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.

Effects of streptozotocin diabetes on the noradrenergic innervation of the rat heart: a longitudinal histofluorescence and neurochemical study.

The effects of the age of induction and total duration of streptozotocin diabetes on the sympathetic noradrenergic innervation of the rat heart was examined with glyoxylic acid induced histofluorescence to demonstrate the distribution of noradrenergic fibers within the heart, and with high performance liquid chromatography with electrochemical detection to measure tissue levels of the neurotransmitter norepinephrine. Diabetes was induced in male Sprague-Dawley rats at 1, 2, and 4 months of age. Within each of these groups, diabetic rats survived for periods of 1, 2, and 4 months. Additional groups of diabetic rats survived to a chronological age of 8 months. Norepinephrine levels in the hearts of diabetic rats were increased over those of control rats in all groups at 1 month duration of diabetes. Ventricles were generally affected to a greater extent than atria. At 2 months duration of diabetes, ventricular levels remained elevated while atrial norepinephrine levels were at or below control levels. At 4 months duration of diabetes, and in all groups at 8 months of age, the norepinephrine levels were at or below control levels, except in the ventricles of rats induced at 4 months of age, which remained elevated. Histofluorescence studies demonstrated the presence of axon bundles and varicose noradrenergic profiles in the diabetic rat hearts, distributed in a pattern similar to that seen in controls. However, at 1 month duration of diabetes in all groups, the density of noradrenergic varicosities in diabetic rat hearts appeared increased with abundant branched profiles. These results are surprising, since studies on genetic models of diabetes have suggested decreased norepinephrine levels in the heart. The present study suggests that during the early phases of streptozotocin induced diabetes, noradrenergic nerves are still intact and may be susceptible to pharmacologic manipulation. The later fall of norepinephrine levels back to or below control levels may indicate actual neuronal damage, suggesting that early intervention may be necessary to protect these nerves from degeneration. This issue is potentially important in view of the reported toxic effects of high NE levels on the heart, and the high incidence of death from myocardial infarct in diabetic humans with autonomic neuropathy.

Effect of experience on medical students' attitudes toward animal laboratories in pharmacology education.

BACKGROUND: Medical students' attitudes toward the use of animal laboratories in pharmacology courses may form a useful source of evaluative information about the laboratories' educational effectiveness. METHOD: In 1992-93, 144 second-year students at the Indiana University School of Medicine were surveyed--before and after completing four hands-on laboratories using dogs--for their assessments of educational and moral aspects of animal laboratories. Statistical analysis involved chi-square and Student's t test. RESULTS: Of the 144 students in the course, 143 responded to the first survey and 86 responded to the second. From before to after the lab experiences, the percentage of students who agreed that the labs would reinforce/had reinforced the lecture material increased from 38% to 69%. In both surveys, 10% of the students objected to the use of any animals in labs, and 24% (before) and 21% (after) objected to the use of dogs. Whereas the percentage agreeing that the labs involved a morally wrong use of animals rose from 11% to 22%, the percentage disagreeing with that notion rose from 53% to 61%. Between 50% and 60% of the students in both surveys opposed doing the labs by computer simulation or videotaped demonstration. CONCLUSION: Most students indicated that the laboratory experiences enhanced their understanding of the actions of drugs, were preferable to alternatives that did not use animals, and did not involve an immoral use of animals. On the other hand, the results suggest that the number of students who have negative feelings about the use of animals in laboratories, though small, tends to be larger than the number who express these feelings to faculty.

Cardiotonic steroids II: 3-deoxycardenolides and 3-deoxycardanolides.

A series of compounds related to 3-deoxydigitoxigenin was prepared and assayed for inhibition of myocardial Na+,K+- adenosine triphosphatase. Although the relatively high activity of 3-deoxydigitoxin was confirmed, the corresponding 3beta,4beta-epoxide and a mixture of 2,3-olefins and 3,4-olefins were less active. 3-Deoxy compounds with variations at the 14-position and the butenolide ring were much less active than the corresponding 3beta-hydroxy analogs. Thus the activity of 3-deoxydigitoxigenin appears to be particularly susceptible to structural changes elsewhere in the molecule.

Effects of ryanodine on cardiac subcellular membrane fractions.

Both the rate and the steady-state magnitude of net calcium accumulation by cardiac sarcoplasmic reticulum (SR) vesicles are increased by ryanodine. Sarcolemmal calcium transport mechanisms are not affected. The apparent augmentation of calcium accumulation by membrane vesicles from junctional SR derives not from an increase in the rate at which calcium is pumped into the vesicles, but from a slowing of the rate of calcium efflux. Recent results show that decamethonium blunts these effects of ryanodine, whereas valinomycin potentiates them. The mechanisms for these latter effects are not well understood, but may involve limitation and promotion, respectively, of access of potassium ion to the interior of the membrane vesicles.

