Gamma-methylene Adenosine 5'-triphosphate Research Articles

Evidence for the presence of both P1 and P2 purinoceptors in the rat myometrium.

1. Adenosine, adenosine triphosphate (ATP) and some stable analogues of adenosine inhibited field stimulation-induced contractions of the uterus from rats treated with oestradiol cypionate (20 micrograms/kg, s.c.) 1 day previously. Adenosine was twice as potent as ATP; both were potentiated by dipyridamole (10 mumol/L). 2. The order of agonist potency of adenosine and its analogues was: 5'-N-ethylcarboxamidoadenosine (NECA) > N6-cyclohexyladenosine > or = R-phenylisopropyladenosine = S-phenylisopropyladenosine = 2-chloroadenosine > or = adenosine > or = ATP > > 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine. This order suggests the presence of P1 purinoceptors of the A2B subtype. 3. Responses to agonists were antagonized to differing extents by the P1 purinoceptor antagonist 8-phenyltheophylline (10 mumol/L). 4. In uterine preparations from rats pretreated for 2 days with oestrogen (20 micrograms/kg, s.c.) and for 1 day with progesterone (3 mg/animal, s.c.), the inhibitory potencies of adenosine and NECA were reduced, indicating hormonal regulation of uterine responsiveness to P1 purinoceptor agonists. 5. Stable analogues of ATP caused contractions of unstimulated myometrial preparations from oestrogen-treated animals, indicating activation of a P2 purinoceptor, possibly of the P2X subtype, because of the relative order of potency was alpha, beta-methylene ATP > beta, gamma-methylene ATP = ATP = 2-methylthio ATP.

P2x-purinoceptors of myenteric neurones from the guinea-pig ileum and their unusual pharmacological properties.

1. Whole-cell and outside-out patch clamp recordings were used to characterize the physiological and pharmacological properties of the P2x-purinoceptors of myenteric neurones from the guinea-pig ileum. 2. Adenosine 5'-triphosphate (ATP) and analogues (1-3000 microM) evoked a rapid inward current in > 90% of all recorded neurones. The reversal potential of this current was dependent on the extracellular sodium concentration, at +14 +/- 1.9, 0 +/- 1.6 and -12 +/- 1 mV for 166, 83 and 42 mM of sodium, respectively. The fast activation and inactivation of this current occurred even when guanosine 5'-triphosphate (GTP) was omitted from the pipette solution or substituted with an equimolar concentration of guanosine 5'-o-[2-thiotriphosphate] (GTP-gamma-S). Single channel currents were observed when these outside-out membrane patches were exposed to ATP (10-30 microM). These channels have a unitary conductance of about 17 picosiemens. 3. The rank-order of potency of the agonists used to induce the whole-cell currents was: ATP-gamma-S = ATP = 2-methylthio-ATP (2-Me-S-ATP) > > alpha, beta-methylene ATP = beta, gamma-methylene ATP; adenosine and uridine 5'-triphosphate (UTP) (up to 1 mM) were inactive. 4. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (1-30 microM) antagonized the effects of ATP (1 mM) with an IC50 of 4 microM. alpha, beta-Methylene ATP (100 microM) did not affect the ATP (30 microM)-induced current. Cibacron Blue 3GA increased the ATP activated cationic current whereas Basilen Blue E-3G had a very weak antagonistic effect (IC50 > or = 3 mM). Suramin potentiated the currents induced by ATP through a mechanism that was independent of its inhibitory effect on ectonucleotidase activity, as suramin also potentiated the effect of alpha, beta-methylene ATP (an ATP analogue that is resistant to nucleotidases). 5. In conclusion, the myenteric P2x-purinoceptor shares some properties with other purinoceptors in particular with the P2x4- and P2x6-purinoceptors. This receptor has also some unusual pharmacological properties suggesting that myenteric neurones express a novel subtype of P2x-purinoceptors. The properties of this receptor, however, might be a result of the combination of two or more of the homomeric purinoceptors so far characterized.

Evidence for a P2-purinoceptor mediating vasoconstriction by UTP, ATP and related nucleotides in the isolated pulmonary vascular bed of the rat.

