Endothelin-1-induced contraction of mesenteric small arteries is mediated by ryanodine receptor Ca2+ channels and cyclic ADP-ribose.

Contraction of vascular smooth muscle by endothelin-1 is dependent on extracellular and intracellular Ca2+. However, the role of ryanodine-sensitive Ca2+ stores in endothelin-1-induced contraction is unknown. Vascular contraction was measured in mesenteric small arteries (200-300 microm intraluminal diameter) isolated from Sprague-Dawley rats and maintained at a constant intraluminal pressure of 40 mm Hg. The presence of functional ryanodine receptor Ca2+ release channels (RyRC) was demonstrated by the finding that ryanodine (10 microM), which locks the RyRC in a subconductance state, produced significant contraction of small arteries in the presence of 15 mM KCl. This effect was inhibited by dantrolene (10 microM), a RyRC inhibitor. Dantrolene significantly reduced the ET(A) receptor-mediated contraction to endothelin-1 (10(-11)-10(-9) M). The ability of dantrolene to reverse contraction induced by endothelin-1 was also determined. Dantrolene (1-10 microM) produced concentration-dependent relaxation of vessels precontracted to 38+/-3% of resting diameter with endothelin-1 but had no effect in vessels precontracted to a similar degree with phenylephrine or KCl. Because activation of RyRC may be dependent on production of cyclic ADP-ribose, the effect of nicotinamide (2 mM), an inhibitor of ADP-ribosyl cyclase, on contraction to endothelin-1 was determined. Nicotinamide had an inhibitory effect similar to that produced by dantrolene. A combination of nicotinamide and dantrolene had no greater effect than either agent alone, suggesting a common pathway for cyclic ADP-ribose and RyRC. In summary, endothelin-1 induces contraction of small mesenteric arteries through ET(A) receptor-mediated production of cyclic ADP-ribose and activation of RyRC.

Voltage change-induced gating transitions of the rabbit skeletal muscle Ca2+ release channel.

1. We used the planar lipid bilayer method to study single ryanodine receptor Ca2+ release channels (RyRCs) from fast skeletal muscle of the rabbit. We found that changes in membrane voltage directly induced gating transitions of the RyRC: (i) in the steady state, even at activating Ca2+ concentrations (20 microM), at a constant membrane potential the channels resided in a low open probability (Po) state (inactivated-, I-mode), and (ii) upon abrupt changes of voltage, the apparent inactivation of the RyRCs was relieved, resulting in a rapid and transient increase in Po. 2. The magnitude of the Po increase was a function of both the duration and the amplitude of the applied prepulse, but was independent of the channel activity during the prepulse. 3. The voltage-induced Po increase probably involved major conformational changes of the channel, as it resulted in substantial alterations in the gating pattern of the channels: the voltage change-induced increase in Po was accompanied by the rapid appearance of two types of channel activity (high (H) and low (L) open probability modes). 4. The response of the RyRC to voltage changes raises the interesting possibility that the activation of RyRC in situ might involve electrical events, i.e. a possible dipole-dipole coupling between the release channel and the voltage sensor.

Kinetic basis of quantal calcium release from intracellular calcium stores.

The kinetics of Ca2+ release from canine cerebellum and rabbit skeletal muscle microsomes, mediated by the inositol 1,4,5-trisphosphate (IP3) receptor (IRC) and the ryanodine receptor (RyRC), respectively, were analyzed by a model, which considers that Ca2+ release channels undergo spontaneous inactivation. We found that: (i) both the initial rate of release (Vo) and the rate of inactivation (Vi) were saturable functions of the activating ligand concentration (CL); and (ii) the ratio of Vi/Vo, termed the relative tendency for inactivation, decreased with increasing CL. Equilibrium [3H]-IP3 binding studies, on the other hand, revealed the presence of one single class of non-co-operative IP3 sites in cerebellum membranes (Kdeq = 47 nM and Hill coefficient = 1.1). Based on the above Vi-Vo relationship and the IP3-binding data, we propose that quantal Ca2+ release through IRCs might be a result of spontaneous channel inactivation, whose rate is controlled by the ratio of IP3-occupied/free monomers in the tetrameric release channel units. Furthermore, because of the kinetic similarities between the IRC- and RyRC-mediated Ca2+ release processes, as well as between quantal Ca2+ release and channel adaptation, the same mechanism is also proposed to apply to the RyRC-mediated Ca2+ release as well as to constitute the basis of release channel adaptation.

