Sadaharu Usune

3 possible coupling of postjunctional ATP release and transmitters' receptor stimulation in smooth muscles

The nature of ATP release from mainly smooth muscles of guinea-pig was evaluated with KCl and agonists for different kinds of receptors. In ileal longitudinal muscles, amounts of net ATP release by ACh and bethanechol (1-10 microM) were much larger (about 10 fold) than that by other drugs, e.g., histamine, 5-hydroxytryptamine, prostaglandin-F2 alpha, substance P and bradykinin, including KC1, although differences between contractions of the tissue evoked by test drugs were approximately 1.5 times at most. The ATP release, as well as the contraction, evoked by ACh or bethanechol was markedly reduced by atropine (0.3 microM), thus, indicating primarily postjunctional release of ATP. The remarkable ATP release from vas deferens by norepinephrine (NE), but not by substance P, was abolished almost completely by prazosin (0.3 microM). Increases in intracellular Ca2+ and subsequent contraction in the ileal tissue were produced by ATP and these responses were fully antagonized by nifedipine (0.1 microM). These findings provide evidence that the drugs-stimulated ATP release from smooth muscles does not result from contractility of muscles, but is substantially elicited only by stimulation of neurotransmitter (NE or ACh) receptors, suggesting the existence of the receptor-stimulus-postjunctional ATP release coupling. The released ATP may contribute, in part, to the muscle contractility via increase of Ca2(+)-influx, presumably, in a manner related to the voltage-gated Ca2(+)-channels.

Antagonism by nifedipine of contraction and Ca2+ ‐influx evoked by ATP in guinea‐pig urinary bladder

1 The effects of Ca2+‐antagonists, especially nifedipine, on contraction and increase of intracellular Ca2+ (Fura‐2/AM method) evoked by ATP were evaluated in a thin outer layer segment of guinea‐pig urinary bladder. 2 The ATP‐evoked contraction was markedly inhibited by dihydropyridine‐type Ca2+‐antagonists, such as nifedipine and nitrendipine, but not by D‐600, ω‐conotoxin and tetramethrin. 3 This antagonism by nifedipine of ATP‐evoked contractions was competitive from the Schild plot analysis, the pA2 value being 8.23. The reduction of ATP‐evoked contraction by nifedipine (0.1 μm) was fully reversed by administration of Bay K 8644 (0.1 μm). 4 ATP (100 μm) caused an increase of fluorescence brightness after loading Fura‐2/AM, which was coupled with a contraction of the bladder. Both the contraction and the elevation of intracellular Ca2+ evoked by the nucleotide were completely antagonized by nifedipine. 5 These results suggest that ATP may activate the dihydropyridine‐sensitive, voltage‐dependent Ca2+‐channels in a direct or indirect fashion and, thereby, elicit a contraction of the bladder.

Effects of PPADS and suramin on contractions and cytoplasmic Ca2+ changes evoked by AP4A, ATP and α,β‐methylene ATP in guinea‐pig urinary bladder

1 The contraction and intracellular Ca2+ change evoked by diadenosine tetraphosphate (AP4A) were studied in the outer longitudinal muscle of the guinea‐pig urinary bladder and compared with those evoked by ATP and αβ‐methylene ATP (a P2‐purinoceptor agonist). 2 AP4A, ATP and α,β‐methylene ATP produced concentration‐dependent transient contractions. These contractions were inhibited by PPADS (pyridoralphosphate‐6‐azophenyl‐ 2′‐4′‐disulphonic acid), 0.3–30 μm, a P2X‐purinoceptor antagonist, and suramin, 1–300 μm, a P2‐purinoceptor antagonist in a concentration‐dependent manner. From Schild plot analysis, the apparent pA2 values for PPADS for contractions evoked by AP4A, ATP and α,β‐methylene ATP were 6.86, 6.56, 6.74, and those for suramin were 6.01, 4.59 and 5.12, respectively; the Schild slopes for PPADS were 1.07, 1.14 and 1.06, and, those for suramin 0.75, 1.05 and 1.16, respectively. 3 AP4A (10 μm) and ATP (100 μm) failed to elicit any contraction of the tissue after a desensitization produced by repeated application of α,β‐methylene ATP (1 μm). 4 In fluorescence experiments with fura‐2, the increases in [Ca2+]i and contraction evoked by AP4A were suppressed by suramin and nifedipine, an L‐type Ca2+ channel blocker. 5 These findings suggest that P2X‐purinoceptors, which are more sensitive to PPADS than suramin, exist on the outer longitudinal muscles of guinea‐pig urinary bladder, and that the AP4A‐evoked contraction results from Ca2+ influx.

