Non-sense mutations in the dihydropyridine receptor beta1 gene, CACNB1, paralyze zebrafish relaxed mutants

Contractions by skeletal muscle require proper excitation-contraction (EC) coupling, whereby depolarization of the muscle membrane leads to an increase in cytosolic Ca(2+) and contraction. Changes in membrane voltage are detected by dihydropyridine receptors (DHPR) that directly interact with and activate ryanodine receptors to release Ca(2+) from the sarcoplasmic reticulum into the cytosol. A genetic screen for motility mutations isolated a new allele of the immotile zebrafish mutant relaxed. Muscles in relaxed embryos do not contract in response to potassium chloride (KCl) thus appear unresponsive to membrane depolarization, but do contract when stimulated by caffeine, an agonist of ryanodine receptors. This suggests that relaxed mutant muscles are defective in EC coupling. Indeed, immunohistochemical analysis demonstrated that mutants lack DHPRs in skeletal muscles. The mutant phenotype results from non-sense mutations in the zebrafish CACNB1 gene that encodes the DHPR beta1 subunit. The zebrafish CACNB1 gene is expressed in skeletal muscles, PNS and CNS. Electrophysiological recordings showed no obvious abnormalities in the motor output of relaxed mutants, presumably due to redundancy provided by other beta subunits. The structural and functional homology of CACNB1 in zebrafish and mammals, suggests that zebrafish can be useful for studying EC coupling and potential neuronal function of CACNB1.     

Developmental transition of touch response from slow muscle-mediated coilings to fast muscle-mediated burst swimming in zebrafish

It is well known that slow and fast muscles are used for long-term sustained movement and short bursts of activity, respectively, in adult animal behaviors. However, the contribution of the slow and fast muscles in early animal movement has not been thoroughly explored. In wild-type zebrafish embryos, tactile stimulation induces coilings consisting of 1–3 alternating contractions of the trunk and tail at 24 hours postfertilization (hpf) and burst swimming at 48 hpf. But, embryos defective in flightless I homolog (flii), which encodes for an actin-regulating protein, exhibit normal coilings at 24 hpf that is followed by significantly slower burst swimming at 48 hpf. Interestingly, actin fibers are disorganized in mutant fast muscle but not in mutant slow muscle, suggesting that slower swimming at 48 hpf is attributable to defects of the fast muscle tissue. In fact, perturbation of the fast muscle contractions by eliminating Ca²⁺ release only in fast muscle resulted in normal coilings at 24 hpf and slower burst swimming at 48 hpf, just as flii mutants exhibited. In contrast, specific inactivation of slow muscle by knockdown of the slow muscle myosin genes led to complete loss of coilings at 24 hpf, although normal burst swimming was retained by 48 hpf. These findings indicate that coilings at 24 hpf is mediated by slow muscle only, whereas burst swimming at 48 hpf is executed primarily by fast muscle. It is consistent with the fact that differentiation of fast muscle follows that of slow muscle. This is the first direct demonstration that slow and fast muscles have distinct physiologically relevant contribution in early motor development at different stages.     

accordion, a zebrafish behavioral mutant, has a muscle relaxation defect due to a mutation in the ATPase Ca2+ pump SERCA1

When wild-type zebrafish embryos are touched at 24 hours post-fertilization (hpf), they typically perform two rapid alternating coils of the tail. By contrast, accordion (acc) mutants fail to coil their tails normally but contract the bilateral trunk muscles simultaneously to shorten the trunk, resulting in a pronounced dorsal bend. Electrophysiological recordings from muscles showed that the output from the central nervous system is normal in mutants, suggesting a defect in muscles is responsible. In fact, relaxation in acc muscle is significantly slower than normal. In vivo imaging of muscle Ca2+ transients revealed that cytosolic Ca2+ decay was significantly slower in acc muscle. Thus, it appears that the mutant behavior is caused by a muscle relaxation defect due to the impairment of Ca2+ re-uptake. Indeed, acc mutants carry a mutation in atp2a1 gene that encodes the sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA1), a Ca2+ pump found in the muscle sarcoplasmic reticulum (SR) that is responsible for pumping Ca2+ from the cytosol back to the SR. As SERCA1 mutations in humans lead to Brody disease, an exercise-induced muscle relaxation disorder, zebrafish accordion mutants could be a useful animal model for this condition.     