Fluorescent probing with felodipine of the dihydropyridine receptor and its interaction with the ryanodine receptor calcium release channel.

The intensity of the fluorescence emission of the fluorescent 1,4-dihydropyridine (DHP) derivative felodipine increased upon binding to both isolated cardiac sarcolemma (SLM) and skeletal muscle sarcoplasmic reticulum (SR) preparations, the latter containing SR-transversal tubule junctional diads and triads. The fluorescence enhancement was due to the binding of felodipine to high-affinity (Kd's of 0.35 and 1.25 nM in cardiac SLM and skeletal SR, respectively) 1,4-dihydropyridine sites of the dihydropyridine receptor (DHPR), as evidenced in competition experiments with the DHP analog isradipine. In both cardiac SLM and SR, the felodipine fluorescence was sensitive to conformational changes of the DHPR, as diltiazem that binds to DHPR at a separate site altered the values of both the Kd and the Hill coefficient characteristic for felodipine binding. In skeletal muscle membranes containing intact TT-SR junctions, ryanodine, a specific ligand of the ryanodine receptor calcium release channel (RyRC), also induced changes in felodipine fluorescence, which was eliminated by detergent and high-salt treatment to solubilize the RyRC. These results suggest that i) felodipine fluorescence is useful to probe conformational changes of the DHPR and ii) coupled conformational changes between the DHPR and the RyRC in skeletal muscle indeed occur and could be monitored by measuring felodipine fluorescence.

Sarcoplasmic reticulum-associated and protein kinase C-regulated ADP-ribosyl cyclase in cardiac muscle.

Two types of ADP-ribosyl cyclase activity were distinguished in dog and rat cardiac muscles by measuring the enzymatic conversion of NGD (as an NAD analog) into the fluorescent product cyclic GDP-ribose in cardiac muscle subcellular fractions. Both types of activity were confined to membrane fractions isolated from microsomes by sucrose gradient centrifugation. One of the activities co-purified with fractions that were enriched in sarcolemma (SLM), as evidenced by immunodetection of the dihydropyridine receptor, while the other activity was found to co-precipitate with the sarcoplasmic reticulum (SR), that was identified on the basis of its immuno-staining with a ryanodine receptor monoclonal antibody. In certain aspects, the plasma membrane-bound ADP-ribosyl cyclase activity resembled the characteristics of CD38 or CD38-like proteins: it was sensitive to thiols and lectins and was recognized by a monoclonal anti CD38 antibody. The SR enzyme had apparently distinct properties, as it was insensitive to both thiols and lectins and was not recognized by the CD38 antibody. In addition, the SR-associated ADP-ribosyl cyclase was inhibited by endogenous protein kinase C (PKC)-dependent phosphorylation in both dog and rat cardiac SR. The PKC-modulated SR ADP-ribosyl cyclase we describe here might be a principal component of the signal transduction machinery that is responsible for regulation of the intracellular levels of cADPR.

Inactivation of the cardiac ryanodine receptor calcium release channel by nitric oxide.

We have recently reported [Mészáros L.G., Minarovic I., Zahradníková A. Inhibition of the skeletal muscle ryanodine receptor calcium release channel by nitric oxide. FEBS Lett 1996; 380: 49-52] that nitric oxide (NO) reduces the activity of the skeletal muscle ryanodine receptor Ca2+ release channel (RyRC), a principal component of the excitation-contraction coupling machinery in striated muscles. Since (i) as shown here, we have obtained evidence which indicates that the NO synthase (eNOS) of cardiac muscle origin co-purified with RyRC-containing sarcoplasmic reticulum (SR) fractions; and (ii) the effects of NO donors on the release channel, as well as on cardiac function, appear somewhat contradictory, we have made an attempt to investigate the response of the cardiac RyRC to NO that is generated in situ from L-arginine in the NOS reaction. We found that L-arginine-derived NO inactivates Ca2+ release from cardiac SR and reduces the steady-state activity (i.e. open probability) of single RyRCs fused into a planar lipid bilayer. This reduction was prevented by NOS inhibitors and the NO quencher hemoglobin and was reversed by 2-mercaptoethanol. We thus conclude that: (i) in isolated SR preparations, it is possible to assess the effects of NO that is generated from L-arginine in the NOS reaction; and (ii) cardiac RyRc responds to NO in a manner which is identical to that we have previously found with the skeletal channel. These findings suggest that the direct modulation of the RyRC by NO is a signaling mechanism which likely participates in earlier demonstrated NO-induced myocardial contractility changes.