Two phases of the prostaglandin F2α-induced contraction in guinea-pig taenia coli involve different Ca2+ channels

SummaryProperties of the contraction produced by PGF2α in the guinea-pig taenia coli were compared to those produced by ACh. Prostaglandin (PG) F2α (3 × 10−7 M) and acetylcholine (ACh, 10−5 M) induced an initial transient contraction (phasic contraction) and a subsequent late contraction (tonic contraction). Both phasic and tonic contractions produced by PGF2α or ACh were abolished in Ca2+-free Krebs solution containing 0.5 mM EGTA. The tonic contractions caused by PGF2α and ACh were markedly suppressed by α-[3-[[2-(3,4-dimethoxy-phenyl)-ethyl]methylamino]-propyl]-3,4,5-trimethoxy-α-(1-methylethyl) benzeneacetonitrile hydrochloride (D600, > 10−7 M) as well as nifedipine (5 × 10−9 M), a Ca2+-antagonist. However, the phasic contraction produced by PGF2α, but not by ACh, was greatly inhibited by Mn2+ (> 10−4 M). Furthermore, the phasic contraction caused by PGF2α was abolished in 18 mM K+ Krebs solution with D600 (2 × 10−7 M), whereas that induced by ACh and the tonic contractions produced by PGF2α as well as by ACh were unaffected in this high K+ solution without D600. Membrane potentials of the tissue in normal (K+, 5.9 mM) and 18 mM K+ Krebs solution containing D600 were about −55 mV and −43 mV, respectively. In a fluorescence study which used Fura-2 an intracellular free Ca2+ indicator in the presence of D600, PGF2α and ACh increased fluorescence intensity in the tissue, which coupled with the magnitude of contractions. Both the enhanced fluorescence intensity and tension development evoked by PGF2α, but not by ACh, were markedly decreased in 18 mM K+ Krebs solution. It is, thus, suggested that the phasic and tonic contractions evoked by PGF2α, unlike those by ACh, may be mediated via certain different Ca2+ channels from extracellular Ca2+-source.

Mechanism of extraneuronal adrenaline release induced by K+ -free medium and ouabain, but not by high KCl in vas deferens of guinea-pig and rat

The effects of K+-free medium, ouabain or high KCl on the contractile response and spontaneous efflux of adrenaline were analyzed in denervated guinea-pig or rat vasa deferentia preloaded with 3 x 10(-5) M (+/-)-adrenaline. The K+-free medium-induced contraction of guinea-pig tissues was related to the concentration of adrenaline used during preloading and was suppressed by the addition of 2.5 mM Rb+ but not by 2.5 mM Cs+. The contractile effects of K+-free medium and 10(-5) M ouabain, but not the response to high KCl, were prevented by 5 x 10(-6) M phentolamine or 3 x 10(-5) M deoxycorticosterone. These contractions, therefore, appear to be mediated by adrenaline released from the extraneuronal compartment. The K+-free medium- or ouabain-induced contractions of denervated rat vasa deferentia were small in comparison with those of the guinea-pig tissue. The amount of spontaneous efflux of adrenaline from the guinea-pig preparation was significantly enhanced by omission of K+ or by 10(-4) M ouabain but not by 80 mM KCl. K+-free medium and 3 x 10(-5) M ouabain merely elicited a small membrane depolarization accompanied by a slight decrease of membrane resistance in the guinea-pig vas deferens, whereas 30 mM KCl markedly altered these electrical parameters. From these findings, it is suggested that the rapid increase in extraneuronal amine release following inhibition of Na+-K+-ATPase may be due to some direct effect of K+ deficiency or of ouabain on the binding between the enzyme and the catecholamine rather than to alterations of membrane properties as a consequence of enzyme inhibition.

Ins(1,4,5)P3 facilitates ATP accumulation via phosphocreatine/creatine kinase in the endoplasmic reticulum extracted from MDCK cells

So far, the content and accumulation of ATP in isolated endoplasmic reticulum (ER) are little understood. First, we confirmed using electron microscopic and Western blotting techniques that the samples extracted from MDCK cells are endoplasmic reticulum (ER). The amounts of ATP in the extracted ER were measured from the filtrate after a spinning down of ultrafiltration spin column packed with ER. When the ER sample (5mug) after 3days freezing was suspended in intracellular medium (ICM), 0.1% Triton X and ultrapure water (UPW), ATP amounts from the ER with UPW were the highest and over 10 times compared with that from the control with ICM, indicating that UPW is the most effective tool in destroying the ER membrane. After a 10-min-incubation with ICM containing phosphocreatine (PCr)/creatine kinase (CK) of the fresh ER. ATP amounts in the filtrate obtained by spinning down were not changed from that in the control (no PCr/CK). However, ATP amounts in the filtrate from the second spinning down of the ER (treated with PCr/CK) suspended in UPW became over 10-fold compared with the control. When 1muM inositol(1,4,5)trisphosphate (Ins(1,4,5)P(3)) was added in the incubation medium (ICM with PCr/CK), ATP amounts from the filtrate after the second spinning down were further enhanced around three times. This enhancement was almost canceled by Ca(2+)-removal from ICM and by adding thapsigargin, a Ca(2+)-ATPase inhibitor, but not by 2-APB and heparin, Ins(1,4,5)P(3) receptor antagonists. Administration of 500muM adenosine to the incubation medium (with PCr/CK) failed to enhance the accumulation of ATP in the ER. These findings suggest that the ER originally contains ATP and ATP accumulation in the ER is promoted by PCr/CK and Ins(1,4,5)P(3).

