Heterogeneous function of ryanodine receptors, but not IP3 receptors, in hamster cremaster muscle feed arteries and arterioles

The roles played by ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP?Rs) in vascular smooth muscle in the microcirculation remain unclear. Therefore, the function of both RyRs and IP?Rs in Ca(2+) signals and myogenic tone in hamster cremaster muscle feed arteries and downstream arterioles were assessed using confocal imaging and pressure myography. Feed artery vascular smooth muscle displayed Ca(2+) sparks and Ca(2+) waves, which were inhibited by the RyR antagonists ryanodine (10 μM) or tetracaine (100 μM). Despite the inhibition of sparks and waves, ryanodine or tetracaine increased global intracellular Ca(2+) and constricted the arteries. The blockade of IP?Rs with xestospongin D (5 μM) or 2-aminoethoxydiphenyl borate (100 μM) or the inhibition of phospholipase C using U-73122 (10 μM) also attenuated Ca(2+) waves without affecting Ca(2+) sparks. Importantly, the IP?Rs and phospholipase C antagonists decreased global intracellular Ca(2+) and dilated the arteries. In contrast, cremaster arterioles displayed only Ca(2+) waves: Ca(2+) sparks were not observed, and neither ryanodine (10-50 μM) nor tetracaine (100 μM) affected either Ca(2+) signals or arteriolar tone despite the presence of functional RyRs as assessed by responses to the RyR agonist caffeine (10 mM). As in feed arteries, arteriolar Ca(2+) waves were attenuated by xestospongin D (5 μM), 2-aminoethoxydiphenyl borate (100 μM), and U-73122 (10 μM), accompanied by decreased global intracellular Ca(2+) and vasodilation. These findings highlight the contrasting roles played by RyRs and IP?Rs in Ca(2+) signals and myogenic tone in feed arteries and demonstrate important differences in the function of RyRs between feed arteries and downstream arterioles.     

ET-1 activates Ca2+ sparks in PASMC: local Ca2+ signaling between inositol trisphosphate and ryanodine receptors

Ca+ sparks originating from ryanodine receptors (RyRs) are known to cause membrane hyperpolarization and vasorelaxation in systemic arterial myocytes. By contrast, we have found that Ca2+ sparks of pulmonary arterial smooth muscle cells (PASMCs) are associated with membrane depolarization and activated by endothelin-1 (ET-1), a potent vasoconstrictor that mediates/modulates acute and chronic hypoxic pulmonary vasoconstriction. In this study, we characterized the effects of ET-1 on the physical properties of Ca2+ sparks and probed the signal transduction mechanism for spark activation in rat intralobar PASMCs. Application of ET-1 at 0.1-10 nM caused concentration-dependent increases in frequency, duration, and amplitude of Ca2+ sparks. The ET-1-induced increase in spark frequency was inhibited by BQ-123, an ETA-receptor antagonist; by U-73122, a PLC inhibitor; and by xestospongin C and 2-aminoethyl diphenylborate, antagonists of inositol trisphosphate (IP3) receptors (IP3Rs). However, it was unrelated to sarcoplasmic reticulum Ca2+ content, activation of L-type Ca2+ channels, PKC, or cADP ribose. Photorelease of caged-IP3 indicated that Ca2+ release from IP3R could cross-activate RyRs to generate Ca2+ sparks. Immunocytochemistry showed that the distributions of IP3Rs and RyRs were similar in PASMCs. Moreover, inhibition of Ca2+ sparks with ryanodine caused a significant rightward shift in the ET-1 concentration-tension relationship in pulmonary arteries. These results suggest that ET-1 activation of Ca2+ sparks is mediated via the ETA receptor-PLC-IP3 pathway and local Ca2+ cross-signaling between IP3Rs and RyRs; in addition, this novel signaling mechanism contributes significantly to the ET-1-induced vasoconstriction in pulmonary arteries.     