Positive inotropic effect of thyrotropin-releasing hormone on isolated rat hearts.

The effects of thyrotropin releasing hormone (TRH) on the contractility of electrically stimulated and perfused isolated rat hearts were investigated. TRH in the range of 0.1-10 mumol/l was found to exert a positive inotropic effect on cardiac contractility, which however qualitatively differed at lower vs. higher concentrations of the hormone: at 1 mumol/l, TRH was found to significantly enhance the rate of contraction as well as that of relaxation (by 23.2 +/- 3.7 and 27.8 +/- 7.7%, respectively), which culminated in an increased peak contractile force. However, at 10 mumol/l, the positive inotropic effect of TRH (i.e. the increase in peak contractile force) was smaller than at 1 mumol/l, which apparently was due to both a reduced TRH-induced elevation in the rate of contraction (12.4 +/- 3.2%) and a TRH-induced decrease in relaxation rate (11.1 +/- 8.1%). Since TRH is expressed in the heart, the above findings suggest that, in addition to its CNS-mediated cardiovascular effects, TRH modulates cardiac contractility as an autocrine regulator in a concentration-dependent manner, which likely involves more than one TRH receptor and associated signaling pathway.

Inhibition of the skeletal muscle ryanodine receptor calcium release channel by nitric oxide.

NO donors were found to reduce the rate of Ca2+ release from isolated skeletal muscle sarcoplasmic reticulum (SR) and the open probability of single ryanodine receptor Ca2+ release channels (RyRCs) in planar lipid bilayers, and these effects were prevented by the NO quencher hemoglobin and reversed by 2-mercaptoethanol. Ca2+ release assessed in skeletal muscle homogenates was also reduced by NO that was generated in situ from L-arginine by endogenous, nitro-L-arginine methylester-sensitive NO-synthase. The effect of NO on the RyRC might explain NO-induced depression of contractile force in striated muscles and, since both RyRC isoforms and NOS isoenzymes aer ubiquitous, may represent a wide-spread feedback mechanism in Ca2+ signaling; i.e. Ca-dependent activation of NO production and NO-evoked reduction of Ca2+ release from intracellular Ca2+ stores.

The kinetics of cyclic ADP-ribose formation in heart muscle.

The kinetics of NAD glycohydrolase activity in cardiac muscle homogenates were investigated using HPLC and TLC techniques to identify reaction products. NAD was found to undergo enzymatic hydrolysis yielding ADP-ribose (ADPR) as well as a cyclase reaction resulting in the formation of cyclic ADP-ribose (cADPR). Both ADPR and cADPR were further converted into adenine and ADPR, respectively. The kinetics of the formation reactions, that manifested a clearly distinguishable initial lag phase in the time course of cADPR formation, suggest that the hydrolytic reaction yielding ADPR and the cyclase reaction yielding cADPR probably represent alternative catalytic functions of the same enzyme, which might be controlled by endogenous ADP-ribosylation reactions.

Heterogeneity of the cardiac calcium release channel as assessed by its response to ADP-ribose.

