Beta-methylene Adenosine 5'-triphosphate Research Articles

MP08-08 NERVE TRANSFER FOR RESTORATION OF LOWER MOTOR NEURON-LESIONED BLADDER FUNCTION. PART 2: ATTENUATION OF PURINERGIC BLADDER SMOOTH MUSCLE CONTRACTIONS

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology (MP08)1 Sep 2021MP08-08 NERVE TRANSFER FOR RESTORATION OF LOWER MOTOR NEURON-LESIONED BLADDER FUNCTION. PART 2: ATTENUATION OF PURINERGIC BLADDER SMOOTH MUSCLE CONTRACTIONS Nagat Frara, Dania Giaddui, Alan S. Braverman, Danielle S. Porecca, Justin M. Brown, Michael Mazzei, Ida J. Wagner, Michel A. Pontari, Ekta Tiwari, Courtney L. Testa, Daohai Yu, Locas J. Hobson, Mary F. Barbe, and Michael R. Ruggieri Nagat FraraNagat Frara More articles by this author , Dania GiadduiDania Giaddui More articles by this author , Alan S. BravermanAlan S. Braverman More articles by this author , Danielle S. PoreccaDanielle S. Porecca More articles by this author , Justin M. BrownJustin M. Brown More articles by this author , Michael MazzeiMichael Mazzei More articles by this author , Ida J. WagnerIda J. Wagner More articles by this author , Michel A. PontariMichel A. Pontari More articles by this author , Ekta TiwariEkta Tiwari More articles by this author , Courtney L. TestaCourtney L. Testa More articles by this author , Daohai YuDaohai Yu More articles by this author , Locas J. HobsonLocas J. Hobson More articles by this author , Mary F. BarbeMary F. Barbe More articles by this author , and Michael R. RuggieriMichael R. Ruggieri More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001981.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: This study determined the effect of pelvic organ long-term decentralization and reinnervation one year later on the contribution of muscarinic and purinergic receptors to ex-vivo, nerve-evoked, bladder smooth muscle contractions. METHODS: Nineteen canines underwent decentralization by bilateral transection of all coccygeal and sacral (S) spinal roots, dorsal roots of lumbar (L)7 and hypogastric nerves. After exclusions, 8 were reinnervated 12 months post-decentralization with obturator-to-pelvic and sciatic-to-pudendal nerve transfers then euthanized 8-12 months later; four served as long-term decentralized only animals. Controls included six sham-operated and three unoperated animals. Bladder tissues were assessed for contractile responses to potassium chloride (KCl) and electric field stimulation (EFS) before and after purinergic receptor desensitization with alpha, beta-methylene adenosine triphosphate (α,β-mATP), muscarinic receptor antagonism with atropine, or sodium channel blockade with tetrodotoxin. RESULTS: Atropine inhibition of EFS-induced contractions increased by 26%in decentralized and 34%in the reinnervated animals compared to controls. Maximal contractile responses to α,β-mATP did not differ between groups. In strips from decentralized and reinnervated animals, the contractile response to EFS was enhanced by 52% at lower frequencies and the response to KCl was increased compared to normal controls. CONCLUSIONS: The observation of increased blockade of nerve-evoked contractions by muscarinic antagonist with no change in responsiveness to purinergic agonist suggests either decreased ATP release or increased ectoATPase activity in detrusor muscle as a consequence of the long-term decentralization. The reduction in the frequency required to produce maximum contraction following decentralization may be due to enhanced nerve sensitivity to EFS or a change in the effectiveness of the neurotransmission. Source of Funding: NINDS R01NS070267 © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e153-e153 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nagat Frara More articles by this author Dania Giaddui More articles by this author Alan S. Braverman More articles by this author Danielle S. Porecca More articles by this author Justin M. Brown More articles by this author Michael Mazzei More articles by this author Ida J. Wagner More articles by this author Michel A. Pontari More articles by this author Ekta Tiwari More articles by this author Courtney L. Testa More articles by this author Daohai Yu More articles by this author Locas J. Hobson More articles by this author Mary F. Barbe More articles by this author Michael R. Ruggieri More articles by this author Expand All Advertisement Loading ...

Area 3a neuron response to skin nociceptor afferent drive.