1. The vasoconstrictor effects of uridine 5'-triphosphate (UTP), uridine 5'-diphosphate (UDP), uridine 5'-monophosphate (UMP) and uridine were tested in the isolated pulmonary vascular bed of the rat. Comparison was made with the effects of adenine nucleotides, adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) and with adenosine. The effect of P2x-purinoceptor desensitization and blockade was compared on the vascular responses to uracil and adenine nucleotides. 2. At doses ranging from 10(-8) to 10(-5) mol, UTP elicited dose-dependent vasoconstriction. UDP was equiactive to UTP, while UMP and uridine did not show vasomotor activity. Similarly, ATP showed dose-related vasoconstrictor activity. ADP was less potent than ATP in eliciting vasoconstriction, while AMP was active only at the higher doses tested and adenosine was ineffective. 3. Vasoconstriction was produced by ATP analogues with the following order of potency: alpha, beta-methylene ATP > ATP gamma S > beta, gamma-methylene ATP > 2-methylthio ATP > or = ATP. 4. Desensitization of P2x-purinoceptors by the selective agonist alpha, beta-methylene ATP did not modify the vasoconstrictor activity of UTP and UDP and only partially reduced vasoconstrictor responses to ATP, while it abolished vascular responses to alpha, beta-methylene ATP itself. 5. The antagonists of P2-purinoceptors, suramin and pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS), did not affect vascular responses to UTP and UDP, but reduced vasoconstriction evoked by beta, gamma-methylene ATP and ATP by about 70 and 30%, respectively. 6. This study demonstrates that uracil nucleotides, UTP and UDP, elicit vasoconstriction in the rat pulmonary vascular bed. In addition to confirming the presence of classical P2x-purinoceptors, these results also suggest the presence of a distinct purinoceptor subtype which mediates UTP- and ATP- evoked vasoconstriction in the rat pulmonary circulation.

Evidence for two distinct P2-purinoceptors subserving contraction of the rat anococcygeus smooth muscle.

1. The effects of the P2-purinoceptor agonists, adenosine 5'-triphosphate (ATP), alpha, beta-methylene ATP (alpha, beta-MeATP), beta, gamma-methylene ATP (beta, gamma-MeATP), L-beta, gamma-methylene ATP (L-beta, gamma-MeATP), adenosine-5'-O-(2-thiodiphosphate) (ADP beta S), and 2-methylthio ATP (2-MeSATP) were investigated on the isometric tension of the rat anococcygeus muscle. 2. Non-cumulative additions of ATP (100-1500 microM), alpha, beta-MeATP (1-300 microM), beta, gamma-MeATP (10-300 microM), L-beta, gamma-MeATP (3-100 microM) and ADP beta S (1-100 microM) produced concentration-dependent contractions, whereas 2-MeSATP (1-100 microM) had no effect. The rank order of potency was alpha, beta-MeATP > L-beta, gamma-MeATP > or = ADP beta S > beta, gamma-MeATP > > ATP > 2-MeSATP. 3. Contractions to cumulative additions of ATP, alpha, beta-MeATP, beta, gamma-MeATP and L-beta, gamma-MeATP were subject to desensitization whilst those to ADP beta S were unaffected. 4. Contractions to ATP, alpha, beta-MeATP, beta, gamma-MeATP and ADP beta S were abolished by the non-selective P2X/. P2Y-purinoceptor antagonist, suramin (100 microM). In contrast, contractions to ATP, alpha, beta-MeATP and beta, gamma-MeATP were not affected by the non-selective P1-purinoceptor antagonist 8-(p-sulphophenyl)-theophylline (8SPT, 30 microM). Blockade of P2X-purinoceptors with the selective P2X-purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM) or desensitization with L-beta, gamma-MeATP (10 microM) abolished contractions to alpha, beta-MeATP, but enhanced those to ADP beta S. The P2Y-purinoceptor antagonist, reactive blue 2 (RB2, 100 microM) enhanced contractions to ATP and alpha, beta-MeATP but abolished those to ADP beta S. 5. Simultaneous addition of alpha, beta-MeATP and ADP beta S produced an additive contraction. 6. The findings suggest that in the rat anococcygeus, smooth muscle cells are endowed with two distinct P2-purinoceptors which subserve contractions: a P2X-purinoceptor activated by ATP and its analogues, and another type of P2-purinoceptor activated by ADP beta S.

Molecular cloning and functional characterization of a rat pituitary G protein-coupled adenosine triphosphate (ATP) receptor.