ADP-ribose (ADPR) was found to decrease the rate of Ca2+ release from isolated cardiac sarcoplasmic reticulum (SR) vesicles, which was limited to a maximum of 46 +/- 8% inhibition and was in accordance with our results obtained with single cardiac ryanodine receptor Ca2+ release channels (RyRC) incorporated into planar lipid bilayers: Out of 23 separate single channels, 9 responded to ADPR by a complete closure, while 14 channels showed no response at all, resulting in a reduction in overall open probability in the presence of ADPR (relative to control channels) by 39.7%. Although the ADPR-responsive and unresponsive single channels showed no differences in their respective open times, current amplitudes or relative occurrences of dwell levels, the bare existence of two types of response to ADPR together with the 50%-limited inhibition of cardiac SR Ca2+ release by ADPR indicates a heterogeneity of RyRCs in cardiac SR, which is likely due to protein(s) that interact(s) with the channel and are present in substoichiometric mole ratios.

In situ Ca(2+)-induced Ca2+ release from a ryanodine-sensitive intracellular Ca2+ store in corneal epithelial cells.

1. In a number of tissues, Ca2+ signaling involves Ca(2+)-induced Ca2+ release (CICR) from ryanodine- and caffeine-sensitive intracellular Ca2+ stores. We sought evidence for such a mechanism in bovine corneal epithelial cells (BCE). 2. We have identified a microsomal fraction of BCE which possesses high-affinity [3H]-ryanodine binding sites indicating the presence of the ryanodine receptor Ca2+ channel. 3. Functional evidence for CICR is that in fura-2 loaded BCE the magnitude of Ca2+ transients induced by the addition of either the adenylate cyclase activator, forskolin, or the L-type Ca2+ channel agonist, BAY-K 8644, were both enhanced by preincubation with 5 microM ryanodine. This ryanodine enhancement provides evidence that Ca2+ release from a ryanodine-sensitive intracellular Ca2+ store also contributes to the Ca2+ transients. Therefore, Ca(2+)-induced Ca2+ release is a component of Ca2+ signaling in BCE.

Coexistence of high- and low-affinity Ca2+ binding sites of the sarcoplasmic reticulum calcium pump.

We have recently shown [Mészáros, L. G., & Bak, J. (1992) Biochemistry 31, 1195-1200] that, during the rapid phase of Ca2+ uptake into sarcoplasmic reticulum (SR), internalization and binding of Ca2+ to the cytoplasmic high-affinity binding sites of the Ca2+ ATPase occur simultaneously, resulting in a transient supernumerary Ca/ATP stoichiometry. Here we address the question of whether the cytoplasmic high-affinity and the luminal low-affinity Ca2+ binding sites of the SR Ca2+ ATPase also coexist. SR vesicles were loaded with Ca2+ (0-10 mM), and then the kinetics of EP formation and decomposition as well as the maximum level of EP formed from radiolabeled ATP were determined at conditions which only allow single-cycle reactions to occur: empty or Ca-loaded SR vesicles (in micromolar extravesicular Ca2+) were either mixed with ATP plus millimolar EGTA or added in amounts that set a Ca2+ ATPase/ATP ratio of 80-85 at the initiation of the reaction. The rates of EP formation and decomposition were both significantly reduced in Ca-loaded, compared to empty (ionomycin-treated), vesicles. However, the value of EPmax was unaltered by Ca-loading, suggesting the existence of the enzyme intermediate, E.Ca2(cyt).Ca2(lum), i.e., the coexistence of the cytoplasmic and the luminal Ca2+ binding sites of the Ca-pump. These results suggest that the uphill transport of Ca2+ might not be based on an alternating relocation and conversion of the Ca2+ binding sites of the Ca2+ ATPase.

Cyclic ADP-ribose as an endogenous regulator of the non-skeletal type ryanodine receptor Ca2+ channel.

The skeletal and cardiac isoforms of the ryanodine receptor Ca2+ channel (RyRC) constitute the Ca2+ release pathway in sarcoplasmic reticulum of skeletal and cardiac muscles, respectively. A direct mechanical and a Ca(2+)-triggered mechanism (Ca(2+)-induced Ca2+ release) have been respectively proposed to explain the in situ activation of Ca2+ release in skeletal and cardiac muscle. In non-muscle cells, however, where the RyRC also participates in Ca2+ signalling, the mechanism of RyRC activation is unknown. Cyclic adenosine 5'-diphosphoribose (cADPR), which is present in many mammalian tissues, has been reported to induce Ca2+ release from ryanodine-sensitive intracellular Ca2+ stores in sea urchin eggs. Here we provide evidence that cADPR directly activates the cardiac but not the skeletal isoform of the RyRC. This, together with results on sea urchin eggs, suggests that cADPR is an endogenous activator of the non-skeletal type of RyRC and may thus have a role similar to inositol 1,4,5-trisphosphate in Ca2+ signalling.

