Beta-methylene ATP Research Articles

ATP-induced chondrocalcinosis.

To determine whether adult articular cartilage mineralizes in the presence of ATP. Intact adult porcine articular cartilage and monolayers of chondrocytes were cultured in physiologic media containing ATP, and mineralization was measured as retention of 45Ca. Cartilage was analyzed by electron microscopy. Articular cartilage sequestered 45Ca when incubated with 100 microM ATP: Use of the ATP analog alpha, beta-methylene ATP did not promote mineralization and addition of pyrophosphatase inhibited mineralization, indicating that hydrolysis of ATP to AMP and inorganic pyrophosphate is necessary for the process to occur. Mineral was concentrated in articular cartilage vesicles in the perichondral area. Adult articular cartilage mineralizes in the presence of ATP, in a manner similar to that found with isolated matrix or articular cartilage vesicles. This supports the notion that these structures have a role in chondrocalcinosis.

Changes in purinergic responses of the rabbit isolated central ear artery after chronic electrical stimulation in vivo.

1. The effect of chronic (4-16 days) electrical stimulation (5 Hz, 0.3 ms, 4-10 V) of the great auricular nerve in vivo on sympathetic cotransmission in the rabbit isolated central ear artery was examined. 2. Chronic stimulation had no significant effect on frequency-dependent (4-60 Hz) neurogenic contractions or contractile responses induced by exogenous noradrenaline (0.1-300 microM). 3. In contrast, contractions induced by exogenous alpha, beta-methylene ATP (10.0 microM) were significantly decreased in preparations from 16-day stimulated animals in comparison with sham-operated, 4-day and 8-day chronically stimulated animal groups. 4. It is concluded that chronic electrical stimulation of nerves supplying the ear artery may lead to the selective alteration of postjunctional P2x-purinoceptor mechanisms, while the effects mediated by post-junctional alpha 1-adrenoceptors remain unchanged.

Effect of nucleotides on the cytosolic free calcium activity and inositol phosphate formation in human glomerular epithelial cells.

1. Glomerular epithelial cells (GEC) were cultured from human kidneys and immunologically characterized. 2. The effect of extracellular nucleotides on the cytosolic free calcium activity [Ca2+]i was investigated with the fura-2 microfluorescence method. Extracellular UTP, UDP, UMP, ATP, adenosine 5'-O-(3-thio)-trisphosphate (ATP-gamma-S), inosine-triphosphate (ITP), guanyltriphosphate (GTP), 2-methylthio-ATP, AMP, alpha,beta-methylene-ATP and adenosine led to a rapid, transient, concentration-dependent increase of [Ca2+]i, followed by a plateau above the baseline level. 3. In a calcium-free extracellular solution, the rapid increase of [Ca2+]i was still present, whereas the plateau level was abolished. 4. ATP and UTP (ED50 both: 10(-5) M) stimulated inositol trisphosphate (InsP3) formation in GEC. 5. The order of potency for the purine nucleotides in stimulating InsP3 formation was ATP = ATP-gamma-S greater than ADP greater than 2-methylthio-ATP greater than AMP = a,beta methylene-ATP = adenosine. 6. The increase of InsP3 induced by ATP (10(-5) M) could be inhibited by the P2 receptor blocker suramin (greater than 10(-4) M). Reactive blue 2 exhibited a weak stimulating effect on the InsP3 formation and only a weak inhibitory effect at a concentration of 10(-3) M was observed. 7. Protein kinase C activation by preincubation of GEC with phorbol 12-myristate 13-acetate (PMA, 100 ng ml-1, 15 min) abolished the effect of ATP (10(-5) M) on InsP3 formation. Downregulation of protein kinase C by long term incubation (18 h) with PMA had no significant effect on the phosphoinositol turnover induced by ATP.8. The results indicate that an increase of [Ca2+]i and inositol phosphate breakdown can be mediated via activation of a P2 receptor in human GEC.

Effect of [D-Phe6] bombesin (6-13) methylester, a bombesin receptor antagonist, towards bombesin-induced contractions in the guinea-pig and rat isolated urinary bladder.