There is increasing evidence that pituitary ATP receptors may play a novel role in modulating pituitary function. This work reports the isolation and expression of a pituitary ATP receptor gene clone from a rat pituitary complementary DNA library. The isolated clone (rpP2U) has a 1125-bp coding sequence flanked by 483 bp of 5' - and 422 bp of 3'-untranslated sequences. The deduced 374-amino acid product shows structural features common to other G protein-coupled receptors, and when stably transfected into a glioma cell line lacking endogenous ATP receptors, is functionally characterized as a P2U purinoceptor. Specifically, the ATP-induced intracellular Ca2+ mobilization in the transfected cells was inhibited by suramin, 2-methylthio-ATP had a modest stimulatory effect on intracellular Ca2+ mobilization, and beta, gamma-methylene ATP and alpha, beta-methylene ATP had no effect. The cloned receptor exhibited the agonist potency and efficacy profile of ATP approximately equal to uridine triphosphate > ADP approximately equal to uridine diphosphate > GTP. Such characteristics very closely mimic the pharmacologically defined P2U purinoceptor of primary rat gonadotropes and mixed sheep pituitary cells, and Southern blot analysis further indicates that there is only one allele in rat genome for the P2U purinoceptor. These findings suggest that the P2U purinoceptor is the predominant G protein-linked ATP receptor found in the pituitary.

Responses of the aorta of the garter snake (Thamnophis sirtalis parietalis) to purines.

1. Isolated aortic rings from the garter snake (Thamnophis sirtalis parietalis) were investigated in order to identify and classify responses to adenosine and adenosine 5'-triphosphate (ATP) and their analogues as part of a comparative study of vertebrate purinoceptors. 2. Adenosine, D-5'-(N-ethylcarboxamide) adenosine (NECA), R- and S-N6-(2-phenylisopropyl) adenosine (R- and S-PIA) and 2-chloroadenosine (2-CA) all concentration-dependently relaxed aorta preconstricted with noradrenaline (NA). The order of potency was: NECA > R-PIA = 2-CA > adenosine > S-PIA. Individual pD2 values for the analogues were: NECA 7.12 +/- 0.13 (9), R-PIA 5.93 +/- 0.25 (7), 2-CA 5.64 +/- 0.40 (5), adenosine 5.04 +/- 0.10 (13) and S-PIA 4.26 +/- 0.10 (7). The order of potency has characteristics of both A1 and A2 receptors and cannot satisfactorily be classified according to the P1-(adenosine) purinoceptor subtypes established in mammalian preparations. 3. ATP, alpha, beta-methylene ATP (alpha, beta-MeATP), 2-methylthio ATP (2MeSATP), beta, gamma-methylene ATP (beta, gamma,-MeATP) and uridine 5'-triphosphate (UTP) all concentration-dependently constricted the isolated aorta. The order of potency was alpha, beta-MeATP = 2MeSATP > ATP > beta, gamma-MeATP > UTP. Only ATP, alpha, beta-MeATP and 2MeSATP consistently produced a maximum response; pD2 values were: ATP 3.98 +/- 0.07 (10), alpha, beta-MeATP 5.86 +/- 0.15 (12) and 2MeSATP 6.06 +/- 0.23 (9). In vessels preconstricted with NA neither ATP nor 2MeSATP caused relaxation in the presence or absence of the endothelium. 4. Suramin (0.1 mM) inhibited vasoconstriction to ATP, alpha,beta-MeATP, 2MeSATP and beta,upsilon-MeATP;however, since contractions to ATP and analogues did not reach a maximum response in the presence of this and other antagonists, pD2 values could not be calculated.5. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 30 micro M), a P2x-purinoceptor antagonist,antagonized constrictions to alpha, beta-MeATP only. Reactive blue 2 (RB2; 30 micro M), a P2Y-purinoceptor antagonist, inhibited vasoconstrictions to 2MeSATP only.6. Indomethacin (30 micro M) inhibited vasoconstriction in response to ATP and 2MeSATP, but not alpha,beta,-MeATP, suggesting that the presence of an unaltered phosphate chain on the ATP analogue was necessary to stimulate the production of a prostanoid.7. Repeated administration of alpha,beta-MeATP (3 microM) caused desensitization of the receptor responsible for the constriction due to alpha,beta-MeATP whereas the responses to ATP and 2MeSATP were unaltered.8. In summary, both P1-purinoceptors mediating vasodilatation and P2-purinoceptors mediating vasoconstriction are present on the garter snake aorta. However, in contrast to mammalian vessels, bothP2x and P2Y subtypes mediate vasoconstriction. There was no evidence for vasodilatation to ATP or analogues. Stimulation of the P2-purinoceptor by ATP and 2MeSATP caused the synthesis of a prostanoid. In addition, the possibility of a receptor activated by ATP, separate from P2X and P2Y subtypes is discussed since contractions to ATP proved to be insensitive to both PPADS and RB2. A comparison is made of purinoceptors in the garter snake aorta with those in other vertebrate vessels.