Micromechanical function of myofibrils isolated from skeletal and cardiac muscles of the zebrafish

The zebrafish is a potentially important and cost-effective model for studies of development, motility, regeneration, and inherited human diseases. The object of our work was to show whether myofibrils isolated from zebrafish striated muscle represent a valid subcellular contractile model. These organelles, which determine contractile function in muscle, were used in a fast kinetic mechanical technique based on an atomic force probe and video microscopy. Mechanical variables measured included rate constants of force development (k(ACT)) after Ca(2+) activation and of force decay (τ(REL)(-1)) during relaxation upon Ca(2+) removal, isometric force at maximal (F(max)) or partial Ca(2+) activations, and force response to an external stretch applied to the relaxed myofibril (F(pass)). Myotomal myofibrils from larvae developed greater active and passive forces, and contracted and relaxed faster than skeletal myofibrils from adult zebrafish, indicating developmental changes in the contractile organelles of the myotomal muscles. Compared with murine cardiac myofibrils, measurements of adult zebrafish ventricular myofibrils show that k(ACT), F(max), Ca(2+) sensitivity of the force, and F(pass) were comparable and τ(REL)(-1) was smaller. These results suggest that cardiac myofibrils from zebrafish, like those from mice, are suitable contractile models to study cardiac function at the sarcomeric level. The results prove the practicability and usefulness of mechanical and kinetic investigations on myofibrils isolated from larval and adult zebrafish muscles. This novel approach for investigating myotomal and myocardial function in zebrafish at the subcellular level, combined with the powerful genetic manipulations that are possible in the zebrafish, will allow the investigation of the functional primary consequences of human disease-related mutations in sarcomeric proteins in the zebrafish model.     

ADP-ribose stimulates the calcium release channel RyR1 in skeletal muscle of rat

The Ca(2+) mobilizing metabolite cyclic ADP-ribose has been shown to release Ca(2+) from intracellular ryanodine sensitive stores in many cells. However, the activation of the ryanodine receptor of skeletal muscle by cADP-ribose (cADPr) and its precursor and metabolite (beta-NAD(+) and ADPr) remains to be discussed. We studied the effect of ADPr on the Ca(2+) release channel of skeletal muscle RyR1 after incorporation of microsomes isolated from fast muscles of rat in planar lipid bilayers. We observed an increase in the electrophysiological activity of the channel after addition of ADPr (10 microM) at micromolar Ca(2+) concentrations, characterized by a time-lag. The increase in P(o) is mainly due to an increase in the open frequency. The long time course observed for the development of the ADPr effect may indicate that this activation induces a change in the conformation of the RyR1 channel, which increases its sensitivity to calcium.     

Excitation-contraction coupling in airway smooth muscle

Excitation-contraction (EC) coupling in striated muscles is mediated by the cardiac or skeletal muscle isoform of voltage-dependent L-type Ca(2+) channel (Ca(v)1.2 and Ca(v)1.1, respectively) that senses a depolarization of the cell membrane, and in response, activates its corresponding isoform of intracellular Ca(2+) release channel/ryanodine receptor (RyR) to release stored Ca(2+), thereby initiating muscle contraction. Specifically, in cardiac muscle following cell membrane depolarization, Ca(v)1.2 activates cardiac RyR (RyR2) through an influx of extracellular Ca(2+). In contrast, in skeletal muscle, Ca(v)1.1 activates skeletal muscle RyR (RyR1) through a direct physical coupling that negates the need for extracellular Ca(2+). Since airway smooth muscle (ASM) expresses Ca(v)1.2 and all three RyR isoforms, we examined whether a cardiac muscle type of EC coupling also mediates contraction in this tissue. We found that the sustained contractions of rat ASM preparations induced by depolarization with KCl were indeed partially reversed ( approximately 40%) by 200 mum ryanodine, thus indicating a functional coupling of L-type channels and RyRs in ASM. However, KCl still caused transient ASM contractions and stored Ca(2+) release in cultured ASM cells without extracellular Ca(2+). Further analyses of rat ASM indicated that this tissue expresses as many as four L-type channel isoforms, including Ca(v)1.1. Moreover, Ca(v)1.1 and RyR1 in rat ASM cells have a similar distribution near the cell membrane in rat ASM cells and thus may be directly coupled as in skeletal muscle. Collectively, our data implicate that EC-coupling mechanisms in striated muscles may also broadly transduce diverse smooth muscle functions.     

