Adenosine Triphosphate Response Research Articles

A receptor that is highly specific for extracellular ATP in developing chick skeletal muscle in vitro

1. Extracellular adenosine 5'-triphosphate (ATP) activated an early excitatory conductance followed by a late potassium conductance in developing chick skeletal muscle. A series of ATP analogues were tested for their ability to activate these two conductances. All compounds tested were either agonists for both responses or for neither. Furthermore, the potency of agonists was similar for the two responses. 2. The order of potency for agonists was ATP approximately adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) approximately 2-methylthio-ATP (2-CH3S-ATP) greater than 2'-deoxy-ATP approximately 3'-deoxy-ATP greater than adenosine 5'-tetraphosphate (ATP-OPO3) approximately adenosine 5'-diphosphate (ADP). Many other ATP analogues were not agonists. 3. Activation of the excitatory response did not require divalent cations. Furthermore, the concentration-response relation of the excitatory response was similar when ATP was applied as the free anion of ATP (ATP4-) or complexed with a divalent cation (M.ATP2-). 4. Three antagonists of the ATP response were characterized. 8-Br-ATP was a weak antagonist, while 2',3'-dialdehyde-ATP and DIDS (4,4'-diisocyanatostilbene-2,2'-disulphonic acid) were potent irreversible inhibitors. The two conductances were equally affected by these antagonists. 5. These results suggest that both ATP responses are activated through the same receptor type, or two very similar receptors.

Adenosine triphosphate‐evoked currents in cultured neurones dissociated from rat parasympathetic cardiac ganglia.

1. The excitatory response of cultured neurones of rat parasympathetic cardiac ganglia to extracellular adenosine 5'-triphosphate (ATP) was examined using the whole-cell isolated membrane patch recording configurations of the patch clamp technique. The short latency between ATP application and activation of the membrane current (less than 20 ms) suggests a direct coupling between purinergic receptor and ion channel. The response was maintained during exposure to ATP suggesting that receptor desensitization is not a factor in current decay. 2. The current-voltage (I-V) relationship for macroscopic ATP-evoked currents showed strong inward rectification in the presence and absence of external divalent cations and a reversal potential of +10 mV (NaCl outside, CsCl inside). Unitary ATP-activated currents in cell-attached membrane patches exhibited a linear (ohmic) I-V relationship with a slope conductance of approximately 60 pS. 3. The order of agonist potency for the purinergic receptor-mediated response was 2-methylthioATP = ATP greater than ADP greater than AMP greater than adenosine = alpha,beta-methylene ATP greater than beta,gamma-methylene ATP, a sequence consistent with a P2y receptor subtype. ATP-evoked currents were attenuated by alpha,beta-methylene ATP (IC50 approximately 10 microM) and reversibly inhibited in a dose-dependent manner by Reactive Blue 2 (Kd = 1 microM). 4. The amplitude of the ATP-evoked current was dependent on the extracellular Na+ concentration. The direction of the shift in reversal potential when NaCl was replaced with mannitol indicated that the purinergic receptor channel is cation selective. The cation permeability relative to Na+ followed the ionic selectivity sequence Ca2+ (1.48) greater than Na+ (1.0) greater than Cs+ (0.67). Anions were not measurably permeant. 5. ATP and ACh-evoked responses in rat intracardiac neurones are mediated by distinct receptor channels. The ATP-activated channels in cardiac neurones may contribute to non-cholinergic, non-adrenergic neurotransmission and mediate, in part, the vagal innervation of the mammalian heart.

Irreversible desensitization of ATP responses in developing chick skeletal muscle.