Simultaneous internalization and binding of calcium during the initial phase of calcium uptake by the sarcoplasmic reticulum Ca pump.

The kinetics of Ca2+ transport mediated by the sarcoplasmic reticulum (SR) Ca-ATPase were investigated by rapid kinetic techniques that either measure the disappearance of Ca2+ from the medium [stopped-flow photometry of Ca2+ indicators or rapid filtration (method 1)] or directly detect the changes in the accessibility of Ca2+ to the exterior of the membrane, i.e., occlusion of Ca2+ within the Ca pump and Ca2+ transport into the lumen of SR vesicles [EGTA quench (method 2)]. SR vesicles were preincubated in micromolar Ca2+ to form the E.2Cacyt intermediate of the Ca-ATPase, and then Ca2+ transport was initiated by addition of ATP. It was found that Ca2+ uptake measured by method 1 began with no lag phase, in spite of the prediction of kinetic models of the Ca-ATPase. Instead, the time course of Ca2+ uptake was found to have two components: a fast and a slow phase, similar to that obtained using method 2, although the rate constant of the fast phase determined by method 1 was considerably lower than that measured by method 2. The fast phase of Ca2+ uptake measured by method 1 was not influenced by either Ca2+ ionophore or detergent treatment, whereas the slow phase was diminished.(ABSTRACT TRUNCATED AT 250 WORDS)

Caffeine- and ryanodine-sensitive Ca2+ stores of canine cerebrum and cerebellum neurons.

[3H]ryanodine binding to and Ca2+ release from microsomal fractions derived from canine cerebrum (CBR) and cerebellum (CBL) were investigated. High-affinity ryanodine binding sites were detected in both cerebrum and cerebellum microsomes [CBR: maximal binding capacity (Bmax) = 446 fmol/mg protein, dissociation constant (Kd) = 9 nM, Hill coefficient (n) = 0.95; CBL: Bmax = 650, Kd = 12, n = 1.8]. Ryanodine binding in both fractions was increased by millimolar concentrations of ATP [or its nonhydrolyzable analogue beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP)] and micromolar concentrations of Ca2+ but was decreased by micromolar concentrations of ruthenium red, similar to that found in sarcoplasmic reticulum (SR) of striated muscle. The addition of caffeine or the sudden elevation of extravesicular Ca2+ induced a rapid La(3+)-sensitive Ca2+ release from both CBR and CBL microsomal fractions with rate constants of approximately 100 s-1, as determined by stopped-flow photometry of the Ca2+ indicator arsenazo III. The release of Ca2+ was activated by either millimolar ATP or AMP-PCP, blocked by micromolar concentrations of La3+, and significantly inhibited by 50 microM ryanodine. Mg2+ and ruthenium red in millimolar and micromolar concentrations, respectively, caused only a slight inhibition of Ca2+ release. These results indicate that rapid Ca2+ release occurs from caffeine-, Ca2+- and ryanodine-sensitive Ca2+ stores in both CBR and CBL microsomal fractions.

Intravesicular calcium transient during calcium release from sarcoplasmic reticulum.

The time course of changes in the intravesicular Ca2+ concentration ([Ca2+]i) in terminal cisternal sarcoplasmic reticulum vesicles upon the induction of Ca2+ release was investigated by using tetramethylmurexide (TMX) as an intravesicular Ca2+ probe. Upon the addition of polylysine at the concentration that led to the maximum rate of Ca2+ release, [Ca2+]i decreased monotonically in parallel with Ca2+ release. Upon induction of Ca2+ release by lower concentrations of polylysine, [Ca2+]i first increased above the resting level, followed by a decrease well below it. The release triggers polylysine, and caffeine brought about dissociation of calcium that bound to a nonvesicular membrane segment consisting of the junctional face membrane and calsequestrin bound to it, as monitored with TMX. No Ca2+ dissociation from calsequestrin-free junctional face membranes or from the dissociated calsequestrin was produced by release triggers, but upon reassociation of the dissociated calsequestrin and the junctional face membrane, Ca2+ dissociation by triggers was restored. On the basis of these results, we propose that the release triggers elicit a signal in the junctional face membrane, presumably in the foot protein moiety, which is then transmitted to calsequestrin, leading to the dissociation of the bound calcium; and in SR vesicles, to the transient increase of [Ca2+]i, and subsequently release across the membrane.