1. The effect of [D-Phe6] bombesin (6-13) methylester (OMe), a newly developed potent antagonist of bombesin receptors, has been investigated against bombesin-induced contractions of the guinea-pig and rat isolated urinary bladder. 2. Bombesin (0.1 nM-10 microM) produced a concentration-dependent contraction of the guinea-pig isolated bladder which approached the same maximum response as KCl (80 mM). The response to bombesin was antagonized in a competitive manner (rightward shift of the concentration-response curve without depression of the maximal response) by [D-Phe6] bombesin (6-13) OMe (0.3-10 microM). Degree of antagonism was concentration-dependent between 0.3 and 3 microM (dose ratios = 2.4, 9 and 39 in the presence of 0.3, 1, 3 microM of the antagonist). However, a larger concentration (10 microM) of the antagonist was not more effective (dose ratio = 36) than 3 microM. 3. Neither the action of bombesin nor the activity of the antagonist was influenced by peptidase inhibitors (bestatin, captopril and thiorphan 3 microM each) or by atropine, indomethacin, chlorpheniramine and desensitization of P2x purinoceptors by alpha, beta methylene ATP. 4. The bombesin antagonist was ineffective against contraction of the guinea-pig urinary bladder produced by the NK-1 tachykinin receptor-selective agonist, [Sar9] substance P sulphone. The action of the NK-1 receptor agonist was antagonized by L 668, 169 (3 microM), a cyclic peptide tachykinin antagonist. L 668, 169 had no effect toward bombesin-induced contraction. 5. The bombesin antagonist (1-10 microM) had no effect against the non-adrenergic non-cholinergic response of the guinea-pig isolated urinary bladder to electrical field stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrical field stimulation causes endothelium-dependent and nitric oxide-mediated relaxation of pulmonary artery.

The objective of this study was to ascertain the mechanism by which electrical field stimulation (EFS) of bovine pulmonary arterial rings causes endothelium-dependent smooth muscle relaxation. Like acetylcholine-elicited relaxation, EFS-elicited relaxation was endothelium-dependent and accompanied by accumulation of guanosine 3',5'-cyclic monophosphate (cGMP) in the vascular smooth muscle. Relaxation in response to EFS was unaltered by tetrodotoxin, guanethidine, atropine, propranolol, chlorpheniramine, cimetidine, indomethacin, aminophylline, alpha, beta-methylene ATP, nifedipine, capsaicin, and certain antioxidants and free radical scavengers. Thus the relaxation was not neurogenically mediated and was not attributed to free radical formation during EFS. Like nitric oxide-elicited relaxation, EFS-elicited relaxation was antagonized by oxyhemoglobin and methylene blue. Relaxation was also antagonized by the three NG-substituted L-arginine analogues: NG-methyl-L-arginine, NG-nitro-L-arginine, and NG-amino-L-arginine. NG-amino-L-arginine also inhibited the tissue cGMP accumulation in response to EFS. The inhibitory effect of the NG-substituted L-arginine analogues was reversed by addition of excess L-arginine but not D-arginine. Relaxation in response to EFS was dependent on the presence of extracellular calcium and intracellular calmodulin, as removal of extracellular calcium or addition of trifluoperazine nearly abolished relaxation. EFS-elicited relaxation was inhibited also by tetraethylammonium chloride and elevated extracellular potassium concentration. These observations indicate that EFS-elicited relaxation of bovine pulmonary artery is mediated by neuronally independent, but endothelium- and calcium-dependent, stimulation of nitric oxide and cGMP formation.

P2 purinoceptors stimulate inositol phosphate release in the organ of Corti.

We examined the effects of purinoceptor agonists on inositol phosphate (IP) release in the guinea-pig organ of Corti. The P2y receptor agonist ATP-gamma-S (200 microM) increased IPs 4-fold; identical concentrations of alpha, beta-methylene ATP, a P2x agonist, and adenosine, a P1 agonist, did not significantly affect IP release. In calcium-free incubations, simulated IP release decreased by 35% indicating partial dependence of ATP-mediated phosphoinositide hydrolysis on calcium influx. ATP-stimulated IP release was not enhanced by the cholinergic agonist carbachol known to increase IPs via muscarinic receptors in the organ of Corti. This is consistent with the notion that ATP and carbachol have a common target, most likely outer hair cells. P2 purinoceptors coupled to the phosphoinositide cascade suggest ATP as an afferent neuromodulator or efferent neurotransmitter in the cochlea.

