alpha,beta-mATP Research Articles

A novel calcium‐sensitive potassium conductance is coupled to P2X3 subunit containing receptors in myenteric neurons of guinea pig ileum

This study characterized P2X receptors in guinea pig ileum myenteric S neurons (n = 124) in vitro using electrophysiological methods. ATP or alpha,beta-methylene ATP (alpha,beta-mATP), an agonist at P2X(1) and P2X(3) subunit containing receptors, depolarized 103 neurons (85%). Pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid (10 micromol L(-1)) blocked ATP- and alpha,beta-mATP-induced depolarizations. ATP-induced depolarizations and fast excitatory postsynaptic potentials (fEPSPs) were reduced by trinitrophenyl-ATP (10 micromol L(-1)), an antagonist that can block P2X(3) receptors. Ivermectin (10 micromol L(-1)), a modulator of P2X(4) and P2X(4/6) receptors, had no effect on alpha,beta-mATP-induced depolarizations. In 58% of neurons, the alpha,beta-mATP induced-depolarization was followed by an afterhyperpolarization (AHP) (P2X-AHP). Under voltage clamp, alpha,beta-mATP induced an inward current followed by an outward current which reversed polarity at 0 and -80 mV respectively. The P2X-AHP was reduced in low extracellular Ca(2+) solutions. Blockers of large, intermediate and small conductance Ca(2+)-activated K(+) channels or voltage-gated K(+) channels did not inhibit the P2X-AHP. Half of the neurons exhibiting the P2X-AHP contained nitric oxide synthase (NOS)-immunoreactivity (ir). In summary, NOS-ir S neurons express P2X(3) subunit containing P2X receptors. P2X receptors couple to activation of a Ca(2+)-activated K(+) conductance that mediates an AHP. As P2X receptors contribute to fEPSPs, the P2X-AHP may modulate S neuron excitability during purinergic synaptic transmission.

Modulation of autonomous contractile activity in the isolated whole bladder of the guinea pig.

To investigate the actions of the nonhydrolysable analogue of ATP, alpha,beta-methylene ATP (alpha,beta-MATP) and the sensory peptide, substance P, on the phasic activity generated by muscarinic stimulation in the isolated whole bladder. Isolated bladder can generate complex contractions resulting in phasic rises in intravesical pressure (the autonomous bladder): activity thought to underlie nonmicturition activity in vivo and which may be important in generating bladder sensations. Experiments were conducted on whole isolated bladders from female guinea pigs (270-300 g). Bladders were cannulated via the urethra, suspended in a heated chamber containing oxygenated solution at 33-36 degrees C and intravesical pressure recorded. All drugs were added to the solution bathing the abluminal surface of the bladder. When alpha,beta-MATP (30-3000 nmol/L) or substance P (30-300 nmol/L) was added to resting bladders there were small rises in intravesical pressure (<2 cmH2O). However, in the presence of phasic activity generated by exposing the bladder to the muscarinic agonist arecaidine (100-300 nmol/L) or the nicotinic ligand lobeline (10-30 micromol/L) similar or lower concentrations of alpha,beta-MATP or substance P produced more dramatic effects: alpha,beta-MATP and substance P (both at 100 nmol/L) activated a rise in basal pressure of > 15 cmH2O and increased the frequency of the phasic activity. On removing alpha,beta-MATP or substance P, there was a slowing of phasic activity indicative of an inhibitory mechanism. In addition to direct effects on smooth muscle the agonists alpha,beta-MATP and substance P appear to be potent regulators of the mechanisms generating phasic activity. A developing concept is that the mechanisms responsible for generating phasic activity underlie nonmicturition activity are the target for excitatory and inhibitory inputs. Regulating such activity may be a factor in generating or modifying bladder sensation. Inappropriate or exaggerated phasic activity could underpin the pathological changes which cause the overactive bladder, thus adding another hypothesis to the neurogenic and myogenic hypotheses of bladder overactivity, i.e. that of the autonomous bladder.

P2X2 subunits contribute to fast synaptic excitation in myenteric neurons of the mouse small intestine.