Calcium handling by cardiac sarcoplasmic reticulum.

Over the past ten years knowledge of the biochemistry of cardiac sarcoplasmic reticulum vesicles has been considerably extended. In almost all respects the Ca2+ pump of cardiac sarcoplasmic reticulum shows striking similarities to the Ca2+ pump of skeletal muscle sarcoplasmic reticulum. On the other hand, Ca2+ release mechanisms seem to be more complex in cardiac than in skeletal sarcoplasmic reticulum. Future research undoubtedly will involve characterizing the function of the many additional proteins present in cardiac sarcoplasmic reticulum, and determining possible roles for these proteins in regulating Ca2+ uptake and Ca2+ release by these membranes.

The relationship between adenosine 3',5'-monophosphate levels and systolic transmembrane calcium flux.

The effects of inotropic agents on systolic transmembrane Ca++ flux were studied in isolated perfused guinea pig hearts depolarized with 22 mM K+. After depolarization, inotropic agents were added to the perfusion medium in an attempt to resotre excitability and contractions to the heart. Electrophysiological studies have shown that if excitability can be restored to the heart under these conditions, Ca++ carries the inward current of the action potential. Agents which putatively act by increasing intracellular levels of adenosine 3',5'-monophosphate (cyclic AMP) (isoproterenol, histamine, theophylline, papaverine, dibutyryl cyclic AMP) and Ca++ were found to restore excitability and contractions to K+-depolarized hearts. Threshold concentration for restoration by isoproterenol was 2 nM, and the strength of contractions developed by the restored heart were directly related to the dose of catecholamine used. Ca++ restoration was abolished by Ca++ antagonists (D 600, verapamil) but unaffected by beta blockade, whereas isoproterenol restoration was abolished by both. Ouabain, glucagon, and the divalent cation ionophore, A32187, failed to restore excitability to depolarized hearts. Correlative studies of tissue cyclic AMP levels were done. These data suggest that cyclic AMP can activate action potential-dependent Ca++ influx channels in myocardial cells.

Role of free calcium and ATP in calcium release from cardiac sarcoplasmic reticulum fragments.

Using both isotopic and spectrophotometric techniques to monitor the overall Ca++ binding and release reactions, we have studied the effects of the relative free concentrations of Ca++, sarcoplasmic reticulum fragments (SRF) protein, and ATP on spontaneous Ca++ release by SRF isolated from normal canine hearts. Low free [Ca++] was achieved by using a Ca-EGTA buffer; higher (micromolar) values were obtained by direct Ca++ additions throughout the reactions. ATP concentrations were stabilized by an ATP-regenerating system or an ATP trap. Concentrations of nucleotides in the reaction mixtures were also determined. Release depended in part on the [Ca++]/[ATP] and on the [ATP]. Spontaneous Ca++ release was prevented or slowed when the [ATP] was rapidly lowered with the ATP trap. ATP regeneration delayed, but did not prevent Ca++ release. Ca++-induced Ca++ release was found in the presence of ADP and ATP. Verapamil and D 600 did not affect release. The results may provide a basis for the slowed Ca++ release in SRF from failing hearts.

Structure-function relationships among ryanodine derivatives. Pyridyl ryanodine definitively separates activation potency from high affinity.

Ryanodine derivatives are differentially effective on the two limbs of the ryanodine concentration-effect curve. This study comparing ryanodine, ryanodol, and pyridyl ryanodine and nine C10Oeq esters of them focuses on structure-function relations underlying their differential effectiveness. Ryanodol and pyridyl ryanodine had significantly lower affinities than ryanodine, but their EC50act values (concentration of ryanoid that induces one-half of full efficacy), potencies, and efficacies were not diminished in like fashion. Ryanodine and ryanodol were partial agonists, whereas pyridyl ryanodine was a full agonist, having a diminished deactivation potency. C10Oeq esterifications enhanced affinities and efficacies of the base ryanoids. The C10-Oeq ester derivatives of ryanodine and pyridyl ryanodine, but not those of ryanodol, lost their capacity to deactivate RyR1s. Thus, affinity differences among ryanoids clearly do not predicate functional differences as regards activation of Ca2+ release channels. The pyrrole carboxylate on the C3 of ryanodine is dispensable to ryanoid activation of Ca2+ release channels. Ryanodol lacks this ring, but it nevertheless effects substantial activation. Moreover, its C10-Oeq esters display full efficacy. The increased ability of all the C10-Oeq derivatives to release Ca2+ from the vesicles strengthens their role in directly impeding deactivation of RyR1, perhaps by interaction with some component within the transmembrane ionic flux pathway.