Both ATP and the peptide VIP are inhibitory neurotransmitters in guinea-pig ileum circular muscle.

1. Intracellular membrane potential recordings were made from circular smooth muscle cells of the guinea-pig ileum in the presence of atropine (1 microM) and nifedipine (0.1 microM) at 30 degrees C. 2. Perfusion with adenosine triphospate (ATP, 100 microM) and vasoactive intestinal peptide (VIP, 2 microM) resulted in membrane hyperpolarizations of 6.4 +/- 0.3 and 6.8 +/- 0.3 mV, respectively. Picospritzes of ATP (10 mM in pipette) and VIP (100 microM in pipette) resulted in membrane hyperpolarizations of 6.9 +/- 0.4 and 6.3 +/- 0.4 mV, respectively. 3. The ATP-induced hyperpolarizations were antagonized by alpha, beta-methylene ATP desensitization (100 microM for 30 min) and the ATP antagonist Reactive Blue 2 (200 microM), but were unaffected by the VIP antagonist VIP 10-28 (1 microM). 4. The VIP-induced hyperpolarizations were antagonized by VIP 10-28, but unaffected by alpha, beta-methylene ATP desensitization and Reactive Blue 2. 5. A single pulse of transmural nerve stimulation (2 ms, 15 mA) resulted in an inhibitory junction potential (IJP) that reached a maximal amplitude of 12.9 +/- 0.5 mV at 378 +/- 20 ms from the stimulus. This fast IJP was abolished by apamin (2 microM) or tetrodotoxin (1 microM), antagonized by alpha, beta-methylene ATP desensitization or Reactive Blue 2, but unaffected by VIP 10-28. 6. In the presence of apamin (1 microM), four pulses of transmural stimulation (2 ms, 20 Hz, 15 mA) resulted in an IJP that reached a maximal amplitude of 4.8 +/- 0.2 mV at 1.4 +/- 0.1 s from the stimulus. This slow IJP was antagonized by tetrodotoxin (1 microM) or VIP 10-28 (1 microM), augmented by Reactive Blue 2 (200 microM), and unaffected by alpha, beta-methylene ATP desensitization. 7. These findings provide evidence that both ATP and VIP are inhibitory neurotransmitters in the circular muscle layer of the ileum and that ATP may be the neurotransmitter responsible for the fast IJP and VIP the neurotransmitter responsible for the slow IJP.

Neurotransmission in human resistance arteries: contribution of alpha 1- and alpha 2-adrenoceptors but not P 2-purinoceptors.

The contribution of postjunctional alpha 1- and alpha 2-adrenoceptors and P2-purinoceptors to the neuroeffector response was examined in isolated segments of human subcutaneous resistance arteries. Electrical field stimulation (EFS, 20 V, 0.2 ms, 1-25 Hz) elicited a maximum contractile response which was 38.2 +/- 1.6% of that elicited by exogenously applied (5 microM) noradrenaline (n = 56). Tetrodotoxin (1 microM), used to inhibit neurotransmission, reduced the electrically evoked response to 24.7 +/- 4.4% (n = 10) of the control response. The alpha 1-adrenoceptor antagonist prazosin (1 microM) reduced the maximum EFS contractile response to 64.8 +/- 5.5% of the control response (n = 17). Application of the alpha 2-adrenoceptor antagonist yohimbine (0.1 microM) reduced the maximum EFS response to 68.2 +/- 8.2% of the control response (n = 9). In the presence of prazosin plus yohimbine at the above-mentioned concentrations the maximum response to EFS was reduced to 47.6 +/- 6.7% (n = 11). Responses following alpha-blockade were not statistically different from those in the presence of tetrodotoxin, but the mean responses indicate that a non-adrenergic component to the EFS response cannot be discounted. Desensitisation of P 2-purinoceptors with alpha, beta-methylene ATP had no effect on responses to EFS; therefore under the conditions studied these receptors do not appear to be involved in neurotransmission. These results confirm the presence of postjunctional alpha 1- and alpha 2-adrenoceptors in human resistance arteries and for the first time demonstrate that the postjunctional alpha 2-adrenoceptor is important in modulating vascular responses elicited by intramural sympathetic nerve fibres.

