Ry Receptors Research Articles

Fluorescent probes for insect ryanodine receptors: candidate anthranilic diamides.

Diamide insecticides with high efficacy against pests and good environmental safety are broadly applied in crop protection. They act at a poorly-defined site in the very complex ryanodine (Ry) receptor (RyR) potentially accessible to a fluorescent probe. Two N-propynyl analogs of the major anthranilic diamide insecticides chlorantraniliprole (Chlo) and cyantraniliprole (Cyan) were accordingly synthesized and converted into two fluorescent ligands by click reaction coupling with 3-azido-7-hydroxy-2H-chromen-2-one. The new diamide analogs and fluorescent ligands were shown to be nearly as potent as Chlo and Cyan in inhibition of [3H]Chlo binding and stimulation of [3H]Ry binding in house fly thoracic muscle RyR. Although the newly synthesized compounds had only moderate activity in insect larvicidal activity assays, their high in vitro potency in a validated insect RyR binding assay encourages further development of fluorescent probes for insect RyRs.

Species differences in chlorantraniliprole and flubendiamide insecticide binding sites in the ryanodine receptor

Anthranilic and phthalic diamides exemplified by chlorantraniliprole (Chlo) or cyantraniliprole (Cyan) and flubendiamide (Flu), respectively, are the newest major chemotype of insecticides with outstanding potency, little or no cross resistance with other classes and low mammalian toxicity. They are activators of the ryanodine (Ry) receptor (RyR)-Ca2+ channel, based on Ca2+ flux and electrophysiology investigations. The goal of this study is to define species differences in the degree and mechanisms of diamide selective action by radioligand specific binding studies at the [3H]Ry, [3H]Chlo and [3H]Flu sites. The [3H]Ry site is observed in muscle of lobster, rabbit and four insect species (Musca domestica, Apis mellifera, Heliothis virescens and Agrotis ipsilon) whereas the [3H]Chlo site is evident in the four insects and the [3H]Flu site in only the two lepidoptera (Agrotis and Heliothis). [3H]Ry binding is significantly stimulated by Chlo, Cyan and Flu with the insects (except Flu with Musca) but not the lobster and rabbit. [3H]Chlo binding is stimulated by Ry and Flu in Musca and Apis but not in the lepidoptera, while Flu and Cyan are inhibitory. [3H]Flu binding is strongly inhibited by Chlo and Cyan in Agrotis and Heliothis. [3H]Chlo and [3H]Flu binding are not dependent on added Ca2+ or ATP in Heliothis and Agrotis whereas the other radioligand-receptor combinations are usually enhanced by Ca2+ and ATP. More generally, there are species differences in the Ry, Chlo and Flu binding sites of the RyR that may confer selective toxicity and determine target site cross resistance mechanisms.

Functional and Biochemical Properties of Ryanodine Receptor Type 1 Channels from Heterozygous R163C Malignant Hyperthermia-Susceptible Mice

Mutations in ryanodine receptor type 1 (RyR1) confer malignant hyperthermia susceptibility. How inherent impairments in Ca(2+) channel regulation affect skeletal muscle function in myotubes and adult fibers under basal (nontriggering) conditions are not understood. Myotubes, adult flexor digitorum brevis (FDB) fibers, and sarcoplasmic reticulum skeletal membranes were isolated from heterozygous knockin R163C and wild-type (WT) mice. Compared with WT myotubules, R163C myotubes have reduced Ca(2+) transient amplitudes in response to electrical field pulses; however, R163C FDB fibers do not differ in their responses to electrical stimuli, despite heightened cellular cytoplasmic resting Ca(2+) ([Ca(2+)](rest)) and sensitivity to halothane. Immunoblotting of membranes from each genotype shows similar expression of RyR1, FK506 binding protein 12 kDa, and Ca(2+)-ATPase, but RyR1 (2844)Ser phosphorylation in R163C muscle is 31% higher than that of WT muscle (p < 0.001). RyR1 channels reconstituted in planar lipid bilayers reveal ∼65% of R163C channels exhibit ≥2-fold greater open probability (P(o)) than WT, with prolonged mean open dwell times and shortened closed dwell times. [(3)H]Ryanodine (Ry) binding and single-channel analyses show that R163C-RyR1 has altered regulation compared with WT: 1) 3-fold higher sensitivity to Ca(2+) activation; 2) 2-fold greater [(3)H]Ry receptor occupancy; 3) comparatively higher channel activity, even in reducing glutathione buffer; 4) enhanced RyR1 activity both at 25 and 37°C; and 5) elevated cytoplasmic [Ca(2+)](rest). R163C channels are inherently more active than WT channels, a functional impairment that cannot be reversed by dephosphorylation with protein phosphatase. Dysregulated R163C channels produce a more overt phenotype in myotubes than in adult fibers in the absence of triggering agents, suggesting tighter negative regulation of R163C-RyR1 within the Ca(2+) release unit of adult fibers.

