Biomedical subjects

J L Sutko

Publications and source records attributed to J L Sutko.

At least 19 recordsLinked to original sources

PubMed1998

Interactions of a reversible ryanoid (21-amino-9alpha-hydroxy-ryanodine) with single sheep cardiac ryanodine receptor channels.

The binding of ryanodine to a high affinity site on the sarcoplasmic reticulum Ca2+-release channel results in a dramatic alteration in both gating and ion handling; the channel enters a high open probability, reduced-conductance state. Once bound, ryanodine does not dissociate from its site within the time frame of a single channel experiment. In this report, we describe the interactions of a synthetic ryanoid, 21-amino-9alpha-hydroxy-ryanodine, with the high affinity ryanodine binding site on the sheep cardiac sarcoplasmic reticulum Ca2+-release channel. The interaction of 21-amino-9alpha-hydroxy-ryanodine with the channel induces the occurrence of a characteristic high open probability, reduced-conductance state; however, in contrast to ryanodine, the interaction of this ryanoid with the channel is reversible under steady state conditions, with dwell times in the modified state lasting seconds. By monitoring the reversible interaction of this ryanoid with single channels under voltage clamp conditions, we have established a number of novel features of the ryanoid binding reaction. (a) Modification of channel function occurs when a single molecule of ryanoid binds to the channel protein. (b) The ryanoid has access to its binding site only from the cytosolic side of the channel and the site is available only when the channel is open. (c) The interaction of 21-amino-9alpha-hydroxy-ryanodine with its binding site is influenced strongly by transmembrane voltage. We suggest that this voltage dependence is derived from a voltage-driven conformational alteration of the channel protein that changes the affinity of the binding site, rather than the translocation of the ryanoid into the voltage drop across the channel.

Animals↗

PubMed1997

Differential distribution and subcellular localization of ryanodine receptor isoforms in the chicken cerebellum during development.

The distribution of ryanodine receptor (RyR) isoforms was examined using isoform-specific monoclonal antibodies in the developing chicken brain, from E18 through adulthood, using light and electron microscopic immunocytochemistry. Monoclonal antibody 110F is specific for the alpha-skeletal muscle form of RyR, while monoclonal antibody 110E recognizes both the beta-skeletal muscle and cardiac isoforms, but does not distinguish between the two. Significant differences in the distribution of the alpha- and beta/cardiac forms were observed. Labeling for the alpha-form was restricted to cerebellar Purkinje neurons while the beta/cardiac form was observed in neurons throughout the brain. A major finding was the presence of labeling for the beta/cardiac in presynaptic terminals of the parallel fibers in the molecular layer and the mossy fiber terminals in the granular layer glomeruli in late development and during adulthood. Labeling for the beta/cardiac, but not the alpha-form, underwent a major redistribution in the cerebellum during the course of development. At 1 day of age, beta/cardiac labeling was present mainly in Purkinje neurons. From 1 day to 4 weeks, immunolabeling for the beta/cardiac form gradually disappeared from Purkinje neurons, but increased in granule cells. Within the molecular layer, the labeling pattern changed from being primarily within Purkinje dendrites to a more diffuse pattern. Electron microscopic examination of the cerebellar molecular layer of 2-week-old chicks revealed that beta/cardiac-labeling was mainly present in the axons and presynaptic processes of the parallel fibers. No developmental changes were observed in other brain regions. This study represents the first demonstration of ryanodine receptor immunoreactivity in presynaptic boutons and suggests that the ryanodine receptor may modulate neurotransmitter release through local regulation of intracellular calcium in the parallel fiber synapse.

Animals↗

PubMed1997

Structural components of ryanodine responsible for modulation of sarcoplasmic reticulum calcium channel function.