Eugenol-induced contractions of saponin-skinned fibers are inhibited by heparin or by a ryanodine receptor blocker

The effects of eugenol on the sarcoplasmic reticulum (SR) and contractile apparatus of chemically skinned skeletal muscle fibers of the frog Rana catesbeiana were investigated. In saponin-skinned fibers, eugenol (5 mmol/L) induced muscle contractions, probably by releasing Ca(2+) from the SR. The Ca(2+)-induced Ca(2+) release blocker ruthenium red (10 micromol/L) inhibited both caffeine- and eugenol-induced muscle contractions. Ryanodine (200 micromol/L), a specific ryanodine receptor/Ca(2+) release channel blocker, promoted complete inhibition of the contractions induced by caffeine, but only partially blocked the contractions induced by eugenol. Heparin (2.5 mg/mL), an inositol 1,4,5-trisphosphate (InsP3) receptor blocker, strongly inhibited the contractions induced by eugenol but had only a small effect on the caffeine-induced contractions. Eugenol neither altered the Ca(2+) sensitivity nor the maximal force in Triton X-100 skinned muscle fibers. These data suggest that muscle contraction induced by eugenol involves at least 2 mechanisms of Ca(2+) release from the SR: one related to the activation of the ryanodine receptors and another through a heparin-sensitive pathway.     

Mutation of divergent region 1 alters caffeine and Ca(2+) sensitivity of the skeletal muscle Ca(2+) release channel (ryanodine receptor)

Replacement of amino acids 4187-4628 in the skeletal muscle Ca(2+) release channel (skeletal ryanodine receptor (RyR1)), including nearly all of divergent region 1 (amino acids 4254-4631), with the corresponding cardiac ryanodine receptor (RyR2) sequence leads to increased sensitivity of channel activation by caffeine and Ca(2+) and to decreased sensitivity of channel inactivation by elevated Ca(2+) (Du, G. G., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 26120-26126). In further investigations, this region was subdivided by the construction of new chimeras, and alterations in channel function were detected by measurement of the caffeine dependence of in vivo Ca(2+) release and the Ca(2+) dependence of [(3)H]ryanodine binding. Chimera RF10a (amino acids 4187-4381) had a lower EC(50) value for activation by caffeine, and RF10c (4557-4628) had a higher EC(50) value, whereas the EC(50) value for chimera RF10b (4382-4556) was unchanged. Chimeras RF10b and RF10c were more sensitive to activation by Ca(2+), whereas RF10a was less sensitive to inactivation by Ca(2+), implicating RF10b and RF10c in Ca(2+) activation and RF10a in Ca(2+) inactivation. Deletion of much of divergent region 1 sequence to create mutant Delta4274-4535 led to higher caffeine and Ca(2+) sensitivity of channel activation and to lower Ca(2+) sensitivity for inactivation. Thus, deletion results demonstrate that caffeine, Ca(2+), and ryanodine binding sites are not located in amino acids 4274-4535. Nevertheless, the properties of the deletion and chimeric mutants demonstrate that amino acids 4274-4535 and three shorter sequences in this region (F10a, amino acids 4187-4381; F10b, 4382-4556; and F10c, 4557-4628) in RyR1 modulate Ca(2+) and caffeine sensitivity of the Ca(2+) release channel.     

Single channel properties of heterotetrameric mutant RyR1 ion channels linked to core myopathies

Skeletal muscle excitation-contraction coupling involves activation of homotetrameric ryanodine receptor ion channels (RyR1s), resulting in the rapid release of Ca(2+) from the sarcoplasmic reticulum. Previous work has shown that Ca(2+) release is impaired by mutations in RyR1 linked to Central Core Disease and Multiple Minicore Disease. We studied the consequences of these mutations on RyR1 function, following their expression in human embryonic kidney 293 cells and incorporation in lipid bilayers. RyR1-G4898E, -G4898R, and -DeltaV4926/I4927 mutants in the C-terminal pore region of RyR1 and N-terminal RyR1-R110W/L486V mutant all showed negligible Ca(2+) permeation and loss of Ca(2+)-dependent channel activity but maintained reduced K(+) conductances. Co-expression of wild type and mutant RyR1s resulted in Ca(2+)-dependent channel activities that exhibited intermediate Ca(2+) selectivities compared with K(+), which suggested the presence of tetrameric RyR1 complexes composed of wild type and mutant subunits. The number of wild-type subunits to maintain a functional heterotetrameric channel differed among the four RyR1 mutants. The results indicate that homozygous RyR1 mutations associated with core myopathies abolish or greatly reduce sarcoplasmic reticulum Ca(2+) release during excitation-contraction coupling. They further suggest that in individuals, expressing wild type and mutant alleles, a substantial portion of RyR1 channels is able to release Ca(2+) from sarcoplasmic reticulum.     

