Jean-Luc Morel

Crucial Role of Type 2 Inositol 1,4,5-Trisphosphate Receptors for Acetylcholine-Induced Ca2+ Oscillations in Vascular Myocytes

Objective—The aim of this study was to correlate the expression of InsP3R subtypes in native vascular and visceral myocytes with specific Ca2+-signaling patterns. Methods and Results—By Western blot and immunostaining, we showed that rat portal vein expressed InsP3R1 and InsP3R2 but not InsP3R3, whereas rat ureter expressed InsP3R1 and InsP3R3 but not InsP3R2. Acetylcholine induced single Ca2+ responses in all ureteric myocytes but only in 50% of vascular myocytes. In the remaining vascular myocytes, the first transient peak was followed by Ca2+ oscillations. By correlating Ca2+ signals and immunostaining, we revealed that oscillating vascular cells expressed both InsP3R1 and InsP3R2 whereas nonoscillating vascular cells expressed only InsP3R1. Acetylcholine-induced oscillations were not affected by inhibitors of ryanodine receptors, Ca2+-ATPases, Ca2+ influx, and mitochondrial Ca2+ uniporter but were inhibited by intracellular infusion of heparin. Using specific antibodies against InsP3R subtypes, we showed that acetylcholine-induced Ca2+ oscillations were specifically blocked by the anti-InsP3R antibody. These data were supported by antisense oligonucleotides targeting InsP3R2, which selectively inhibited Ca2+ oscillations. Conclusions—Our results suggest that in native smooth muscle cells, a differential expression of InsP3R subtypes encodes specific InsP3-mediated Ca2+ responses and that the presence of the InsP3R2 subtype is required for acetylcholine-induced Ca2+ oscillations in vascular myocytes.

Angiotensin II-activated Ca2+ entry-induced release of Ca2+ from intracellular stores in rat portal vein myocytes

1 . The action of angiotensin II (AII) was studied in single myocytes from rat portal vein in which the cytoplasmic Ca2+ concentration was estimated by emission from dyes Fura‐2 or Indo‐1 and the Ca2+ channel current was measured with the whole‐cell mode of the patch‐clamp technique. 2 . Most of the AII‐evoked increases in [Ca2+]i were reduced by about 60% after pretreatment with ryanodine and caffeine to deplete intracellular Ca2+ stores. However, in some cells the AII‐induced Ca2+ responses were of small amplitude and resembled those obtained in the presence of ryanodine and caffeine. Both types of Ca2+ responses induced by AII were selectively inhibited by losartan, suggesting that the AII effects resulted from activation of the angiotensin AT1 receptors. 3 . The concentration‐response curve to AII had an EC50 value close to 1 nM for the increase in [Ca2+]i obtained after depletion of intracellular Ca2+ stores. This value was increased to around 18 nM in experiments where the intracellular Ca2+ stores were not depleted. 4 . AII‐evoked Ca2+ responses were abolished in the absence of external Ca2+ and in the presence of 1 μm oxodipine to block L‐type Ca2+ channels. 5 . Intracellular applications of the InsP3 receptor antagonist, heparin or an anti‐PdtIns antibody did not modify AII‐induced Ca2+ responses. 6 . Our results show that AII releases Ca2+ from intracellular stores without involving InsP3 but through a Ca2+ release mechanism activated by Ca2+ influx through L‐type Ca2+ channels.

Ca(2+) signals mediated by Ins(1,4,5)P(3)-gated channels in rat ureteric myocytes.

