline as an inhibitor of elongation.The application of adenosine triphosphate effectively counteracted the hydroxy-proline-induced inhibition of elongation; completely in water, and partially in indole-3-acetic acid. Optimal ATP concentrations were between 0.25 and 0.75 mm. Similarly, guanine-HCL, l-glutamic acid, and l-ornithine-HCL were capable of reversing the hydroxyproline inhibition. Other compounds that were tested but which proved to be less effective are tabulated.It is suggested that hydroxyproline may exert its inhibitory effect on the protein and/or RNA synthesis necessary for elongation of the oat coleoptile by interference with the metabolism of adenosine triphosphate or other high energy compounds.

Reversal of ethionine-induced inhibition of elongation of Avena coleoptiles by adenosine triphosphate

Ethionine inhibition of elongation of Avena coleoptiles has been measured in water and in 3-indoleacetic acid. In the presence of 0.01 M 1-ethionine the inhibition amounts to about 70%. The application of adenosine triphosphate (ATP) effectively counteracts this inhibition; optimal ATP concentrations are between 2.5 and 5 × 10 −4 M. In the absence of ethionine ATP still exhibits a stimulatory effect on elongation. It is suggested that ethionine may exert its inhibiting effect on the protein synthesis necessary for elongation of the oat coleoptile by interference with ATP metabolism.

Effects of thioctic acid and adenosine triphosphate on liver diseases. I. Influences of thioctic acid and adenosine triphosphate on the liver of the mice damaged with carbon tetrachloride.

It was observed that metabolic abnormalities in the liver damaged by CCl4 were definitely improved by application of adenosine triphosphate or thioctic acid, particularly the former was found to be more effective than the latter in the action toward glycolysis disturbance as represented by the changes of acid-soluble phosphate, copper, copper-protein, transaminase and dehydrogenase activities.

Interaction between Acetylcholine and Adenosine Triphosphate in Normal, Curarised and Denervated Muscle

Summary. Application of adenosine triphosphate (ATP) preceding the intra‐arterial injection of acetylcholine increases the sensitivity of striated amphibian and mammalian muscle to acetylcholine. This sensitization is not specifically connected with the neuromuscular transmission, for there is also in curarised muscle an increase of the mechanical response produced by electrical stimulation after previous application of ATP. This increase is, however, restricted to the actual period of contraction. In denervated muscle intra‐arterial injection of acetylcholine renders the muscle insensitive to subsequent application of ATP. When small amounts of acetylcholine are applied, the block is only present when ATP is administered through the artery, intramuscular application of this substance still being highly effective in the release of contraction. Larger amounts of acetylcholine also prevent intramuscularly applied ATP from being active. The electrical excitability remains unaltered. The threshold for intra‐artefially injected acetylcholine was ound to be ten times higher in winterfrogs than in summer‐frogs. In the former acetylcholine produces a contracture‐like response. Previous application of ATP changes it to a contraction of short duration. When twitches are produced by electrical stimulation during the acetylcholine response, they are enhanced in summerfrogs and reduced in height in winterfrogs. Intra‐arterial injection of acetylcholine decreases the electrical excitability and changes the strength‐duration curve of muscle for a considerable time after its application. The effect is especially pronounced in denervated muscle. The electrical excitability of denervated muscle is considerably higher than that of curarised muscle, while strength‐duration curves of denervated muscle are unaffected by curarisation. Previous application of ATP does not affect the strength‐duration curve of a muscle.

Application of Adenosine Triphosphate and Related Compounds to Mammalian Striated and Smooth Muscle

IN a former note1 an account was given of the stimulating effect of adenosine triphosphate and related substances on the isolated striated frog muscle fibre. When adenosine triphosphate is applied to striated mammalian muscle (m. tib. ant. of the decerebrated cat) by close arterial injection2 in amounts of 0·05–0·53 mgm. per gm. muscle (1·46–14·6 × 10-6 mol./ml. = 0·1–1·0 × 10-6 mol./gm. muscle) a rapid, tetanic contraction is released which is accompanied by interfering electrical activity (see accompanying record). Threshold dose and mechanical response are identical in non-curarized and curarized preparations, the effect of total curarization being insured by inexcitability of the sciatic nerve towards maximal stimuli and by insensitiveness of the muscle to intra-arterial injection of 50 μgm. acetylcholine. Intra-arterial injection of 5 μgm. acetylcholine after previous treatment of the non-curarized preparation with adenosine triphosphate releases a mechanical response with a considerably longer duration and higher tension than the same dose of acetylcholine does to a muscle without previous application of adenosine triphosphate.

Release of Contraction and Changes in Birefringence Caused by Adenosine Triphosphate in Isolated Cross Striated Muscle Fibres.1

Summary. 1 Micro-application of adenosine triphospbate (a 99 per cent preparation) in iso-osmotic Ringer solution releases in threshold amounts of 1–2 times 10-5μg a brief tetanus-like activity in non-curarised and curarised isolated striated muscle fibres. 2 A mechanical response following electric stimulation lasts considerably longer when a small fibre bundle has previously been treated with adenosine triphosphate than it does before application of the substance. 3 Application of adenosine triphosphate causes a reversible fall in birefringence. This decrease is abolished by poisoning with iodoacetate. 4 The fall in birefringence amounts to 10–30 per cent, increasing with increasing concentration of adenosine triphosphate, and decreases with increasing stretch of the fibre. 5 While the initial fall in birefringence following electrical stimulation occurs more rapidly than after application of adenosine triphosphate, the time of restitution is 10–20 min, being identical in both cases. The fibre has regained its resting condition, when birefringence reaches its minimum, and the protracted return is regarded as an expression of restitutional processes in muscle proteins. 6 Adenosine diphosphate and adenosine diphosphate + ortho-phosphate act like adenosine triphosphate both as regards initiation of contraction and effect on birefringence. In the case of adenosine diphosphate × orthophosphate the decrease in birefringenee is, however, essentially less than after application of adenosine triphosphate and adenosine diphosphate. 7 Adenylic acid has no effect on birefringence or contraction. Adenylic acid + pyrophosphate release contraction and cause a decrease of birefringence which reaches its minimum more rapidly than after application of adenosine diphosphate. 8 Inorganic sodium triphosphate releases contraction, while its influence on birefringence is absent or slight. 9 Sodium pyrophosphate causes contracture-like mechanical responses. Threshold amounts for the release of contraction are accompanied by irreversible changes in birefringence. 10 Sodium orthophosphate has no influence on birefringence and acts, even in large concentrations inconstantly as a chemical stimulus; the response, if present, is protracted. Birefringence remains unchanged. 11 The energy-rich phosphate bond in the form of adenosine-triphosphate is not only the immediate source of energy recharge, but also the agent for the release of contraction.