Nathalie Macrez

Gβγ dimers stimulate vascular L-type Ca2+ channels via phosphoinositide 3-kinase

We have previously reported that, in venous myocytes, G²γ scavengers inhibit angiotensin AT1A receptor‐induced stimulation of L‐type Ca2+ channels (1). Here, we demonstrate that intracellular infusion of purified G²γ complexes stimulates the L‐type Ca2+ channel current in a concentration‐dependent manner. Additional intracellular dialysis of GDP‐bound inactive Gαo or of a peptide corresponding to the G²γ binding region of the ²‐adrenergic receptor kinase completely inhibited the G²γ‐induced stimulation of Ca2+ channel currents. The gating properties of the channel were not affected by intracellular application of G²γ, suggesting that G²γ increased the whole‐cell calcium conductance. In addition, both the angiotensin AT1A receptor‐ and the G²γ‐induced stimulation of L‐type Ca2+ channels were blocked by pretreatment of the cells with wortmannin, at nanomolar concentrations. Correspondingly, intracellular infusion of an enzymatically active purified recombinant G²γ‐sensitive phosphoinositide 3‐kinase, PI3Kγ, mimicked G²γ‐induced stimulation of Ca2+ channels. Both G²γ‐ and PI3Kγ‐induced stimulations of Ca2+ channel currents were reduced by protein kinase C inhibitors suggesting that the G²γ/PI3Kγ‐activated transduction pathway involves a protein kinase C. These results indicate for the first time that G²γ dimers stimulate the vascular L‐type Ca2+ channels through a G²γ‐sensitive PI3K.— Viard, P., Exner, T., Maier, U., Mironneau, J., Nürnberg, B., Macrez, N. G²γ dimers stimulate vascular L‐type Ca2+ channels via phosphoinositide 3‐kinase γ. FASEB J. 13, 685–694 (1999)

Regulation of Vascular L-type Ca2+ Channels by Phosphatidylinositol 3,4,5-Trisphosphate

Modulation of voltage-gated L-type Ca2+ channels by phosphoinositide 3-kinase (PI3K) regulates Ca2+ entry and plays a crucial role in vascular excitation–contraction coupling. Angiotensin II (Ang II) activates Ca2+ entry by stimulating L-type Ca2+ channels through G&bgr;&ggr;-sensitive PI3K&ggr; in portal vein myocytes. Moreover, PI3K and Ca2+ entry activation have been reported to be necessary for receptor tyrosine kinase-coupled and G protein-coupled receptor-induced DNA synthesis in vascular cells. We have previously shown that tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks are able to modulate vascular L-type Ca2+ channels. PI3Ks display 2 enzymatic activities: a lipid-kinase activity leading to the formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3 or PIP3] and a serine-kinase activity. Here we show that exogenous PIP3 applied into the cell through the patch pipette is able to reproduce the Ca2+ channel-stimulating effect of Ang II and PI3Ks. Moreover, the Ang II–induced PI3K-mediated stimulation of Ca2+ channel and the resulting increase in cytosolic Ca2+ concentration are blocked by the anti-PIP3 antibody. Mutants of PI3K&ggr; transfected into vascular myocytes also revealed the essential role of the lipid-kinase activity of PI3K&ggr; in Ang II–induced Ca2+ responses. These results suggest that PIP3 is necessary and sufficient to activate a Ca2+ influx in vascular myocytes stimulated by Ang II.

Inositol 1,4,5-trisphosphatem and ryanodinemsensitive Ca2+ release channel-dependent Ca2+ signalling in rat portal vein myocytes