Interaction between cyclic adenosine monophosphate and cyclic gunaosine monophosphate in guinea pig ventricular myocardium.

The purpose of this study was to examine the mechanisms underlying adrenergic-cholinergic antagonism in ventricular myocardium. Myocardial contractility, cyclic adenosine monophosphate (AMP) levels, and cyclic guanosine monophosphate (GMP) levels were measure in isolated guinea pig ventricles after treatment with various inotropic agents given alone and simultaneously with acetylcholine. Acetylcholine alone markedly elevated cyclic GMP levels but did not substantially change myocardial contractility. However, the same concentration of acetylcholine significantly attenuated the inotropic effect of isoproterenol and histamine, two drugs that may act by increasing myocardial levels of cyclic AMP. The decrease in the inotropic response to isoproterenol did not appear to be due to a decrease in the generation of cyclic AMP, because cyclic AMP levels were similar in hearts receiving isoproterenol alone and those receiving isoproterenol with acetylcholine. Dibutyryl cyclic GMP also antagonized the intropic action of isoproterenol. Acetylcholine did not alter the inotropic effects of ouabain, an agent that increases myocardial contractility without changing cyclic AMP levels. These results suggest that cyclic GMP mediates the antiadrenergic effects of acetylcholine by specifically antagonizing the inotropic actions of cyclic AMP.

Binding and effect of tritiated quinidine on cardiac subcellular enzyme systems: sarcomplasmic reticulum vesicles, mitochondria and Na+, K+-adenosine triphosphatase.

A tritium-labeled derivative of quinidine (D3HQ) was used to assess binding and effect of this drug on isolated membrane preparations from myocardium. D3HG bound to sarcoplasmic reticulum vesicles (SRV) and diminished both Ca++ binding and Ca++ uptake activity. Binding of more than 19 nmol of D3HQ were required to displace 1 nmol of Ca++. Dual-wavelength spectrophotometric methods for monitoring the alterations in Ca++ binding showed that D3HQ depressed maximal Ca++ binding and hastened the onset of Ca++ release from Ca++-loaded SRV, but did not alter the maximal rate of Ca++ release. D3HG also diminished Ca++ sequestration by isolated cardiac mitochondria but the level of D3HQ binding did not correlate with the degree of inhibition. Binding of D3HQ to Na+, K+-adenosine triphosphatase also occurred to a limited extent and a partial inhibition of enzyme activity resulted. A reciprocal relationship between D3HQ binding and a decrease in functional activity of the subcellular membrane systems could be demonstrated only for SRV. The results suggest that cinchona alkaloids might affect myocardial contractility by their effects on Ca++ handling by SRV.

Unmasking effect of alamethicin on the (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles.

A mechanism for the activating effect of alamethicin on membrane enzymes was investigated, using a purified preparation of cardiac sarcolemmal vesicles. (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities were measured. alamethicin increased ouabain-sensitive (Na+,K+)-ATPase activity of sarcolemmal vesicles 5- to 7-fold and adenylate cyclase activity 2.5- to 4-fold. Adenylate cyclase retained its sensitivity to the beta-adrenergic agonist isoproterenol after membranes were treated with alamethicin. Alamethicin caused a 4- to 6-fold increase in the number of detectable (Na+,K+)-ATPase enzymic sites, but no increase ws noted for the number of muscarinic-cholinergic receptor-binding sites. Phosphorylation of endogenous proteins of sarcolemmal vesicles by an intrinsic cAMP-dependent protein kinase activity was stimulated 5- to 7-fold by alamethicin. The regulatory subunit of the membrane-bound cAMP-dependent protein kinase was labeled with the photoaffinity probe 8-azido-adenosine 3':5'[32P]monophosphate (8-N3-[32P]cAMP), and it migrated with an apparent molecular weight of 55,000 in sodium dodecyl sulfate polyacrylamide gels. Alamethicin stimulated autophosphorylation of the regulatory subunit by [gamma-32P]ATP 6-fold and incorporation of of 8-N3-[32P]cAMP into the subunit 2.6-fold. The results suggest that alamethicin disrupts membrane barriers of sarcolemmal vesicles, which are mostly right side out, giving substrates and activators access to enzymic sites in the interior of the vesicles, while preserving functional coupling of enzymes to their effectors.