P2X receptors are ATP-gated cation channels composed of one or more of seven different subunits. ATP acts at P2X receptors to contribute to fast excitatory postsynaptic potentials (fEPSPs) in myenteric neurons but the subunit composition of enteric P2X receptors is unknown. These studies used tissues from P2X2 wild-type (P2X2+/+) and P2X2 gene knockout (P2X2-/-) mice to investigate the role of this subunit in enteric neurotransmission. Intracellular electrophysiological methods were used to record synaptic and drug-induced responses from ileal myenteric neurons in vitro. Drug-induced longitudinal muscle contractions and peristaltic contractions of ileal segments were also studied in vitro. Gastrointestinal transit was measured as the progression in 30 min of a liquid radioactive marker administered by gavage to fasted mice. RT-PCR analysis of mRNA from intestinal tissues and data from immunohistochemical studies verified P2X2 gene deletion. The fEPSPs recorded from S neurons in tissues from P2X2+/+ mice were reduced by mecamylamine (nicotinic cholinergic receptor antagonist) and PPADS (P2X receptor antagonist). The fEPSPs recorded from S neurons from P2X2-/- mice were unaffected by PPADS but were blocked by mecamylamine. ATP depolarized S and AH neurons from P2X2+/+ mice. ATP depolarized AH but not S neurons from P2X2-/- mice. alpha,beta-Methylene ATP (alpha,beta-mATP)(an agonist at P2X3 subunit-containing receptors) did not depolarize S neurons but it did depolarize AH neurons in P2X2+/+ and P2X2-/- mice. Peristalsis was inhibited in ileal segments from P2X2-/- mice but longitudinal muscle contractions caused by nicotine and bethanechol were similar in segments from P2X2+/+ and P2X2-/- mice. Gastrointestinal transit was similar in P2X2+/+ and P2X2-/- mice. It is concluded that P2X2 homomeric receptors contribute to fEPSPs in neural pathways underlying peristalsis studied in vitro.

Purinoceptor-mediated contractility of the perfused uterine vasculature of the guinea-pig: influence of oestradiol and pregnancy.

1. The effects of ATP, the stable ATP analogues alpha,beta-methylene ATP (alpha,beta-mATP), 2-methylthioATP (2meSATP) and adenosine tetraphosphate (ATP4), the pyrimidine nucleotide uridine 5'-triphosphate (UTP) and the alpha1-adrenoceptor agonist phenylephrine were examined on the isolated perfused uterine vasculature of dioestrous, oestradiol-treated, dexamethasone-treated and late-pregnant guinea-pigs. 2. The alpha1-adrenoceptor agonist phenylephrine elicited concentration-dependent vasoconstriction from preparations of perfused uterine vasculature from dioestrous, estradiol-treated and late-pregnant guinea-pigs. The mean maximal response to phenylephrine was unaffected by treatment of dioestrus guinea-pigs with oestradiol or dexamethasone, but was reduced in preparations from late-pregnant animals. 3. In perfused uterine arteries from dioestrous animals, the pyrimidine UTP, but not ATP4 and ATP, elicited vasoconstrictor responses. In preparations from oestradiol-treated animals, all three agonists elicited vasoconstriction, with a rank order of potency of ATP4 = UTP >> ATP, whereas in preparations from late-pregnant animals this order of potency was ATP4 >> UTP = ATP. In preparations from dexamethasone-treated animals, the vasoconstriction was similar to that seen in dioestrous animals. Vasoconstrictor responses to ATP4 were significantly greater in preparations of uterine vasculature from oestradiol-treated and pregnant animals than in preparations from dioestrous animals or dexamethasone-treated animals. 4. In preparations from dioestrous, oestradiol-treated, pregnant and dexamethasone-treated animals, alpha,beta-mATP was approximately two to three orders of magnitude more potent than 2meSATP. Compared with preparations from dioestrous animals, the maximal responses to alpha,beta-mATP were significantly greater in tissues from oestradiol-treated and pregnant animals. In preparations from dioestrous animals, the P2 purinoceptor antagonist suramin (100 micro mol/L) inhibited the responses to alpha,beta-mATP, but not those to ATP4. 5. The present study has demonstrated that pregnancy, but not the steroid treatment of dioestrous guinea-pigs with oestradiol or dexamethasone, reduces the sensitivity of the guinea-pig isolated perfused uterine vasculature to phenylephrine. In contrast, preparations from pregnant or oestradiol-treated guinea-pigs respond to ATP4 and to alpha,beta-mATP with significantly greater constrictions than those of dioestrous or dexamethasone-treated animals. These data indicate that the sensitivity of the uterine vasculature to adrenoceptor and purinoceptor agonists is differentially regulated by oestradiol and pregnancy, but not by the synthetic glucocorticoid dexamethasone.