Alkaline pH shifts Ca2+ sparks to Ca2+ waves in smooth muscle cells of pressurized cerebral arteries

The effects of external pH (7.0-8.0) on intracellular Ca(2+) signals (Ca(2+) sparks and Ca(2+) waves) were examined in smooth muscle cells from intact pressurized arteries from rats. Elevating the external pH from 7.4 to 7.5 increased the frequency of local, Ca(2+) transients, or "Ca(2+) sparks," and, at pH 7.6, significantly increased the frequency of Ca(2+) waves. Alkaline pH-induced Ca(2+) waves were inhibited by blocking Ca(2+) release from ryanodine receptors but were not prevented by inhibitors of voltage-dependent Ca(2+) channels, phospholipase C, or inositol 1,4,5-trisphosphate receptors. Activating ryanodine receptors with caffeine (5 mM) at pH 7.4 also induced repetitive Ca(2+) waves. Alkalization from pH 7.4 to pH 7.8-8.0 induced a rapid and large vasoconstriction. Approximately 82% of the alkaline pH-induced vasoconstriction was reversed by inhibitors of voltage-dependent Ca(2+) channels. The remaining constriction was reversed by inhibition of ryanodine receptors. These findings indicate that alkaline pH-induced Ca(2+) waves originate from ryanodine receptors and make a minor, direct contribution to alkaline pH-induced vasoconstriction.     

Termination of cardiac Ca(2+) sparks: an investigative mathematical model of calcium-induced calcium release

A Ca(2+) spark arises when a cluster of sarcoplasmic reticulum (SR) channels (ryanodine receptors or RyRs) opens to release calcium in a locally regenerative manner. Normally triggered by Ca(2+) influx across the sarcolemmal or transverse tubule membrane neighboring the cluster, the Ca(2+) spark has been shown to be the elementary Ca(2+) signaling event of excitation-contraction coupling in heart muscle. However, the question of how the Ca(2+) spark terminates remains a central, unresolved issue. Here we present a new model, "sticky cluster," of SR Ca(2+) release that simulates Ca(2+) spark behavior and enables robust Ca(2+) spark termination. Two newly documented features of RyR behavior have been incorporated in this otherwise simple model: "coupled gating" and an opening rate that depends on SR lumenal [Ca(2+)]. Using a Monte Carlo method, local Ca(2+)-induced Ca(2+) release from clusters containing between 10 and 100 RyRs is modeled. After release is triggered, Ca(2+) flux from RyRs diffuses into the cytosol and binds to intracellular buffers and the fluorescent Ca(2+) indicator fluo-3 to produce the model Ca(2+) spark. Ca(2+) sparks generated by the sticky cluster model resemble those observed experimentally, and Ca(2+) spark duration and amplitude are largely insensitive to the number of RyRs in a cluster. As expected from heart cell investigation, the spontaneous Ca(2+) spark rate in the model increases with elevated cytosolic or SR lumenal [Ca(2+)]. Furthermore, reduction of RyR coupling leads to prolonged model Ca(2+) sparks just as treatment with FK506 lengthens Ca(2+) sparks in heart cells. This new model of Ca(2+) spark behavior provides a "proof of principle" test of a new hypothesis for Ca(2+) spark termination and reproduces critical features of Ca(2+) sparks observed experimentally.     