Systemic ablation of RyR3 alters Ca2+ spark signaling in adult skeletal muscle

Ca2+ sparks are localized intracellular Ca2+ release events from the sarcoplasmic reticulum in muscle cells that result from synchronized opening of ryanodine receptors (RyR). In mammalian skeletal muscle, RyR1 is the predominant isoform present in adult skeletal fibers, while some RyR3 is expressed during development. Functional studies have revealed a differential role for RyR1 and RyR3 in the overall Ca2+ signaling in skeletal muscle, but the contribution of these two isoforms to Ca2+ sparks in adult mammalian skeletal muscle has not been fully examined. When enzyme-disassociated, individual adult skeletal muscle fibers are exposed to an osmotic shock, the resting fiber converts from a quiescent to a highly active Ca2+ release state where Ca2+ sparks appear proximal to the sarcolemmal membrane. These osmotic shock-induced Ca2+ sparks occur in ryr3(-/-) muscle with a spatial distribution similar to that seen in wild type muscle. Kinetic analysis reveals that systemic ablation of RyR3 results in significant changes to the initiation, duration and amplitude of individual Ca2+ sparks in muscle fibers. These changes may reflect the adaptation of the muscle Ca2+ signaling or contractile machinery due to the loss of RyR3 expression in distal tissues, as biochemical assays identify significant changes in expression of myosin heavy chain protein in ryr3(-/-) muscle.     

Comparative analysis of the isoform expression pattern of Ca(2+)-regulatory membrane proteins in fast-twitch, slow-twitch, cardiac, neonatal and chronic low-frequency stimulated muscle fibers

Although all muscle cells generate contractile forces by means of organized filament systems, isoform expression patterns of contractile and regulatory proteins in heart are not identical compared to developing, conditioned or mature skeletal muscles. In order to determine biochemical parameters that may reflect functional variations in the Ca(2+)-regulatory membrane systems of different muscle types, we performed a comparative immunoblot analysis of key membrane proteins involved in ion homeostasis. Cardiac isoforms of the alpha(1)-dihydropyridine receptor, Ca(2+)-ATPase and calsequestrin are also present in skeletal muscle and are up-regulated in chronic low-frequency stimulated fast muscle. In contrast, the cardiac RyR2 isoform of the Ca(2+)-release channel was not found in slow muscle but was detectable in neonatal skeletal muscle. Up-regulation of RyR2 in conditioned muscle was probably due to degeneration-regeneration processes. Fiber type-specific differences were also detected in the abundance of auxiliary subunits of the dihydropyridine receptor, the ryanodine receptor and the Ca(2+)-ATPase, as well as triad markers and various Ca(2+)-binding and ion-regulatory proteins. Hence, the variation in innervation of different types of muscle appears to have a profound influence on the levels and pattern of isoform expression of Ca(2+)-regulatory membrane proteins reflecting differences in the regulation of Ca(2+)-homeostasis. However, independent of the muscle cell type, key Ca(2+)-regulatory proteins exist as oligomeric complexes under native conditions.     

Ca2+ signaling in HEK-293 and skeletal muscle cells expressing recombinant ryanodine receptors harboring malignant hyperthermia and central core disease mutations

Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca(2+) release channel. Rabbit RyR1 mutant cDNAs carrying mutations corresponding to those in human RyR1 that cause MH and CCD were expressed in HEK-293 cells, which do not have endogenous RyR, and in primary cultures of rat skeletal muscle, which express rat RyR1. Analysis of intracellular Ca(2+) pools was performed using aequorin probes targeted to the lumen of the endo/sarcoplasmic reticulum (ER/SR), to the mitochondrial matrix, or to the cytosol. Mutations associated with MH caused alterations in intracellular Ca(2+) homeostasis different from those associated with CCD. Measurements of luminal ER/SR Ca(2+) revealed that the mutations generated leaky channels in all cases, but the leak was particularly pronounced in CCD mutants. Cytosolic and mitochondrial Ca(2+) transients induced by caffeine stimulation were drastically augmented in the MH mutant, slightly reduced in one CCD mutant (Y523S) and completely abolished in another (I4898T). The results suggest that local Ca(2+) derangements of different degrees account for the specific cellular phenotypes of the two disorders.     