Sympathetic neurotransmission in the Watanabe heritable hyperlipidaemic rabbit mesentery artery

The aim was to examine sympathetic neurotransmission in the mesenteric artery of atherosclerotic (Watanabe heritable hyperlipidaemic, WHHL) rabbits. Contractile responses to perivascular nerve stimulation (8-64 Hz) and exogenous application of the cotransmitters noradrenaline (0.1-100 microM) and adenosine triphosphate (1 microM) were measured. Catecholamine containing nerves were localised using fluorescence histochemistry. Female WHHL rabbits at 4, 6, and 12 months of age were used, with New Zealand white (NZW) rabbits as controls. The contractile responses to nerve stimulation were significantly smaller in 12 month old WHHL compared to NZW rabbits, although there was no difference at 4 and 6 months of age. Contractile responses of WHHL vessels to noradrenaline (0.1-100 microM) were greater than NZW controls at 4 months, but no differences were seen at 6 and 12 months. Contractile responses to alpha, beta-methylene ATP (0.1 microM) were greater in WHHL rabbits at 6 months but were unchanged at 4 and 12 months. There was no difference in the contractile responses to potassium chloride (120 mM) between 4, 6, and 12 month old WHHL and NZW rabbits. There was no variation in either strain of rabbit in the density of catecholamine containing nerves between 4 and 12 months of age. The reduction of sympathetic neurotransmission in WHHL rabbits at 12 months of age may largely be due to a reduction in the release of the cotransmitters noradrenaline and ATP. The reduced contractile response to nerve stimulation in 12 month old WHHL rabbits is discussed in terms of "protection" of the mesenteric artery from potential vasospasm and atherogenesis.

Omega conotoxin and prejunctional modulation of the biphasic response of the rat isolated urinary bladder to single pulse electrical field stimulation

1. Single pulse electrical field stimulation (EFS) produces a biphasic response of muscle strips of the rat isolated urinary bladder consisting of an early and a late contraction which were atropine-resistant and atropine-sensitive, respectively. Repeated application of desensitizing doses of the P2 purinoceptor agonist, alpha, beta-methylene ATP (mATP) inhibited the early response while leaving unaffected the late component. 2. Omega conotoxin (CTX, 0.1 microM) inhibited both the early and the late response either in control conditions or after enhancement by physostigmine (0.1 microM). The effect of CTX was, in both cases, more pronounced on the late than the early response to EFS. CTX (0.1 microM) failed to affect contraction produced by ATP or acetylcholine at concentrations (0.3 mM and 0.5 microM) which produced a response similar to that to EFS. 3. The effect of physostigmine was more intense for the late than the early response and was abolished by atropine. In the presence of CTX, physostigmine enhanced both the early and the late components of the mechanical response to EFS. 4. Nifedipine (0.1-1 microM) reduced to a similar extent both the early and late responses. Bay K 8644 (1 microM) produced a marked enhancement of the response to EFS, which, however, did not have a distinct late peak. In the presence of Bay K 8644, either atropine (3 microM) or tetrodotoxin (1 microM) had minor inhibitory effects indicating the myogenic origin of the response. 5. Neurokinin A (0.1-1 nM) enhanced both the early and late responses to EFS without affecting the contraction produced by exogenous acetylcholine or ATP. A consistent potentiation was evident also in the presence of CTX and for the early response, in the presence of atropine. Clonidine (3 microM) inhibited the response to EFS either in the absence or the presence of physostigmine. The inhibitory effect of clonidine, shown previously to depend upon activation of prejunctional alpha 2-adrenoceptors, was still observed in presence of CTX or atropine. 6. It is concluded that CTX-sensitive voltage dependent calcium channels play a more important role in determining the cholinergic rather than the non-cholinergic, putatively purinergic, component of the biphasic response of the rat bladder to single pulse EFS. The action of CTX is likely to be exerted on N-type rather than L-type (dihydropyridine-sensitive) calcium channels. Prejunctional modulation (enhancement by neurokinin A, inhibition by clonidine) occurs even in the presence of CTX-sensitive channels blockade.