Area 3a neurons are identified that respond weakly or not at all to skin contact with a 25-38 degrees C probe, but vigorously to skin contact with the probe at > or =49 degrees C. Maximal rate of spike firing associated with 1- to 7-s contact at > or =49 degrees C occurs 1-2 s after probe removal from the skin. The activity evoked by 5-s contact with the probe at 51 degrees C remains above-background for approximately 20 s after probe retraction. After 1-s contact at 55-56 degrees C activity remains above-background for approximately 4 s. Magnitude of spike firing associated with 5-s contact increases linearly as probe temperature is increased from 49-51 degrees C. Intradermal capsaicin injection elicits a larger (approximately 2.5x) and longer-lasting (100x) increase in area 3a neuron firing rate than 5-s contact at 51 degrees C. Area 3a neurons exhibit enhanced or novel responsivity to 25-38 degrees C contact for a prolonged time after intradermal injection of capsaicin or alpha, beta methylene adenosine triphosphate. Their 1) delayed and persisting increase in spike firing in response to contact at > or =49 degrees C, 2) vigorous and prolonged response to intradermal capsaicin, and 3) enhanced and frequently novel response to 25-38 degrees C contact following intradermal algogen injection or noxious skin heating suggest that the area 3a neurons identified in this study contribute to second pain and mechanical hyperalgesia/allodynia.

Muscarinic and Alpha 1-Adrenergic Receptor Binding Characteristics of Saw Palmetto Extract in Rat Lower Urinary Tract

To elucidate the in vitro and ex vivo effects of saw palmetto extract (SPE) on autonomic receptors in the rat lower urinary tract. The in vitro binding affinities for alpha 1-adrenergic, muscarinic, and purinergic receptors in the rat prostate and bladder were measured by radioligand binding assays. Rats received vehicle or SPE (0.6 to 60 mg/kg/day) orally for 4 weeks, and alpha 1-adrenergic and muscarinic receptor binding in tissues of these rats were measured. Saw palmetto extract inhibited specific binding of [3H]prazosin and [N-methyl-3H]scopolamine methyl chloride (NMS) but not alpha, beta-methylene adenosine triphosphate [2,8-(3)H]tetrasodium salt in the rat prostate and bladder. The binding activity of SPE for muscarinic receptors was four times greater than that for alpha 1-adrenergic receptors. Scatchard analysis revealed that SPE significantly reduced the maximal number of binding sites (Bmax) for each radioligand in the prostate and bladder under in vitro condition. Repeated oral administration of SPE to rats brought about significant alteration in Bmax for prostatic [3H]prazosin binding and for bladder [3H]NMS binding. Such alteration by SPE was selective to the receptors in the lower urinary tract. Saw palmetto extract exerts significant binding activity on autonomic receptors in the lower urinary tract under in vitro and in vivo conditions.

Bladder Volume Alters Cholinergic Responses of the Isolated Whole Mouse Bladder

The isolated bladder expresses autonomous activity, which may contribute to the generation of lower urinary tract sensation or pathophysiology. We evaluated how the effect of a cholinergic agonist on autonomous activity alters with increasing volume and in the presence of substances known to modulate functional bladder capacity. The bladder of 22 adult female C57 black mice were mounted in whole organ tissue baths. Recordings of intravesical pressure were performed under standardized conditions at different bladder volumes. At low volume the muscarinic agonist arecaidine elicited an initial peak response, which subsided to a sustained steady state pressure. At high volume phasic pressure fluctuations were also apparent. An M2-receptor antagonist caused a significantly greater decrease in peak and steady state responses than in pressure fluctuations. An M3-receptor antagonist decreased all 3 components. Alpha, beta-methylene adenosine triphosphate markedly decreased fluctuations, in contrast to norepinephrine, which eliminated the steady state response while preserving fluctuations. The response to cholinergic stimulation of the isolated bladder has 3 components. The initial tonic peak response increases with bladder distention and it is inhibited by M2 and M3 muscarinic receptor antagonists. The tonic steady state response does not vary with bladder volume and it is inhibited by M2 and M3-receptor antagonists, and by beta3-adrenergic receptor agonists. Phasic fluctuations are minimal at low bladder volume, and with alpha, beta-methylene adenosine triphosphate or an M3-receptor antagonist. Thus, the response to cholinergic stimulation varies with bladder volume. It can be differentially modulated by muscarinic antagonists and also by agents acting through nonmuscarinic receptors.

Electromyographic detection of purinergic activity in Guinea pig detrusor smooth muscle.