Ca2+ handling is altered when arterial myocytes progress from a contractile to a proliferative phenotype in culture

Phenotypic modulation of vascular myocytes is important for vascular development and adaptation. A characteristic feature of this process is alteration in intracellular Ca(2+) handling, which is not completely understood. We studied mechanisms involved in functional changes of inositol 1,4,5-trisphosphate (IP(3))- and ryanodine (Ry)-sensitive Ca(2+) stores, store-operated Ca(2+) entry (SOCE), and receptor-operated Ca(2+) entry (ROCE) associated with arterial myocyte modulation from a contractile to a proliferative phenotype in culture. Proliferating, cultured myocytes from rat mesenteric artery have elevated resting cytosolic Ca(2+) levels and increased IP(3)-sensitive Ca(2+) store content. ATP- and cyclopiazonic acid [CPA; a sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor]-induced Ca(2+) transients in Ca(2+)-free medium are significantly larger in proliferating arterial smooth muscle cells (ASMCs) than in freshly dissociated myocytes, whereas caffeine (Caf)-induced Ca(2+) release is much smaller. Moreover, the Caf/Ry-sensitive store gradually loses sensitivity to Caf activation during cell culture. These changes can be explained by increased expression of all three IP(3) receptors and a switch from Ry receptor type II to type III expression during proliferation. SOCE, activated by depletion of the IP(3)/CPA-sensitive store, is greatly increased in proliferating ASMCs. Augmented SOCE and ROCE (activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol) in proliferating myocytes can be attributed to upregulated expression of, respectively, transient receptor potential proteins TRPC1/4/5 and TRPC3/6. Moreover, stromal interacting molecule 1 (STIM1) and Orai proteins are upregulated in proliferating cells. Increased expression of IP(3) receptors, SERCA2b, TRPCs, Orai(s), and STIM1 in proliferating ASMCs suggests that these proteins play a critical role in an altered Ca(2+) handling that occurs during vascular growth and remodeling.

Store-operated Ca2+ Influx Causes Ca2+ Release from the Intracellular Ca2+ Channels That Is Required for T Cell Activation

The precise control of many T cell functions relies on cytosolic Ca(2+) dynamics that is shaped by the Ca(2+) release from the intracellular store and extracellular Ca(2+) influx. The Ca(2+) influx activated following T cell receptor (TCR)-mediated store depletion is considered to be a major mechanism for sustained elevation in cytosolic Ca(2+) concentration ([Ca(2+)](i)) necessary for T cell activation, whereas the role of intracellular Ca(2+) release channels is believed to be minor. We found, however, that in Jurkat T cells [Ca(2+)](i) elevation observed upon activation of the store-operated Ca(2+) entry (SOCE) by passive store depletion with cyclopiazonic acid, a reversible blocker of sarco-endoplasmic reticulum Ca(2+)-ATPase, inversely correlated with store refilling. This indicated that intracellular Ca(2+) release channels were activated in parallel with SOCE and contributed to global [Ca(2+)](i) elevation. Pretreating cells with (-)-xestospongin C (10 microM) or ryanodine (400 microM), the antagonists of inositol 1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR), respectively, facilitated store refilling and significantly reduced [Ca(2+)](i) elevation evoked by the passive store depletion or TCR ligation. Although the Ca(2+) release from the IP3R can be activated by TCR stimulation, the Ca(2+) release from the RyR was not inducible via TCR engagement and was exclusively activated by the SOCE. We also established that inhibition of IP3R or RyR down-regulated T cell proliferation and T-cell growth factor interleukin 2 production. These studies revealed a new aspect of [Ca(2+)](i) signaling in T cells, that is SOCE-dependent Ca(2+) release via IP3R and/or RyR, and identified the IP3R and RyR as potential targets for manipulation of Ca(2+)-dependent functions of T lymphocytes.