Comparative molecular field analysis (CoMFA) was used to analyze the relationship between the structure of a group of ryanoids and the modulation of the calcium channel function of the ryanodine receptor. The conductance properties of ryanodine receptors purified from sheep heart were measured using the planar, lipid bilayer technique. The magnitude of the ryanoid-induced fractional conductance was strongly correlated to specific structural loci on the ligand. Briefly, electrostatic effects were more prominent than steric effects. The 10-position of the ryanoid had the greatest influence on fractional conductance. Different regions of the ligand have opposing effects on fractional conductance. For example, steric bulk at the 10-position is correlated with decreased fractional conductance, whereas steric bulk at the 2-position (isopropyl position) is correlated with increased fractional conductance. In contrast to fractional conductance, the 3-position (the pyrrole locus) had the greatest influence on ligand binding, whereas the 10-position had comparatively little influence on binding. Two possible models of ryanodine action, a direct (or channel plug) mechanism and an allosteric mechanism, were examined in light of the CoMFA. Taken together, the data do not appear to be consistent with direct interaction between ryanodine and the translocating ion. The data appear to be more consistent with an allosteric mechanism. It is suggested the ryanoids act by inducing or stabilizing a conformational change in the ryanodine receptor that results in the observed alterations in cation conductance.

Allosteric Regulation↗

PubMed1997

The pharmacology of ryanodine and related compounds.

The goal of this review has been to describe the current state of the pharmacology of ryanodine and related compounds relative to the vertebrate RyRs. Resolution of questions concerning the molecular properties of RyR channel function and the contributions made by the RyR isoforms to cellular signaling in a variety of tissues will require the production of new pharmacological agents directed against these proteins. Novel naturally occurring ryanodine congeners have been identified, and significant advances have been made in developing chemical approaches that permit the structure of ryanodine to be derivatized in selective ways. Moreover, several of these changes have yielded compounds that differ in their binding affinities and in their abilities to modify the properties of the RyR channels. These advances give substance to the possibility of designing the required pharmacological agents based on rational design changes of the structure ryanodine.

Acylation↗

PubMed1996

The pyrrole locus is the major orienting factor in ryanodine binding.

Ryanodine, a natural product, is a complex modulator of a class of intracellular Ca2+ release channels commonly called the ryanodine receptors. Ryanodine analogs can cause the channel to persist in long-lived, subconductance states or, at high ligand concentrations, in closed, nonconducting states. In this paper, we further explore the relationship between structure and ryanodine binding to striated muscle. Ryanodine, 3-epiryanodine, and 10-ryanodine are three structural isomers of ryanodine. The dissociation constants of these compounds were measured using rabbit skeletal muscle ryanodine receptors. Placing the pyrrole carbonyl group at the 3-epi- and 10-positions of ryanodol largely restores the large loss of binding energy observed when ryanodine is hydrolyzed to ryanodol. Comparative molecular field analysis successfully predicts the enhanced binding and indicates that the pyrrole group controls the orientation of ligand binding. We propose that the ryanoids are reorientated in the binding site of the ryanodine receptors such that the pyrrole always occupies the same subsite. By applying this model, the binding constants of other ryanoids are predicted.

Animals↗

PubMed1996

Electrophysiological effects of ryanodine derivatives on the sheep cardiac sarcoplasmic reticulum calcium-release channel.

We have examined the effects of a number of derivatives of ryanodine on K+ conduction in the Ca2+ release channel purified from sheep cardiac sarcoplasmic reticulum (SR). In a fashion comparable to that of ryanodine, the addition of nanomolar to micromolar quantities to the cytoplasmic face (the exact amount depending on the derivative) causes the channel to enter a state of reduced conductance that has a high open probability. However, the amplitude of that reduced conductance state varies between the different derivatives. In symmetrical 210 mM K+, ryanodine leads to a conductance state with an amplitude of 56.8 +/- 0.5% of control, ryanodol leads to a level of 69.4 +/- 0.6%, ester A ryanodine modifies to one of 61.5 +/- 1.4%, 9,21-dehydroryanodine to one of 58.3 +/- 0.3%, 9 beta,21beta-epoxyryanodine to one of 56.8 +/- 0.8%, 9-hydroxy-21-azidoryanodine to one of 56.3 +/- 0.4%, 10-pyrroleryanodol to one of 52.2 +/- 1.0%, 3-epiryanodine to one of 42.9 +/- 0.7%, CBZ glycyl ryanodine to one of 29.4 +/- 1.0%, 21-p-nitrobenzoyl-amino-9-hydroxyryanodine to one of 26.1 +/- 0.5%, beta-alanyl ryanodine to one of 14.3 +/- 0.5%, and guanidino-propionyl ryanodine to one of 5.8 +/- 0.1% (chord conductance at +60 mV, +/- SEM). For the majority of the derivatives the effect is irreversible within the lifetime of a single-channel experiment (up to 1 h). However, for four of the derivatives, typified by ryanodol, the effect is reversible, with dwell times in the substate lasting tens of seconds to minutes. The effect caused by ryanodol is dependent on transmembrane voltage, with modification more likely to occur and lasting longer at +60 than at -60 mV holding potential. The addition of concentrations of ryanodol insufficient to cause modification does not lead to an increase in single-channel open probability, such as has been reported for ryanodine. At concentrations of > or = 500 mu M, ryanodine after initial rapid modification of the channel leads to irreversible closure, generally within a minute. In contrast, comparable concentrations of beta-alanyl ryanodine do not cause such a phenomenon after modification, even after prolonged periods of recording (>5 min). The implications of these results for the site(s) of interaction with the channel protein and mechanism of the action of ryanodine are discussed. Changes in the structure of ryanodine can lead to specific changes in the electrophysiological consequences of the interaction of the alkaloid with the sheep cardiac SR Ca2+ release channel.