Effects of divalent cations on the potency of ATP and related agonists in the rat isolated vagus nerve: implications for P2 purinoceptor classification

1. By use of a 'grease-gap' technique, the depolarizing effects of adenosine 5'-triphosphate (ATP) and ATP analogues on the rat isolated vagus nerve were determined in normal and in Ca2+/Mg(2+)-free (+ 1 x 10(-3) M ethylenediamine tetraacetic acid) physiological salt solution (PSS). 2. In normal PSS, ATP produced concentration-dependent depolarization responses but the concentration-effect curve to ATP was incomplete and a maximum effect was not achieved. The threshold concentration for depolarization was 1 x 10(-5) M and at the highest concentration tested (1 x 10(-3) M) the peak amplitude of the response to ATP only amounted to 71% of the depolarization produced by a near maximal response to 5-hydroxytryptamine (5-HT, 1 x 10(-5) M). 3. In Ca2+/Mg(2+)-free PSS, ATP produced depolarization responses at much lower concentrations and of markedly larger amplitude. Under these conditions, the threshold concentration for depolarization was 1-3 x 10(-7) M and the maximal response to ATP amounted to 526% of the response to 5-HT (1 x 10(-5) M) in normal PSS. The concentration-effect curve to ATP was sigmoid, with a defined maximum effect and a mean EC50 value of 1.2 x 10(-6) M. 4. In contrast to the effects on responses to ATP, the absence of divalent cations in the PSS did not modify the effective concentrations of either alpha, beta-methylene ATP or 5-HT. However, the maximum responses to both alpha, beta-methylene ATP and 5-HT were significantly increased in Ca2+/Mg(2+)-free PSS. 5. The depolarizing effects of several analogues of ATP were determined in Ca2+/Mg2+-free PSS.ATP-gamma-S and 2-methylthioATP were of similar potency to ATP (respective equi-effective molar ratios(EMRs) of 1.9 and 1.3, where ATP = 1) and similar maximum responses were obtained. Alpha, beta-MethyleneATP, beta, gamma-methylene ATP and ,beta, gamma-imido ATP were considerably less potent than ATP, analysis yielding mean EMRs of 48.9, 85.0 and 60.0, respectively. Maximum responses to these latter three agonists were not obtained at the highest concentrations tested (1 x 10-4-3 X 10- M). Benzoyl ATP, adenosine 5'-0-(2-thiodiphosphate) and adenosine diphosphate produced only small depolarizing responses at high concentrations (>1 x 10-4 M). Adenosine monophosphate, adenosine and uridine S'-triphosphate each had little or no depolarizing effect in Ca2+/Mg2+-free PSS.6. These data demonstrate that in the absence of divalent cations the excitatory actions of some, but not all, purine nucleotides in the rat vagus nerve are markedly potentiated. In Ca2+/Mg2+-free PSS, the rank order of agonist potencies was ATP = 2-methylthioATP = ATP-gamma-S>> alpha,beta-methylene ATP = beta, gamma imido ATP = P,y-methylene ATP. These findings are in stark contrast to our previous observations in normal PSS where the rank order of agonist potencies for these nucleotides was alpha,beta-methyleneATP> ATP-gamma-S > beta,gamma-imido ATP = beta,gamma-methylene ATP> 2-methylthioATP> ATP.7. We suggest that the two different rank orders of potency can be explained by differential metabolism involving Ca2+/Mg2+-dependent ectonucleotidases. If so, these data indicate that ATP and 2-methylthioATP are inherently more potent than alpha,beta-methylene ATP as agonists at neuronal P2X purinoceptors in the rat vagus nerve. The possible implications of these findings to the present system for subclassifying P2 purinoceptors are profound.

The effects of the purinergic system on digitalis-induced epileptiform activity.

It has been suggested that endogenous chemical substances, such as adenosine, released during a seizure attack, may act as anticonvulsants in vivo. We have investigated electrophysiologically the effects of purinoceptor agonists and antagonists on the epileptiform activity induced by intracortical digitalis in anesthetized rats. Intracortical injections of 1, 2, or 4 micrograms digitalis (desacetyl lanatocid C) caused an epileptiform electrocorticogram (ECoG). The application of adenosine (25 or 100 microM) or adenosine triphosphate (ATP) (3 mM) after desacetyl lanatocid C blocked the epileptiform activity. beta, gamma-Methylene ATP (0.1-0.8 mM), a stable analog of ATP, produced inhibition and then death. The epileptogenic effect of desacetyl lanatocid C was enhanced by theophylline (1 mM); however, suramin (1 mM) changed the pattern of epilepsy. These results indicate that the purinergic system may be involved in the mechanism of action of digitalis glycosides.

ATP modulates the efferent function of capsaicin-sensitive neurones in guinea-pig isolated atria.