We recorded nerve mediated extracellular electrical activity from guinea pig detrusor smooth muscle strips using suction electrodes and determined the electrophysiological origins of this signal and its relationship to contractile activity. Mucosa-free detrusor strips were prepared from male guinea pigs sacrificed under Home Office license, physiologically superfused, attached to a pressure transducer and electrically stimulated (0.1 millisecond pulses). Electrical signals recorded using a bipolar reversible suction electrode were processed and recorded simultaneously with changes in strip tension. The effect of superfusion with alpha, beta-methylene adenosine triphosphate (ATP), atropine, extracellular [CaCl(2)] depletion and pharmacological Ca2+ channel blockade on the electrical and mechanical signals was determined. A biphasic electrical signal was consistently recorded from 37 detrusor strips. The signal was sensitive to graded reduction in [CaCl(2)] of the superfusate and abolished by tetrodotoxin in 7 preparations. The signal was also abolished in 12 preparations by alpha, beta-methylene ATP in association with an attenuated contraction but not significantly reduced in amplitude (p = 0.77) despite a significant reduction in tension with atropine (mean plus or minus SD 74% +/- 14% of control, p <0.001). The signal was attenuated to a mean maximum of 9% +/- 3% of control by pharmacological Ca2+ channel blockade and the remaining signal was abolished by alpha, beta-methylene ATP. The extracellular electrical signal recorded from guinea pig detrusor strips using suction electrodes originates from a purinergic mechanism. Although an atropine sensitive component may be present, the signal does not depend on cholinergic neuromuscular transmission and would not be expected to be generated by normal human detrusor. Provided that the electrophysiological basis of purinergic neurotransmission in guinea pig and human bladders is similar suction electrodes may be a valuable tool with which to evaluate in vitro and clinically by electromyography the pathological purinergic neuromuscular transmission that can be expressed in addition to normal cholinergic mechanisms in detrusor from dysfunctional human bladders.

Smooth Muscle and Purinergic Contraction of the Human, Rabbit, Rat, and Mouse Testicular Capsule1

The smooth-muscle cells of the testicular capsule (tunica albuginea) of man, rat, and mouse were examined by electron microscopy. They were characteristically flattened, elongated, branching cells and diffusely incorporated into the collagenous matrix and did not form a compact muscle layer. Contractile and synthetic smooth-muscle cell phenotypes were identified. Nerve varicosities in close apposition to smooth muscle were seen in human tissue. Contractions induced by adenosine 5'-triphosphate (ATP), alpha, beta-methylene ATP, noradrenaline (NA), acetylcholine (ACh), and electrical field stimulation (EFS) of autonomic nerves were investigated. Nerve-mediated responses of the rabbit and human tunica albuginea were recorded. The EFS-induced human responses were completely abolished by prazosin. In the rabbit, EFS-induced contractile responses were reduced by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid by 36% and by prazosin by 77%. Both antagonists together almost completely abolished all EFS-induced contractions. The human tunica albuginea was contracted by NA, ATP, and alpha, beta-methylene ATP, but not by ACh. The rabbit and rat tunica albuginea were contracted by NA, ATP, alpha, beta-methylene ATP, and ACh. The mouse tunica albuginea was contracted by ACh, ATP, and alpha, beta-methylene ATP, but relaxed to NA. Immunohistochemical studies showed that P2X1 (also known as P2RX1) and P2X2 (also known as P2RX2) receptors were expressed on the smooth muscle of the rodent testicular capsule, expression being less pronounced in man. The testicular capsule of the rat, mouse, rabbit, and man all contain contractile smooth muscle. ATP, released as a cotransmitter from sympathetic nerves, can stimulate the contraction of rabbit smooth muscle. Human, rat, and mouse testicular smooth muscle demonstrated purinergic responsiveness, probably mediated through the P2X1 and/or P2X2 receptors.

Effects of adenosine and ATP on the membrane potential and synaptic transmission in neurons of the rat locus coeruleus.