Intact vesicles of canine cardiac sarcolemma: evidence from vectorial properties of Na+, K+-ATPase.

Most biological membranes are functionally asymmetric. To study biochemical control of cardiac transsarcolemmalion fluxes, it would be of obvious advantage to use isolated vesicles of sarcolemma which retains the low passive permeability characteristics of intact sarcolemma because in such vesicles the membrane should exhibit its normal asymmetric character with respect to enzymic activities. The purpose of this investigation was to attempt identify such vesicles in a cardiac microsomal (membrane vesicular) preparation. We studied activation by Na+ and K+ of Na+, K+-ATPase and its associated K+-phosphatase activities, using as substrates ATP or p-nitrophenylphosphate (pNPP) in the presence of Mg2+. Optimal concentrations of K+ alone (10 mM) stimulated p-nitrophenylphosphatase (pNPPase) activity 1.8-fold, and over 80% of the increase could be inhibited by ouabain. Optimal Na+ plus K+ concentrations (100 mM and 10 mM, respectively) stimulated the rate of ATP hydrolysis 2-fold, but only 11 +/- 1.1% of the increased activity was ouabain-sensitive. Optimal pretreatment with sodium dodecyl sulfate (SDS) (0.3 mg/ml) rendered both activities completely sensitive to inhibition by ouabain and reduced the basal Mg2+-ATPase activity by 70-90%. The K+-stimulated pNPPase activity doubled after preincubation in SDS, but the ATPase activity stimulated by Na+ plus K+ fell by 50% under these conditions. A similar pattern of apparent activation was produced by preincubation with deoxycholate (DOC), except that basal Mg2+-dependent activities were resistant to destruction by this detergent. The incremental responses to activation by ions and substrates, and inhibition by oubain, are consistent with the hypothesis that permeability-intact vesicles of sarcolemma are present in the isolated preparation, and that detergent activation renders the vesicles highly permeable to the ions, substrates, and ouabain.

Characterization of [3H](+/-)carazolol binding to beta-adrenergic receptors. Application to study of beta-adrenergic receptor subtypes in canine ventricular myocardium and lung.

[3H](+/-)Carazolol, a newly available beta-adrenergic receptor antagonist, can be used to characterize beta-adrenergic receptor subtypes present in membrane vesicles derived from canine ventricular myocardium and canine lung. [3H](+/-)Carazolol binding is saturable, of high affinity, and is displaceable by beta-adrenergic agents in accordance with their known pharmacological potencies. The interaction of carazolol with beta-adrenergic receptors is stereospecific; the (-) stereoisomer demonstrates greater potency than the (+) stereoisomer. Kinetic analysis of [3H](+/-)carazolol interaction with beta-adrenergic receptors suggests that presence of two phases of interaction, consistent with initial rapidly reversible "low" affinity association of ligand with receptor, followed by isomerization to form a high affinity, slowly reversible complex. Through use of a [3H](+/-)carazolol binding assay based on the high affinity complex, pharmacological specificities of beta-adrenergic receptor populations of canine myocardium and lung were quantified. Analysis using computer-assisted techniques suggests a beta 1/beta 2 receptor ratio of approximately 85%/15% for canine myocardium and 5%/95% for canine lung. In the absence of added guanine nucleotides, comparison of potencies of beta-adrenergic agonists in the two membrane systems suggests significant beta 2 selectivity of l-isoproterenol and l-epinephrine, and non-selectivity of norepinephrine. In the presence of saturating levels of guanine nucleotides, comparison of agonist potencies confirms the non-selectivity of l-isoproterenol and l-epinephrine, and beta 1 selectivity of norepinephrine. These results demonstrate that the state of guanine nucleotide regulation of receptors should be defined when examining agonist selectivities for beta-adrenergic receptor subtypes in vitro.

Effects of ryanodine on calcium sparks in cut twitch fibres of Rana temporaria.