Properties of ionic currents induced by external ATP in a mouse mesodermal stem cell line.

1. ATP was puff applied to cells of a mesodermal stem cell line, C3H10T1/2, and the responses were studied by whole-cell patch clamp recording. 2. In 91% of the cells (90/99), K+ current lasting for tens of seconds was observed after several seconds latency. The current showed outward rectification. In 10% of the cells (9/99), ATP induced Cl- current which also lasted for tens of seconds after several seconds latency, but showed little rectification. In 6% of these cells (5/99), both K+ and Cl- currents were induced by ATP. 3. The K+ current induced by ATP was dose dependent, with a Kd of 0.4 microM. The effects of ATP analogues were tested at a concentration of 20 microM. ADP and ATP-gamma-S induced the K+ current, while AMP and adenosine did not. alpha, beta-Methylene ATP produced a diminished K+ current. 4. The ATP-induced K+ current was not observed when EGTA in the internal solution was raised from 0.1 to 5 mM. In Fluo-3-loaded cells, an increase in intracellular Ca2+ concentration induced by the application of ATP was observed, and the time course was similar to the induced K+ current. Both the increase in intracellular Ca2+ and the K+ current were induced by ATP even in Ca(2+)-free external solution. Ryanodine (50 microM) in the external solution did not affect the ATP response, and application of 10 mM-caffeine alone to the external solution did not induce any response. 5. The variance of the steady-state fluctuations in the course of the ATP-induced slow K+ current was analysed. The single-channel conductance was estimated as 2.7 pS at 0 mV with external and internal K+ concentrations of 5 and 140 mM respectively. The K+ current was not affected by apamin at concentrations of up to 1 microM but was reduced to one-third by 140 mM-tetraethylammonium (TEA). 6. It was concluded that puff-applied ATP has two main effects in the mesodermal stem cells: an increase in the intracellular Ca2+ concentration and a succeeding hyperpolarization due to the Ca(2+)-activated K+ conductance which is present in this cell. The significance of the increase in intracellular Ca2+ caused by ATP is discussed.

Comparison of neurotransmission with nerve trunk and transmural field stimulation in guinea-pig mesenteric artery.

1. Intracellular electrical and contractile responses to sympathetic nerve trunk stimulation (NTS) and transmural electrical field stimulation (TMS) were compared in the guinea-pig mesenteric artery in vitro. 2. Step increases in voltage with NTS gave rise to excitatory junctional potentials (EJPs) which reached a plateau amplitude of 5-10 mV, whereas with TMS larger amplitude EJPs and sometimes action potentials were obtained. 3. EJPs of equal amplitude (1-7 mV) elicited with TMS and NTS had the same rise time, duration and decay half-time. 4. Slow depolarization obtained with repetitive stimulation was significantly greater in amplitude with TMS than with NTS. 5. Equal amplitude EJPs were obtained throughout the preparation with NTS. With TMS, the amplitude of responses declined substantially with distance from the stimulating electrodes. 6. Tetrodotoxin (TTX) completely blocked EJPs, slow depolarization and contraction with NTS; however, with TMS a TTX-resistant component was observed. The TTX-resistant response to TMS was abolished in the presence of a low-Ca2+ superfusion solution but was not affected by endothelium removal. 7. Phentolamine or prazosin abolished slow depolarization but not EJPs with NTS or TMS. Prazosin abolished contraction with NTS and reduced but did not abolish contraction with TMS. 8. alpha, beta-Methylene ATP abolished EJPs with NTS, whereas with TMS only EJPs obtained with low stimulus intensities were abolished. alpha, beta-Methylene ATP did not block contraction with either NTS or TMS. 9. Combined TTX, prazosin and alpha, beta-methylene ATP abolished EJPs initiated with TMS at all but the highest stimulus intensities (12-20 V, 0.3 ms duration). 10. It is concluded that responses obtained with NTS can be reliably attributed to the release of transmitter by the conduction of action potentials in paravascular nerves, whereas activation by TMS is a more complex phenomenon dependent upon stimulus strength and probably involving multiple forms of activation.