Calcium waves in intact guinea pig gallbladder smooth muscle cells

Intracellular Ca(2+) waves and spontaneous transient depolarizations were investigated in gallbladder smooth muscle (GBSM) whole mount preparations with intact mucosal layer [full thickness (FT)] by laser confocal imaging of intracellular Ca(2+) and voltage recordings with microelectrodes, respectively. Spontaneous Ca(2+) waves arose most often near the center, but sometimes from the extremities, of GBSM cells. They propagated regeneratively by Ca(2+)-induced Ca(2+) release involving inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptors and were not affected by TTX and atropine (ATS). Spontaneous Ca(2+) waves and spontaneous transient depolarizations were more prevalent in FT than in isolated muscularis layer preparations and occurred with similar pattern in GBSM bundles. Ca(2+) waves were abolished by the Ins(1,4,5)P(3) receptor inhibitors 2-aminoethoxydiphenyl borate and xestospongin C and by caffeine and cyclopiazonic acid. These events were reduced by voltage-dependent calcium channels (VDCCs) inhibitors diltiazem and nifedipine, by PLC inhibitor U-73122, and by thapsigargin and ryanodine. ACh, CCK, and carbachol augmented Ca(2+) waves and induced Ca(2+) flashes. The actions of these agonists were inhibited by U-73122. These results indicate that in GBSM, discharge and propagation of Ca(2+) waves depend on sarco(endo)plasmic reticulum (SR) Ca(2+) release via Ins(1,4,5)P(3) receptors, PLC activity, Ca(2+) influx via VDCCs, and SR Ca(2+) concentration. Neurohormonal enhancement of GBSM excitability involves PLC-dependent augmentation and synchronization of SR Ca(2+) release via Ins(1,4,5)P(3) receptors. Ca(2+) waves likely reflect the activity of a fundamental unit of spontaneous activity and play an important role in the excitability of GBSM.     

Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry

Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.     

Inositol 1,4,5-trisphosphate and ryanodine receptors mobilize calcium from a common functional pool in human U373 MG cells

This investigation concentrates on the change in Ca(2+) concentration ([Ca(2+)]) caused by ryanodine in U373 MG cells. This cell type from a human astrocytoma is a unique cellular model because it only expresses the type 3 ryanodine receptor (RyR3), which is generally the least abundant isoform. In the presence of physiological [Ca(2+)] in the extracellular medium, U373 MG cells are caffeine-insensitive, even after forskolin treatment, and ryanodine-sensitive only when an unusually high concentration (30 microM) is applied. Xestospongin C behaves like thapsigargin and therefore cannot be used as a selective antagonist of inositol 1,4,5-trisphosphate receptors (InsP(3)Rs). After ryanodine challenge, addition of an analog of Substance P (SP), which should deplete InsP(3)-sensitive stores, has no effect on [Ca(2+)](i). After thapsigargin treatment, which unmasks the calcium leak from intracellular stores, neither ryanodine nor SP change [Ca(2+)](i), suggesting that thapsigargin completely depletes the ryanodine-sensitive and the InsP(3)-sensitive stores of U373 MG cells. Finally, in experiments monitoring the [Ca(2+)] in intracellular stores, InsP(3) stimulation of permeabilized cells causes a decrease in [Ca(2+)] that is not affected by subsequent ryanodine treatment. Our results support the conclusion that U373 MG cells express both InsP(3)Rs and RyRs that can individually or in combination mobilize only one functional Ca(2+) pool.     

Functional coupling between ryanodine receptors, mitochondria and Ca(2+) ATPases in rat submandibular acinar cells

Agonist stimulation of exocrine cells leads to the generation of intracellular Ca(2+) signals driven by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) that rapidly become global due to propagation throughout the cell. In many types of excitable cells the intracellular Ca(2+) signal is propagated by a mechanism of Ca(2+)-induced Ca(2+) release (CICR), mediated by ryanodine receptors (RyRs). Expression of RyRs in salivary gland cells has been demonstrated immunocytochemically although their functional role is not clear. We used microfluorimetry to measure Ca(2+) signals in the cytoplasm, in the endoplasmic reticulum (ER) and in mitochondria. In permeabilized acinar cells caffeine induced a dose-dependent, transient decrease of Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)). This decrease was inhibited by ryanodine but was insensitive to heparin. Application of caffeine, however, did not elevate cytosolic Ca(2+) concentration ([Ca(2+)](i)) suggesting fast local buffering of Ca(2+) released through RyRs. Indeed, activation of RyRs produced a robust mitochondrial Ca(2+) transient that was prevented by addition of Ca(2+) chelator BAPTA but not EGTA. When mitochondrial Ca(2+) uptake was blocked, activation of RyRs evoked only a non-transient increase in [Ca(2+)](i) and substantially smaller Ca(2+) release from the ER. Upon simultaneous inhibition of mitochondrial Ca(2+) uptake and either plasmalemmal or ER Ca(2+) ATPase, activation of RyRs caused a transient rise in [Ca(2+)](i). Collectively, our data suggest that Ca(2+) released through RyRs is mostly "tunnelled" to mitochondria, while Ca(2+) ATPases are responsible for the fast initial sequestration of Ca(2+). Ca(2+) uptake by mitochondria is critical for maintaining continuous CICR. A complex interplay between RyRs, mitochondria and Ca(2+) ATPases is accomplished through strategic positioning of mitochondria close to both Ca(2+) release sites in the ER and Ca(2+) pumping sites of the plasmalemma and the ER.     