1. Localized calcium release events (calcium sparks) were studied in voltage-clamped cut twitch fibres of Rana temporaria. 2. A histogram of thousands of spontaneous sparks displayed a monotonically decreasing amplitude distribution from the low to the high limit of > 7 DeltaF/F(0) units. 3. Several effects of low micromolar concentrations of ryanodine (0.4-2 microM) on spontaneous sparks, reproducing the agent's effects on single ryanodine receptor channel current in bilayers, were observed collectively for the first time in live fibres, namely (a) increases in spark frequency followed by (b) conversions of sparks into steady glows lasting tens of seconds, (c) occasional interruptions of the glows by brief gaps of darkness, and (d) abolition of sparks at the locations of the glows. The glow could reflect the incessant Ca(2+) flux through a single (or a few) calcium release channel locked in the semi-open state, which was allowed to make occasional transitions to the closed state but not to the fully open state. 4. Higher concentrations of ryanodine (> or = 20 microM) suppressed the spontaneous sparks effectively and permanently, presumably by deactivating the ryanodine receptors. 5. Depolarization-evoked sparks elicited with small pulses had higher frequencies and larger amplitudes than spontaneous sparks and were abolished by both concentrations of ryanodine. 6. With 1-2 microM ryanodine, however, a uniform non-sparking calcium release persisted during the pulse, with the globally averaged increase in fluorescence intensity being about half that of the control. A possible origin of this non-sparking release may be related to the structural coupling between the voltage sensors and the ryanodine receptors that can exist only in live fibres but not in the bilayer preparation.

Ryanodine receptor dysfunction in hearts of streptozotocin-induced diabetic rats.

Studies have shown that evoked calcium release from sarcoplasmic reticulum is compromised in diabetic rat hearts. The present study was undertaken to determine whether this decrease might be ascribed to a reduction in expression and/or alteration in function of ryanodine receptor (RyR2) and whether changes could be minimized with insulin treatment. Hearts were isolated from 4- and 6-week streptozotocin (STZ)-induced diabetic, 4-week diabetic/2-week insulin-treated, and age-matched control rats. RyR2 mRNA and protein levels were determined using reverse transcription-polymerase chain reactions and polyacrylamide gel electrophoresis, respectively, whereas the functional integrity of RyR2 was assessed from their ability to bind [3H]ryanodine. RyR2 protein was unchanged with up to 6 weeks of untreated STZ-induced diabetes. Two weeks of insulin treatment initiated after 4 weeks of diabetes increased RyR2 mRNA levels by 42% and RyR2 protein levels by 45 to 61%. At equivalent amounts, RyR2 protein from 4-week STZ-induced diabetic rat hearts bound 9% less [3H]ryanodine than age-matched control rats (74.1 +/- 3.9 versus 67.4 +/- 3.4 fmol/microg RyR2), whereas that from 6-week STZ-diabetic rats bound 36% less than control rats (47.9 +/- 4.8 versus 74.2 +/- 4.5 fmol/microg RyR2, p < 0.05). RyR2 from insulin-treated animals bound significantly less [3H]ryanodine than control rats (65.2 +/- 4.9 fmol/microg RyR2, p < 0.05). Apparent affinity of ryanodine for RyR2 was similar among all groups (K(d) approximately 1.04 +/- 0.08 nM). Because expression did not change significantly but ryanodine binding decreased, these data suggest that the functional integrity of RyR2 is compromised in diabetic rat hearts, and these changes can be attenuated with 2 weeks of insulin treatment.

Dual effect of suramin on calcium fluxes across sarcoplasmic reticulum vesicle membranes.

Suramin is a polysulfonated naphthylurea developed originally to treat trypanosomiasis. This drug has gained considerable attention recently as an effective anticancer agent. Previous studies have demonstrated that suramin also is an antagonist of ATP at P2x purinergic receptors. In the present study suramin was shown to evoke Ca++ release from skeletal muscle sarcoplasmic reticulum (SR) vesicles in a concentration-dependent manner. Ca++ release was inducable from vesicles derived from junctional SR but not from those derived from longitudinal SR. This subcellular site-dependent specificity suggests that suramin's actions on muscle involve the Ca++ release channel (CRC), a protein unique to terminal cisternae. This channel has been established as the site of action of ryanoid alkaloids such as ryanodine and dehydroryanodine. Suramin did not mimic ryanoid actions on the SR CRC, nor did it competitively diminish ryanodine binding. Instead, suramin actually increased [3H]ryanodine binding to junctional SR membranes. In this respect, suramin exhibited agonist effects like those of the adenine nucleotide, beta,gamma-methyleneadenosine 5'-triphosphate. Suramin's mechanism of action did not involve oxidation of sulfhydryl groups on the SR CRC, because dithiothreitol (1 mM) had no effect on suramin-induced Ca++ release. Independently of its effects on the CRC, suramin inhibited the Ca(++)-adenosine triphosphatase (EC 3.6.1.38, SERCA1) of SR membrane vesicles. The ability of suramin to diminish ATP-dependent Ca++ accumulation by SR vesicles therefore reflects two distinct actions: 1) activation (opening) of the SR Ca++ release channel and 2) inhibition of the Ca++ pump.