1. The effect of adenosine triphosphate (ATP) and its stable analogues, alpha, beta-methylene-ATP and beta, gamma-methylene-ATP, on the efferent function of capsaicin-sensitive non-adrenergic, non-cholinergic (NANC) nerves was tested in guinea-pig isolated atria. 2. Transmural nerve stimulation of atria isolated from reserpine-pretreated guinea-pigs, in the presence of 1 microM atropine and 0.3 microM CGP 20712A, induced a transient positive inotropic effect attributable to calcitonin gene-related peptide (CGRP) release from NANC nerve endings. 3. ATP (1-30 microM) concentration-dependently reduced the cardiac response to transmural nerve stimulation, without affecting the inotropic response to 10 nM exogenous CGRP. The inhibitory effect of ATP was competitively antagonized by the P1-purinoceptor antagonist, 8-phenyltheophylline (8-PT, 1 microM), but was unaffected by the P2-purinoceptor antagonist, suramin (100 microM). 4. beta, gamma-methylene-ATP in the same concentration range as ATP, inhibited the cardiac response to transmural nerve stimulation. The inhibitory effect of beta, gamma-methylene ATP was antagonized by 1 microM 8-PT. The desensitizing agonist for P2-purinoceptors, alpha, beta-methylene ATP did not induce any inhibitory effect either on the cardiac response to transmural nerve stimulation or on the inhibitory effect curve for ATP. 5. The inhibitory effect of ATP on the NANC neurotransmission was inconsistently modified in the presence of 10 microM alpha, beta-methylene-adenosine diphosphate, an inhibitor of the 5'-ectonucleotidases. 6. These results demonstrate that ATP modulates the efferent function of cardiac NANC nerve endings through prejunctional inhibitory receptors belonging to the P1 type. The metabolic conversion of ATP to adenosine does not seem to be a pre-requisite for the ATP agonist activity.

Characterization of P2x‐receptors in rabbit isolated ear artery

1. The isolated central ear artery of the rabbit contracts in response to adenosine 5'-triphosphate (ATP) and analogues, effects proposed to be mediated by stimulation of P2x-receptors. We have extended the characterization of the purinoceptor in this tissue by examining the effects of a series of receptor agonists. The study was designed in such a way as to avoid factors which normally limit attempts to classify receptors on the basis of agonist potency orders. 2. D-alpha, beta-methylene ATP (D-alpha, beta-meATP), D-beta, gamma-methylene ATP (D-beta, gamma-meATP), L-beta, gamma-methylene ATP (L-beta, gamma-meATP), 2-methylthio-D-ATP (2-MeSATP) and ATP produced concentration-related contractions of the ear artery with similar maximum responses, suggesting that they were full agonists. Selective desensitization of P2x-receptors abolished or greatly reduced responses to D-alpha, beta-meATP, L-beta, gamma-meATP, D-beta, gamma-meATP. Responses to ATP were inhibited by by desensitization but a significant resistant component was still apparent. 3. D-alpha, beta-meATP was the most potent agonist tested (pA50 6.47 +/- 0.04) being 2138 times more potent than ATP and approximately 9 times more potent than L-beta, gamma-meATP. The agonist potency order was: D-alpha, beta-meATP greater than L-beta, gamma-meATP greater than D-beta, gamma-meATP greater than or equal to 2-MeSATP greater than ATP. This is generally consistent with the order proposed for P2x-receptors. The relative potencies of P2x-agonists in the rabbit ear artery show both similarities to and differences from data obtained in other smooth muscle preparations.

Histamine secretion from mast cells stimulated with ATP

Adenosine 5'-triphosphate (ATP) induced a non-cytotoxic, calcium-dependent release of histamine and prostaglandin D2 from rat serosal mast cells. The effect was tissue and species specific. In particular, tissue mast cells from the guinea pig and man were totally unresponsive. The release evoked by the nucleotide was unaffected by antiasthmatic chromones but was inhibited by structurally related flavonoids and by cAMP-active drugs. Secretion was blocked both by the P2Y-purinoceptor antagonist reactive blue 2 and the P2X-antagonist suramin, whereas the P2-agonists 2-methylthio ATP, alpha, beta- and beta, gamma-methylene ATP were inactive. These findings indicate that ATP acts through a novel purinoceptor or binding site which is present on certain types of mast cells.

Dynein as a microtubule translocator in ciliary motility: current studies of arm structure and activity pattern.

