Chiemi Sato

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 α,β‐methylene ATP (α,β‐mATP) and other P2‐agonists in guinea‐pig ileum. 2 α,β‐mATP 100 μM 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 μM, respectively. The maximum release of ATP by α,β‐mATP was markedly reduced by 250 μM suramin, 30 μM pyridoxal‐phosphate‐6‐azophenyl‐2′,5′‐disulphonic acid (PPADS) and 30 μM 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 α,β‐mATP was strongly diminished by Ca2+‐removal and nifedipine, and also by tetrodotoxin and atropine at 0.3 μM, the release of ATP was virtually unaffected by these procedures. 4 UTP, β,γ‐methylene ATP (β,γ‐mATP) and ADP at 100 μM 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 α,β‐mATP>UTP>β,γ‐mATP>ADP. 6 In superfusion experiments with cultured smooth muscle cells from the ileum, α,β‐mATP (100 μM) 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 α,β‐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.

ATP release caused by bradykinin, substance P and histamine from intact and cultured smooth muscles of guinea-pig vas deferens.

Abstract Histamine (60 μM) produced ATP release from segments of guinea-pig vas deferens which was blocked by pyrilamine and triprolidine, H1-blockers, but not by ranitidine, an H2-blocker. The evoked-release was inhibited by the mitochondrial inhibitors, carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and oligomycin. Bradykinin (BK) and substance P (SP) also caused substantial and moderate release of ATP, respectively. The BK-evoked release of ATP was inhibited by HOE140, a B2-antagonist, but not by [Des-Arg10] HOE140, a B1-antagonist. On the other hand, VIP, angiotensin II (AII) and cholecystokinin-octapeptide (CCK-8) failed to elicit a measurable release of ATP. Histamine and BK also enhanced the release of ATP from superfused cultured smooth muscle cells. These results suggest that ATP may be released as an autacoid from the smooth muscles in the presence of these chemical mediators.

Inositol(1,4,5)trisphosphate signal triggers a receptor-mediated ATP release

Intracellular signal transduction pathways involved in ATP release evoked by angiotensin II (Ang II) were investigated in cultured guinea pig Taenia coli smooth muscle cells. Ang II (0.3-1 microM) elicited substantial release of ATP from the cells, but not from a human fibroblast cell line. However, Ang II even at 10 microM failed to cause a leakage of lactate dehydrogenase (LDH) from the smooth muscle cells. The release of ATP by Ang II was suppressed by 10 microM SC52458, an AT1 receptor antagonist, not by 10 microM PD123319, an AT2 receptor antagonist. The evoked release of ATP was almost completely inhibited in the presence of 10 microM U73122, a phospholipase C inhibitor, and 0.5 microM thapsigargin, a Ca2+-ATPase inhibitor. Furthermore, the release was hampered by 50 microM BAPTA/AM, an intracellular Ca2+ chelator, but not by 0.1 microM nifedipine, a voltage gated Ca2+ channel inhibitor. The basal release of ATP was increased by BAPTA/AM, but was reduced by U-73122. Ang II enhanced instantaneously inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) accumulation in the cells. The enhancing effect was perfectly antagonized by SC52458. These findings suggest that intracellular Ca2+ signals activated via stimulation of Ins(1,4,5)P3 receptor are involved in the release of ATP evoked by Ang II.