Animals↗

PubMed1996

Ryanodine receptor Ca2+ release channels: does diversity in form equal diversity in function?

Complexities in calcium signaling in eukaryotic cells require diversity in the proteins involved in generating these signals. In this review, we consider the ryanodine receptor (RyR) family of intracellular calcium release channels. This includes species, tissue, and cellular distributions of the RyRs and mechanisms of activation, deactivation, and inactivation of RyR calcium release events. In addition, as first observed in nonmammalian vertebrate skeletal muscles, it is now clear that more than one RyR isoform is frequently coexpressed within many cell types. How multiple ryanodine receptor release channels are used to generate intracellular calcium transients is unknown. Therefore, a primary focus of this review is why more than one RyR is required for this purpose, particularly in a tissue, such as vertebrate fast-twitch skeletal muscles, where a relatively simple and straightforward change in calcium would appear to be required to elicit contraction. Finally, the roles of the RyR isoforms and the calcium release events they mediate in the development of embryonic skeletal muscle are considered.

Animals↗

PubMed1995

Embryonic chicken skeletal muscle cells fail to develop normal excitation-contraction coupling in the absence of the alpha ryanodine receptor. Implications for a two-ryanodine receptor system.

Two ryanodine receptor (RyR), sarcoplasmic reticulum Ca2+ release channels, alpha and beta, co-exist in chicken skeletal muscles. To investigate a two-RyR Ca2+ release system, we compared electrically evoked Ca2+ transients in Crooked Neck Dwarf (cn/cn) cultured muscle cells, which do not make alpha RyR, and normal (+/?) cells. At day 3 in culture, Ca2+ release in +/? cells required extracellular Ca2+ (Ca2+o), and Ca2+ transients had slow kinetics. At day 5, Ca2+ release was Ca2+o-independent in 40% of the cells, and transients were more rapid. By day 7, all +/? cells had Ca2+o-independent Ca2+ release. Contractions were observed in +/? cells on all days. Ca2+ transients were observed in cn/cn cells on days 3, 5, and 7, but in each case they were Ca2+o-dependent and exhibited slow kinetics. Localized vesiculations, not contractions, occurred in cn/cn cells. By day 10, Ca2+ transients were no longer observed in cn/cn cells even in Ca2+o. Sarcoplasmic reticulum Ca2+ was not depleted, as caffeine induced Ca2+ transients. Thus, in the absence of alpha RyR there is a failure to develop Ca2+o-independent Ca2+ release and contractions and to sustain Ca2+o-dependent release. Moreover, contributions by the alpha RyR cannot be duplicated by the beta RyR alone.

Animals↗

PubMed1995

Interaction between ryanodine receptor function and sarcolemmal Ca2+ currents.