Effects of adenosine (Ado) and adenosine 5'-triphosphate (ATP) on the membrane potential and synaptic transmission in neurons of the rat locus coeruleus (LC) were examined, in vitro. Ado (30-300 microM) produced a hyperpolarizing response and inhibited spontaneous firing activity in neurons of the rat LC. Ado decreased input resistance of LC neurons. The Ado-induced hyperpolarization reversed polarity near the equilibrium potential of K+ (EK). Ado (100-300 microM) depressed both excitatory postsynaptic potential (EPSP) and inhibitory postsynaptic potential (IPSP). Ado (300 microM) did not alter the hyperpolarization induced by norepinephrine (30 microM). N6-Cyclopentyladenosine (CPA, 100 microM), an A1 receptor agonist, also produced a hyperpolarizing response and depressed both the EPSP and IPSP. Another A1 receptor agonist, adenosine amine congener (ADAC, 30 microM) also produced a hyperpolarizing response and consistently depressed the EPSP and IPSP. Application of ATP (100 microM) to LC neurons caused a depolarizing response associated with an increase in the firing rate of spontaneous action potential in LC neurons. The ATP-induced depolarization was accompanied by an increased input resistance and reversed polarity at--91 mV. ATP (100 microM) consistently depressed the IPSP, while it did not change the amplitude of the EPSP in a majority of neurons. alpha, beta-Methylene ATP (alpha, beta-meATP, 30 micro/M), a P2 receptor agonist, mimicked these effects of ATP. Adenosine 5'-(beta, gamma-imido) triphosphate (AMP-PNP, 100 microM), a non-metabolizable analogue of ATP, produced a depolarizing response in LC neurons, but it produced no obvious depression of the EPSP and IPSP. These results suggest that Ado and ATP cause inhibitory and excitatory modulation, respectively, of neuronal activity and synaptic transmission in the rat LC.

Electromyographic Detection of Purinergic Activity in Guinea Pig Detrusor Smooth Muscle

We recorded nerve mediated extracellular electrical activity from guinea pig detrusor smooth muscle strips using suction electrodes and determined the electrophysiological origins of this signal and its relationship to contractile activity. Mucosa-free detrusor strips were prepared from male guinea pigs sacrificed under Home Office license, physiologically superfused, attached to a pressure transducer and electrically stimulated (0.1 millisecond pulses). Electrical signals recorded using a bipolar reversible suction electrode were processed and recorded simultaneously with changes in strip tension. The effect of superfusion with alpha, beta-methylene adenosine triphosphate (ATP), atropine, extracellular [CaCl(2)] depletion and pharmacological Ca2+ channel blockade on the electrical and mechanical signals was determined. A biphasic electrical signal was consistently recorded from 37 detrusor strips. The signal was sensitive to graded reduction in [CaCl(2)] of the superfusate and abolished by tetrodotoxin in 7 preparations. The signal was also abolished in 12 preparations by alpha, beta-methylene ATP in association with an attenuated contraction but not significantly reduced in amplitude (p = 0.77) despite a significant reduction in tension with atropine (mean plus or minus SD 74% +/- 14% of control, p <0.001). The signal was attenuated to a mean maximum of 9% +/- 3% of control by pharmacological Ca2+ channel blockade and the remaining signal was abolished by alpha, beta-methylene ATP. The extracellular electrical signal recorded from guinea pig detrusor strips using suction electrodes originates from a purinergic mechanism. Although an atropine sensitive component may be present, the signal does not depend on cholinergic neuromuscular transmission and would not be expected to be generated by normal human detrusor. Provided that the electrophysiological basis of purinergic neurotransmission in guinea pig and human bladders is similar suction electrodes may be a valuable tool with which to evaluate in vitro and clinically by electromyography the pathological purinergic neuromuscular transmission that can be expressed in addition to normal cholinergic mechanisms in detrusor from dysfunctional human bladders.

Age-related changes in cholinergic and purinergic neurotransmission in human isolated bladder smooth muscles.

We evaluated the correlation among age, cholinergic and purinergic neurotransmissions in the electrical field stimulation-induced contractions in human isolated urinary bladder smooth muscles, using the muscle bath technique. Human bladder specimens were divided into three groups (G1, under 50years; G2, 51-70years; G3, over 70years old), and each muscle strip was suspended in a thermostatically controlled organ bath filled with oxygenated Krebs-Henseleit solution, connected to an isometric force displacement transducer, and an isometric tension development was recorded. The contractile responses induced by KCl, carbachol, adenosine triphosphate (ATP) and electrical field stimulation, and the effects of atropine and alpha, beta methylene ATP on electrical field stimulation-induced contractions were observed. The contractile response to KCl and concentration-response curves for carbachol and ATP, and frequency-response curves for electrical field stimulation were not significantly different among the three groups. The atropine sensitive and resistant parts of contraction induced by electrical field stimulation were decreased and increased with age, respectively. There are significant positive and negative correlations between age and the purinergic, and age and the cholinergic neurotransmissions in human isolated bladder smooth muscles, respectively. The age-related changes in neurotransmissions may contribute to the changes in bladder function in the elderly.