Adrenergic and purinergic cotransmission in nicotine-evoked vasoconstriction in rabbit ileocolic arteries

The possible involvement of ATP, in addition to noradrenaline, in nicotine-evoked vasoconstriction was studied in branches of the ileocolic artery of the rabbit. For measurement of vasoconstrictor responses, the arteries were simultaneously incubated and perfused. For measurement of the release of [3H]-noradrenaline, they were preincubated with [3H]-noradrenaline and then superfused. Prazosin (0.1 mumol/l) antagonized the constrictor effect of exogenous noradrenaline but not that of exogenous ATP. Desensitization of P2X-receptors by alpha, beta-methylene ATP markedly attenuated the effect of exogenous ATP but not that of noradrenaline. The presumed P2-purinoceptor antagonist suramin (100 mumol/l) reduced the maximal contraction obtainable with noradrenaline and shifted the concentration-response curve for the constrictor effect of alpha, beta-methylene ATP to the right, but did not change the effect of ATP. Nicotine elicited monophasic vasoconstrictions which faded while nicotine was still in the medium. The concentration-response curve was bell-shaped with an EC50 of 50 mumol/l and a maximal effect at 180 mumol/l, and the exposure time-response curve indicated that responses were maximal after 5 s of contact of nicotine (180 mumol/l) with the tissue. Neither prazosin 0.1 mumol/l nor desensitization by alpha,beta-methylene ATP changed the time course of the response to nicotine, but both depressed the magnitude of the responses over the whole concentration- and exposure time-response curves. The depression was greater with prazosin than with alpha,beta-methylene ATP. Desensitization by alpha,beta-methylene ATP or addition of suramin 100 mumol/l practically abolished the prazosin-resistant part of the response. The effect of nicotine was blocked by hexamethonium as well as by sympathetic denervation by 6-hydroxydopamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of presynaptic P2 receptor stimulation on transmitter release.

Because ATP is degraded to adenosine, its effect could be mediated by both P1 and P2 receptors. Hence, the actions of an ATP analogue, resistant to enzymatic breakdown (alpha, beta-methylene ATP), were studied on the resting and electrically evoked release of radioactivity from longitudinal muscle strips of guinea pig ileum, preloaded either with [3H]choline or with [3H]noradrenaline. Their effects were compared with the actions of adenosine and ATP. Although adenosine and ATP markedly decreased the [3H]acetylcholine release evoked by field stimulation, alpha,beta-methylene-ATP, a potent and selective agonist of P2x receptors, enhanced this release. However, 2-methyl-2-thio-ATP, an agonist of the P2y receptors, neither enhanced nor inhibited the [3H]-acetylcholine release. 8-Phenyltheophylline, an antagonist of P1 receptors, increased the stimulation-evoked release of acetylcholine, indicating that the release of acetylcholine is tonically controlled by endogenous adenosine via P1 receptors. When alpha,beta-methylene-ATP and 8-phenyltheophylline were added together, their potentiating effect on the acetylcholine release proved to be additive. Because alpha,beta-methylene-ATP failed to antagonize the presynaptic effect of adenosine on P1 purinoceptors, it seems very likely that its effect to enhance transmitter release is mediated via separate receptors, i.e., via P2x receptors, located on the axon terminals. Similarly, the stimulation-evoked release of [3H]noradrenaline was enhanced slightly by alpha,beta-methylene-ATP. Our results suggest that both cholinergic and noradrenergic axon terminals are equipped with P2 receptors through which the stimulation-evoked release of transmitter can be modulated by ATP in a positive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

P2 purinoceptor regulation of mucin release by airway goblet cells in primary culture