The swimming performance of brown trout and whitefish: the effects of exercise on Ca2 + handling and oxidative capacity of swimming muscles

The swimming performance of two fish species, the brown trout and whitefish, having initially different swimming strategies, was measured after nine different training programs in order to relate the effects of exercise on Ca(2+) handling and oxidative capacity of swimming muscles. The time to 50% fatigue was measured during the training period, and compared with the density of dihydropyridine (DHP) and ryanodine (Ry) receptors and succinate dehydrogenase (SDH) and phosphorylase activity determined by histochemical analysis of the swimming muscles. Overall, both trained brown trout and whitefish had superior swimming performance as compared to control ones. Interestingly, the training programs had different effect on the two species studied since brown trout achieved the highest swimming performance, swimming against the water flow velocity of 2 BL s(-1) while among whitefish the best efficiency was seen after training with lower swimming velocities. Training also induced a significant increase in DHP and Ry receptor density in both species. Generally, in brown trout the most notable increase in the receptor densities was observed in red muscle sections from the fish swimming for 6 weeks against water currents of 1 BL s(-1) (DHPR 176.5 +/- 7.7% and RyR 231.4 +/- 11.8%) and white muscle sections against 2 BL s(-1) (DHPR 129.6 +/- 12.4% and RyR 161.9 +/- 15.5%). In whitefish the most prominent alterations were noted in samples from both muscle types after 6 weeks of training against water current of 1.5 BL s(-1) (DHPR 167.1 +/- 16.9% and RyR 190.4 +/- 19.4%). Finally, after all the training regimens the activity of SDH increased but the phosphorylase activity decreased significantly in both the species. To conclude, our findings demonstrate an improved swimming performance and enhanced Ca(2+) regulation and oxidative capacity after training. Moreover, there seems to be a connection between the swimming performance and receptor levels, especially in white swimming muscles of different fish species, regardless of their initially deviant swimming behaviours. However, depending on the training regimen the divergent swimming behaviours do cause a different response, resulting in the most prominent adaptational changes in the receptor levels of red muscle samples with lower swimming velocities in brown trout and with higher ones in whitefish.

Expression of dihydropyridine and ryanodine receptors in type IIA fibers of rat skeletal muscle.

In this study, the fiber type specificity of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs) in different rat limb muscles was investigated. Western blot and histochemical analyses provided for the first time evidence that the expression of both receptors correlates to a specific myosin heavy chain (MHC) composition. We observed a significant (p=0.01) correlation between DHP as well as Ry receptor density and the expression of MHC IIa (correlation factor r=0.674 and r=0.645, respectively) in one slow-twitch, postural muscle (m. soleus), one mixed, fast-twitch muscle (m. gastrocnemius) and two fast-twitch muscles (m. rectus femoris, m. extensor digitorum longus). The highest DHP and Ry receptor density was found in the white part of m. rectus femoris (0.058±0.0060 and 0.057±0.0158 ODu, respectively). As expected, the highest relative percentage of MHC IIa was also found in the white part of m. rectus femoris (70.0±7.77%). Furthermore, histochemical experiments revealed that the IIA fibers stained most strongly for the fluorophore-conjugated receptor blockers. Our data clearly suggest that the expression of DHPRs and RyRs follows a fiber type-specific pattern, indicating an important role for these proteins in the maintenance of an effective Ca2+ cycle in the fast contracting fiber type IIA.

Effects of low-intensity training on dihydropyridine and ryanodine receptor content in skeletal muscle of mouse

To evaluate low-intensity exercise training induced changes in the expression of dihydropyridine (DHP) and ryanodine (Ry) receptors both mRNA and protein levels were determined by quantitative RT-PCR and immunoblot analysis from gastrocnemius (GAS) and rectus femoris (RF) muscles of mice subjected to a 15-week aerobic exercise program. The level of muscular work was assayed by changes in myosin heavy chain (MHC) content, myoglobin (Mb) expression and muscle size. The mRNA expression and optical density of DHP receptor increased significantly in GAS by 66.8 and 39.5%, respectively. The expression of Ry receptor, on the other hand, was not up-regulated. In RF, there was a significant increase of 38.4% in the mRNA expression of DHP receptor, although the protein level remained the same. No changes in Ry receptor expression was observed. The training resulted in a 1.58% increase in the amount of MHC IIa and a 2.34% decrease in that of IIb and IId in GAS. A significant 8.3% increase in the Mb content was observed. In RF, no significant changes in MHC or in Mb content were noted. Our results show that an evident increase in the mRNA and protein expression of DHP receptor was induced in GAS even by a relatively low-intensity exercise. Surprisingly, contrast to DHP receptor expression, no changes in Ry receptor mRNA, or protein levels were found, indicating more abundant demand for DHP receptor after increased muscle activity.

Effects of different training protocols on Ca2+ handling and oxidative capacity in skeletal muscle of Atlantic salmon (Salmo salarL.)