Localized Ca(2+)-release signals (puffs) and propagated Ca(2+) waves were characterized in rat ureteric myocytes by confocal microscopy. Ca(2+) puffs were evoked by photorelease of low concentrations of Ins(1,4,5)P(3) from a caged precursor and by low concentrations of acetylcholine; they were also observed spontaneously in Ca(2+)-overloaded myocytes. Ca(2+) puffs showed some variability in amplitude, time course and spatial spread, suggesting that Ins(1,4,5)P(3)-gated channels exist in clusters containing variable numbers of channels and that within these clusters a variable number of channels can be recruited. Immunodetection of Ins(1,4,5)P(3) receptors revealed the existence of several spots of fluorescence in the confocal cell sections, supporting the existence of clusters of Ins(1,4,5)P(3) receptors. Strong Ins(1,4,5)P(3) photorelease and high concentrations of acetylcholine induced Ca(2+) waves that originated from an initiation site and propagated in the whole cell by spatial recruitment of neighbouring Ca(2+)-release sites. Both Ca(2+) puffs and Ca(2+) waves were blocked selectively by intracellular applications of heparin and an anti-Ins(1,4,5)P(3)-receptor antibody, but were unaffected by ryanodine and intracellular application of an anti-ryanodine receptor antibody. mRNAs encoding for the three subtypes of Ins(1,4,5)P(3) receptor and subtype 3 of ryanodine receptor were detected in these myocytes, and the maximal binding capacity of [(3)H]Ins(1,4,5)P(3) was 10- to 12-fold higher than that of [(3)H]ryanodine. These results suggest that Ins(1,4,5)P(3)-gated channels mediate a continuum of Ca(2+) signalling in smooth-muscle cells expressing a high level of Ins(1,4,5)P(3) receptors and no subtypes 1 and 2 of ryanodine receptors.

Ryanodine receptor subtype 2 encodes Ca2+ oscillations activated by acetylcholine via the M2 muscarinic receptor/cADP-ribose signalling pathway in duodenum myocytes

In this study, we characterized the signalling pathway activated by acetylcholine that encodes Ca2+ oscillations in rat duodenum myocytes. These oscillations were observed in intact myocytes after removal of external Ca2+, in permeabilized cells after abolition of the membrane potential and in the presence of heparin (an inhibitor of inositol 1,4,5-trisphosphate receptors) but were inhibited by ryanodine, indicating that they are dependent on Ca2+ release from intracellular stores through ryanodine receptors. Ca2+ oscillations were selectively inhibited by methoctramine (a M2 muscarinic receptor antagonist). The M2 muscarinic receptor-activated Ca2+ oscillations were inhibited by 8-bromo cyclic adenosine diphosphoribose and inhibitors of adenosine diphosphoribosyl cyclase (ZnCl2 and anti-CD38 antibody). Stimulation of ADP-ribosyl cyclase activity by acetylcholine was evaluated in permeabilized cells by measuring the production of cyclic guanosine diphosphoribose (a fluorescent compound), which resulted from the cyclization of nicotinamide guanine dinucleotide. As duodenum myocytes expressed the three subtypes of ryanodine receptors, an antisense strategy revealed that the ryanodine receptor subtype 2 alone was required to initiate the Ca2+ oscillations induced by acetylcholine and also by cyclic adenosine diphosphoribose and rapamycin (a compound that induced uncoupling between 12/12.6 kDa FK506-binding proteins and ryanodine receptors). Inhibition of cyclic adenosine diphosphoribose-induced Ca2+ oscillations, after rapamycin treatment, confirmed that both compounds interacted with the ryanodine receptor subtype 2. Our findings show for the first time that the M2 muscarinic receptor activation triggered Ca2+ oscillations in duodenum myocytes by activation of the cyclic adenosine diphosphoribose/FK506-binding protein/ryanodine receptor subtype 2 signalling pathway.

Specific Gq protein involvement in muscarinic M3 receptor‐induced phosphatidylinositol hydrolysis and Ca2+ release in mouse duodenal myocytes

Cytosolic Ca2+ concentration ([Ca2+]i) during exposure to acetylcholine or caffeine was measured in mouse duodenal myocytes loaded with fura‐2. Acetylcholine evoked a transient increase in [Ca2+]i followed by a sustained rise which was rapidly terminated after drug removal. Although L‐type Ca2+ currents participated in the global Ca2+ response induced by acetylcholine, the initial peak in [Ca2+]i was mainly due to release of Ca2+ from intracellular stores. Atropine, 4‐diphenylacetoxy‐N‐methylpiperidine (4‐DAMP, a muscarinic M3 antagonist), pirenzepine (a muscarinic M1 antagonist), methoctramine and gallamine (muscarinic M2 antagonists) inhibited the acetylcholine‐induced Ca2+ release, with a high affinity for 4‐DAMP and atropine and a low affinity for the other antagonists. Selective protection of muscarinic M2 receptors with methoctramine during 4‐DAMP mustard alkylation of muscarinic M3 receptors provided no evidence for muscarinic M2 receptor‐activated [Ca2+]i increase. Acetylcholine‐induced Ca2+ release was blocked by intracellular dialysis with a patch pipette containing either heparin or an anti‐phosphatidylinositol antibody and by external application of U73122 (a phospholipase C inhibitor). Acetylcholine‐induced Ca2+ release was insensitive to external pretreatment with pertussis toxin, but concentration‐dependently inhibited by intracellular dialysis with a patch pipette solution containing an anti‐αq/α11 antibody. An antisense oligonucleotide approach revealed that only the Gq protein was involved in acetylcholine‐induced Ca2+ release. Intracellular applications of either an anti‐βcom antibody or a peptide corresponding to the Gβγ binding domain of the β‐adrenoceptor kinase 1 had no effect on acetylcholine‐induced Ca2+ release. Our results show that, in mouse duodenal myocytes, acetylcholine‐induced release of Ca2+ from intracellular stores is mediated through activation of muscarinic M3 receptors which couple with a Gq protein to activate a phosphatidylinositol‐specific phospholipase C.