1. In developing chick skeletal muscle, extracellular adenosine 5'-triphosphate (ATP) elicits an early excitatory conductance increase followed by a late potassium conductance increase. Both of these responses desensitize profoundly. Intracellular recordings and whole-cell voltage-clamp recordings were made in order to examine the time course and mechanism of desensitization and the recovery from desensitization. 2. Most of the loss of responsiveness to ATP occurred during the first minute of exposure to ATP. For the excitatory conductance, the loss of responsiveness to ATP resulted in part from long-lasting activation of the ATP-sensitive channels and in part from entrance into an inactive (non-conducting) state. In contrast, desensitization of the potassium conductance was entirely the result of a relatively fast transition to an inactive state. 3. Recovery from desensitization took many hours for both responses and was quite sensitive to temperature. 4. Recovery from desensitization for both responses was prevented by preincubation with the glycosylation inhibitor, tunicamycin. Several lines of evidence suggest that tunicamycin treatment blocked the delivery of new ATP receptors to the cell surface. 5. The recovery of the early response to ATP following exposure to two non-competitive inhibitors of the ATP response was also examined. These two compounds are thought to covalently modify the receptor. After exposure to either of these inhibitors, responsiveness to ATP returned over a time course that was similar to the time course of recovery from desensitization. 6. These results indicate that, following activation, ATP receptors do not become available for reactivation, and that recovery from desensitization is due to the insertion of newly synthesized receptors into the plasma membrane.

Permeation of both cations and anions through a single class of ATP-activated ion channels in developing chick skeletal muscle.

Micromolar concentrations of extracellular adenosine 5'-triphosphate (ATP) elicit a rapid excitatory response in developing chick skeletal muscle. Excitation is the result of a simultaneous increase in membrane permeability to sodium, potassium, and chloride ions. In the present study we quantify the selectivity of the ATP response, and provide evidence that a single class of ATP-activated ion channels conducts both cations and anions. Experiments were performed on myoballs using the whole-cell patch-clamp technique. We estimated permeability ratios by measuring the shift in reversal potential when one ion was substituted for another. We found that monovalent cations, divalent cations, and monovalent anions all permeate the membrane during the ATP response, and that there was only moderate selectivity between many of these ions. Calcium was the most permeant ion tested. To determine if ATP activates a single class of channels that conducts both cations and anions, or if ATP activates separate classes of cation and anion channels, we analyzed the fluctuations about the mean current induced by ATP. Ionic conditions were arranged so that the reversal potential for cations was +50 mV and the reversal potential for anions was -50 mV. Under these conditions, if ATP activates a single class of channels, ATP should not evoke an increase in noise at the reversal potential of the ATP current. However, if ATP activates separate classes of cation and anion channels, ATP should evoke a significant increase in noise at the reversal potential of the ATP current. At both +40 and -50 mV ATP elicited a clear increase in noise, but at the reversal potential of the ATP current (-5 mV), no increase in noise above background was seen. These results indicate that there is only a single class of excitatory ATP-activated channels, which do not select by charge. Based on analysis of the noise spectrum, the conductance of individual channels is estimated to be 0.2-0.4 pS.

The relation of ATP response in CaNT tumours after X-irradiation to varying tumour volumes.

The response of adenosine-5'-triphosphate (ATP) levels in implanted murine CaNT tumours, 2.5 h after 10 Gy X-rays (100 kVp) was measured in vivo using various tumour volumes. It is probable that the concentration of ATP, which increased after X-irradiation, plays a major role in energy supply associated with cellular repair processes. Nevertheless, when ATP levels in irradiated tumours were plotted against tumour volume in the range 150-550 mm3, a monotonically decreasing relation was found. This is interpreted as reflecting the degree of hypoxia and changes in the tumour metabolic status. In addition effects of vascular occlusion and artificial hypoxia have been studied after clamping the tumour for 15 min, followed by a radiation dose of 10 Gy and maintaining the clamp for a further 2.5 h. This showed a drastic decrease in the ATP levels 2.5 h after the irradiation, compared with controls in normoxia. It is thought this is due to induction of hypoxia which is produced throughout the tumour volume, resulting in the depletion of cellular ATP. This implies that the clamped tumour cells are less capable of generating a metabolic response following radiation damage.

Co-transmission in the rat vas deferens: Postjunctional synergism of noradrenaline and adenosine 5′-triphosphate

In the isolated prostatic half of the rat vas deferens, joint application of noradrenaline (NA) and adenosine 5′-triphosphate (ATP) produced a contractile response whose magnitude was greatly larger than the addition of the tension generated by the application of each agent alone. The effect of ATP was mimicked by two non-hydrolyzable ATP analogs, but not by GTP, AMP or adenosine. In sympathectomized rats. ATP potentiated NA effects, increasing both the peak tension and the duration of the vas deferens contractile response. The synergism was concentration related. Prazosin antagonized the NA synergism but not the ATP response. Likewise, desensitization of the P 2-purinoceptor blocked the ATP synergism without modifying the NA-induced contraction.