A mutation in serca underlies motility dysfunction in accordion zebrafish

Zebrafish acquire the ability for fast swimming early in development. The motility mutant accordion (acc) undergoes exaggerated and prolonged contractions on both sides of the body, interfering with the acquisition of patterned swimming responses. Our whole cell recordings from muscle indicate that the defect is not manifested in neuromuscular transmission. However, imaging of skeletal muscle of larval acc reveals greatly prolonged calcium transients and associated contractions in response to depolarization. Positional cloning of acc identified a serca mutation as the cause of the acc phenotype. SERCA is a sarcoplasmic reticulum transmembrane protein in skeletal muscle that mediates calcium re-uptake from the myoplasm. The mutation in SERCA, a serine to phenylalanine substitution, is likely to result in compromised protein function that accounts for the observed phenotype. Indeed, direct evidence that mutant SERCA causes the motility dysfunction was provided by the finding that wild type fish injected with an antisense morpholino directed against serca, exhibited accordion-like contractions and impaired swimming. We conclude that the motility dysfunction in embryonic and larval accordion zebrafish stems directly from defective calcium transport in skeletal muscle rather than defective CNS drive.     

Pregnant rat myometrial cells show heterogeneous ryanodine- and caffeine-sensitive calcium stores

IntracellularCa²⁺ release channels such asryanodine receptors play crucial roles in theCa²⁺-mediated signaling thattriggers excitation-contraction coupling in muscles. Although theexistence and the role of these channels are well characterized inskeletal and cardiac muscles, their existence in smooth muscles, andmore particularly in the myometrium, is very controversial. We have nowclearly demonstrated the expression of ryanodine receptorCa²⁺ release channels in ratmyometrial smooth muscle, and for the first time, intracellularCa²⁺ concentration experimentswith indo 1 on single myometrial cells have revealed the existence of afunctional ryanodine- and caffeine-sensitive Ca²⁺ release mechanism in 30% ofrat myometrial cells. RT-PCR and RNase protection assay on wholemyometrial smooth muscle demonstrate the existence of all threeryr mRNAs in the myometrium:ryr3 mRNA is the predominant subtype,with much lower levels of expression forryr1 andryr2 mRNAs, suggesting that theryanodine Ca²⁺ release mechanismin rat myometrium is largely encoded byryr3. Moreover, using intracellularCa²⁺ concentration measurementsand RNase protection assays, we have demonstrated that the expression,the percentage of cells responding to ryanodine, and the function ofthese channels are not modified during pregnancy.

     

Role of amino-terminal half of the S4-S5 linker in type 1 ryanodine receptor (RyR1) channel gating

The type 1 ryanodine receptor (RyR1) is a Ca(2+) release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca(2+) itself, i.e. Ca(2+)-induced Ca(2+) release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr(4825)-Ser(4829)) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca(2+) release, single-channel current recordings, and [(3)H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca(2+) sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.     

The zebrafish ennui behavioral mutation disrupts acetylcholine receptor localization and motor axon stability

The zebrafish ennui mutation was identified from a mutagenesis screen for defects in early behavior. Homozygous ennui embryos swam more slowly than wild-type siblings but normal swimming recovered during larval stages and homozygous mutants survived until adulthood. Electrophysiological recordings from motoneurons and muscles suggested that the motor output of the CNS following mechanosensory stimulation was normal in ennui, but the synaptic currents at the neuromuscular junction were significantly reduced. Analysis of acetylcholine receptors (AChRs) in ennui muscles showed a marked reduction in the size of synaptic clusters and their aberrant localization at the myotome segment borders of fast twitch muscle. Prepatterned, nerve-independent AChR clusters appeared normal in mutant embryos and dispersed upon outgrowth of motor axons onto the muscles. Genetic mosaic analysis showed that ennui is required cell autonomously in muscle fibers for normal synaptic localization of AChRs. Furthermore, exogenous agrin failed to induce AChR aggregation, suggesting that ennui is crucial for agrin function. Finally, motor axons branched more extensively in ennui fast twitch muscles especially in the region of the myotome borders. These results suggest that ennui is important for nerve-dependent AChR clustering and the stability of axon growth.     

o-Phthalaldehyde activates the Ca(2+) release mechanism from skeletal muscle sarcoplasmic reticulum.