Interaction between Bay-K-8644 and various relaxant agents on K+-depolarized contracture of guinea pig taenia coli.

1. Interaction between Bay-K-8644 and nifedipine, D600, diltiazem, papaverine or W-7 on a K+-contracture of the guinea pig taenia coli have been investigated. 2. These relaxing drugs except Bay-K-8644 inhibited the K+-contracture in a concentration-dependent manner. 3. Complete inhibition by nifedipine (1 x 10(-7) M) of the contracture was absolutely antagonized by Bay-K-8644 (1 x 10(-6) M), a Ca2+-agonist, an apparent pA2 value of Bay-K-8644 as an antagonist for nifedipine obtained from Schild plot being 7.94 and its slope 0.99. 4. Marked inhibitions by D600 (1 x 10(-5) M), diltiazem (1 x 10(-5) M), papaverine (1 x 10(-4) M) and W-7 (1 x 10(-4) M) were unaffected but mild inhibitions by around IC50 of these relaxing agents, except W-7, were fully antagonized by Bay-K-8644. 5. These results suggest that Bay-K-8644 is a competitive antagonist for nifedipine, but not for other agents, on common Ca2+ channel sites.

Effects of SC-52458, a new nonpeptide angiotensin II receptor antagonist, on increase in cytoplasmic Ca2+ concentrations and contraction induced by angiotensin II and K+-depolarization in guinea-pig taenia coli

1. The effects of angiotensin (Ang) II receptor antagonist, SC-52458, on peak and plateau components of Ang II-induced contraction were evaluated in the guinea-pig taenia coli. 2. SC-52458 suppressed both the components of and increases in cytoplasmic Ca2+ concentrations, [Ca2+]i, coupled with the contraction by Ang II; tetrodotoxin and atropine did not affect the contractions. 3. SC-52458 inhibited a plateau component of the contraction induced by K(+)-depolarization to some extent, without affecting a peak component. 4. SC-52458 suppressed both the contraction and increase of [Ca2+]i by antagonizing AT1 receptors in the smooth muscle.

Inhibition by ouabain and veratridine of acetylcholine-evoked phasic contraction in the guinea-pig taenia coli.

SummaryInhibitory action of ouabain and veratridine on acetylcholine (ACh)-evoked phasic contraction was examined in the guinea-pig taenia coli. ACh (5×10−4m) produced a biphasic (phasic and tonic) contraction. As the phasic contraction, but not the tonic contraction, was resistant to gallopamil (D600, 2 × 10−7m), a blocker of voltage-dependent Ca2+ channels, all experiments were carried out in the presence of gallopamil at this concentration. Addition of high Ca2+ (30 mm) to the solution containing ACh induced a sustained contraction, which increased in the presence of ouabain (10−5m), an inhibitor of the Na+-K+ exchange system. On the other hand, the ACh-evoked phasic contraction was suppressed by ouabain 10−7−(10−5m) in a time- and dose-dependent manner. The suppression by ouabain (10−6m) of the phasic contraction was transiently potentiated by the addition of veratridine (10−6m), an activator of Na+ channel. In contrast, the greater suppression by ouabain (10−5m) of the contraction was antagonized by amiloride (10−4m), a blocker of Na+ channel. This antagonism by amiloride was transiently inhibited in the presence of veratridine. In the absence of ouabain, the amplitude of the phasic contraction was transiently reduced by adding veratridine but was increased by amiloride. In addition, the phasic contraction by ACh increased 80 min after exposure to Na+-free isotonic high-K+ solution (K+, 143 mm), which elicited greater depolarization of the cell membrane. In a fluorescence study with Fura-2, an intracellular free-Ca2+ indicator, ACh increased the fluorescence intensity from the tissue by excitative light at 340 nm, which coupled with the phasic contraction. Both the increased fluorescence and tension development were reduced by ouabain. These findings suggest that the ACh-evoked phasic contraction is induced by an increase in intracellular free-Ca2+ levels and seems to be inhibited by an elevation of intracellular free-Na+ levels.