We used the whole cell voltage-clamp technique to investigate the effects of disruption of Ca2+ release from the sarcoplasmic reticulum (SR) on sarcolemmal Ca2+ currents of chick myotubes kept in culture for 7 or 8 days. Ca2+ currents were recorded in 145 mM tetraethylammonium chloride and 10 mM Ca2+ with pipettes containing cesium and 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. We found two components of Ca2+ current: 1) relatively large T-type currents that were activated near -50 mV and inactivated during 100-ms depolarizations to potentials positive to -60 mV (they were of similar magnitude in Ba2+ or Ca2+ and were insensitive to nifedipine) and 2) L-type currents that were activated near 0 mV and showed little or no inactivation during 100-ms depolarizations (they were larger when Ba2+ was the charge carrier and were blocked by 10 microM nifedipine). Addition of 1 or 100 microM ryanodine to the culture medium for 6-7 days caused a modest but significant increase in the L-type Ca2+ current density (pA/pF). Ryanodine (1 or 100 microM) exposure for 1-7 days reduced the T-type Ca2+ current density to < 10% of control. In contrast, exposure to 1 microM ryanodine for 0.5-3 h had no significant effect on either component of Ca2+ current. These data indicate that ryanodine has no direct action on Ca2+ currents in chick myotubes. However, disruption of SR Ca2+ release for > 24 h changes sarcolemmal Ca2+ channel expression or function.

Animals↗

PubMed1994

High affinity C10-Oeq ester derivatives of ryanodine. Activator-selective agonists of the sarcoplasmic reticulum calcium release channel.

The plant alkaloids ryanodine and dehydroryanodine are specific and potent modulators of the sarcoplasmic reticulum calcium release channel. In the present study, acidic, basic, and neutral side chains esters of these diterpene compounds were prepared and their pharmacologic activities were assessed. Binding affinities of the novel C10-Oeq ester derivatives for the sarcoplasmic reticulum Ca2+ release channel were evaluated with sarcoplasmic reticular vesicles prepared from rabbit skeletal muscle. Kd values of the derivatives varied 500-fold, ranging from 0.5 to 244 nM. In comparison, Kd values for ryanodine and dehydroryanodine were 4.4 nM and 5.4 nM, respectively. Basic substituents at the C10-Oeq side chain terminus produced the highest affinity derivatives (Kd values from 0.5 to 1.3 nM). Neutral and/or hydrophobic side chain derivatives exhibited intermediate affinities for the high affinity ryanodine receptor site (Kd values from 2.5 to 39 nM), whereas a derivative with a terminal acidic group had the lowest affinity (Kd value > 100 nM). Certain of the higher affinity C10-Oeq derivatives were evaluated more extensively for their pharmacologic activity on the sarcoplasmic reticular Ca2+ release channel. Both channel activating (opening) and deactivating (closing) actions were assessed from the ability of the ryanoids to alter Ca2+ efflux rates from skeletal junctional sarcoplasmic reticular vesicles that had been passively loaded with Ca2+. The natural Ryania secondary metabolites ryanodine, dehydroryanodine and esters E and F, all exhibit antithetical concentration-effect curves, indicating both activator and deactivator actions. In contrast, the semi-synthetic C10-Oeq esters selectively activate the Ca2+ release channel. Half-maximal concentrations for such activation (EC50 act) ranged from 0.87 microM to 4.2 microM, compared with an EC50 act of 1.3 microM for ryanodine. These derivatives were also evaluated for their ability to augment ATP-dependent CA2+ accumulation by cardiac junctional sarcoplasmic reticular vesicles, an effect that results from deactivation of the Ca2+ release channels. None of the derivatives tested was able to significantly augment Ca2+ accumulation, further substantiating their inability to deactivate the sarcoplasmic reticular Ca2+ release channel. Additionally, these derivatives functionally antagonized the action of ryanodine to close the Ca2+ release channel. The results presented demonstrate that these C10-Oeq ester derivatives of ryanodine and dehydroryanodine bind specifically to the SR Ca2+ release channel, selectively activate the channel, and, although they fail to effect channel closure, they nevertheless functionally compete with ryanodine at its low affinity (deactivator) site(s).

Adenosine Triphosphate↗

PubMed1994

Structural determinants of high-affinity binding of ryanoids to the vertebrate skeletal muscle ryanodine receptor: a comparative molecular field analysis.