Identification and function of ryanodine receptor subtype 3 in non-pregnant mouse myometrial cells

Subtype 3 of the ryanodine receptor (RYR3) is a ubiquitous Ca2+ release channel which is predominantly expressed in smooth muscle tissues and certain regions of the brain. We show by reverse transcription‐polymerase chain reaction (RT‐PCR) that non‐pregnant mouse myometrial cells expressed only RYR3 and therefore could be a good model for studying the role of endogenous RYR3. Expression of RYR3 was confirmed by Western blotting and immunostaining. Confocal Ca2+ measurements revealed that in 1.7 mm extracellular Ca2+, neither caffeine nor photolysis of caged Ca2+ were able to trigger any Ca2+ responses, whereas in the same cells oxytocin activated propagated Ca2+ waves. However, under conditions of increased sarcoplasmic reticulum (SR) Ca2+ loading, brought about by superfusing myometrial cells in 10 mm extracellular Ca2+, all the myometrial cells responded to caffeine and photolysis of caged Ca2+, indicating that it was possible to activate RYR3. The caffeine‐induced Ca2+ responses were inhibited by intracellular application of an anti‐RYR3‐specific antibody. Immunodetection of RYR3 with the same antibody revealed a rather homogeneous distribution of fluorescence in confocal cell sections. In agreement with these observations, spontaneous or triggered Ca2+ sparks were not detected. In conclusion, our results suggest that under conditions of increased SR Ca2+ loading, endogenous RYR3 may contribute to the Ca2+ responses of myometrial cells.

Beta-3 adrenergic stimulation of L-Type Ca2+ channels in rat portal vein myocytes

The effects of β3‐adrenergic stimulation were studied on the L‐type Ca2+ channel in single myocytes from rat portal vein using the whole‐cell mode of the patch‐clamp technique. Reverse transcription‐polymerase chain reaction showed that β1‐, β2‐ and β3‐adrenoceptor subtypes were expressed in rat portal vein myocytes. Application of both propranolol (a non‐selective β1‐ and β2‐adrenoceptor antagonist) and SR59230A (a β3‐adrenoceptor antagonist) were needed to inhibit the isoprenaline‐induced increase in L‐type Ca2+ channel current. L‐type Ca2+ channels were stimulated by CGP12177A (a β3‐adrenoceptor agonist with potent β1‐ and β2‐adrenoceptor antagonist property) in a manner similar to that of isoprenaline. The CGP12177A‐induced stimulation of Ca2+ channel current was blocked by SR59230A, cyclic AMP‐dependent protein kinase inhibitors, H‐89 and Rp 8‐Br‐cyclic AMPs, but was unaffected by protein kinase C inhibitors, GF109203X and 19‐31 peptide. This stimulation was mimicked by forskolin and 8‐Br‐cyclic AMP. In the presence of okadaic acid (a phosphatase inhibitor), the β3‐adrenoceptor‐induced stimulation was maintained after withdrawal of the agonist. The β3‐adrenoceptor stimulation of L‐type Ca2+ channels was blocked by a pretreatment with cholera toxin and by the intracellular application of an anti‐Gαs antibody. This stimulation was unaffected by intracellular infusion of an anti‐Gβcom antibody and a βARK1 peptide. These results show that activation of β3‐adrenoceptors stimulates L‐type Ca2+ channels in vascular myocytes through a Gαs‐induced stimulation of the cyclic AMP/protein kinase A pathway and the subsequent phosphorylation of the channels.