Implication of ATP released from atrial, but not papillary, muscle segments of guinea pig by isoproterenol and forskolin

Effects of isoproterenol and forskolin, which increases intracellular cyclic AMP, on contraction and ATP release in atrial and papillary muscles of guinea pig were evaluated. In the electrically driven-left atrial muscle segments, isoproterenol and forskolin at 0.1 microM produced an ATP release coupled with a positive inotropic effect, the values of net ATP release at 5 min after these drugs being 5.20 +/- 0.59 and 5.37 +/- 0.55 nmoles/g wet weight, respectively. The forskolin evoked-ATP release was unaffected by prazosin plus propranolol or by guanethidine, implying that ATP is released from non-neuronal origin. In contrast, in papillary muscle segments, the test cardiotonics did not elicit any ATP release despite producing contractile response similar to that in atrial preparations. However, there is no difference in ectoATPase activities of both tissues. Adenosine added exogenously inhibited electrically evoked-contraction of the atrium, but not that of the papillary, although inhibitions by verapamil of the contractions were approximately equal in these preparations. These findings suggest that cardiotonics such as isoproterenol produce a liberation of ATP from auricle muscles, but not from ventricle muscles, and that the liberated ATP may mainly be catabolized to adenosine by ectoenzymes and the resultant nucleoside may serve as a functional modulator through stimulation of pre or postsynaptic A1-receptors.

Possible transsynaptic cholinergic neuromodulation by ATP released from ileal longitudinal muscles of guinea pigs

The effects of alpha, beta-methylene ATP (alpha, beta-mATP) and beta, gamma-methylene ATP (beta, gamma-mATP) on endogenous acetylcholine (ACh) release evoked by electrical nerve stimulation were evaluated in guinea-pig ileal longitudinal muscles. Release of ACh was measured with an HPLC-electrochemical detector system and release of ATP by luciferin-luciferase assay. Electrically evoked endogenous ACh release was reduced by both alpha, beta-mATP and beta, gamma-mATP at concentrations of 3 and 30 microM. The inhibitory effect of alpha, beta-mATP (30 microM) on ACh release was not detectable in the presence of theophylline (100 microM), a P1-purinoceptor antagonist, that itself enhanced ATP release. When exogenous ATP (0.1 microM) was added to the bath in which the ileal segment was suspended, it was rapidly metabolized, presumably by ecto-ATPase, and disappeared from the medium within 15 min. At 30 microM, alpha, beta-mATP induced ATP release in a suramin-sensitive but Ca(2+)- and atropine-insensitive manner, suggesting P2-receptor-mediated release of ATP from the smooth muscle. We conclude from these findings that alpha, beta-mATP and, probably, also beta, gamma-mATP, do not reduce ACh release by direct stimulation of presynaptic P1-purinoceptors, and that endogenous ATP released postjunctionally by these ATP analogs is decomposed metabolically to adenosine in the synapse and this adenosine triggers P1-purinoceptor sensitive neuromodulation of cholinergic transmission.

ATP release by angiotensin II from segments and cultured smooth muscle cells of guinea-pig taenia coil

Effects of angiotensin II on ATP release were evaluated in segments and cultured smooth muscle cells from the guinea-pig taenia coli. In the segments, angiotensin II (0.3–3 μM) elicited release of ATP which was blocked by losartan and SC-52458, non-peptide angiotensin II type-1 receptor (AT1)-antagonists, but not by PD-123319, an AT2-antagonist. In superfused cultured cells, 10μM angiotensin II likewise elicited release of ATP Again the response was blocked by losartan and SC-52458 but not by PD-123319. These findings suggest that angiotensin II releases ATP from the smooth muscles by activation of angiotensin II-, presumably ATE1-, receptors.

ATP release evoked by isoprenaline from adrenergic nerves of guinea pig atrium

Mode and site of release of ATP evoked by isoprenaline were evaluated in the electrically driven left atrial segment of guinea pig. The peak release of ATP 5 min after 1 microM isoprenaline was inhibited by 1 microM propranolol and 1 microM butoxamine, but not by 1 microM atenolol, showing that the ATP release is due to stimulation of the presynaptic beta 2-adrenoceptor by isoprenaline. The maximum ATP release was markedly reduced by Ca2+/calmodulin antagonists, W-7 and trifluoperazine, and by a mitotic inhibitor, vinblastine. Further, the release was similarly inhibited by myosin light chain kinase inhibitors, ML-7 and wortmannin. Nifedipine, a Ca(2+)-channel blocker, decreased the release of ATP evoked by isoprenaline. By contrast, Bay K 8644, a Ca(2+)-channel opener, tended to enhance the ATP release. These findings suggest that isoprenaline produces ATP release from adrenergic nerve terminals of atrium, implying that ATP serves as a co-transmitter.