The modulation of calcium channel density and oxidative capacity in skeletal muscle after different training protocols were studied in 3-year-old Atlantic salmon smolts. The effect of endurance exercise on dihydropyridine (DHP) and ryanodine (Ry) receptor densities as well as on muscle metabolism were determined by immunoblot and histochemical analysis from swimming muscles of fish subjected to nine different training protocols varying in duration and water current velocity. In general, exercise training caused a significant increase in the density of both DHP and Ry receptors in both muscle types studied. In red muscle, the most notable increase in DHP and Ry receptor expression was observed in muscle sections from fish swimming against intermediate current velocity for a 2-week period (182.3+/-16.3%, 234.6+/-30.3%, respectively). In white muscle, the expression of DHP and Ry receptors was most upregulated after a 6-week swimming period also at intermediate water current velocity (270.4+/-23.9%, 114.4+/-15.3%, respectively). As with the activity of enzymes involved in muscle energy supply, endurance exercise resulted in a significant increase in succinate dehydrogenase (SDH) activity, but a significant decrease in phosphorylase activity. We conclude that the expression of both DHP and Ry receptors was upregulated in the swimming muscles of salmon as a consequence of exercise training. This, along with the increased oxidative enzyme activity, provides benefits to the contraction efficiency of fish muscles while swimming. However, it was also observed that optimal oxidative swimming capacity is achieved only with a proper exercise program, since the most relevant changes in DHP and Ry receptor expression, as well as in oxidative capacity, were seen in the group training with the intermediate swimming velocity.

Intracellular calcium changes trigger connexin 32 hemichannel opening

Connexin hemichannels have been proposed as a diffusion pathway for the release of extracellular messengers like ATP and others, based on connexin expression models and inhibition by gap junction blockers. Hemichannels are opened by various experimental stimuli, but the physiological intracellular triggers are currently not known. We investigated the hypothesis that an increase of cytoplasmic calcium concentration ([Ca2+]i) triggers hemichannel opening, making use of peptides that are identical to a short amino-acid sequence on the connexin subunit to specifically block hemichannels, but not gap junction channels. Our work performed on connexin 32 (Cx32)-expressing cells showed that an increase in [Ca2+]i triggers ATP release and dye uptake that is dependent on Cx32 expression, blocked by Cx32 (but not Cx43) mimetic peptides and a calmodulin antagonist, and critically dependent on [Ca2+]i elevation within a window situated around 500 nM. Our results indicate that [Ca2+]i elevation triggers hemichannel opening, and suggest that these channels are under physiological control.

Temperature and Ca2+dependence of [3H]ryanodine binding in the burbot (Lota lotaL.) heart

Opening and closing of the cardiac ryanodine (Ry) receptor (RyR) are coordinated by the free intracellular Ca2+ concentration, thus making the Ca2+ binding properties of the RyR important for excitation-contraction coupling. Unlike mammalian cardiac RyRs, which lose their normal function at low temperatures, RyRs of ectothermic vertebrates remain operative at 2-4 degrees C, as indicated by Ry sensitivity of contractile force. To investigate the mechanisms of low temperature adaptation of ectothermic RyRs, we compared Ca2+-dependent kinetics of [3H]ryanodine binding in cardiac preparations of a fish (burbot, Lota lota) and a mammal (rat). The number of ventricular [3H]ryanodine binding sites determined at 20 degrees C was 1.54 times higher in rat than burbot heart (0.401 +/- 0.039 and 0.264 +/- 0.019 pmol/mg protein, respectively) (P < 0.02), while the binding affinity (Kd) for [3H]ryanodine was similar (3.38 +/- 0.63 and 4.38 +/- 1.14 nM for rat and burbot, respectively) (P = 0.47). The high-affinity [3H]ryanodine binding to burbot and rat cardiac preparations was tightly coordinated by the free Ca2+ concentration at both 20 degrees C and 2 degrees C and did not differ between the two species. Half-maximal [3H]ryanodine binding occurred at 0.191 +/- 0.027 microM and 0.164 +/- 0.034 microM Ca2+ for rat and at 0.212 +/- 0.035 microM and 0.188 +/- 0.039 microM Ca2+ for burbot (P = 0.65), at 2 degrees C and 20 degrees C, respectively. In two other fish species, rainbow trout (Oncorhynchus mykiss) and crucian carp (Carassius carassius), the Ca2+-binding affinity at 20 degrees C was 4.4 and 5.9 times lower, respectively, than in the burbot. At 20 degrees C, the rate of [3H]ryanodine binding to the high-affinity binding site was similar in rat and burbot but was drastically slowed in rat at 2 degrees C. At 2 degrees C, [3H]ryanodine failed to dissociate from rat cardiac RyRs, and at 10 degrees C and 20 degrees C, the rate of dissociation was two to three times slower in rat than burbot preparations. The latter finding is compatible with a channel gating mechanism, where the closing of the Ca2+ release channel is impaired or severely retarded by low temperature in rat but less so in burbot preparations. The stronger effect of low temperature on association and dissociation rate of [3H]ryanodine binding in rat compared with burbot suggests that RyRs of the ectothermic fish, unlike those of endothermic rat, are better able to open and close at low temperatures.