Ryanodine binds to specific membrane proteins, altering the calcium permeability of intracellular membranes. In this study 19 ryanoids were isolated or synthesized and the structures correlated to the strength of binding to vertebrate skeletal muscle ryanodine receptors. Global minima were determined by employment of molecular mechanics and dynamics augmented by systematic searching of conformational space. Overall, steric and electrostatic factors contribute about equally to the differences in the experimentally determined dissociation constants. The dominant electrostatic interaction is localized to a hydroxyl group in an apolar region of the molecule. The pyrrole and isopropyl groups located together at one pole of the molecule have the greatest effect on steric interactions between ligand and receptor. We suggest ryanodine binds to the receptor with the pyrrole and isopropyl groups buried deep inside a cleft in the protein. This arrangement places special importance on the conformation of the pyrrole and isopropyl groups. In contrast, the opposite pole appears to be positioned at the entrance of the binding pocket because bulky adducts placed in the 9 position of ryanodine alter binding minimally. For example, a fluorescent ryanodine adduct was synthesized which has a dissociation constant close to that of ryanodine. Detailed examination reveals subtle interactions between ryanoid and receptor. In many cases, the major factors altering the strength of binding were found to be conformational alterations in the molecule remote from the site of covalent modification.

Animals↗

PubMed1994

Chicken skeletal muscle ryanodine receptor isoforms: ion channel properties.

To define the roles of the alpha- and beta-ryanodine receptor (RyR) (sarcoplasmic reticulum Ca2+ release channel) isoforms expressed in chicken skeletal muscles, we investigated the ion channel properties of these proteins in lipid bilayers. alpha- and beta RyRs embody Ca2+ channels with similar conductances (792, 453, and 118 pS for K+, Cs+ and Ca2+) and selectivities (PCa2+/PK+ = 7.4), but the two channels have different gating properties. alpha RyR channels switch between two gating modes, which differ in the extent they are activated by Ca2+ and ATP, and inactivated by Ca2+. Either mode can be assumed in a spontaneous and stable manner. In a low activity mode, alpha RyR channels exhibit brief openings (tau o = 0.14 ms) and are minimally activated by Ca2+ in the absence of ATP. In a high activity mode, openings are longer (tau o1-3 = 0.17, 0.51, and 1.27 ms), and the channels are activated by Ca2+ in the absence of ATP and are in general less sensitive to the inactivating effects of Ca2+. beta RyR channel openings are longer (tau 01-3 = 0.34, 1.56, and 3.31 ms) than those of alpha RyR channels in either mode. beta RyR channels are activated to a greater relative extent by Ca2+ than ATP and are inactivated by millimolar Ca2+ in the absence, but not the presence, of ATP. Both alpha- and beta RyR channels are activated by caffeine, inhibited by Mg2+ and ruthenium red, inactivated by voltage (cytoplasmic side positive), and modified to a long-lived substate by ryanodine, but only alpha RyR channels are activated by perchlorate anions. The differences in gating and responses to channel modifiers may give the alpha- and beta RyRs distinct roles in muscle activation.

Adenosine Triphosphate↗

PubMed1993

Distribution of ryanodine receptors in the chicken central nervous system.

The ryanodine receptor (RR), an intracellular calcium release channel, has been identified in the nervous system but its contributions to neuronal function are unknown. We have utilized immunohistochemical techniques to establish the distribution of RRs in the central nervous system (CNS) of the chick as a step toward elucidating the function of RRs in this system. RR immunoreactivity is observed throughout the brain, most prominently in large neurons. The strongest immunoreactivity is found in cerebellar Purkinje neurons, but nuclei in the motor, visual and vestibular systems are also intensely labeled, and immunoreactive neurons are observed the olfactory bulb and the hippocampus. In these neurons, labeling is prominent in cell bodies, dendrites and axons, but is not observed in the dendritic spines or in plasma membranes. The neuronal RRs bind [3H]ryanodine with high affinity and this activity is regulated by calcium, caffeine, MgCl2/ATP and ionic strength. Multiple forms of the RRs are found in the chicken CNS. Immunoprecipitation and localization studies using RR isoform specific monoclonal antibodies reveal major differences in their distribution. The predominant species in the cerebellum is similar to the skeletal muscle isoform while there is a lower level of expression of either the cardiac or beta skeletal isoforms. In the remainder of the brain, the predominant isoform is similar to the cardiac or beta skeletal muscle isoforms. The broad distribution of RRs in the CNS suggests that calcium release events mediated by these proteins may have a functional role in a diverse array of neurons. Moreover within the populations of neurons expressing RR's, the presence of specific RR isoforms may correlate with specialization in the calcium release events mediated by these proteins.

Animals↗

PubMed1993

Three ryanodine receptor isoforms exist in avian striated muscles.