Hyperreactivity of α1-adrenoceptors, but not of P2X-purinoceptors, in vas deferens of spontaneously hypertensive rats

We evaluated the contractile reactivity to various stimuli, and the content and release of noradrenaline (NA) from a non-vascular tissue, the vas deferens, isolated from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). The concentration-contraction curves for NA in tissue from animals of two ages (10-25 weeks and 30-45 weeks) were shifted to the left in SHR as compared with in age-matched WKY, with significant differences at 1.0 and/or 10 microM of NA. Similarly, the amplitude of contraction produced by electrical stimulation at 4, 8 and 16 Hz in the tissue was much larger in SHR than in WKY. However, ATP (10-100 microM) evoked contractions of the tissue to a similar extent in both SHR and WKY. The electrically evoked contractions of vas deferens from both strains were inhibited by isoprenaline in an approximate dose-dependent and equipotent manner. The tissue NA content, determined by HPLC-ECD, was nearly same in both SHR and WKY. In addition, the same amount of NA was released from the vas deferens of both strains by electrical stimulation in the presence of 4-aminopyridine. The present findings indicate that the contractile response of vas deferens to stimulation of alpha 1-adrenoceptors, but not of beta-adrenoceptors or P2X-purinoceptors, is more pronounced in SHR than in WKY and that a response indicative of hypertension may also occur in non-vascular tissue as it does in vascular tissue.

In vitro responses of neurofibroma fibroblasts, mast cells and Schwann cells obtained from patients with neurofibromatosis 1 to 308-nm excimer light and/or vitamin D3

Fibroblasts, mast cells and Schwann cells were isolated from neurofibromas of patients with neurofibromatosis 1, and their responses to 308‐nm excimer light irradiation and/or vitamin D3 or an analog (tacalcitol; 1,24‐dihydroxyvitamin D3) were examined in vitro. Excimer light irradiation (300 mJ/cm2) suppressed the growth of all three cell types. Exposure to 10−7 mol/L of 1α,25(OH)2D3 (VD3) or tacalcitol suppressed the growth of fibroblasts and mast cells, but not Schwann cells. These results suggest that the different neurofibroma cell types show different responses to VD3. A combination of excimer light irradiation and VD3 is necessary to suppress the growth of neurofibroma cells in vivo.

Possible neuronal origin of ATP release evoked by forskolin and ouabain from guinea-pig atrial segments

The characteristics of ATP release evoked by forskolin and ouabain from atrial segments of guinea-pig were evaluated under electrical stimulation. Forskolin (1 microM) produced a massive release of ATP together with a positive inotropic response. Both 30 microM W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide.HCI), a calmodulin antagonist, and 30 microM vinblastine, a mitotic inhibitor, markedly inhibited the evoked release of ATP without affecting the evoked contraction. However, 100 microM N-ethylmaleimide abolished completely the basal and drug-evoked ATP release and further the evoked contraction. Both the ATP release and contraction evoked by ouabain (3 microM) were similarly affected by W-7, vinblastine and n-ethylmaleimide. The release of ATP, but not the contraction, evoked by forskolin was strongly suppressed by 10 microM okadaic acid, a protein phosphatase inhibitor. The suppression by okadaic acid of the evoked release was thoroughly antagonized in the presence of 0.01 microM PMA (phorbol 12-myristate 13-acetate), but not 10 microM H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine). These results suggest that forskolin, like ouabain, may dominantly cause the neuronal release of ATP from cardiac adrenergic nerves, although the possible participation of release from muscular sources cannot be ignored.