Functionally separate intracellular Ca2+ stores in smooth muscle

In smooth muscle, release via the inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) controls oscillatory and steady-state cytosolic Ca(2+) concentrations ([Ca(2+)](c)). The interplay between the two receptors, itself determined by their organization on the SR, establishes the time course and spatial arrangement of the Ca(2+) signal. Whether or not the receptors are co-localized or distanced from each other on the same store or whether they exist on separate stores will significantly affect the Ca(2+) signal produced by the SR. To date these matters remain unresolved. The functional arrangement of the RyR and Ins(1,4,5)P(3)R on the SR has now been examined in isolated single voltage-clamped colonic myocytes. Depletion of the ryanodine-sensitive store, by repeated application of caffeine, in the presence of ryanodine, abolished the response to Ins(1,4,5)P(3), suggesting that Ins(1,4,5)P(3)R and RyR share a common Ca(2+) store. Ca(2+) release from the Ins(1,4,5)P(3)R did not activate Ca(2+)-induced Ca(2+) release at the RyR. Depletion of the Ins(1,4,5)P(3)-sensitive store, by the removal of external Ca(2+), on the other hand, caused only a small decrease ( approximately 26%) in caffeine-evoked Ca(2+) transients, suggesting that not all RyR exist on the common store shared with Ins(1,4,5)P(3)R. Dependence of the stores on external Ca(2+) for replenishment also differed; removal of external Ca(2+) depleted the Ins(1,4,5)P(3)-sensitive store but caused only a slight reduction in caffeine-evoked transients mediated at RyR. Different mechanisms are presumably responsible for the refilling of each store. Refilling of both Ins(1,4,5)P(3)-sensitive and caffeine-sensitive Ca(2+) stores was inhibited by each of the SR Ca(2+) ATPase inhibitors thapsigargin and cyclopiazonic acid. These results may be explained by the existence of two functionally distinct Ca(2+) stores; the first expressing only RyR and refilled from [Ca(2+)](c), the second expressing both Ins(1,4,5)P(3)R and RyR and dependent upon external Ca(2+) for refilling.     

Phosphorylation of inositol 1,4,5-trisphosphate receptors in parotid acinar cells. A mechanism for the synergistic effects of cAMP on Ca2+ signaling.

Acetylcholine-evoked secretion from the parotid gland is substantially potentiated by cAMP-raising agonists. A potential locus for the action of cAMP is the intracellular signaling pathway resulting in elevated cytosolic calcium levels ([Ca(2+)](i)). This hypothesis was tested in mouse parotid acinar cells. Forskolin dramatically potentiated the carbachol-evoked increase in [Ca(2+)](i), converted oscillatory [Ca(2+)](i) changes into a sustained [Ca(2+)](i) increase, and caused subthreshold concentrations of carbachol to increase [Ca(2+)](i) measurably. This potentiation was found to be independent of Ca(2+) entry and inositol 1,4,5-trisphosphate (InsP(3)) production, suggesting that cAMP-mediated effects on Ca(2+) release was the major underlying mechanism. Consistent with this hypothesis, dibutyryl cAMP dramatically potentiated InsP(3)-evoked Ca(2+) release from streptolysin-O-permeabilized cells. Furthermore, type II InsP(3) receptors (InsP(3)R) were shown to be directly phosphorylated by a protein kinase A (PKA)-mediated mechanism after treatment with forskolin. In contrast, no evidence was obtained to support direct PKA-mediated activation of ryanodine receptors (RyRs). However, inhibition of RyRs in intact cells, demonstrated a role for RyRs in propagating Ca(2+) oscillations and amplifying potentiated Ca(2+) release from InsP(3)Rs. These data indicate that potentiation of Ca(2+) release is primarily the result of PKA-mediated phosphorylation of InsP(3)Rs, and may largely explain the synergistic relationship between cAMP-raising agonists and acetylcholine-evoked secretion in the parotid. In addition, this report supports the emerging consensus that phosphorylation at the level of the Ca(2+) release machinery is a broadly important mechanism by which cells can regulate Ca(2+)-mediated processes.     