Effects of a selective neuropeptide Y Y(1) receptor antagonist BIBP 3226 on double peaked vasoconstrictor responses to periarterial nerve stimulation in canine splenic arteries.

The periarterial electrical nerve stimulation (30 s trains of pulses at a frequency of 1, 4 or 10 Hz) induced a double peaked vasoconstriction consisting of an initial transient constriction (first peak) followed by a prolonged response (second peak) in the isolated, perfused canine splenic artery. At low frequencies (1 and 4 Hz), a neuropeptide Y (NPY) Y(1) receptor antagonist BIBP 3226 (0.1-1 microM) produced a dose-dependent inhibitory effect on the second peak, but did not modify the first peak. At a high frequency (10 Hz), 1 microM BIBP 3226 induced a slight, but significant inhibition on both the first and second peaked responses. At a low frequency (1 Hz), the first peak was not influenced by blockade of alpha(1)-adrenoceptors or NPY Y(1) receptors with prazosin (0.1 microM) or BIBP 3226 (1 microM), respectively, but abolished by P2X receptor desensitization with alpha,beta-methylene ATP (alphabeta-m ATP, 1 microM). At a high frequency (10 Hz), the first peak was mostly inhibited by alphabeta-m ATP and partially by prazosin and BIBP 3226. On the other hand, the second peak at a low frequency was largely decreased by BIBP 3226 and partially by prazosin and alphabeta-m ATP, whereas at a high frequency, it was largely attenuated by prazosin and partially by alphabeta-m ATP and BIBP 3226. The results suggest that at a low frequency, the firstly transient constriction of double peaked responses is mainly induced via an activation of P2X-receptors, whereas at a high frequency, it is mostly mediated by the P2X-receptors, and partially by alpha(1)-receptors and NPY Y(1)-receptors. The secondary prolonged vasoconstriction at frequencies used is predominantly mediated via both alpha(1)-receptor and NPY Y(1) receptor activations, and in part by P2X-receptors. Furthermore, an activation of NPY Y(1) receptors may play an important role in evoking the prolonged vasoconstrictor response to longer pulse trains of stimulation at a low frequency, whereas an alpha(1)-adrenoceptor activation exerts a main vasomotor effect for the prolonged response at a high frequency.

ATP release and contraction mediated by different P2-receptor subtypes in guinea-pig ileal smooth muscle.

1. The present study was addressed to clarify the subtypes of P2-purinoceptor involved in ATP release and contraction evoked by alpha, beta-methylene ATP (alpha, beta-mATP) and other P2-agonists in guinea-pig ileum. 2. alpha, beta-mATP 100 microM produced a transient and steep contraction followed by ATP release from tissue segments. These maximum responses appeared with different time-courses and their ED50 values were 5 and 25 microM, respectively. The maximum release of ATP by alpha, beta-mATP was markedly reduced by 250 microM suramin, 30 microM pyridoxal-phosphate-6-azophenyl-2',5'-disulphonic acid (PPADS) and 30 microM reactive blue 2 (RB-2), P2-receptor antagonists. However, the contractile response was inhibited by suramin, tetrodotoxin and atropine, but not by PPADS and RB-2. 3. Although the contraction caused by alpha, beta-mATP was strongly diminished by Ca(2+)-removal and nifedipine, and also by tetrodotoxin and atropine at 0.3 microM, the release of ATP was virtually unaffected by these procedures. 4. UTP, beta, gamma-methylene ATP (beta, gamma-mATP) and ADP at 100 microM elicited a moderate release of ATP. The release caused by UTP was virtually unaffected by RB-2. However, these P2-agonists failed to elicit a contraction of the segment. 5. The potency order of all the agonists tested for the release of ATP was alpha, beta-mATP > UTP > beta, gamma-mATP > ADP. 6. In superfusion experiments with cultured smooth muscle cells from the ileum, alpha, beta-mATP (100 microM) enhanced the release of ATP 5 fold above the basal value. This evoked release was inhibited by RB-2. 7. These findings suggest that ATP release and contraction induced by P2-agonists such as alpha, beta-mATP in the guinea-pig ileum result mainly from stimulation of different P2-purinoceptors. P2Y-like purinoceptors on the smooth muscles and, probably, P2X-purinoceptors on cholinergic nerve terminals, respectively. However, the ATP release may also be mediated, in part, by P2U-receptors, because UTP caused RB-2-insensitive ATP release.