Diet Ingestion and Destination in Culiseta inornata (Diptera: Culicidae): Effects of Mouthpart Deployment and Contact of the Fascicle and Labellum with Sucrose, Water, Saline, or Atp

Diet ingestion and destination in female Culiseta inornata (Williston) is controlled by a complex sequence of stimuli which include the phagostimulants in the diet. Attached animals with their mouthparts separated were stimulated by having combinations of solutions applied to the labellum and the end of the fascicle. A solution on the fascicle contacts the labral chemoreceptors and, if there was any ingestion, receptors in the cibarial region. An amount of 1 M sucrose, saline, adenosine triphosphate (ATP) in saline, or water applied to the fascicle had a much greater effect on diet ingestion and destination than did the same solutions applied to the labellum. ATP in saline (1 mM) at 20°C was applied to the fascicle and induced 70% of the test insects to gorge. Consequently, separation of mouthparts is sufficient to facilitate the ATP response and a heat signal is not necessary. Artificial mouthpart separation produces different results than normal mouthpart deployment through a membrane. It enhances the ingestion of saline and biases the diet destination of 1 M sucrose. Normal mouthpart deployment through a membrane reduces the response to sugar, and blocks the ingestion of water completely. The results illustrate the complexity and the integration of the various stimulus-response events involved in the feeding behaviors of mosquitoes.

Purine receptors in the rat anococcygeus muscle.

Adenosine triphosphate (ATP) caused contraction of the resting isolated rat anococcygeus muscle. Non-phosphorylated purines did not cause contraction of the resting muscle but did so in muscles in which the tone was raised by carbachol or guanethidine. Adenosine, (-)N6-phenylisopropyladenosine (PIA) and 5'-N-ethyl-carboxamide adenosine (NECA) were approximately equipotent, and these responses were not prevented by theophylline, quinidine, 2,2'-pyridylisatogen tosylate, phentolamine, methysergide, dipyridamole, hexobendine or indomethacin. The contractions became smaller as muscle tone progressively declined, and it is suggested that this effect may explain the apparent blockade of ATP responses by indomethacin reported previously. Adenosine, 2-chloroadenosine, ATP, PIA and NECA inhibited contractile responses of the anococcygeus to field stimulation of the excitatory adrenergic innervation. This inhibitory action was blocked by theophylline, and as PIA was easily the most potent purine tested, it may involve activation of an A1/Ri receptor. It is also argued, however, that the A/R scheme of classification may be inappropriate for the description of responses of intact tissues. As response to noradrenaline were not changed by the purines, the inhibitory effect on stimulation-evoked contractions is probably mediated at a presynaptic site. None of the purines tested had any effect on the neurally mediated inhibition of the anococcygeus which is seen when intrinsic tone is raised and the excitatory adrenergic nerves are blocked.

The effects of adenyl compounds on the heart of the axolotl ( Ambystoma mexicanum)

1. In the spontaneously beating axolotl atrium, adenosine 5'-triphosphate (ATP) produced initial excitation followed by inhibition and then by a secondary excitation. 2. This third phase of the ATP response was only seen in electrically driven preparations in the presence of 8-phenyltheophylline (8-PT), an adenosine receptor antagonist. 3. α,β-Methylene ATP (APCPP), a stable analogue of ATP, produced only excitatory effects, while adenosine and β,γ -methylene ATP (APPCP), a slowly degradable ATP analogue, produced inhibition or inhibition followed by excitation. 4. 2-Chloroadenosine produced inhibition. 5. The excitatory effects were not antagonized by 8-PT, indomethacin or propranolol and phentolamine. 6. The negative inotropic responses of these compounds were antagonized by 8-PT and, with the exception of 2-chloroadenosine, potentiated by dipyridamole, an adenosine uptake blocker. 7. In the ventricle, ATP, APCPP and APPCP produced positive inotropic effects, which were not affected by 8-PT, indomethacin or propranolol and phentolamine. Adenosine produced a negative inotropic effect which was not antagonized by 8-PT nor atropine nor potentiated by dipyridamole. 8. The effects of adenyl compounds on the axolotl (urodele) heart suggest that, like the frog (anuran) heart, both P 1- and P 2-purinoceptors are present in the axolotl atrium and that only P 2-purinoceptors are present in the axolotl ventricle, although adenosine does produce an inhibitory effect on the ventricle which is probably mediated via the release of a neurotransmitter other than acetylcholine.