1. The effects of adenine analogues on mucin release by airway goblet cells have been examined in a hamster primary tracheal epithelial cell culture. 2. Adenosine, a P1 receptor agonist, had no effect on mucin release even at 2 mM, while ATP, a P2 receptor agonist, stimulated mucin release in a dose-dependent fashion with an apparent EC50 of 20 microM. The relative potency order among adenine nucleotides was ATP greater than ADP greater than AMP = adenosine. 3. ATP gamma S, a non-hydrolyzable analogue of ATP, was equipotent with ATP in stimulating mucin release. The potency order among some ATP analogues was ATP greater than 2-methylthio ATP greater than alpha, beta-methylene ATP greater than beta, gamma-methylene ATP. Reactive blue 2, a putative P2 gamma-purinoceptor antagonist, did not block the ATP-induced mucin release. 4. The present results indicate that mucin release by airway goblet cells is stimulated by extracellular ATP via P2 receptor-mediated mechanism. We suggest that this mechanism may be important in the physiological regulation of airway goblet cell mucin release in vivo.

Nuclear overhauser effect studies of the conformations of MG(α, β-Methylene)ATP bound to E. coli isoleucyl-tRNA synthetase

Internuclear distances obtained from transferred nuclear Overhauser effects were used in combination with distance geometry calculations to define the E. coli isoleucyl-tRNA synthetase bound conformation of Mg(alpha, beta-methylene)ATP both in the absence and in the presence of the cognate and noncognate amino acids L-isoleucine and L-valine, respectively. A single nucleotide structure having an anti adenine-ribose glycosidic torsional angle of -114 degrees was found to satisfy the experimental distance constraints. The nearly identical anti glycosidic torsional angles observed in all three complexes demonstrate that the conformation of the adenosine moiety of the enzyme-bound nucleotide is not sensitive to the presence or to the nature of the amino acid bound at the aminoacyladenylate site. In addition, the acceptable range of Mg(alpha, beta-methylene)ATP conformations bound to the E. coli isoleucyl-tRNA synthetase was found to be nearly identical to that previously determined for the E. coli methionyl-tRNA synthetase (Williams and Rosevear (1991) J. Biol. Chem. 266, 2089-2098). Thus, the predicted structural homology between the isoleucyl- and methionyl-tRNA synthetases, both members of the same class of synthetases on the basis of common consensus sequences, is further supported by consensus enzyme-bound nucleotide conformations.

Sympathetic co‐transmission to the cauda epididymis of the rat: characterization of postjunctional adrenoceptors and purinoceptors

1. Electrical field stimulation (10 Hz, 60 V, 1 ms, for 10 s) produced monophasic contractions of isolated preparations of rat cauda epididymis which could be abolished by guanethidine, and attenuated by prazosin and alpha, beta-methylene ATP. 2. The rank order of potency of adrenoceptor agonists in causing contraction of the preparation in the presence of the neuronal uptake blocker, nisoxetine (0.1 microM) was: adrenaline greater than or equal to phenylephrine greater than or equal to noradrenaline greater than clonidine greater than methoxamine greater than metaraminol greater than dopamine greater than or equal to isoprenaline greater than xylazine. 3. Responses to the agonists were blocked by prazosin but not by propranolol or idazoxan. 4. The rank order of potency of purinoceptor agonists in causing contraction of the cauda epididymis was: alpha,beta-methylene ATP greater than beta,gamma-methylene ATP greater than or equal to 2-methylthio ATP greater than ATP greater than ADP. AMP and adenosine did not cause contractions. 5. Contractile responses to the purine nucleotide analogues were blocked by repeated application of alpha,beta-methylene ATP. 6. It is concluded that both ATP and noradrenaline may act as co-transmitters in the sympathetic nerves supplying the smooth muscle of the rat cauda epididymis, and that alpha 1-adrenoceptors and P2x-purinoceptors are present postjunctionally.