Ca2+ signalling events were analyzed in single myocytes from rat portal vein by using a laser confocal microscope combined with the patch-clamp technique. Increase in inositol 1,4,5-trisphosphate (InsP3) concentration was obtained by photorelease from a caged precursor or intracellular dialysis of 3F-InsP3. Low InsP3 concentrations activated either small elevations of [Ca2+]i or localized Ca2+ transients whereas high InsP3 concentrations activated either homogeneous Ca2+ responses or propagated Ca2+ waves. The InsP3-evoked localized Ca2+ transients had spatio-temporal properties characteristic of Ca2+ sparks. In addition, compounds that blocked Ca2+ sparks and Ca2+ responses activated by Ca2+ jumps reduced the global InsP3-activated Ca2+ responses and suppressed the Ca2+ transients. In contrast, Ca2+ responses evoked by flash-photolytic Ca2+ jumps or caffeine were not affected by heparin (an InsP3 receptor antagonist). These results suggest that the absence of elementary Ca2+ events evoked by InsP3 may be related to the lack of clustered InsP3 receptor units in these cells, as confirmed by immunocytochemistry. Cooperativity between InsP3- and ryanodine-sensitive Ca2+ channels may represent a novel mechanism to amplify Ca2+ release from the same intracellular store and give rise to propagated Ca2+ waves.

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.

T‐type Cav3.3 calcium channels produce spontaneous low‐threshold action potentials and intracellular calcium oscillations

The precise contribution of T‐type Ca2+ channels in generating action potentials (APs), burst firing and intracellular Ca2+ signals needs further elucidation. Here, we show that Cav3.3 channels can trigger repetitive APs, generating spontaneous membrane potential oscillations (MPOs), and a concomitant increase in the intracellular Ca2+ concentration ([Ca2+]i) when overexpressed in NG108‐15 cells. MPOs were dependent on Cav3.3 channel activity given that they were recorded from a potential range of −55 to −70 mV, blocked by nickel and mibefradil, as well as by low external Ca2+ concentration. APs of distinct duration were recorded: short APs (sAP) or prolonged APs (pAP) with a plateau potential near −40 mV. The voltage‐dependent properties of the Cav3.3 channels constrained the AP duration and the plateau potential was supported by sustained calcium current through Cav3.3 channels. The sustained current amplitude decreased when the resting holding potential was depolarized, thereby inducing a switch of AP shape from pAP to sAP. Duration of the [Ca2+]i oscillations was also closely related to the shape of APs. The Cav3.3 window current was the oscillation trigger as shown by shifting the Cav3.3 window current potential range as a result of increasing the external Ca2+ concentration, which resulted in a corresponding shift of the AP threshold. Overall, the data demonstrate that the Cav3.3 window current is critical in triggering intrinsic electrical and [Ca2+]i oscillations. The functional expression of Cav3.3 channels can generate spontaneous low‐threshold calcium APs through its window current, indicating that Cav3.3 channels can play a primary role in pacemaker activity.

L-type and Ca2+ release channel-dependent hierarchical Ca2+ signalling in rat portal vein myocytes

Ca2+ signalling events and whole-cell Ca2+ currents were analyzed in single myocytes from rat portal vein by using a laser scanning confocal microscope combined with the patch-clamp technique. In myocytes in which the intracellular Ca2+ store was depleted or Ca2+ release channels were blocked by 10 microM ryanodine, inward Ca2+ currents induced slow and sustained elevations of [Ca2+]i. These Ca2+ responses were suppressed by 1 microM oxodipine and by depolarizations to +120 mV, a potential close to the reversal potential for Ca2+ ions, suggesting that they reflected Ca2+ influx through L-type Ca2+ channels. With functioning intracellular Ca2+ stores, flash photolysis of caged Ca2+ gave rise to a small increase in [Ca2+]i with superimposed Ca2+ sparks, reflecting the opening of clustered Ca2+ release channels. Brief Ca2+ currents in the voltage range from -30 to +10 mV triggered Ca2+ sparks or macrosparks that did not propagate in the entire line-scan image. Increasing the duration of Ca2+ current for 100 ms or more allowed the trigger of propagating Ca2+ waves which originated from the same initiation sites as the caffeine-activated response. Both Ca2+ sparks and initiation sites of Ca2+ waves activated by Ca2+ currents were observed in the vicinity of areas that excluded the Ca2+ probes, reflecting infoldings of the plasma membrane close to the sarcoplasmic reticulum, as revealed by fluorescent markers. The hierarchy of Ca2+ signalling events, from submicroscopic fundamental events to elementary events (sparks) and propagated waves, provides an integrated mechanism to regulate vascular tone.

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.