The dynein arms of ciliary doublet microtubules cause adjacent axonemal doublets to slide apart with fixed polarity. This suggests that there is a unique mechanochemistry to the dynein arm with unidirectional force generation in all active arms and also that not all arms are active at once during a ciliary beat. Negative stain and thin-section images of arms in axonemes treated with beta, gamma methylene adenosine triphosphate (AMP-PCP) show a consistent subunit construction where the globular head of the arm interacts with subfiber B of doublet N+1. This interpretation differs from that provided by freeze etch and STEM interpretations of in situ arm construction and has implications for the mechanochemical cycle of the arm. A computer model of the arms in relation to other axonemal structures has been constructed to test these interpretations. Attachment of the head of the arm subfiber B is directly demonstrable in splayed axonemes in AMP-PCP. About half of the doublets in an axoneme show such attachments, while half do not. This might imply that about half the doublets in an axoneme are active at any given instant and can be identified as such. This information may be useful in probing questions of how active arms differ biochemically from inactive arms and of how microtubule translocators in general become active.

A study of the post-tetanic twitch depression at the prostatic end of the rat vas deferens.

The effects of different drugs on the post-tetanic twitch responses of the prostatic end of the isolation rat vas deferens were examined. Following repetitive field stimulation (4-5 Hz for 40-60 s), the post-tetanic twitches (0.2-0.5 Hz) were depressed relative to the steady-state pretetanic twitches. Maximum post-tetanic twitch depression (PTD) occurred 15-30 s after the tetanic stimulation. The PTD was not reversed but deepened and prolonged by the presynaptic alpha 2-adrenoceptor antagonists idazoxan (RX781094) or yohimbine (10(-7) M) alone or combined with the neuronal uptake blocker, desipramine (10(-6) M). The PTD was insensitive to the beta-adrenoceptor blocker, propranolol (10(-6) M), the P1-purinoceptor antagonist, 8-phenyltheophylline (10(-5) M) or the prostaglandin E synthesis inhibitor, indomethacin (10(-6) M). Pre- and post-tetanic contractions to superfused noradrenaline (NA), adenosine 5'-triphosphate (ATP), or beta, gamma-methylene ATP (beta, gamma-mATP) (bolus injections into superfusate, 1-8 X 10(-5) M) were not significantly (p less than 0.01) different. The PTD was absent in high magnesium (6.2-11.2 mM) solutions. These results are consistent with the PTD having an intrinsic presynaptic origin. They are discussed in relation to stimulation-induced modulation of transmitter release.

Purinoceptors in the rat heart.

The effects of an intracoronary bolus of adenosine triphosphate (ATP), alpha, beta-methylene ATP (APCPP), beta, gamma-methylene ATP (APPCP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine on coronary tone and ventricular myocardial contraction were investigated in the perfused rat heart. Adenine nucleotides, given by bolus injection were negatively inotropic in amounts greater than 3 X 10(-7) mol. The potency order was ATP greater than ADP greater than AMP. Adenosine (less than 1 X 10(-5)mol) had no effect on ventricular myocardial contraction. Adenine nucleotides and adenosine (1 X 10(-10)-1 X 10(-7) mol) reduced coronary tone. The potency order was ATP greater than ADP greater than AMP = adenosine. The ATP analogue APPCP was less active than ATP at reducing coronary tone, and APCPP had no vasodilator effect. This suggests the presence of a P2-purinoceptor, subclass P2Y, which mediates vasodilation. ATP and ADP increased the concentration of prostacyclin (measured as 6-keto prostaglandin F1 alpha) in the perfusate, but only after injection of greater than 3 X 10(-7) mol, suggesting that the vasodilator responses to ATP and ADP were not mediated by prostacyclin. AMP and adenosine had no effect, even at 1 X 10(-5) mol. At a dose of 3 X 10(-9) mol, approximately 40% of ATP and 70% of ADP was converted to AMP and adenosine whilst passing through the heart. The amounts of AMP and adenosine formed, however, were insufficient to account for the vasodilator effects of ATP and ADP. 6 Vasodilatation mediated by AMP and adenosine was inhibited by an infusion of 8-phenyltheophylline (8-PT; 2 x 10-5 M) indicating interaction with a P1-purinoceptor. Vasodilatation induced by ATP (at doses at which AMP and adenosine had no action) was also depressed by 8-PT indicating either an action of ATP on PI-purinoceptors, or an effect of 8-PT on P2y receptors. 7 Vasodilatation induced by AMP was unaltered during an infusion of alpha,beta-methylene ADP (2 x 10-6 M, which inhibited breakdown of AMP to adenosine by 54.2 +/- 1.5%, n = 4). This suggests that AMP acted directly, and it did not require conversion to adenosine to induce vasodilatation. 8 The ATP analogues APCPP (1 x 10-9_1 x 10-8 mol) and APPCP (1 x 10-8_l x 10-7mol) increased coronary tone, as did high doses (I x 10-5 mol) ofATP and ADP, indicating the presence of an additional P2-purinoceptor, subclass P2X, mediating vasoconstriction.