Effects of ATP and its analogues on [Ca2+]i dynamics in the rabbit corneal epithelium.

Adenosine-5'-triphosphate (ATP) plays a pivotal role in various tissues as an extracellular transmitter. ATP released from nerve endings and/or damaged cells may elicit reactions in adjacent cells. To identify such reactions, we investigated the dynamics of the intracellular calcium ion concentrations ([Ca2+]i) in the rabbit corneal epithelium during ATP-stimulation. Intact epithelial sheets isolated from corneal tissue were loaded with Fura-2, and [Ca2+]i dynamics in each cell layer were analyzed using a digital imaging system (Argus 50/CA). Normal architecture was preserved, suggesting that functional integrity remained intact. Perfusion with HEPES-buffered Ringer's solution containing ATP (10 microM) and uridine-5'-triphosphate (UTP; 10 microM) caused a biphasic [Ca2+]i increase in the superficial layer that manifested itself as a rapid initial spike followed by a long-lasting plateau phase. Adenosine-5'-diphate (10 microM) elevated the [Ca2+]i level, but induced only the initial spike, which was smaller than those induced by ATP and UTP. Adenosine (10 microM) did not elicit any [Ca2+]i changes in the epithelial cells. Suramin (10 microM; a P2 receptor antagonist) blocked the ATP-induced [Ca2+]i increase, whereas the P2X receptor agonists, alpha, beta-methylene ATP (10 microM), 2-methyl-thio ATP (10 microM) and Benzoylbenzoyl ATP (10 microM), did not elicit any increases in [Ca2+]i. In the basal cell layer, ATP-induced [Ca2+]i dynamics were biphasic, while oscillatory fluctuations of [Ca2+]i were induced in the wing cells of the mid layer of the corneal epithelium by ATP stimulation. Ca2+ oscillations were sometimes synchronized among adjacent wing cells, but these waves did not propagate to other cell layers. These results suggest that extracellular ATP elicits a [Ca2+]i increase mainly via P2Y receptors. In addition, synchronized Ca2+ oscillation in the wing cell layer indicates that intracellular events may spread to neighboring cells within the layer.

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.

ATP-gated cation channels (P2X purinoceptors) in trigeminal mesencephalic nucleus neurons of the rat.

1. We have investigated whether receptors for ATP exist on neurones of the trigeminal mesencephalic nucleus (MNV) of the rat using whole-cell and outside-out patch-clamp recording in coronal brainstem slices. 2. With whole-cell recording, the batch application of ATP, adenosine 5'-O-[3-thiotriphosphate] (ATP gamma S) and alpha,beta methylene adenosine triphosphate (alpha beta meATP) caused concentration-dependent inward currents in all cells tested (holding potential, -62 mV), with EC50 values of 437, 15 and 254 microM, respectively. All three agonist-evoked currents developed rapidly (rise time, approximately 10-25 s), desensitized slowly (over approximately 20-50 s), cross-desensitized with each other, were associated with an increase in membrane conductance and were attenuated by the application of suramin (30 microM). 3. The inward current evoked by ATP decreased as the membrane potential was made less negative and had a zero current potential of +1.0 +/- 3.7 mV. The current-voltage relationship showed marked inward rectification. 4. Brief flickery single-channel openings could be resolved in response to ATP (3 microM) in outside-out membrane patches. Unitary current at -82 mV was -1.81 +/- 0.2 pA, which corresponds to a unitary conductance of 22 pS. 5. We conclude that proprioceptive MNV neurones contain ATP-gated cation channels. Such P2X purinoceptors may be involved in the processing of proprioceptive information, thus suggesting a potentially important physiological role of ATP.

Involvement of ATP in the non-adrenergic non-cholinergic inhibitory neurotransmission of lamb isolated coronary small arteries.

