Christine Dettbarn

Pharmacology of calcium release from sarcoplasmic reticulum

Calcium release from sarcoplasmic reticulum (SR) has been elicited in response to additions of many different agents. Activators of Ca2+ release are here tentatively classified as activators of a Ca2+-induced Ca2+ release channel preferentially localized in SR terminal or as likely activators of other Ca2+ efflux pathways. Some of these pathways may be associated with several different mechanisms for SR Ca2+ release that have been postulated previously. Studies of various inhibitors of excitation-contraction coupling and of certain forms of SR Ca2+ release are summarized. The sensitivity of isolated SR to certain agents is unusually affected by experimental conditions. These effects can seriously undermine attempts to anticipate effects of the same pharmacological agentsin situ. Finally, mention is made of a new preparation (“sarcoballs”) designed to make the pharmacological study of SR Ca2+ release more accessible to electrophysiologists, and some concluding speculations on the future of SR pharmacology are offered.

A new promoter for α1C subunit of human L-type cardiac calcium channel CaV1.2

The cardiac Ca channel known as a1C or CaV1:2 is shown to express a new longer first exon equivalent to that formerlyreported in rabbit heart or rat aorta. Ribonuclease protection assayindicates that this exon is found in the majorityof Ca V1:2 transcripts in human heart RNA. The presence of this exon also suggests that expression of this transcript is driven bya promoter immediately upstream of this exon and its 5 0 untranslated region. The putative promoter exhibits 69% homologyto its rat counterpart and displays functional promoter activity when transfected into heart cells in culture in luciferase-expressing constructs. 2002 Elsevier

Arachidonic acid-induced Ca2+ release from isolated sarcoplasmic reticulum

Arachidonic acid has been shown to release Ca2+ from isolated skeletal and cardiac sarcoplasmic reticulum (SR) vesicles. The release took place nearly equally well from all fractions of the SR and was only partially inhibited by ruthenium red, suggesting that some other pathway is involved in addition to the SR Ca2+ release channel. Arachidonic acid increased SR Ca2+ efflux even in the presence of several different SR Ca2+ pump inhibitors. It also had considerably less effect on uptake measured in the presence of oxalate and did not appear to inhibit Ca(2+)-dependent ATPase activity. Thus, the SR Ca2+ pump also appears to be minimally perturbed by arachidonic acid. Arachidonyl CoA was more effective at releasing Ca2+ than the parent compound. Arachidonic acid effects were not inhibited by lipoxygenase or cyclooxygenase inhibitors, suggesting that no eicosanoids are involved in the effects under study here. Flunarizine, cinnarizine and propyl-methylenedioxyindene inhibited the Ca2+ release induced by arachidonic acid. The effects of arachidonic acid appear to depend on the ratio of arachidonic acid to membrane vesicles.

Involvement of ryanodine receptors in sphingosylphosphorylcholine-induced calcium release from brain microsomes.

Sphingosylphosphorylcholine (SPC) releases Ca2+ from brain microsomes. SPC-induced CA2+ release differs from IP3-induced Ca2+ release in that it is more extensive in the cerebrum than in the cerebellum. SPC has little effect on [3H] IP3 binding but enhances [3H] ryanodine binding, as expected for an activator of ryanodine receptors. SPC-induced Ca2+ release is inhibited by ryanodine receptor blockers but not by selective blockers of IP3 receptors. We conclude that SPC releases Ca2+ from brain microsomes by activating ryanodine receptors rather than IP3 receptors. Activation of an additional SPC-sensitive pathway for releasing Ca2+ is not precluded.

Dihydropyridine receptor isoform expression in adult rat skeletal muscle

Abstract The expression of isoform-specific dihydropyridine receptor Ca2+ channel (DHPR) α1-subunit genes in rat diaphragm, soleus and extensor digitorum longus muscles was investigated using RNase protection assays. As expected, mRNA expression levels for the DHPR skeletal muscle isoform were highest in extensor digitorum longus. Unexpectedly, both diaphragm and soleus expressed mRNA for the cardiac isoform at a significant level. Moreover, immunohistochemical experiments provided evidence of the cardiac DHPR isoform at the protein level in muscle fibres. The presence of the cardiac DHPR in the soleus and diaphragm is consistent with a degree of reported cardiac-like excitation-contraction coupling in these muscles, and may be an explanation for some of the therapeutic effects of theophylline in asthmatics, but is likely to serve some other role(s) as well.

