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Characterization of the P2Y-purinoceptor involved in the ATP-induced rise in cytosolic Ca2+ concentration in rat ileal myocytes.

ABSTRACT: 1. The P2-purinoceptor subtype and the intracellular signalling mechanism(s) involved in the rise in the free cytosolic Ca2+ concentration ([Ca2+]i) induced by ATP and analogues were analyzed in myocytes isolated from the longitudinal muscle layer of rat ileum by means of molecular and physiological techniques. 2. The P2-purinoceptor expressed by ileal smooth muscle cells shared 100% amino acid identity with the rat P2Y1-receptor. 3. Short applications of the purinoceptor agonists induced a transient rise in [Ca2+]i in an all-or-nothing manner. The rank order of potency of the analogues of ATP and ADP, determined by measuring the percentage of responding cells was 2-methylthioATP = 2-chloro-ATP > ADP > ATP, with concentrations giving [Ca2+]i response in 50% of cells ranging between 3 nM and 0.6 microM. The concentration-response curves to ADP and ATP were shifted to the right by 10 microM pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). 4. Although the rise in [Ca2+]i induced by stimulation of the ileal P2v-purinoceptor was inhibited by heparin (5 mg ml-1), we were not able to detect stimulation of phospholipase C under conditions (37 degrees C) where muscarinic cholinoceptor activation markedly increased inositol phosphate (InsP) accumulation. However, the carbachol (CCh)-induced increase in InsP accumulation was suppressed when the agonist was applied at 20 degrees C while a CCh-induced [Ca2+]i rise similar to that obtained in response to the P2-purinoceptor agonist was still observed. 5. Our results indicate that the rat ileal myocytes express a PPADS-sensitive P2-purinoceptor similar to the P2Y1-receptor subtype. Although there is no detectable increase in InsP production, stimulation of these receptors leads to a rise in [Ca2+]i by activation of the inositol 1,4,5-trisphosphate receptor-channel of the intracellular Ca2+ store, indicating that they couple to phospholipase C.

SUBMITTER: Pacaud P

PROVIDER: S-EPMC1909881 | biostudies-other | 1996 Aug

REPOSITORIES: biostudies-other

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Characterization of the P2Y-purinoceptor involved in the ATP-induced rise in cytosolic Ca2+ concentration in rat ileal myocytes.

Pacaud P P Feolde E E Frelin C C Loirand G G

British journal of pharmacology 19960801 8

1. The P2-purinoceptor subtype and the intracellular signalling mechanism(s) involved in the rise in the free cytosolic Ca2+ concentration ([Ca2+]i) induced by ATP and analogues were analyzed in myocytes isolated from the longitudinal muscle layer of rat ileum by means of molecular and physiological techniques. 2. The P2-purinoceptor expressed by ileal smooth muscle cells shared 100% amino acid identity with the rat P2Y1-receptor. 3. Short applications of the purinoceptor agonists induced a tran ...[more]