1. The involvement of non-adrenergic non-cholinergic (NANC) transmitters, such as nitric oxide (NO) and adenosine 5'-triphosphate (ATP), in the neurogenic relaxation of lamb coronary small arteries was investigated in vessel segments with an internal lumen diameter of 200-550 microns, isolated from the left ventricle of the heart, and suspended for isometric tension recording in microvascular myographs. 2. In both endothelium-intact and -denuded coronary small arteries treated with phentolamine (3 x 10(-6) M), propranolol (3 x 10(-6) M), and atropine (10(-6) M) and contracted to 3 x 10(-7) M of the thromboxane analogue U46619, electrical field stimulation (EFS) evoked frequency-dependent relaxations, which were markedly reduced in the presence of tetrodotoxin (10(-6) M). 3. Exogenous NO added as acidified sodium nitrite (10(-6)-10(-3) M) and L-nitrosocysteine induced potent relaxations of lamb coronary small arteries. However, both inhibition of NO synthase with NG- nitro-L-arginine (L-NOARG, 3 x 10(-5) M), and mechanical endothelial cell removal increased rather than inhibited relaxations to EFS. In small arteries processed for NADPH-diaphorase histochemistry, activity was only observed within endothelial cells. 4. In arteries contracted to U46619, exogenously added ATP caused concentration-dependent relaxations with pD2 and maximum responses of 4.72 +/- 0.12 and 89.6 +/- 3.8% (n = 12), respectively. ADP and the P2Y-agonist, 2-methylthio-ATP, induced relaxations equipotent to ATP, while the P2x-agonist, alpha, beta-methylene ATP (10(-9)-10(-4) M), and the P2U-agonist, UTP (10(-9)-10(-4) M) only caused small transient relaxations at the highest concentrations (10(-4) and 10(-3) M). 5. ATP and EFS-induced relaxations were unchanged in the presence of the P1-purinoceptor antagonist, 8-phenyltheophylline (10(-5) M), while this antagonist inhibited the concentration-dependent relaxations to adenosine. In contrast, the P2-purinoceptor antagonist, suramin (3 x 10(-5) M), markedly reduced the relaxations to EFS. 6. After desensitization of P2x-purinoceptors with alpha, beta-methylene ATP (2 x 10(-5) M), the relaxations to exogenous added ATP were enhanced, but this procedure did not influence the relaxations to EFS. In contrast, the P2y-purinoceptor antagonist, basilen blue E-3G (3 x 10(-5) M, earlier named reactive blue 2) significantly inhibited the concentration-relaxation curves to ATP and almost abolished the EFS-induced relaxations. 7. Mechanical removal of the endothelium significantly inhibited ATP-induced maximal relaxations without affecting sensitivity, pD2 and maximum relaxations being 4.72 +/- 0.12 and 89.7 +/- 3.8% (n = 10), and 5.45 +/- 0.38 and 48.0 +/- 8.6% (P < 0.05, paired t test, n = 10) in endothelium-intact and -denuded coronary small arteries, respectively. However, incubation with L-NOARG did not change relaxations elicited by ATP. 8. The present study suggests that in NANC conditions neurogenic relaxations of coronary small arteries are mediated by ATP, which relaxes coronary small arteries through P2Y-purinoceptors. A prejunctional modulation of these relaxations by endothelial-derived NO cannot be excluded.

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.

Diadenosine polyphosphates induce transplasma membrane calcium influx in cultured glomerular mesangial cells.

The effects of diadenosine tetraphosphate (AP4A) diadenosine pentaphosphate (AP5A) and diadenosine hexaphosphate (AP6A) on the cytosolic-free calcium concentration ([Ca2+]i) were evaluated in cultured rat glomerular mesangial cells (MCs) using the fluorescent dye technique. The addition of 10 mumol L-1 AP4A, AP5A or AP6A significantly increased [Ca2+]i in MCs by 57 +/- 9 nmol L-1 n = 17; P < 0.01), 76 +/- 27 nmol L-1 (n = 9; P < 0.01) or 65 +/- 12 nmol L-1 (n = 18; P < 0.01) respectively. In the absence of extracellular calcium, there was no significant change in [Ca2+]i in MCs after administration of diadenosine polyphosphates, indicating that these agents induce transplasma membrane Ca2+ influx. AP6A significantly enhanced the angiotensin II-induced changes in [Ca2+]i in MCs. The AP5A-induced transplasma membrane Ca2+ influx was inhibited by the P2 purinoceptor blockers suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), but was not affected by the adenosine A1 receptor blocker 8-cyclopentyl-1.3-dipro-pylzanthine (CPDPX). Adenosine triphosphate (ATP) and adenosine 5'-O-(3-thio)triphosphate (ATP-gamma S) increased [Ca2+]i in MCs, whereas alpha, beta-methylene ATP had no effect on [Ca2+]i in MCs. Measurements of diacylglycerol and phosphatidic acid showed that AP5A and AP6A also stimulated phospholipase C, but had no effect on phospholipase D. The inhibition of phosphatidylcholine-specific phospholipase C significantly reduced the AP5A-induced [Ca2+]i increase. In summary, diadenosine polyphosphates induce Ca2+ influx through P2 purinoceptors and may be involved in the local regulation of vascular resistance evoked by the Ca(2+)-dependent contractile response of mesangial cells.