Effects of ryanodine receptor activation on neurotransmitter release and neuronal cell death following kainic acid-induced status epilepticus

Dynamic changes in intracellular free Ca 2+ concentration play a crucial role in various neural functions. The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and the ryanodine (Ry) receptor (RyR) are involved in Ca 2+-induced Ca 2+-release (CICR). Recent studies have shown that type 3 IP3R is highly expressed in rat hippocampal neurons after kainic acid (KA)-induced seizures and that dantrolene, a RyR antagonist, reduces KA-induced neuronal cell death. We investigated the RyR-associated effects of CICR agents on basal and K +-evoked releases of GABA and glutamate in rat hippocampus and the changes in expression of mRNA for RyRs in mouse brain after KA-induced seizures. The stimulatory effect of Ry on releases of GABA and glutamate was concentration-dependent in a biphasic manner. The inflection point in concentration–response curves for Ry on GABA release was lower than that for glutamate in both basal and K +-evoked conditions, suggesting that hyperactivation of RyR-associated CICR produces the imbalance between GABAergic and glutamatergic transmission. Following KA-induced seizures, transient up-regulation of brain-type RyR mRNA was observed in the hippocampal CA3 region and striatum, and signals for c-Fos mRNA increased transiently in the hippocampus, dentate gyrus and deeper layers of the neocortex. Thereafter, some dead neurons with single-stranded DNA (ssDNA) immunoreactive fragmented nuclei appeared in these areas. These findings suggest that intracellular Ca 2+ release via the RyR might be one of the mechanisms involved in KA-induced neuronal cell death.

Differential functional properties of Ca 2+ stores in pulmonary arterial conduit and resistance myocytes

In smooth muscle cells, oscillations of intracellular Ca 2+ concentration ([Ca 2+] i) are controlled by inositol 1,4,5-trisphosphate (InsP 3) and ryanodine (Ry) receptors on the sarcoplasmic reticulum (SR). Here we show that these Ca 2+ oscillations are regulated differentially by InsP 3 and Ry receptors in cells dispersed from the main trunk of the pulmonary artery (conduit myocytes) or from tertiary and quaternary arterial branches (resistance myocytes). Ry receptor antagonists inhibit either spontaneous or ATP-induced Ca 2+ oscillations in resistance myocytes but they do not affect the oscillations in most conduit myocytes. In contrast, agents that inhibit InsP 3 production or activation of InsP 3 receptors do not alter the oscillations is resistance myocytes but block them in conduit myocytes. We have also examined the degree of overlap of Ry- and InsP 3-sensitive stores in myocytes along the pulmonary arterial tree. In conduit myocytes, depletion of Ry-sensitive stores with repeated application of caffeine in the presence of Ry or in Ca 2+ free solutions did not prevent the ATP-induced Ca 2+ release from InsP 3-dependent stores. However, responsiveness to ATP was completely abolished in resistance myocytes subjected to the same experimental protocol. Thus, InsP 3- and Ry-dependent stores appear to be separated in conduit myocytes but joined in resistance myocytes. These data demonstrate for the first time differential properties of intracellular Ca 2+ stores and receptors in myocytes distributed along the pulmonary arterial tree and help to explain the distinct functional responses of large and small pulmonary vessels to vasoactive agents.

Activation of ryanodine receptors in the nuclear envelope alters the conformation of the nuclear pore complex