PMID: 8864564

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Project description:The origin of the increase in cytosolic free Ca2+ concentration ([Ca2+]i) induced by extracellular ATP was investigated in single isolated cardiac myocytes loaded with indo-1. The nucleotide added at a concentration of 10 microM triggers a few Ca2+ spikes, followed by a cluster of Ca2+ oscillations, increasing [Ca2+]i to around 200 nM from a basal value of 70 nM. Neither caffeine nor ryanodine affects the magnitude of the Ca2+ transient, but both shorten it by preventing the Ca2+ oscillations. This indicates that the latter must be related to the release of Ca2+ from the sarcoplasmic reticulum. Since ATP also induces cell depolarization (as shown by experiments using the potential sensitive dye bis-oxonol), the initial Ca2+ spikes were attributed to the opening of voltage-dependent Ca2+ channels. A small Ca2+ transient still remains under experimental conditions designed to prevent Ca2+ influx from external medium (low-Ca2+ high-Mg2+ medium containing La3+) and after depletion of the sarcoplasmic-reticulum Ca2+ load with caffeine. Under these conditions, when this Ca2+ transient was buffered by 1,2-bis-(O-aminophenoxy)ethane-NNN'N'-tetra-acetic acid, ATP was unable to trigger the initial Ca2+ spikes. These results indicate that ATP mobilizes Ca2+ ions from an intracellular pool other than the sarcoplasmic reticulum and that this Ca2+ release is responsible for the depolarization. The effects of ATP on [Ca2+]i share the same characteristics as the acidification simultaneously induced by the nucleotide (as shown by experiments using the pH-sensitive probe snarf-1). These ionic variations are highly specific to ATP and its hydrolysis-resistant analogues. They both require the presence of Mg2+ and Cl- ions in the extracellular medium, and they are prevented by pretreatment of the cells with 4,4'-di-isothiocyanostilbene or probenecid. These results suggest that: (1) the ATP-induced acidification leads to displacement of Ca2+ ions from or close to the internal face of sarcolemma; (2) the Ca2+ ions activate a non-specific membrane conductance responsible for the depolarization of the cells; (3) the depolarization leads to a Ca2+ influx, owing to the opening of the voltage-dependent Ca2+ channels; (4) this increase in Ca2+ triggers the release of Ca2+ from the sarcoplasmic reticulum, which is facilitated by the increase in inositol trisphosphate following P2-purinergic stimulation.

Project description:Mitochondrial Ca2+ ([Ca2+]m) regulates oxidative phosphorylation and thus contributes to energy supply and demand matching in cardiac myocytes. Mitochondria take up Ca2+ via the Ca2+ uniporter (MCU) and extrude it through the mitochondrial Na+/Ca2+ exchanger (mNCE). It is controversial whether mitochondria take up Ca2+ rapidly, on a beat-to-beat basis, or slowly, by temporally integrating cytosolic Ca2+ ([Ca2+]c) transients. Furthermore, although mitochondrial Ca2+ efflux is governed by mNCE, it is unknown whether elevated intracellular Na+ ([Na+]i) affects mitochondrial Ca2+ uptake and bioenergetics. To monitor [Ca2+]m, mitochondria of guinea pig cardiac myocytes were loaded with rhod-2-acetoxymethyl ester (rhod-2 AM), and [Ca2+]c was monitored with indo-1 after dialyzing rhod-2 out of the cytoplasm. [Ca2+]c transients, elicited by voltage-clamp depolarizations, were accompanied by fast [Ca2+]m transients, whose amplitude (delta) correlated linearly with delta[Ca2+]c. Under beta-adrenergic stimulation, [Ca2+]m decay was approximately 2.5-fold slower than that of [Ca2+]c, leading to diastolic accumulation of [Ca2+]m when amplitude or frequency of delta[Ca2+]c increased. The MCU blocker Ru360 reduced delta[Ca2+]m and increased delta[Ca2+]c, whereas the mNCE inhibitor CGP-37157 potentiated diastolic [Ca2+]m accumulation. Elevating [Na+]i from 5 to 15 mmol/L accelerated mitochondrial Ca2+ decay, thus decreasing systolic and diastolic [Ca2+]m. In response to gradual or abrupt changes of workload, reduced nicotinamide-adenine dinucleotide (NADH) levels were maintained at 5 mmol/L [Na+]i, but at 15 mmol/L, the NADH pool was partially oxidized. The results indicate that (1) mitochondria take up Ca2+ rapidly and contribute to fast buffering during a [Ca2+]c transient; and (2) elevated [Na+]i impairs mitochondrial Ca2+ uptake, with consequent effects on energy supply and demand matching. The latter effect may have implications for cardiac diseases with elevated [Na+]i.

Dataset Information

Characterization of the P2Y-purinoceptor involved in the ATP-induced rise in cytosolic Ca2+ concentration in rat ileal myocytes.

Publications

Characterization of the P2Y-purinoceptor involved in the ATP-induced rise in cytosolic Ca2+ concentration in rat ileal myocytes.

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