Two isoforms of the ryanodine receptor (termed alpha and beta) are coexpressed in avian fast twitch skeletal muscle, whereas a single isoform is expressed in avian cardiac muscle. We have investigated the relationship between these three proteins, comparing several different properties. First, the three receptor isoform subunits have different mobilities on SDS-polyacrylamide gels. Second, monoclonal antibodies against the chicken skeletal muscle receptor isoforms recognize shared and unique epitopes in each receptor protein, indicating there is not a simple antigenic relationship between the isoforms. Third, the three receptor isoforms exhibit different susceptibilities to proteolysis by trypsin, and limited tryptic digestion yields a different peptide map for each isoform. Fourth, in native sarcoplasmic reticulum membranes, the chicken muscle receptor isoforms are phosphorylated to different extents by the multifunctional calcium/calmodulin-dependent protein kinase II (beta > cardiac > alpha). Fifth, the sites phosphorylated by the calcium/calmodulin-dependent protein kinase in the chicken cardiac and skeletal receptor isoforms are not equivalent. A polyclonal serum, produced against a synthetic peptide containing the site phosphorylated by this kinase in the mammalian cardiac muscle receptor, by immunoprecipitation showed markedly different avidities for the receptor isoforms, and recognized only the cardiac receptor isoform on Western blots. Sixth, the chicken ryanodine receptor isoforms differ in the extent to which they bind azido[125I]calmodulin (alpha > beta > cardiac). These results indicate that three distinct ryanodine receptor proteins are expressed in chicken striated muscles.

Animals↗

PubMed1993

Amino- and guanidinoacylryanodines: basic ryanodine esters with enhanced affinity for the sarcoplasmic reticulum Ca(2+)-release channel.

Amino- and guanidinoacyl esters of ryanodine were prepared to evaluate the effect of basicity on the binding affinity of these derivatives for the sarcoplasmic reticulum Ca(2+)-release channel (SR CRC). In the presence of DCC and DMAP Cbz-beta-alanine reacts with ryanodine in CH2Cl2 to give O10eq-Cbz-beta-alanylryanodine (3a), which on hydrogenolysis yields the beta-alanyl ester (4a). N,N'-bis-Cbz-S-methylthiourea reacts with 4a to yield beta-N,N'-bis-Cbz-guanidinopropionylryanodine (5a). O10eq-beta-guanidinopropionylryanodine (6a) is obtained on hydrogenolytic deprotection of 5a. The binding affinity of beta-alanine ester (4a) and its glycyl congener (4b) is 2-3-fold greater, and that of the beta-guanidinopropionyl ester (6a) and its acetyl congener (6b) 3-6-fold greater, than that of ryanodine. The effect of ryanodine on SR Ca2+ flux is of a biphasic nature: nanomolar levels open (activate) the channel, while micromolar levels close (deactivate) it. The base-substituted esters 4a and 6a both display a unidirectional effect: they only open the channel. An understanding of ryanodine's mode of action and the design of effective SR CRC activating and deactivating ryanoids for possible therapeutic application are major research objectives.

Amino Acids↗

PubMed1993

Failure to make normal alpha ryanodine receptor is an early event associated with the crooked neck dwarf (cn) mutation in chicken.

We have investigated the molecular basis of the Crooked Neck Dwarf (cn) mutation in embryonic chickens. Using biochemical and pharmacological techniques we are unable to detect normal alpha ryanodine receptor (RyR) protein in intact cn/cn skeletal muscle. Extremely low levels of alpha RyR immunoreactivity can be observed in mutant muscles, but the distribution of this staining differs from that in normal muscle and colocalizes with the rough endoplasmic reticulum immunoglobulin binding protein, BiP. This suggests the existence of an abnormal alpha RyR protein in mutant muscle. In day E12 cn/cn muscle the levels of RyR mRNA are reduced by approximately 80%, while the levels of other muscle proteins, including the alpha 1 subunit of the dihydropyridine receptor, the SR Ca(2+)-ATPase, calsequestrin, and glyceraldehyde-3-phosphate dehydrogenase, and their associated mRNAs are essentially normal in cn/cn muscle. There is also a failure to express alpha RyR in cn/cn cerebellar Purkinje neurons. Expression of the beta RyR, a second RyR isoform, is not initiated in normal skeletal muscle until day E18. In cn/cn skeletal muscle significant muscle degeneration has occurred by this time and the beta RyR is found at low levels in only a subset of fibers suggesting the reduced levels of this isoform are a secondary consequence of the mutation. The cardiac RyR isoform is found in cn/cn cardiac muscle, which contracts in a vigorous manner. In summary, a failure to make normal alpha RyR receptor appears to be an event closely associated with the cn mutation and one which may be largely responsible for development of the cn/cn phenotype in embryonic skeletal muscle.