Characteristics of the vagally driven non-adrenergic, non-cholinergic inhibitory innervation of ferret gastric corpus.

This paper reports a quantitative in vivo study on the vagal activation of the intramural non-adrenergic, non-cholinergic inhibitory nerves in the ferret gastric corpus. The nature of the inhibitory neurotransmitter was also investigated. In the atropinized, guanethidine-treated, urethane-anaesthetized ferret, electrical stimulation (10 s at 20 V, 1-20 Hz, 0.5 ms pulses) of the cervical vagi produced a prompt fall in intracorpus pressure that was related to the stimulus frequency. The maximal response was achieved at 10 Hz. The time taken for the intracorpus pressure to return to pre-stimulus levels after a 10 s period of stimulation was related to the stimulus frequency; at 10 Hz the pressure took approximately 11 min to recover. In contrast to studies in the cat (Martinson & Muren, 1963), there was no detectable difference in the electrical threshold for activation of the vagal excitatory and vagal inhibitory fibres. The nature of the vagal non-adrenergic, non-cholinergic inhibitory neurotransmitter was investigated using a variety of antagonists and agonists. Adenosine triphosphate (ATP), adenosine, alpha beta-methylene ATP and beta gamma-methylene ATP all contracted the corpus in the presence of vagotomy, atropine, guanethidine and indomethacin. The vagally induced fall in corpus pressure was not blocked by high doses of alpha beta-methylene ATP. A variety of peptides were investigated for their effects on corpus pressure in the presence of atropine, guanethidine and vagotomy. Bombesin, pentagastrin, substance P, cholecystokinin octapeptide (CCK-8) and bradykinin all produced an increase in intracorpus pressure. Neurotensin and vasoactive intestinal polypeptide (VIP) both decreased intracorpus pressure, and of the two VIP most closely mimicked the response to vagal activation of the non-cholinergic, non-adrenergic inhibitory neurones. The results provide support for the involvement of a peptide (possibly VIP) rather than a purine in the vagally driven decrease in intracorpus pressure in the ferret.

Energy-independent protection of the oxidative phosphorylation capacity of mitochondria against anoxic damage by ATP and its non-metabolizable analogs.

Preservation of the oxidative phosphorylation capacity of mitochondria by addition of ATP under anaerobic conditions was analyzed by use of non-metabolizable adenine nucleotide analogs. The capacity was well preserved in the presence of ATP and did not require the hydrolysis of ATP, since ATP analogs, such as beta, gamma-methylene adenosine triphosphate (AMPPCP), alpha, beta-methylene adenosine triphosphate (AMPCPP), and adenylyl imidodiphosphate (AMPPNP), were as effective as ATP. These analogs were incorporated into mitochondria through ATP/ADP translocase to maintain the original level of total adenine nucleotides in the mitochondria. ADP apparently had the same effect as ATP, but its effect was shown to be due to ATP generated from it by adenylate kinase in mitochondria. An analog of ADP, alpha, beta-methylene adenosine diphosphate (AMPCP), which was found to be a substrate of the translocase but not of adenylate kinase, could not replace ADP or ATP. From these results, it was concluded that the oxidative phosphorylation capacity of mitochondria was maintained by ATP, but not ADP, through a process not requiring energy.

Effects of adenosine, adenosine triphosphate and structural analogues on glucagon secretion from the perfused pancreas of rat in vitro.

The effects of adenosine, adenosine triphosphate (ATP) and structural analogues have been studied on glucagon secretion from the isolated perfused pancreas of the rat in the presence of glucose (2.8 mM). Adenosine induced a transient increase of glucagon secretion. This effect was concentration-dependent in the range of 0.165 to 165 microM. ATP also induced an increase, but the effect was no greater at 165 microM than at 16.5 microM. 2-Chloroadenosine, an analogue more resistant to metabolism or uptake systems than adenosine, was more effective. Among the three structural analogues of ATP or ADP studied, beta, gamma-methylene ATP which can be hydrolyzed into AMP and adenosine had an effect similar to adenosine or ATP at the same concentrations (1.65 and 16.5 microM); in contrast alpha, beta-methylene ATP and alpha, beta-methylene ADP (resistant to hydrolysis into AMP and adenosine) were ineffective. Theophylline (50 microM) a specific blocker of the adenosine receptor, suppressed the glucagon peak induced by adenosine, 2-chloroadenosine, ATP and beta, gamma-methylene ATP (1.65 microM). An inhibitor of 5' nucleotidase, alpha, beta-methylene ADP (16.5 microM), reduced the glucagon increase induced by ATP and did not affect the response to adenosine (1.65 microM). These results support the hypothesis of adenosine receptors (P1-purinoceptors) on the pancreatic glucagon secretory cells and indicate that ATP acts after hydrolysis to adenosine.