Calcium and the action of adrenaline, adenosine triphosphate and carbachol on guinea-pig taenia caeci.

1. The action of adrenaline (in the presence of propranolol; 3 x 10(-6) M), adenosine triphosphate (ATP) and carbachol on guinea-pig taenia caeci, and the interaction between these agonists, was studied by measuring changes in membrane potential using the sucrose-gap method in quiescent preparations at 22 degrees C.2. A sustained hyperpolarization was caused by addition of adrenaline (3 x 10(-6) M) and by applying adenosine triphosphate (ATP; 4 x 10(-4) M) for 5 min in Krebs solution. In calcium-free medium containing EGTA (0.4 mM) and high magnesium (6.2 mM), both the alpha-agonist and ATP caused a transient hyperpolarization which passed off within 5 min, although the agonist was still present.3. The transient hyperpolarization evoked by these agonists in the absence of calcium could be evoked only once. The response was restored after exposure to high calcium, (40 mM for 2 s, or 10 mM for 30 s). The maximum amplitudes of the hyperpolarization caused by adrenaline or ATP after exposure to high calcium (40 mM or 10 mM) were similar, while the maximum hyperpolarization after application of 2.5 mM-calcium was smaller.4. The area of the maximal response evoked by adrenaline or ATP was independent of the exposure time to calcium-free solution after removal of the extracellular calcium (20 min). The sum of the areas of a first submaximal response, obtained by applying adrenaline for less than 5 min to the calcium-free solution (20 min), and of the second response (5 min application) elicited after continuing in calcium-free medium for another 8 min, was constant.5. In the presence of the bee toxin apamin (10(-7) M), addition of ATP (4 x 10(-4) M) caused depolarization of the membrane both in the presence and absence of external calcium. These responses were not blocked in low sodium solution (22.7 mM) but were reduced by the calcium antagonist D600 (2 x 10(-5) M).6. In calcium-free conditions the alpha-response to adrenaline was decreased by a preceding addition of ATP and vice versa. Abolition of the ATP response (4 x 10(-4) M) by adrenaline (10(-5) M) was prevented by blocking the alpha-receptors with phentolamine (2 x 10(-5) M).7. Carbachol (5 x 10(-7)-5 x 10(-5) M) depolarized the muscle cells in calcium-free medium; a second addition of carbachol also caused depolarization, the amplitude being lower. The carbachol depolarization was dependent on the exposure time to calcium-free solution.8. The adrenaline response was reduced by about 25% by carbachol if applied previously, independent of the carbachol concentration (5 x 10(-7)-5 x 10(-5) M). The carbachol response, however, was not affected if preceded by the alpha-response.9. It is concluded that ATP and the alpha-agonist, after binding to their receptor sites, activate the same mechanism, which is mobilization of calcium from the same membrane compartment to open potassium channels, causing hyperpolarization of the muscle cell membrane; the hyperpolarization is transient or sustained in nature depending on the availability of external calcium to replenish the calcium compartment localized in the membrane. This adrenaline and ATP-sensitive calcium compartment is distinct from that which is sensitive to carbachol.

Electrical and mechanical activity of the isolated lower urinary tract of the guinea-pig.

1 Strips of urethra taken from guinea-pigs contracted in response to acetylcholine, noradrenaline (via alpha-adrenoceptors) and 5-hydroxytryptamine, and were relaxed by adenosine triphosphate (ATP) if the tone was raised. Isoprenaline produced relaxation of bladder strips (via beta-adrenoceptors) whereas ATP caused contraction. 2 Atropine completely blocked all responses to acetylcholine; quinidine failed to block ATP responses selectively; methysergide blocked responses of the urethra but not the bladder to 5-hydroxytryptamine. 3 Spontaneous electrical activity was recorded with intracellular microelectrodes from all regions: in the urethra infrequent bursts of spikes occurred at 1-7 min intervals; regular spikes at 6-30/min were recorded from the detrusor muscle. In the bladder base, bursts of spikes were superimposed on the regular pattern. 4 Bursts of spikes in the urethra were initiated by noradrenaline, phenylephrine or acetylcholine and inhibited by ATP; regular spikes in the bladder were accelerated by acetylcholine or ATP and slowed by noradrenaline or isoprenaline. 5 The intrinsic electrical activity and pharmacological properties of the urethra therefore differ from those of the bladder. This may account for the different responses of the two regions in normal function.