Nuclear Overhauser effect studies on the conformations of Mg(alpha, beta-methylene)ATP bound to Escherichia coli methionyl-tRNA synthetase

Internuclear distances obtained from nuclear Overhauser effects were used in combination with a distance geometry algorithm to determine the conformation of Mg(alpha,beta-methylene)ATP bound to the Escherichia coli truncated methionyl-tRNA synthetase (delta MTS) both in the absence and presence of cognate and noncognate amino acids. Mg(alpha,beta-methylene)ATP, a nonhydrolyzable analog of ATP, was used to prevent hydrolysis of the nucleotide in the presence of either cognate or noncognate amino acids. Kinetic analysis showed that Mg(alpha,beta-methylene)ATP was a linear competitive inhibitor with respect to ATP in the ATP-pyrophosphate exchange reaction with a Ki = 1.2 mM. The pattern of internuclear Overhauser effects on Mg(alpha,beta-methylene)ATP bound to delta MTS was qualitatively consistent only with an anti glycosidic torsional angle, suggesting that the adenosine portion of the nucleotide is uniquely oriented in the binary enzyme-nucleotide complex. Nearly identical patterns of nuclear Overhauser effects were also observed in ternary complexes containing either cognate L-methionine or noncognate L-homocysteine amino acids. Distance geometry calculations permitted the range and conformational space of the allowed adenine-ribose glycosidic torsional angles in each of the complexes to be better defined and compared. Average adenine-ribose glycosidic torsional angles for enzyme-bound Mg(alpha,beta-methylene)ATP of -106 +/- 9 degrees, -99 +/- 11 degrees, and -97 +/- 11 degrees were determined for the delta MTS.Mg(alpha,beta-methylene)ATP, delta MTS.Mg(alpha,beta-methylene)ATP.L-methionine, and delta MTS.Mg(alpha,beta-methylene)ATP.L-homocysteine complexes, respectively. Comparison of the three enzyme-bound conformations showed that a single nucleotide structure having an adenine-ribose glycosidic torsional angle of -98 degrees with a 3'-endo to O4'-exo ribose sugar pucker was, within error, consistent with the experimental internuclear distances obtained in all three complexes. The nearly identical anti glycosidic torsional angles observed in all three complexes demonstrates that the conformation of the adenosine moiety of the enzyme-bound nucleotide is not sensitive to the presence or the nature of the amino acid bound at the aminoacyladenylate site. Therefore, conformational changes known to occur in the methionyl-tRNA synthetase upon ligand binding appear not to alter the bound conformation of the nucleotide. Information on the conformation and arrangement of substrates bound at the aminoacyladenylate site of delta MTS is necessary for understanding the molecular mechanisms involved in amino acid activation and discrimination.

Sympathetic neurotransmission in the rabbit isolated central ear artery is affected as early as one week following a single dose of X-irradiation.

1. The short-term effect of a single dose of 4500 rad X-irradiation on sympathetic neurotransmission (involving both noradrenergic and purinergic components) was assessed in the rabbit central ear artery, 1, 4 and 6 weeks post-irradiation. 2. Neurally mediated contractions were reduced as early as 1 week post-irradiation, with responses to lower frequency stimulation being initially most affected. This suggest that the purinergic component of the contractile response is affected earlier than the adrenergic component. 3. There was no change in the amplitude or sensitivity of treated preparations to the cumulative application of noradrenaline when compared with untreated preparations. In contrast, contractions to alpha, beta-methylene ATP (1 microM), a P2-purinoceptor agonist, were significantly increased at 4 and 6 weeks post-irradiation, although not at 1 week post-irradiation. 4. There were no apparent changes in the pattern of catecholamine fluorescence as a result of irradiation. However, the tissue content of noradrenaline was significantly reduced 6 weeks post-irradiation when compared with control preparations. 5. It is concluded that damage to sympathetic cotransmission is one of the early effects of irradiation, with initial impairment predominantly of the purinergic component.

Modulation by prostaglandin E2 of ATP and noradrenaline co‐transmission in the guinea‐pig vas deferens

1. In the guinea-pig vas deferens, prostaglandin E2 (100 nM) enhanced the overflow of ATP, whereas it inhibited [3H]-noradrenaline overflow due to field stimulation at 2 Hz. At 20 Hz, prostaglandin E2 still inhibited the overflow of [3H]-noradrenaline, whereas it was without effect on ATP overflow. 2. Prostaglandin E2 enhanced contractions to exogenously added noradrenaline and alpha, beta-methylene ATP. 3. These results provide evidence for pre- and postjunctional modulation of purinergic and adrenergic transmission by PGE2 in the guinea-pig vas deferens. The importance of these findings in relation to co-transmission is discussed.