Calcium releasing action of quercetin on sarcoplasmic reticulum from frog skeletal muscle.

The release of Ca by quercetin from the sarcoplasmic reticulum has been claimed to be a result of the well-known inhibition of Ca2+-ATPase activity, or to be due to an intrinsic property of quercetin. To get a clearer understanding of the effect of quercetin, we examined it using fragmented sarcoplasmic reticulum (FSR) from bullfrog skeletal muscle. The rapid phase of Ca release (hereafter simply referred to as "Ca release") from loaded FSR was almost completed within 5 s after addition of quercetin in the presence of ATP. It cannot be ascribed to the inhibition of Ca2+-ATPase activity on the basis of following findings. First, when Ca uptake was driven by carbamylphosphate, no or little Ca release was observed in marked contrast to a stronger reduction in the rate of Ca uptake. Secondly, procaine reverses the Ca releasing action of quercetin, whereas it show a synergistic action in the inhibition of Ca2+-ATPase activity. Thirdly, HFSR released more Ca than LFSR, while the Ca2+-ATPase activities of both fractions were inhibited to a similar extent. The Ca release by quercetin is enhanced by ATP or beta, gamma-methylene adenosine triphosphate, and decreased by procaine or a high concentration of Mg2+. In the presence of 2.5 mM caffeine, the amount of Ca2+ released by quercetin was decreased, and the dose-effect relationship was shifted to higher doses of quercetin. This indicates that quercetin and caffeine probably overlap in the site(s) of the action, but that quercetin is dissimilar from halothane in the mode of its Ca-releasing action.

The effect of adenyl compounds on the rat heart.

1 The effects of adenyl compounds were examined on the rat atrium and ventricle. 2 Adenosine, adenosine 5'-monophosphate, adenosine 5'-diphosphate, adenosine 5'-triphosphate (ATP) and beta, gamma-methylene ATP (APPCP) produced negative inotropic effects on the rat atrium. These inhibitory effects were antagonized by 8-phenyltheophylline (8-PT), a P1-purinoceptor antagonist, and potentiated by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), an adenosine deaminase inhibitor, but were not affected by dipyridamole, which blocks adenosine uptake. 3 alpha, beta-Methylene ATP (APCPP), which is resistant to degradation, did not produce a similar inhibitory response in the rat atrium. 4 Adenosine did not affect the basal developed force of the rat ventricle nor did it affect the beta-adrenoceptor-mediated positive inotropic effect. 5 Very high concentrations of ATP (0.1-3 mM) produced negative inotropic effects in the rat ventricle. APPCP (0.3 mM) also produced an inhibitory response, which was significantly smaller than that produced by ATP (0.3 mM). APCPP elicited excitation rather than the expected inhibitory response. 6 The inhibitory effect of ATP in the rat ventricle was not blocked by indomethacin, 8-PT or atropine. 7 It is possible that the action of ATP in the rat ventricle is mediated via P2-purinoceptors and that the lack of inhibitory action of APCPP on the rat ventricle is due to the difference in structural conformation between ATP and APCPP. 8 It seems likely that inhibitory P1-purinoceptors are present in the rat atrium and that the inhibitory responses produced by ATP in the rat ventricle may be mediated via P2-purinoceptors.

Effects of phosphate-modified adenine nucleotide analogues on insulin secretion from perfused rat pancreas.

1 The effects of three methylene analogues of adenosine 5'-triphosphate (ATP) or 5'-diphosphate (ADP) have been studied on insulin secretion from the isolated perfused pancreas of the rat: 5'-adenylmethylene diphosphonate or beta, gamma-methylene ATP, adenosine 5'-alpha, beta-methylene triphosphate or alpha, beta-methylene ATP and adenosine 5'-alpha, beta-methylene diphosphonate or alpha, beta-methylene ADP. 2 beta, gamma-Methylene ATP did not elicit any increase of insulin release: alpha, beta-methylene ATP and alpha, beta-methylene ADP induced a biphasic stimulation of insulin secretion; this effect was dose-related between 1.65 and 165 mumol/l. Relative potency ATP/alpha, beta-methylene ATP was 1.2 and ATP/alpha, beta-methylene ADP was 0.31. 3 Our results point to the importance of the steric and electronic characteristics of the polyphosphate chain of the analogues of ATP and ADP in inducing an insulin secretory effect. They support the hypothesis of a purine receptor for ATP and ADP.