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.

P2U receptor is linked to cytosolic Ca2+ transient and release of vasorelaxing factor in bovine endothelial cells in situ.

1. With the use of front-surface fluorimetry and fura-2-loaded strips of bovine aortic valve, we characterized the [Ca2+]i transients induced in endothelial cells in situ using a non-selective purinergic agonist (adenosine 5'-triphosphate (ATP)), and selective agonists for P2X (alpha, beta-methylene ATP), P2Y (2-methylthio-ATP (2MeSATP)) and P2U (uridine 5'-triphosphate (UTP)) purinoceptors and an unrelated agonist bradykinin (BK). 2. Double staining with fura-2 and acetylated low-density lipoprotein labelled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indo-carbocyanine perchlorate showed that the fura-2 fluorescence arose exclusively from a single monolayer of endothelial cells covering the surface of the valvular strips. 3. All nucleotides (ATP, UTP and 2MeSATP) induced an elevation of the intracellular Ca2+ concentration ([Ca2+]i), with an initial transient peak and a subsequent lower sustained elevation. Blockade of the Ca2+ influx with 1 mM Ni2+ did not affect the peak levels of the [Ca2+]i transients, whereas it abolished the sustained increases in [Ca2+]i induced by these nucleotides. 4. The potency order of these nucleotides was 2MeSATP > ATP > UTP, while the order of the maximum responses was UTP = ATP > 2MeSATP. alpha, beta-Methylene ATP (up to 1 mM) had only a minimal effect. 5. Prolonged exposure to ATP or UTP, at concentrations giving a maximum response, desensitized the responses to ATP, UTP and 2MeSATP, but not to BK. Prolonged exposure to 2MeSATP at concentrations giving a maximum response did not desensitize the responses to UTP or BK, but did desensitize those to ATP and 2MeSATP. Prolonged exposure to BK did not induce heterologous desensitization to any of the three nucleotides. 6. [Ca2+]i elevation in valvular endothelial cells induced by UTP was associated with the relaxation of adjacent vascular medial strips precontracted with U-46619, the stable analogue of thromboxane A2. 7. We conclude that: (1) the peak elevation of the [Ca2+]i transient induced by these nucleotides is independent of extracellular Ca2+, which therefore suggests the release of intracellular Ca2+ and, (2) mature endothelial cells in situ, in a valvular preparation, have a common receptor for ATP and UTP (nucleotide or P2U receptor), which coexists with the P2Y receptor. Thus we propose that the activation of the nucleotide receptor, P2U, induces [Ca2+]i elevation in endothelial cells in situ, and thus leads to the release of vasorelaxing factors.

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.

ATP-activated cation conductance in a Xenopus renal epithelial cell line.

1. Using a whole-cell voltage-clamp technique and fura-2 fluorescence measurements, the actions of extracellular adenosine 5'-triphosphate (ATP) in single cells of an epithelial cell line (A6), were investigated. 2. ATP (0.1-1000 microM) induced currents in cells held under voltage clamp. The sequence of purinergic agonist potency in activating the currents (2-methylthio ATP > adenosine 5'-diphosphate (ADP) > ATP > alpha, beta-methylene ATP) was consistent with that of P2y receptors. 3. Reversal potentials (Erev) of the currents under various ionic conditions suggest that potassium channels and non-selective cation channels were responsible for the ATP-activated conductance, which was permeable to calcium. 4. ATP activated the currents in a calcium-free extracellular solution. In the presence of extracellular calcium, the currents were completely inhibited with 10 mM EGTA in the pipette. 5. ATP (10 microM) increased the intracellular calcium concentration ([Ca2+]i) whether cells were bathed in a solution containing calcium or not. 6. These results indicate that ATP evoked a calcium-dependent cation conductance, permeable to calcium, through P2y receptors by releasing calcium from intracellular stores in A6 cells.