Calcium signalling through nucleotide receptor P2X1 in rat portal vein myocytes

1 ATP‐mediated Ca2+ signalling was studied in freshly isolated rat portal vein myocytes by means of a laser confocal microscope and the patch‐clamp technique. 2 In vascular myocytes held at −60 mV, ATP induced a large inward current that was supported mainly by activation of P2X1 receptors, although other P2X receptor subtypes (P2X3, P2X4 and P2X5) were revealed by reverse transcription‐polymerase chain reaction. 3 Confocal Ca2+ measurements revealed that ATP‐mediated Ca2+ responses started at initiation sites where spontaneous or triggered Ca2+ sparks were not detected, whereas membrane depolarizations triggered Ca2+ waves by repetitive activation of Ca2+ sparks from a single initiation site. 4 ATP‐mediated Ca2+ responses depended on Ca2+ influx through non‐selective cation channels that activated, in turn, Ca2+ release from the intracellular store via ryanodine receptors (RYRs). Using specific antibodies directed against the RYR subtypes, we show that ATP‐mediated Ca2+ release requires, at least, RYR2, but not RYR3. 5 Our results suggest that, in vascular myocytes, Ca2+ influx through P2X1 receptors may trigger Ca2+‐induced Ca2+ release at intracellular sites where RYRs are not clustered.

Effect of a 14-day hindlimb suspension on cytosolic Ca2+ concentration in rat portal vein myocytes.

Effects of a 14-day hindlimb suspension were examined on increases in cytosolic Ca2+ concentration ([Ca2+]i) evoked by vasoactive compounds and on Ca2+ channels in rat portal vein myocytes. The maximal increases in [Ca2+]ielicited by caffeine, norepinephrine, and angiotensin II were reduced by 30-50% in suspended rats, and complete recovery was obtained 4 days after suspension removal. In contrast, voltage-gated Ca2+ channels were unaffected by hindlimb suspension. Using both confocal microscopy and the patch-clamp technique, we measured local increases in [Ca2+]iwhich corresponded to activation of a small number of ryanodine-sensitive Ca2+-release channels (Ca2+ sparks) andd- myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-gated Ca2+ channels. After hindlimb suspension, these local Ca2+events, as well as the Ca2+sensitivity of ryanodine-sensitive Ca2+ release channels, remained unchanged. In contrast, the propagated Ca2+ responses (Ca2+ waves) were significantly reduced in parallel with a noticeable inhibition of [3H]ryanodine binding to vascular membranes. Taken together, these results suggest that inhibition of the vasoconstrictor-induced increases in [Ca2+]iduring long-term suspension may be related to a reduction of the number of functional ryanodine-sensitive and Ins(1,4,5)P3-gated channels in the sarcoplasmic reticulum of rat portal vein myocytes.

Spaceflight regulates ryanodine receptor subtype 1 in portal vein myocytes in the opposite way of hypertension.

Gravity has a structural role for living systems. Tissue development, architecture, and organization are modified when the gravity vector is changed. In particular, microgravity induces a redistribution of blood volume and thus pressure in the astronaut body, abolishing an upright blood pressure gradient, inducing orthostatic hypotension. The present study was designed to investigate whether isolated vascular smooth muscle cells are directly sensitive to altered gravitational forces and, second, whether sustained blood pressure changes act on the same molecular target. Exposure to microgravity during 8 days in the International Space Station induced the decrease of ryanodine receptor subtype 1 expression in primary cultured myocytes from rat hepatic portal vein. Identical results were found in portal vein from mice exposed to microgravity during an 8-day shuttle spaceflight. To evaluate the functional consequences of this physiological adaptation, we have compared evoked calcium signals obtained in myocytes from hindlimb unloaded rats, in which the shift of blood pressure mimics the one produced by the microgravity, with those obtained in myocytes from rats injected with antisense oligonucleotide directed against ryanodine receptor subtype 1. In both conditions, calcium signals implicating calcium-induced calcium release were significantly decreased. In contrast, in spontaneous hypertensive rat, an increase in ryanodine receptor subtype 1 expression was observed as well as the calcium-induced calcium release mechanism. Taken together, our results shown that myocytes were directly sensitive to gravity level and that they adapt their calcium signaling pathways to pressure by the regulation of the ryanodine receptor subtype 1 expression.