Low [Mg(2+)](o) induces contraction and [Ca(2+)](i) rises in cerebral arteries: roles of ca(2+), PKC, and PI3

Removal of extracellular Ca(2+) concentration ([Ca(2+)](o)) and pretreatment of canine basilar arterial rings with either an antagonist of voltage-gated Ca(2+) channels (verapamil), a selective antagonist of the sarcoplasmic reticulum Ca(2+) pump [thapsigargin (TSG)], caffeine plus a specific antagonist of ryanodine-sensitive Ca(2+) release (ryanodine), or a D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]- mediated Ca(2+) release antagonist (heparin) markedly attenuates low extracellular Mg(2+) concentration ([Mg(2+)](o))-induced contractions. Low [Mg(2+)](o)-induced contractions are significantly inhibited by pretreatment of the vessels with G?-6976 [a protein kinase C-alpha (PKC-alpha)- and PKC-betaI-selective antagonist], bisindolylmaleimide I (Bis, a specific antagonist of PKC), and wortmannin or LY-294002 [selective antagonists of phosphatidylinositol-3 kinases (PI3Ks)]. These antagonists were also found to relax arterial contractions induced by low [Mg(2+)](o) in a concentration-dependent manner. The absence of [Ca(2+)](o) and preincubation of the cells with verapamil, TSG, heparin, or caffeine plus ryanodine markedly attenuates the transient and sustained elevations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) induced by low-[Mg(2+)](o) medium. Low [Mg(2+)](o)-produced increases in [Ca(2+)](i) are also suppressed markedly in the presence of G?-6976, Bis, wortmannin, or LY-294002. The present study suggests that both Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+) release from intracellular stores [both Ins(1,4,5)P(3) sensitive and ryanodine sensitive] play important roles in low-[Mg(2+)](o) medium-induced contractions of isolated canine basilar arteries. Such contractions are clearly associated with activation of PKC isoforms and PI3Ks.     

Role of FKBP12.6 in hypoxia- and norepinephrine-induced Ca2+ release and contraction in pulmonary artery myocytes

The cellular and molecular processes underlying the regulation of ryanodine receptor (RyR) Ca(2+) release in smooth muscle cells (SMCs) are incompletely understood. Here we show that FKBP12.6 proteins are expressed in pulmonary artery (PA) smooth muscle and associated with type-2 RyRs (RyR2), but not RyR1, RyR3, or IP(3) receptors (IP(3)Rs) in PA sarcoplasmic reticulum. Application of FK506, which binds to FKBPs and dissociates these proteins from RyRs, induced an increase in [Ca(2+)](i) and Ca(2+)-activated Cl(-) and K(+) currents in freshly isolated PASMCs, whereas cyclosporin, an agent known to inhibit calcineurin but not to interact with FKBPs, failed to induce an increase in [Ca(2+)](i). FK506-induced [Ca(2+)](i) increase was completely blocked by the RyR antagonist ruthenium red and ryanodine, but not the IP(3)R antagonist heparin. Hypoxic Ca(2+) response and hypoxic vasoconstriction were significantly enhanced in FKBP12.6 knockout mouse PASMCs. FK506 or rapamycin pretreatment also enhanced hypoxic increase [Ca(2+)](i), but did not alter caffeine-induced Ca(2+) release (SR Ca(2+) content) in PASMCs. Norepinephrine-induced Ca(2+) release and force generation were also markedly enhanced in PASMCs from FKBP12.6 null mice. These findings suggest that FKBP12.6 plays an important role in hypoxia- and neurotransmitter-induced Ca(2+) and contractile responses by regulating the activity of RyRs in PASMCs.     

