While the roles of subtypes 1 and 2 of the ryanodine receptors (RYRs) have been studied in cellular systems expressing specifically one or the other of these subtypes (i.e. skeletal and cardiac muscle), the function of these receptors has not been evaluated in smooth muscles. We have previously reported RYR-mediated elementary (Ca²⁺ sparks) and global Ca²⁺ responses in rat portal vein myocytes. Here, we investigated the respective roles of all three RYR subtypes expressed in these cells as revealed by reverse transcriptase-polymerase chain reaction. Antisense oligonucleotides targeting each one of the three RYR subtypes were shown to specifically inhibit the expression of the corresponding mRNA and protein without affecting the other RYR subtypes. Confocal Ca²⁺ measurements revealed that depolarization-induced Ca²⁺ sparks and global Ca²⁺ responses were blocked when either RYR1 or RYR2 expression was suppressed. Caffeine-induced Ca²⁺ responses were partly inhibited by the same antisense oligonucleotides. Neither the corresponding scrambled oligonucleotides nor the antisense oligonucleotides targeting RYR3 affected depolarization- or caffeine-induced Ca²⁺ responses. Our results show that, in vascular myocytes, the two RYR1 and RYR2 subtypes are required for Ca²⁺ release during Ca²⁺ sparks and global Ca²⁺ responses, evoked by activation of voltage-gated Ca²⁺ channels.

The subunit composition of angiotensin AT₁ receptor-activated G protein was identified by using antisense oligonucleotide injection into the nucleus of rat portal vein myocytes. In these cells, we have previously shown that increases in the cytoplasmic calcium concentration ([Ca²⁺]_i) induced by activation of angiotensin AT₁ receptors were dependent on extracellular Ca²⁺ entry by L-type Ca²⁺ channels and subsequent Ca²⁺-induced Ca²⁺ release from the intracellular stores. The angiotensin AT₁ receptor-activated increases in [Ca²⁺]_i were selectively inhibited by injection of antisense oligonucleotides directed against the mRNAs coding for the α₁₃, β₁, and γ₃ subunits. A correlating reduction in Gα₁₃, Gβ₁, and Gγ₃ protein expression was confirmed by immunocytochemistry. In addition, anti-α₁₃ antibody and synthetic peptide corresponding to the carboxyl terminus of the Gα₁₃ subunit inhibited, in a concentration-dependent manner, the angiotensin AT₁ receptor-mediated Ca²⁺ response. Reverse transcription-polymerase chain reaction analysis showed that only the angiotensin AT_1A receptor was expressed in rat portal vein smooth muscle. Furthermore, injection of anti-AT_1A oligonucleotides selectively inhibited the angiotensin II-induced increase in [Ca²⁺]_i. We conclude that the receptor-activated signal leading to increases in [Ca²⁺]_i is transduced by the heterotrimeric G₁₃ protein composed of α₁₃/β₁/γ₃ subunits and that the carboxyl terminus of the Gα₁₃ subunit interacts with the angiotensin AT_1A receptor.

A G protein composed of α₁₃, β₁, and γ₃ subunits selectively couples the angiotensin AT_1A receptors to increase cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in rat portal vein myocytes (Macrez-Leprêtre, N., Kalkbrenner, F., Morel, J. L., Schultz, G., and Mironneau, J. (1997) J. Biol. Chem. 272, 10095–10102). We show here that Gβγ transduces the signal leading to stimulation of L-type Ca²⁺channels. Intracellular dialysis through the patch pipette of a carboxyl-terminal anti-β_com antibody and a peptide corresponding to the Gβγ binding region of the β-adrenergic receptor kinase 1 inhibited the stimulation of Ca²⁺channels and the increase in [Ca²⁺]_i evoked by angiotensin II. The Gβγ binding peptide did not prevent the dissociation of the heterotrimeric G protein into its subunits, as it did not block activation of phospholipase C-β by Gα_q in response to stimulation of α₁-adrenoreceptors. Transient overexpression of the β-adrenergic receptor kinase 1 fragment and of Gα subunits also inhibited the angiotensin II-induced increase in [Ca²⁺]_i. Both anti-α₁₃ antibody and carboxyl-terminal α₁₃ peptide abrogated the angiotensin II-induced stimulation of Ca²⁺ channels. We conclude that activation of angiotensin AT₁ receptors requires all three α, β, and γ subunits of G₁₃ for receptor-G protein interaction, whereas the transduction of the signal to L-type Ca²⁺ channels is mediated by Gβγ.

Modifications of gravity levels induce generalized adaptation of mammalian physiology, including vascular, brain, muscle, bone and immunity functions. As a crucial interface between the vascular system and the brain, the blood–brain barrier (BBB) acts as a filter to protect neurons from pathogens and inflammation. Here we compare the effects of several protocols of hypergravity induced by centrifugation and whole-body vibrations (WBV) on BBB integrity. The immunohistochemistry revealed immunoglobulin G (IgG) extravasation from blood to hippocampal parenchyma of mice centrifuged at 2 × g during 1 or 50 days, whereas short exposures to higher hypergravity mimicking the profiles of spaceflight landing and take-off (short exposures to 5 × g) had no effects. These results suggest prolonged centrifugation (>1 days) at 2 × g induced a BBB leakage. Moreover, WBV were similarly tested. The short exposure to +2 × g vibrations (900 s/day at 90 Hz) repeated for 63 days induced IgG extravasation in hippocampal parenchyma, whereas the progressive increase of vibrations from +0.5 to +2 × g for 63 days was not able to affect the IgG crossing through the BBB. Overall, these results suggest that the BBB permeability is sensitive to prolonged external accelerations. In conclusion, we advise that the protocols of WBV and centrifugation, proposed as countermeasure to spaceflight, should be designed with progressively increasing exposure to reduce potential side effects on the BBB.

Objective— The aim of this study was to correlate the expression of InsP ₃ R subtypes in native vascular and visceral myocytes with specific Ca ²⁺ -signaling patterns. Methods and Results— By Western blot and immunostaining, we showed that rat portal vein expressed InsP ₃ R1 and InsP ₃ R2 but not InsP ₃ R3, whereas rat ureter expressed InsP ₃ R1 and InsP ₃ R3 but not InsP ₃ R2. Acetylcholine induced single Ca ²⁺ responses in all ureteric myocytes but only in 50% of vascular myocytes. In the remaining vascular myocytes, the first transient peak was followed by Ca ²⁺ oscillations. By correlating Ca ²⁺ signals and immunostaining, we revealed that oscillating vascular cells expressed both InsP ₃ R1 and InsP ₃ R2 whereas nonoscillating vascular cells expressed only InsP ₃ R1. Acetylcholine-induced oscillations were not affected by inhibitors of ryanodine receptors, Ca ²⁺ -ATPases, Ca ²⁺ influx, and mitochondrial Ca ²⁺ uniporter but were inhibited by intracellular infusion of heparin. Using specific antibodies against InsP ₃ R subtypes, we showed that acetylcholine-induced Ca ²⁺ oscillations were specifically blocked by the anti-InsP ₃ R antibody. These data were supported by antisense oligonucleotides targeting InsP ₃ R2, which selectively inhibited Ca ²⁺ oscillations. Conclusions— Our results suggest that in native smooth muscle cells, a differential expression of InsP ₃ R subtypes encodes specific InsP ₃ -mediated Ca ²⁺ responses and that the presence of the InsP ₃ R2 subtype is required for acetylcholine-induced Ca ²⁺ oscillations in vascular 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.