o-Phthalaldehyde (OPA) is a bifunctional reagent that forms an isoindole derivative by reacting with cysteine and lysine residues separated by approximately 0.3 nm. OPA inhibits sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity at low micromolar concentrations and induces Ca(2+) release from actively loaded SR vesicles by activating the ryanodine receptor from fast twitch skeletal muscle. Both ryanodine binding and single-channel activity show a biphasic concentration dependence. At low OPA concentrations (<100 microM), ryanodine binding and single channel activity are stimulated, while at higher concentrations, a time-dependent sequential activation and inhibition of receptor binding is observed. Activation is characterized by a Ca(2+)-independent increase in maximal receptor occupancy. Data are presented to support a model in which Ca(2+) channel and ryanodine binding activity are enhanced due to an intramolecular cross-linking of nearby lysine and nonhyperreactive cysteine residues. OPA complexation with endogenous lysine residue(s) is critical for receptor activation.     

Complex effects of ryanodine on the sarcoplasmic reticulum Ca2+ levels in smooth muscle cells

We have studied the effects of ryanodine and inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) with thapsigargin, on both [Ca(2+)](i) and the sarcoplasmic reticulum (SR) Ca(2+) level during caffeine-induced Ca(2+) release in single smooth muscle cells. Incubation with 10 microM ryanodine did not inhibit the first caffeine-induced [Ca(2+)](i) response, although it abolished the [Ca(2+)](i) response to a second application of caffeine. To assess whether ryanodine was inducing a permanent depletion of the internal Ca(2+) stores, we measured the SR Ca(2+) level with Mag-Fura-2. The magnitude of the caffeine-induced reduction in the SR Ca(2+) level was not augmented by incubating cells with 1 microM ryanodine. Moreover, on removal of caffeine, the SR Ca(2+) levels partially recovered in 61% of the cells due to the activity of thapsigargin-sensitive SERCA pumps. Unexpectedly, 10 microM ryanodine instead of inducing complete depletion of SR Ca(2+) stores markedly reduced the caffeine-induced SR Ca(2+) response. It was necessary to previously inhibit SERCA pumps with thapsigargin for ryanodine to be able to induce caffeine-triggered permanent depletion of SR Ca(2+) stores. These data suggest that the effect of ryanodine on smooth muscle SR Ca(2+) stores was markedly affected by the activity of SERCA pumps. Our data highlight the importance of directly measuring SR Ca(2+) levels to determine the effect of ryanodine on the internal Ca(2+) stores.     

Different roles of ryanodine receptors and inositol (1,4,5)-trisphosphate receptors in adrenergically stimulated contractions of small arteries

The functions of ryanodine receptors (RyRs) and inositol (1,4,5)-trisphosphate receptors [Ins(1,4,5)P(3)Rs] in adrenergically activated contractions of pressurized rat mesenteric small arteries were investigated. Caffeine (20 mM) but not phenylephrine (PE; 10 microM) facilitated the depletion of smooth muscle sarcoplasmic reticulum (SR) Ca(2+) stores by ryanodine (40 microM). In ryanodine-treated SR-depleted arteries, 1) Ca(2+) sparks were absent, 2) low concentrations of PE failed to elicit either vasoconstriction or normal asynchronous propagating Ca(2+) waves, and 3) high [PE] induced abnormally slow oscillatory contractions (vasomotion) and synchronous Ca(2+) oscillations. In ryanodine-treated SR-depleted arteries denuded of endothelium, high [PE] induced steady contraction and steady elevation of intracellular [Ca(2+)]. In contrast, 2-aminoethyl diphenylborate (2-APB), a putative blocker of Ins(1,4,5)P(3)Rs, produced opposite effects to ryanodine: 1) Ca(2+) sparks were present; 2) Ca(2+) waves were absent; 3) caffeine-releasable Ca(2+) stores were intact; and 4) PE, even at high concentrations on endothelial-denuded arteries, failed to elicit contraction, asynchronous Ca(2+) waves, or synchronous Ca(2+) oscillations or maintained elevated [Ca(2+)]. We conclude that 1) Ins(1,4,5)P(3)Rs are essential for adrenergically induced asynchronous Ca(2+) waves and the associated steady vasoconstriction, 2) RyRs are not appreciably opened during adrenergic activation (because PE did not facilitate the development of the effects of ryanodine), and 3) Ins(1,4,5)P(3)Rs are not essential for Ca(2+) sparks. This provides an explanation of the fact that adrenergic stimulation decreases the frequency of Ca(2+) sparks (previously reported) while simultaneously increasing the frequency of asynchronous propagating Ca(2+) waves; different SR Ca(2+)-release channels are involved.