Bile acids induce Ca2+ release from both the endoplasmic reticulum and acidic intracellular calcium stores through activation of inositol trisphosphate receptors and ryanodine receptors

Gallstones can cause acute pancreatitis, an often fatal disease in which the pancreas digests itself. This is probably because of biliary reflux into the pancreatic duct and subsequent bile acid action on the acinar cells. Because Ca(2+) toxicity is important for the cellular damage in pancreatitis, we have studied the mechanisms by which the bile acid taurolithocholic acid 3-sulfate (TLC-S) liberates Ca(2+). Using two-photon plasma membrane permeabilization and measurement of [Ca(2+)] inside intracellular stores at the cell base (dominated by ER) and near the apex (dominated by secretory granules), we have characterized the Ca(2+) release pathways. Inhibition of inositol trisphosphate receptors (IP(3)Rs), by caffeine and 2-APB, reduced Ca(2+) release from both the ER and an acidic pool in the granular area. Inhibition of ryanodine receptors (RyRs) by ruthenium red (RR) also reduced TLC-S induced liberation from both stores. Combined inhibition of IP(3)Rs and RyRs abolished Ca(2+) release. RyR activation depends on receptors for nicotinic acid adenine dinucleotide phosphate (NAADP), because inactivation by a high NAADP concentration inhibited release from both stores, whereas a cyclic ADPR-ribose antagonist had no effect. Bile acid-elicited intracellular Ca(2+) liberation from both the ER and the apical acidic stores depends on both RyRs and IP(3)Rs.     

The initial inositol 1,4,5-trisphosphate response induced by histamine is strongly amplified by Ca(2+) release from internal stores in smooth muscle

We have studied the Ca(2+)-dependence and wortmannin-sensitivity of the initial inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) response induced by activation of either histamine or muscarinic receptors in smooth muscle from guinea pig urinary bladder. Activation of H(1) receptors with histamine (100 microM) produced a significant elevation in Ins(1,4,5)P(3) levels with only 5s stimulation and in the presence of external Ca(2+). However, this response was abolished fully by either the prolonged absence of external Ca(2+) or the depletion of internal Ca(2+) stores with thapsigargin (100nM) or ryanodine (10 microM). In contrast, the same conditions only slightly reduced the initial Ins(1,4,5)P(3) response induced by carbachol. The prolonged incubation of smooth muscle in 10 microM wortmannin to inhibit type III PI 4-kinase abolished both the early histamine-evoked Ins(1,4,5)P(3) and Ca(2+) responses. Conversely, wortmannin did not alter Ca(2+) release induced by carbachol, despite a partial reduction of its Ins(1,4,5)P(3) response. Collectively, these data indicate that the detectable histamine-induced increase in Ins(1,4,5)P(3) is more the consequence of Ca(2+) release from internal stores than a direct activation of phospholipase C by H(1) receptors. In addition, the effect of wortmannin implies the existence of a Ca(2+)-dependent amplification loop for the histamine-induced Ins(1,4,5)P(3) response in smooth muscle.     