Attenuation of contractile responses to sympathetic co-transmitters in veins from subjects with essential hypertension.

Neuropeptide Y (NPY), noradrenaline (NA) and ATP are cotransmitters of the sympathetic nervous system and exert vasocontractile effects. The aim of this study was to determine the role of these sympathetic co-transmitters in human hypertension. Subcutaneous vessels from 12 patients with essential hypertension and 12 matched controls were studied in vitro. Vascular contractile responses to NPY, NA, alpha,beta-methylene ATP (alpha,beta-mATP) and potassium were studied in isolated arteries and veins (diameter 0.1-1.1 mm) with intact endothelium. The dilatory effect of acetylcholine was used to test the endothelial function. There was no difference in potency (pD2) or contractile response to NPY, NA or alpha,beta-mATP between hypertensive and control arteries. In veins, however, the contractile response to NPY was significantly reduced in hypertensives and the responses to NA were unchanged. Furthermore, the sensitivity (pD2) to alpha,beta-mATP was significantly reduced in veins from hypertensives. There was no difference in the dilatory response to acetylcholine between the hypertensives and the controls, neither in the arteries nor in the veins, indicating that the observed changes in vascular reactivity to NPY, NA and alpha,beta-mATP were not endothelium-dependent. In conclusion, the postjunctional contractile effect of NPY and sensitivity (pD2) to alpha,beta-mATP, co-transmitters of the peripheral sympathetic nervous system, are attenuated in veins in essential hypertension.

Design and pharmacology of selective P2-purinoceptor antagonists.

1. The symmetrical 3'-urea of 8-(benzamido)naphthalene-1,3,5-trisulfonic acid (NF023) and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) were investigated for their ability to antagonize responses mediated via P2-purinoceptors. In addition, putative inhibitory effects of these compounds on ecto-nucleotidase activity were studied. 2. In rabbit vas deferens, PPADS antagonized contractions to alpha, beta-methylene ATP (alpha, beta-mATP) in a pseudoirreversible manner (pA2 = 6.34). This P2X-antagonism by PPADS has been confirmed in certain vascular and visceral smooth muscles (pA2 = 6.02-6.41). PPADS inhibited P2Y-receptor-mediated relaxant responses to ADP-beta-S or 2-methylthio ATP (2-MeSATP) in guinea-pig taenia coli (pA2 = 4.59 and 5.26, respectively) as well as in rat duodenum (pA2 = 5.09) and mesenteric artery (pA2 = 5.46). Experiments on the rat mesenteric arterial bed demonstrated the ineffectiveness of PPADS at P2U-purinoceptors. P2T-purinoceptor-mediated platelet aggregation was affected only at high concentrations of PPADS (> 100 microM). PPADS (100 microM) was also weakly active in inhibiting ecto-nucleotidase activity in guinea-pig taenia coli. 3. In rabbit vas deferens, NF023 antagonized P2X-purinoceptor-mediated contractions to alpha, beta-mATP in a competitive manner. The estimated pA2 value of 5.68 was very similar to that obtained at P2X-receptors in rat mesenteric (pA2' = 5.54) and rabbit saphenous artery (pA2 = 5.69). In rat duodenum and guinea-pig taenia coli, NF023 competitively inhibited relaxant responses to the P2Y-selective agonists, ADP-beta-S and 2-MeSATP (pA2 = 4.00-4.25). In rat mesenteric arterial bed, NF023 antagonized vasodilator responses mediated by P2Y-purinoceptors (pA2 = 4.94), but had no effect on vasodilator responses to the P2U-selective agonist, UTP. NF023 was found to inhibit ectonucleotidase activity in guinea-pig taenia coli and rabbit vas deferens (pIC40 = 3.52 and 4.12, respectively). 4. At 100 microM, PPADS and NF023 did not interact with alpha 1- and alpha 2-adrenoceptors, adenosine A1- and A2-, histamine H1- and muscarinic M1-, M2- and M3-receptors. 5. In conclusion, the results demonstrate that PPADS and NF023 are specific P2-receptor antagonists and useful tools in the study of multiple subtypes of P2-purinoceptors.