Nuclear pore complexes (NPCs) are supramolecular protein pores that traverse the nuclear envelope and form the only known direct route of transport between the cytoplasmic and nuclear spaces. Detailed studies have identified both active and passive mechanisms of transport through the NPC and structural studies have revealed its three-dimensional architecture. Under certain conditions, structural studies have found evidence for a mass in the central pore of the NPC whose identity remains unclear. Some studies suggest this mass represents cargo caught in transit, while others suggest it is an integral component of the NPC, the position of which is sensitive to sample conditions. Regardless of its identity, previous studies have shown that the central mass location within the NPC pore is influenced by the presence of calcium in the cisternal spaces of the nuclear membrane. Specific depletion of these calcium stores through inositol 1,4,5-trisphosphate (IP 3) receptor activation leads to the apparent displacement of the central mass towards both the cytoplasmic and nucleoplasmic sides of the NPC. Whether the central mass is cargo or a NPC component, these observations may offer interesting insights linking transport and calcium signaling pathways. Here, we show that ryanodine (Ry) receptors are also present in the nuclear envelope of Xenopus laevis oocytes, and their specific activation can affect the conformational state of the NPC. Although previously undetected, Western blot analysis of isolated oocyte nuclei reveals the presence of Ry receptors in the nuclear envelope, albeit in low abundance. Extensive atomic force microscopy (AFM) studies at the single pore level of isolated, fixed nuclei reveal changes in the NPC conformational state following treatments that stimulate Ry receptor activity. At resting calcium levels (∼200 nM Ca 2+), the central mass within the lumen of the NPC is recessed 5.3 nm below the cytoplasmic rim of the NPC. Following treatment with 10 nM ryanodine, the central mass displaces towards the cytoplasmic face occupying a new position only 2.9 nm below the cytoplasmic rim. Interestingly, at high ryanodine concentrations (20 μM), which are reported to deactivate Ry receptors, the central mass is observed to return to the recessed position, 5.4 nm below the cytoplasmic rim. Treatments with caffeine also lead to large changes in the NPC conformation, confirming the link to specific activation of Ry receptors. These observations are consistent with a new mechanism of NPC regulation in which specific activation of Ry receptors located in the nuclear envelope can modulate cisternal calcium levels, leading to changes in the NPC conformation. Together with previous studies, it now appears that both IP 3 and Ry receptors are present in the nuclear envelope of Xenopus oocytes and are capable, through activation, of indirectly influencing the conformational state of the NPC.

If-dependent modulation of pacemaker rate mediated by cAMP in the presence of ryanodine in rabbit sino-atrial node cells

I f contributes to generation and autonomic control of spontaneous activity of cardiac pacemaker cells through a cAMP-dependent, Ca 2+-independent mechanism of rate regulation. However, disruption of Ca 2+ release from sarcoplasmic reticulum (SR) by ryanodine (Ry) has been recently shown to slow spontaneous rate and inhibit β-adrenergic receptor (βAR)-induced rate acceleration, leading to the suggestion that the target of βAR modulation of pacemaking is the intracellular Ca 2+-regulatory process. We have investigated whether the Ry-induced decrease of βAR rate modulation alternatively involves disruption of the βAR-adenylate-cyclase-cAMP- I f mechanism. Prolonged exposure to Ry (3 μM, >2 min) slowed spontaneous rate of pacemaker cells by 29.8% via a depolarizing shift of take-off potential (TOP) without significantly changing early diastolic depolarization rate. Ry depressed rate acceleration caused by isoproterenol (Iso) (1 μM, 23.6% in control vs. 8.0%), but did not modify that caused by two membrane-permeable cAMP analogs, CPT-cAMP (300 μM, 17.7% vs. 17.3%) and Rp-cAMPs (50 μM, 18.0% vs. 20.6%). Consistent with the rate effect, exposure to Ry decreased the shift induced by Iso, but not that induced by either cAMP analog on the I f-activation curve. We conclude that disruption of Ry receptor function and SR Ca 2+ release depresses βAR-induced modulation of heart rate, but does not impair cAMP-dependent rate acceleration mediated by I f. However, abolishment of normal Ca 2+ homeostasis may result in the failure of βAR agonists to sufficiently elevate cAMP near f-channels. The molecular basis for Ca 2+-dependent interference in β-adrenergic signaling remains to be determined.

Cyclic ADP-Ribose Contributes to Contraction and Ca2+ Release by M1 Muscarinic Receptor Activation in Coronary Arterial Smooth Muscle

The present study determined the role of cyclic ADP-ribose (cADPR) in mediating vasoconstriction and Ca²⁺ release in response to the activation of muscarinic receptors. Endothelium-denuded small bovine coronary arteries were microperfused under transmural pressure of 60 mm Hg. Both acetylcholine (ACh; 1 nmol/L to 1 µmol/L) and oxotremorine (OXO; 2.5–80 µmol/L) produced a concentration-dependent contraction. The vasoconstrictor responses to both ACh and OXO were significantly attenuated by nicotinamide (Nicot; an ADP-ribosyl cyclase inhibitor), 8-bromo-cADPR (8-Br-cADPR; a cADPR antagonist) or ryanodine (Ry; an Ry receptor antagonist). Intracellular Ca²⁺ ([Ca²⁺]_i) was determined by fluorescence spectrometry using fura-2 as a fluorescence indicator. OXO produced a rapid increase in [Ca²⁺]_i in freshly isolated single coronary arterial smooth muscle cells (CASMCs) bathed with Ca²⁺-free Hanks’ solution. This OXO-induced rise in [Ca²⁺]_i was significantly reduced by pirenzepine (PIR; an M₁ receptor-specific blocker), Nicot, 8-Br-cADPR or Ry. The effects of OXO on the activity of ADP-ribosyl cyclase (cADPR synthase) were examined in cultured CASMCs by measuring the rate of cyclic GDP- ribose (cGDPR) formation from β-nicotinamide guanine dinucleotide. It was found that OXO produced a concentration-dependent increase in the production of cGDPR. The stimulatory effect of OXO on ADP-ribosyl cyclase was inhibited by both PIR and Nicot. These results suggest that the cADPR signaling pathway participates in the contraction of small coronary arterial smooth muscle and Ca²⁺ release induced by activation of M₁ muscarinic receptors.