Regulation of muscarinic cationic current in myocytes from guinea-pig ileum by intracellular Ca2+ release: a central role of inositol 1,4,5-trisphosphate receptors

The dynamics of carbachol (CCh)-induced [Ca(2+)](i) changes was related to the kinetics of muscarinic cationic current (mI(cat)) and the effect of Ca(2+) release through ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP(3)Rs) on mI(cat) was evaluated by fast x-y or line-scan confocal imaging of [Ca(2+)](i) combined with simultaneous recording of mI(cat) under whole-cell voltage clamp. When myocytes freshly isolated from the longitudinal layer of the guinea-pig ileum were loaded with the Ca(2+)-sensitive indicator fluo-3, x-y confocal imaging revealed CCh (10 microM)-induced Ca(2+) waves, which propagated from the cell ends towards the myocyte centre at 45.9 +/- 8.8 microms(-1) (n = 13). Initiation of the Ca(2+) wave preceded the appearance of any measurable mI(cat) by 229 +/- 55 ms (n = 7). Furthermore, CCh-induced [Ca(2+)](i) transients peaked 1.22 +/- 0.11s (n = 17) before mI(cat) reached peak amplitude. At -50 mV, spontaneous release of Ca(2+) through RyRs, resulting in Ca(2+) sparks, had no effect on CCh-induced mI(cat) but activated BK channels leading to spontaneous transient outward currents (STOCs). In addition, Ca(2+) release through RyRs induced by brief application of 5 mM caffeine was initiated at the cell centre but did not augment mI(cat) (n = 14). This was not due to an inhibitory effect of caffeine on muscarinic cationic channels (since application of 5 mM caffeine did not inhibit mI(cat) when [Ca(2+)](i) was strongly buffered with Ca(2+)/BAPTA buffer) nor was it due to an effect of caffeine on other mechanisms possibly involved in the regulation of Ca(2+) sensitivity of muscarinic cationic channels (since in the presence of 5 mM caffeine, photorelease of Ca(2+) upon cell dialysis with 5 mM NP-EGTA/3.8 mM Ca(2+) potentiated mI(cat) in the same way as in control). In contrast, IP(3)R-mediated Ca(2+) release upon flash photolysis of "caged" IP(3) (30 microM in the pipette solution) augmented mI(cat) (n = 15), even though [Ca(2+)](i) did not reach the level required for potentiation of mI(cat) during photorelease of Ca(2+) (n = 10). Intracellular calcium stores were visualised by loading of the myocytes with the low-affinity Ca(2+) indicator fluo-3FF AM and consisted of a superficial sarcoplasmic reticulum (SR) network and some perinuclear formation, which appeared to be continuous with the superficial SR. Immunostaining of the myocytes with antibodies to IP(3)R type 1 and to RyRs revealed that IP(3)Rs are predominant in the superficial SR while RyRs are confined to the central region of the cell. These results suggest that IP(3)R-mediated Ca(2+) release plays a central role in the modulation of mI(cat) in the guinea-pig ileum and that IP(3) may sensitise the regulatory mechanisms of the muscarinic cationic channels gating to Ca(2+).     

Biphasic modulation of ryanodine receptors by sulfhydryl oxidation in rat ventricular myocytes

To understand better the modulation of ryanodine receptors (RyRs) during oxidative stress, the effect of 4,4'-dithiodipyridine (DTDP), a cell-permeant and thiol-reactive oxidant, on global Ca(2+) signal and spontaneous Ca(2+) sparks of rat ventricular myocytes was investigated. It was shown that a brief Ca(2+) transient was elicited by DTDP, when its concentration was raised to 100 microM DTDP. In addition a dose-dependent increase of cytoplasmic free Zn(2+) concentration was induced by DTDP. An increase of the frequency of spontaneous Ca(2+) sparks appeared at 3 microM DTDP, whereas higher concentration of DTDP caused a biphasic change of the frequency in both intact and permeabilized myocytes. Consistent with the biphasic effect, caffeine-induced Ca(2+) transients were similarly affected. Because DTDP did not reduce the free Ca(2+) concentration in the sarcoplasmic reticulum lumen, it is likely that the effects of DTDP on the frequency and caffeine-induced Ca(2+) transients are due mainly to sulfhydryl oxidation-induced activation and subsequent inactivation of RyRs. Unlike the frequency, the spatio-temporal properties of Ca(2+) sparks were not influenced by DTDP. The finding that DTDP does not affect the duration of Ca(2+) sparks is inconsistent with that the DTDP-induced increase of the open time of reconstituted RyR channels. The mechanism underlying this discrepancy, especially the possible role of the interaction between arrayed RyRs in myocytes, is discussed. This study suggests that, even if oxidative stress is mild enough not to cause intracellular Ca(2+) accumulation, it may affect signaling pathways through directly modulating the RyR or its complex and in turn changing the frequency of spontaneous Ca(2+) sparks. Thus, the functional importance of moderate oxidative stress should not be overlooked.     