Animals↗

PubMed1993

Crooked neck dwarf (cn) mutant chicken skeletal muscle cells in low density primary cultures fail to express normal alpha ryanodine receptor and exhibit a partial mutant phenotype.

The Crooked Neck Dwarf (cn) mutation in chickens causes marked changes in intact embryonic skeletal muscle. We have investigated whether the cn/cn phenotype develops in vitro, and if cultured muscle cells are suitable for studies of this mutation. The properties of cn/cn muscle cells maintained in low density primary cultures (6.25 x 10(3) cells/cm2) are described in this report. In normal muscle cells, the alpha ryanodine receptor (RyR) isoform appears prior to, and at greater levels than, the beta RyR, and is detected in mononucleated myocytes. The beta RyR isoform appears within 24 hr after the initiation of myotube formation, which is earlier than anticipated from studies with intact embryonic muscle. Normal alpha RyR protein is not detected in cultured cn/cn muscle cells, whereas the beta RyR, the alpha 1-subunit of the dihydropyridine receptor, the sarcoplasmic reticulum Ca(2+)-ATPase, and calsequestrin are expressed at comparable levels in normal and mutant muscle cells. Calcium transients elicited by electrical stimulation, acetylcholine, and caffeine are similar in normal and cn/cn cultured myotubes and are blocked by ryanodine in both cell types. In addition, comparable L- and T-type calcium currents are observed in normal and mutant muscle cells, suggesting that both the alpha 1-subunit of the dihydropyridine receptor and the beta RyR in mutant muscle cells are functional. Normal and cn/cn muscle cells proliferate and form myotubes in a similar manner. These latter events do not appear to depend on sarcoplasmic reticulum calcium release, as they also occur in normal muscle cells in which calcium release is prevented by chronic treatment with 100 microM ryanodine. Both cn/cn and ryanodine-treated normal muscle cells exhibit morphological changes similar to those observed in intact cn/cn skeletal muscle. Thus, the mutant phenotype observed in ovo is partially expressed under low density culture conditions, and neither beta RyR protein nor its function appear to be capable of preventing the associated changes.

Animals↗

PubMed1993

Ryanodine receptor expression in embryonic avian cardiac muscle.

The cardiac ryanodine receptor serves as a sarcoplasmic reticulum calcium release channel and contributes to the rise in cytosolic calcium necessary for contraction of the heart. We have investigated the presence and oligomeric form of the ryanodine receptor during embryonic development of the avian heart. A single isoform of the ryanodine receptor was identified both in adult and in embryonic cardiac tissue from Days 4 to 20 of development, using anti-receptor monoclonal antibodies in conjunction with [3H]ryanodine binding. The results of sucrose density gradient sedimentation analysis and [3H]ryanodine binding indicated that the cardiac ryanodine receptor is present in a tetrameric form in both the adult and embryos at Day 6 of development. The observation of specific [3H]ryanodine binding in hearts from Days 4 and 5 of embryonic development also indicates the presence of a tetrameric receptor protein. Although the heart begins to beat at approximately 33-38 hr (Day 1.5) of embryonic development, we were unable to detect the cardiac ryanodine receptor, using biochemical or immunological techniques, prior to Embryonic Day 4. Ryanodine was found to alter the chronotropic state of intact hearts as early as Hamburger and Hamilton stages 15-19 (Embryonic Day 3) suggesting that receptor protein may be present in a limited subset of cells involved in pacemaker activity in the very early embryo. After Embryonic Day 4, ryanodine also exerted a negative inotropic effect on embryonic hearts. Our results suggest that a single isoform of the ryanodine receptor is present from Day 4 to Day 20 of embryonic development in avian cardiac muscle and that the ryanodine receptor assumes a tetrameric structure capable of forming a functional calcium release channel that participates excitation-contraction coupling as early as Embryonic Day 4. In addition, a ryanodine receptor-related function may serve as a determinant of chronotropic effects in the very early embryonic heart.

Animals↗