Toxicity of Bacillus thuringiensis entomocidal protein toward cultured insect tissue

The entomocidal protein from crystalline inclusion bodies of Bacillus thuringiensis can be bioassayed in vitro using cultured insect tissue. Larval cells of the spruce budworm, Choristoneura fumiferana, are damaged by enzyme-digested (activated) protein isolated from B. thuringiensis crystals. Measurement of toxicity is accomplished by detection of adenosine triphosphate (ATP) in treated cultures using firefly bioluminescence. The ATP content of toxin-treated tissue is inversely proportional to the amount of toxin added. Tissue cells from the spruce budworm exhibited maximum susceptibility to activated δ-endotoxin after 120 hr incubation. Probit analysis of tissue ATP response to toxin dose indicated 50% of the cells were damaged by 14.6 μg or less of toxin protein per 2 × 10 5 insect tissue cells. Activated δ-endotoxin was entomocidal to insects as well, as detemined by mortality studies with second-instar larvae of the European corn borer. Electron microscopic observations of insect tissue treated with activated δ-endotoxin protein for 60 min revealed massive outer membrane disruption and subsequent loss of cytoplasmic constituents, accompanied by swelling of the nuclear membrane.

Identification of a purine (P2) receptor linked to ion transport in a cultured renal (MDCK) epithelium.

1 Exogenous adenosine triphosphate (ATP) stimulates the short circuit current (SCC) in a cultured renal-derived epithelium (MDCK). Half-maximal stimulation is achieved at 1.91 X 10(-5) M ATP. 2 It is suggested that ATP interacts with a P2 purine receptor upon the basis of (a) agonist potency (ATP greater than adenosine diphosphate much greater than adenosine monophosphate, adenosine; ATP greater than uridine triphosphate greater than inosine triphosphate much greater than cytosine triphosphate, guanosine triphosphate); (b) the inhibition of the ATP response by quinidine (1 X 10(-3) M) but not by theophylline (1 X 10(-3) M). 3 Indomethacin (1 X 10(-5) M) inhibits the response of the cultured epithelium to ATP. 4 Prostaglandin E1 (PGE1) stimulates SCC but potentiates the effect of ATP on SCC. The divalent cationic ionophore A23187 (1 X 10(-6) M) transiently stimulates SCC itself and abolishes ATP-induced stimulation of the SCC.

Contribution of prostaglandins to the adenosine triphosphate-induced contraction of rabbit urinary bladder.

1 Adenosine 5'-triphosphate (ATP) produced an initial rapid, phasic contraction and a later, slowly developing tonic contraction in the isolated detrusor of the rabbit but mainly a rapid, phasic response in the guinea-pig bladder. 2 Electrical field stimulation elicited only a rapid, phasic contraction in both rabbit and guinea-pig bladders. 3 Prostaglandin synthesis inhibition by means of indomethacin and suprofen abolished the tonic response to ATP in the rabbit detrusor, leaving the phasic part of the contraction almost unaffected. The ATP-induced contraction in guinea-pig bladder was not influenced by indomethacin. 4 The contractile response of rabbit urinary bladder to prostaglandins F2 alpha and E2 and to carbachol were not significantly influenced by indomethacin. The contractions induced by the prostaglandins were similar to the tonic response to ATP. 5 Tetrodotoxin, atropine, phentolamine, and theophylline did not alter the ATP-induced contraction. However, the calcium antagonists, nifedipine and nimodipine, abolished the phasic ATP response and greatly reduced the tonic part of the contraction. 6 Tachyphylaxis occurred on repeated addition of ATP; the response to field stimulation was progressively reduced only after indomethacin pretreatment. 7 ATP and prostaglandins may contribute to the non-adrenergic, non-cholinergic component of the excitation of rabbit and guinea-pig bladder.