Non-identical behavior of the Ca2(+)-ATPase in the terminal cisternae and the longitudinal tubules fractions of sarcoplasmic reticulum.

The kinetics of Ca2+ dissociation from and binding to the Ca2(+)-ATPase and the coupled tryptophan fluorescence changes were compared in the heavy and the light sarcoplasmic reticulum (SR) fractions. It was found that in the light SR both the dissociation of Ca2+ from the Ca2(+)-ATPase and the coupled tryptophan fluorescence change took place in a biphasic fashion, but they were monophasic in the heavy SR. On the other hand, the time courses of both the Ca2+ binding and the coupled tryptophan fluorescence increase were biphasic, and virtually indistinguishable between the heavy and the light SR fractions. Submicromolar ruthenium red altered the kinetics of both the Ca2+ dissociation and the coupled fluorescence change in heavy, but not in light SR; the monophasic time course characteristic of the heavy fraction became biphasic, similar to that found for light SR in the absence of ruthenium red. Extraction of non-ATPase proteins from the heavy SR vesicles also changed the kinetics of the Ca2+ dissociation-coupled fluorescence decrease in the heavy SR from monophasic to biphasic. These results suggest that the difference between the heavy and light SR Ca2(+)-ATPase in the Ca2+ dissociation kinetics is most probably produced by a ruthenium-red-sensitive interaction of the Ca2(+)-ATPase with additional protein(s), which is/are present exclusively in the heavy SR, rather than being due to a difference inherent to the Ca2(+)-ATPase polypeptide itself.

Postulated role of calsequestrin in the regulation of calcium release from sarcoplasmic reticulum.

Ca2+ release from heavy sarcoplasmic reticulum (SR) vesicles was induced by 2 mM caffeine, and the amount (A) and the rate constant (k) of Ca2+ release were investigated as a function of the extent of Ca2+ loading. Under both passive and active loading conditions, the A value increased monotonically in parallel to Ca2+ loading. On the other hand, k sharply increased at partial Ca2+ loading, and upon further loading, it decreased to a lower level. Since most of the intravesicular calcium appears to be bound to calsequestrin both under passive and under active loading conditions, these results suggest that the kinetic properties of induced Ca2+ release show significant variation depending upon how much calcium has been bound to calsequestrin at the time of the induction of Ca2+ release. An SR membrane segment consisting of the junctional face membrane (jfm) and attached calsequestrin (jfm-calsequestrin complex) was prepared. The covalently reacting thiol-specific conformational probe N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide (DACM) was incorporated into several proteins of the jfm, but not into calsequestrin. The fluorescence intensity of DACM increased with Ca2+. Upon dissociation of calsequestrin from the jfm by salt treatment, the DACM fluorescence change was abolished, while upon reassociation of calsequestrin by dilution of the salt it was partially restored. These results suggest that the events occurring in the jfm proteins are mediated via the attached calsequestrin rather than by a direct effect of Ca2+ on the jfm proteins. We propose that the [Ca2+]-dependent conformational changes of calsequestrin affect the jfm proteins and in turn regulate the Ca2+ channel functions.

Kinetic analysis of excitation-contraction coupling.

Recent studies of isolated muscle membrane have enabled induction and monitoring of rapid Ca2+ release from sarcoplasmic reticulum (SR)5 in vitro by a variety of methods. On the other hand, various proteins that may be directly or indirectly involved in the Ca2+ release mechanism have begun to be unveiled. In this mini-review, we attempt to deduce the molecular mechanism by which Ca2+ release is induced, regulated, and performed, by combining the updated information of the Ca2+ release kinetics with the accumulated knowledge about the key molecular components.