TRIC-A channels in vascular smooth muscle contribute to blood pressure maintenance

TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation channels postulated to mediate counter-ion movements facilitating physiological Ca(2+) release from internal stores. Tric-a-knockout mice developed hypertension during the daytime due to enhanced myogenic tone in resistance arteries. There are two Ca(2+) release mechanisms in vascular smooth muscle cells (VSMCs); incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization, while agonist-induced activation of inositol trisphosphate receptors (IP(3)Rs) evokes global Ca(2+) transients causing contraction. Tric-a gene ablation inhibited RyR-mediated hyperpolarization signaling to stimulate voltage-dependent Ca(2+) influx, and adversely enhanced IP(3)R-mediated Ca(2+) transients by overloading Ca(2+) stores in VSMCs. Moreover, association analysis identified single-nucleotide polymorphisms (SNPs) around the human TRIC-A gene that increase hypertension risk and restrict the efficiency of antihypertensive drugs. Therefore, TRIC-A channels contribute to maintaining blood pressure, while TRIC-A SNPs could provide biomarkers for constitutional diagnosis and personalized medical treatment of essential hypertension.     

Isolation, characterization, and localization of the inositol 1,4,5-trisphosphate receptor protein in Xenopus laevis oocytes.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) induces Ca2+ oscillations and waves in Xenopus laevis oocytes. Microsomes from oocytes exhibit high-affinity binding for Ins(1,4,5)P3, and demonstrate Ins(1,4,5)P3-induced Ca2+ release. The Ins(1,4,5)P3 receptor (InsP3R) was purified from oocyte microsomes as a large tetrameric complex and shown to have a monomer molecular mass of 256 kDa, compared with 273 kDa for the brain InsP3R. Binding to the oocyte receptor is highly specific for Ins(1,4,5)P3 and is inhibited by heparin (IC50, 2 micrograms/ml). Immunoblot analysis revealed that an antibody against the C-terminal sequence of the brain receptor recognized the oocyte receptor. These results, in addition to the difference in pattern obtained after limited proteolysis, suggest that the oocyte InsP3R is a new shorter isoform of the mammalian brain type I InsP3R. Immunofluorescence experiments indicated the presence of the InsP3R in the cortical layer and the perinuclear endoplasmic reticulum of the oocyte. However, immunological and biochemical experiments did not reveal the presence of the ryanodine receptor. The presence of an InsP3R and the absence of a ryanodine receptor support the importance of Ins(1,4,5)P3 in Ca2+ handling by oocytes and particularly in the induction of Ca2+ oscillations and waves.     

Characterization of elementary Ca2+ release signals in NGF-differentiated PC12 cells and hippocampal neurons

Elementary Ca2+ release signals in nerve growth factor- (NGF-) differentiated PC12 cells and hippocampal neurons, functionally analogous to the "Ca2+ sparks" and "Ca2+ puffs" identified in other cell types, were characterized by confocal microscopy. They either occurred spontaneously or could be activated by caffeine and metabotropic agonists. The release events were dissimilar to the sparks and puffs described so far, as many arose from clusters of both ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (InsP3Rs). Increasing either the stimulus strength or loading of the intracellular stores enhanced the frequency of and coupling between elementary release sites and evoked global Ca2+ signals. In the PC12 cells, the elementary Ca2+ release preferentially occurred around the branch points. Spatio-temporal recruitment of such elementary release events may regulate neuronal activities.