A gonadotropin-releasing hormone insensitive, thapsigargin-sensitive Ca2+ store reduces basal gonadotropin exocytosis and gene expression: comparison with agonist-sensitive Ca2+ stores.

We examined whether distinct Ca2+ stores differentially control basal and gonadotropin (GTH-II)-releasing hormone (GnRH)-evoked GTH-II release, long-term GTH-II secretion and contents, and GTH-II-beta mRNA expression in goldfish. Thapsigargin (Tg)-sensitive Ca2+ stores mediated neither caffeine-evoked GTH-II release, nor salmon (s)GnRH- and chicken (c)GnRH-II-stimulated secretion; the latter responses were previously shown to involve ryanodine (Ry)-sensitive Ca2+ stores. Surprisingly, Tg decreased basal GTH-II release. This response was attenuated by prior exposure to sGnRH and caffeine, but was insensitive to the phosphatase inhibitor okadaic acid, the inhibitor of constitutive release brefeldin A and cGnRH-II. GTH-II-beta mRNA expression was decreased at 24 h by 2 microm Tg, and by inhibiting (10 microm Ry) and stimulating (1 nm Ry) Ry receptors. Transient increases in GTH-II-beta mRNA were observed at 2 h and 12 h following 10 microm and 1 nm Ry treatment, respectively. Effects of Tg, Ry and GnRH on long-term GTH-II secretion, contents and apparent production differed from one another, and these changes were not well correlated with changes in GTH-II-beta mRNA expression. Our data show that GTH-II secretion, storage and transcription can be independently controlled by distinct Ca2+ stores.

Homogeneous Ca 2+ stores in rat adrenal chromaffin cells

The localization and function of Ca 2+ stores in isolated chromaffin cells of rat adrenal medulla were investigated using confocal laser microscopy and amperometry. Binding sites for BODIPY-inositol 1,4,5-trisphosphate (IP 3), -ryanodine (Ry), and -thapsigargin (Thap) were both perinuclear and at the cell periphery. The endoplasmic reticulum (ER), which was identified by ER Tracker dye, took up fluorescent Ry and IP 3, and the majority of BODIPY-Ry-binding area was bound by fluorescent IP 3. Under Ca 2+-free conditions, the amount of caffeine-induced catecholamine secretion was 33% of that of muscarine-induced secretion, but muscarine induced little or no secretion after exposure to caffeine. Muscarine-induced Ca 2+ increases, as observed with fluo-3, lasted for a few tens of seconds under Ca 2+-free conditions, whereas a caffeine-induced Ca 2+ transient diminished rapidly with a half decay time of 3 s and this spike-like Ca 2+ transient was then followed by a sustained increase with a low level. These results indicate that IP 3 receptors and Ry receptors (RyRs) are present in common ER Ca 2+ storage and the lower potency of caffeine for secretion may be due to a rapid decrease in RyR channel activity to a low level.

Function-specific calcium stores selectively regulate growth hormone secretion, storage, and mRNA level.

Ca(+) stores may regulate multiple components of the secretory pathway. We examined the roles of biochemically independent intracellular Ca(2+) stores on acute and long-term growth hormone (GH) release, storage, and mRNA levels in goldfish somatotropes. Thapsigargin-evoked intracellular Ca(2+) concentration ([Ca(2+)](i)) signal amplitude was similar to the Ca(2+)-mobilizing agonist gonadotropin-releasing hormone, but thapsigargin (2 microM) did not acutely increase GH release, suggesting uncoupling between [Ca(2+)](i) and exocytosis. However, 2 microM thapsigargin affected long-term secretory function. Thapsigargin-treated cells displayed a steady secretion of GH (2, 12, and 24 h), which decreased GH content (12 and 24 h), but not GH mRNA/production (24 h). In contrast to the results with thapsigargin, activating the ryanodine (Ry) receptor (RyR) with 1 nM Ry transiently increased GH release (2 h). Prolonged activation of RyR (24 h) reduced GH release, contents and apparent production, without changing GH mRNA levels. Inhibiting RyR with 10 microM Ry increased GH mRNA levels, production, and storage (2 h). Increasing [Ca(2+)](i) independently of Ca(2+) stores with the use of 30 mM KCl decreased GH mRNA. Collectively, these results suggest that parts of the secretory pathway can be controlled independently by function-specific Ca(2+) stores.