Dihydropyridine receptor gene expression in skeletal muscle from mdx and control mice

The expression of isoform-specific dihydropyrine receptor-calcium channel (DHPR) alpha 1-subunit genes was investigated in mdx and control mouse diaphragm (DIA) and tibialis anterior (TA). RNase protection assays were carried out with a rat DHPR cDNA probe specific for skeletal muscle and a mouse DHPR cDNA probe specific for cardiac muscle. The level of expression of the gene encoding the cardiac DHPR was very weak in TA muscle from both control and mdx mice. Compared to TA, DIA expressed mRNA for the cardiac isoform at significantly higher levels, but mdx and control mouse DIA levels were similar to one another. In contrast, mRNA expression levels for the DHPR skeletal muscle isoform were lower in control DIA than TA. However, there was a dramatic increase in the expression for the DHPR skeletal muscle isoform in mdx DIA compared with control DIA, reaching the TA expression level, whereas dystrophy did not affect TA expression. [3H]-PN200-110 binding was used to further assess DIA DHPR expression at the protein level. The density of binding sites for the probe was not significantly affected in DIA muscles of mdx vs. control mice, but it was reduced in older mdx and control mice. The increase in DHPR mRNA levels without a consequent increase in DHPR protein expression could be secondary to possible enhanced protein degradation which occurs in mdx DIA. The altered DHPR expression levels found here do not appear to be responsible for the severe deficits in contractile function of the mdx DIA.

Potentiation of sarcoplasmic reticulum Ca2+ release by 2,3-butanedione monoxime in crustacean muscle

The effect of the chemical phosphatase 2,3-butanedione monoxime (BDM) on various aspects of excitation/contraction coupling in crustacean muscle was investigated. Despite having a depressant effect on vertebrate skeletal and cardiac muscle, BDM was a potentiator of contraction in crustacean muscle. At concentrations of 1–3 mM BDM caused an increase of potassium contractures in bundles of fibers isolated from crayfish muscle. At higher concentrations BDM caused oscillatory contractions by itself. In single voltage-clamped cut muscle fibers loaded with rhod-2, BDM (0.5–2 mM) potentiated the magnitude and duration of intracellular Ca2+ transients elicited by depolarization. At the same time BDM did not affect the rate of Ca2+ removal from the myoplasm under conditions where Ca2+ release was blocked by tetracaine. Nor did BDM increase Ca2+ entry; in fact it caused a decrease in the amplitude of the inward Ca2+ current (ICa). In microsomes isolated from lobster muscle, BDM also potentiated Ca2+ release induced by caffeine and at higher concentrations (above 3 mM) induced release by itself. At the same time it had little effect on Ca2+ uptake. These results indicate that BDM potentiates Ca2+ release in crustacean muscle possibly by dephosphorylation of the Ca2+-release channel.

Involvement of sarcoplasmic reticulum ‘Ca2+ release channels’ in excitation‐contraction coupling in vertebrate skeletal muscle.

1. Pharmacological blockers of calcium‐induced calcium release from isolated skeletal sarcoplasmic reticulum (SR) vesicles have been introduced into frog skeletal muscle fibres to determine their effects on excitation‐contraction coupling. 2. Among the blockers tested, Ruthenium Red, neomycin, gentamicin and 9‐aminoacridine inhibited the SR Ca2+ release associated with excitation‐contraction (E‐C) coupling as much as they inhibited caffeine potentiation of that release. Protamine, certain of its derivatives, and spermine were ineffective in both in situ tests. 3. Alternative sites of polyamine action on the contractile proteins, SR Ca2+ uptake or charge movements were ruled out. 4. All polyamines tested required considerably higher concentrations to inhibit excitation‐contraction coupling than to block Ca2+ release from isolated SR vesicles. 5. The quantitative pharmacological difference in sensitivity between isolated and intact systems serves as a reminder that results on isolated systems cannot generally be used to predict results of the same substances on more physiological systems. 6. Since caffeine is known to open the SR ‘Ca2+ release channels’ (the ryanodine receptors that mediate Ca(2+)‐induced Ca2+ release), the equal effectiveness of these blockers at inhibiting excitation‐contraction (E‐C) coupling and its potentiation by caffeine suggests that the SR ‘Ca2+ release channels’ are indeed involved in excitation‐concentration coupling in skeletal muscle, although the results do not indicate how the channel is gated open during E‐C coupling.

A Pharmacological Approach to the Physiological Mechanism of Excitation-Contraction Coupling

The use of pharmacologic agents as tools to aid in the characterization and separation of physiological processes is not a new idea. The availability of specific toxins like tetrodotoxin and saxitoxin made it easy to test for the presence of sodium channels in excitable cells and made possible the purification(1) reconstitution(2,3) and cloning(4) of such channels. We hope to utilize a pharmacologic approach here to determine whether a Ca2+ -channel isolated recently from skeletal muscle sarcoplasmic reticulum (SR) is the one of physiological importance. Is it the right channel? We know several methods exist to cause Ca2+ release from SR.(5,6) If we had specific inhibitors for each form of release, we could test whether the releases or channels they blocked were involved in excitation-contraction coupling. We will examine the role of Ca2+ -induced Ca2+ release channels first because these are the channels already isolated.