Inotropic responses of the frog ventricle to adenosine triphosphate and related changes in endogenous cyclic nucleotides.

1. A study has been made of a well documented but poorly understood response of the isolated frog ventricle to treatment with exogenous adenosine 5' triphosphate (ATP). Measurements of membrane potential, isometric twitch tension and levels of endogenous 3',5'-cyclic nucleotides have been made at various times during the ATP-induced response. 2. ATP elicits a characteristic triphasic response, which comprises an initial, abrupt increase in contractility, rising to a maximum within a few beats (first phase); followed by a period when the twitch amplitude falls, sometimes to below the control level (second phase); and superceded by a more slowly developing and longer-lasting increase in contractile force (third phase). The response is unaffected by atropine, propranolol or phentolamine. However, the prostaglandin synthetase inhibitor indomethacin depresses the first phase and entirely suppresses the third phase. 3. The inotropic effects of ATP are accompanied by changes in the shape of the action potential. These effects are dose-related. The duration of the action potential (D-30mV) and its positive overshoot (O) are increased during all phases of the response, for [ATP]o's up to 10(-5) M. However, at higher [ATP]o's, D-30mV and O ar both reduced during the second phase (but not the first or third phase), when isometric twitch tension is also depressed. The relationship between action potential duration and twitch tension (P) for different [ATP]o's is linear for all three phases of the response, but the slopes of the curves (delta P/delta D) are markedly different, indicating that the sensitivity of the contractile system to membrane depolarization is not constant, but varies continuously throughout the response. 4. ATP has a potent stimulatory effect on the metabolism of endogenous 3',5'-cyclic nucleotides. The time courses of the changes in adenosine 3','5-cyclic monophosphate (3',5'-cyclic AMP) and guanosine 3',5'-cyclic monophosphate (3',5'-cyclic GMP) are complex, but the accompanying change in isometric twitch tension is paralleled closely by corresponding changes in the ratio 3',5'cyclic AMP:3',5'-cyclic GMP. 5. It is concluded that ATP exerts a dual effect on the ventricle and that the contractile response is regulated by changes in the metabolism of 3',5'-cyclic nucleotides. The effects of indomethacin indicate a possible involvement of prostaglandins in mediating the ATP response. It is suggested that the initial effect of ATP on the ventricle is to increase the permeability of the fibres to Ca2+. 6. The relationship between 3',5' cyclic nucleotide levels and ventricular contractility is discussed. It is postulated that the antagonistic effects of 3',5'-cyclic AMP and 3',5'-cyclic GMP are expressed at the level of certain phosphoproteins which regulate both the availability of Ca2+ and the sensitivity of the contractile proteins to Ca2+.

Succinate oxidase in Neurospora.

Two kinetically distinct states of succinate oxidase have been detected in the mitochondria of Neruospora crassa. One state has a K(m) for succinate of 4.1 x 10(-3)m, and the other has a K(m) for succinate of 3.5 x 10(-4)m. The high K(m) state was found in freshly extracted mitochondria from either 20- or 72-hr mycelium. However, the succinate oxidase activity in mitochondria from 20-hr mycelium rapidly deteriorated in vitro, leaving a stable residual activity with the lower K(m) for succinate. Adenosine triphosphate (ATP) plus Mg(2+) stabilized the high K(m) state in these preparations. The high K(m) state of succinate oxidase was further characterized by a two- to threefold increase in activity over the pH range 6.6 to 8.0 and by classical competitive inhibition by fumarate and malonate. By contrast, the low K(m) state of succinate oxidase showed a relatively flat response to pH over the range 6.6 to 8.0 and a nonclassical pattern of inhibition by fumarate and malonate, as shown by nonlinear plots of reciprocal velocity versus reciprocal substrate concentration in the presence of inhibitor or reciprocal velocity versus inhibitor concentration at fixed substrate concentrations. The relationship of mycelial age to the in vitro stability of succinate oxidase is considered with reference to probable changes in the relative pool sizes of extra- and intramitochondrial ATP in response to changes in the rate of glycolysis.