Localization of calcium binding proteins and calcium release receptor channels in human oocytes, fertilized eggs and preimplantation embryos.

Objective: There is growing evidence that in both oocytes and embryos complex spatiotemporal calcium (Ca) oscillations are generated, at least partly, by intracellular Ca stores. However, little is known about the essential protein constituents of the Ca stores that are involved in Ca storage and release in mammalian oocytes and embryos (including human). Therefore, the objective of this study was to investigate the presence and localization of Ca binding proteins and Ca release receptor channels in human oocytes, fertilized eggs and preimplantation embryos.Design: A descriptive immunocytochemical study of human gametes and embryos.Materials/Methods: Intact oocytes (n = 149) and embryos (n = 130) were fixed with either formaldehyde or methanol and processed for immunocytochemistry using specific antibodies against the proteins being studied. Immunostaining was assessed using confocal scanning microscopy.Results: Nonrandom distribution and abundant quantities of calreticulin and calsequestrin, the two major Ca storage proteins of somatic cells, were observed in maturing oocytes, pronuclear zygotes and early cleaving embryos (2-8 cell stage). Calreticulin was always found to be confined to the cell cortex, while calsequestrin was dispersed throughout the cytoplasm of the cells. Calreticulin co-localized with another chaperone of the endoplasmic reticulum, calnexin. Direct evidence for the presence of the inositol triphosphate (IP3, type-II) and ryanodine (Ry, type I-III) receptors was also demonstrated. Whereas the arrangement of Ca binding proteins appeared to remain unchanged during oocyte maturation and embryo growth, the IP3 and Ry receptors underwent dynamic developmental changes in density and distribution. The IP3 receptor had a cortical distribution, which was similar to that of calreticulin. The Ry receptor was uniformly distributed throughout the cytoplasm, which was similar to the calsequestrin localization.Conclusions: This is the first report documenting the presence and distribution of Ca storage and release proteins in human oocytes, fertilized eggs and preimplantation embryos. The present findings suggest that human oocytes and embryos possess highly organized and diversified Ca sequestration and release mechanisms. Fine-tuning of these mechanisms may play a crucial role in the regulation of Ca transients during oocyte maturation, fertilization and early embryo development. Objective: There is growing evidence that in both oocytes and embryos complex spatiotemporal calcium (Ca) oscillations are generated, at least partly, by intracellular Ca stores. However, little is known about the essential protein constituents of the Ca stores that are involved in Ca storage and release in mammalian oocytes and embryos (including human). Therefore, the objective of this study was to investigate the presence and localization of Ca binding proteins and Ca release receptor channels in human oocytes, fertilized eggs and preimplantation embryos. Design: A descriptive immunocytochemical study of human gametes and embryos. Materials/Methods: Intact oocytes (n = 149) and embryos (n = 130) were fixed with either formaldehyde or methanol and processed for immunocytochemistry using specific antibodies against the proteins being studied. Immunostaining was assessed using confocal scanning microscopy. Results: Nonrandom distribution and abundant quantities of calreticulin and calsequestrin, the two major Ca storage proteins of somatic cells, were observed in maturing oocytes, pronuclear zygotes and early cleaving embryos (2-8 cell stage). Calreticulin was always found to be confined to the cell cortex, while calsequestrin was dispersed throughout the cytoplasm of the cells. Calreticulin co-localized with another chaperone of the endoplasmic reticulum, calnexin. Direct evidence for the presence of the inositol triphosphate (IP3, type-II) and ryanodine (Ry, type I-III) receptors was also demonstrated. Whereas the arrangement of Ca binding proteins appeared to remain unchanged during oocyte maturation and embryo growth, the IP3 and Ry receptors underwent dynamic developmental changes in density and distribution. The IP3 receptor had a cortical distribution, which was similar to that of calreticulin. The Ry receptor was uniformly distributed throughout the cytoplasm, which was similar to the calsequestrin localization. Conclusions: This is the first report documenting the presence and distribution of Ca storage and release proteins in human oocytes, fertilized eggs and preimplantation embryos. The present findings suggest that human oocytes and embryos possess highly organized and diversified Ca sequestration and release mechanisms. Fine-tuning of these mechanisms may play a crucial role in the regulation of Ca transients during oocyte maturation, fertilization and early embryo development.