Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle

Abstract

Thapsigargin has been reported to inhibit ATP-dependent Ca2+ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca2+ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca2+ content of the SR. The SR was first depleted of Ca2+ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 microM was required to inhibit Ca2+ loading by 50%. In contrast, another SR Ca2+ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 microM and total inhibition at 50 microM. When cyclopiazonic acid (100 microM) was applied after, rather than during, Ca2+ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 microM), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca2+ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.

Endoplasmic reticulum Ca2+-ATPase pump is up-regulated in calcium-transporting dental enamel cells: a non-housekeeping role for SERCA2b

“…Thapsigargin is considered to be highly selective for SERCA, subject to use at low nanomolar concentrations [16]. However, many reports of poor sensitivity to thapsigargin appear in the literature and several contributing factors have been recognized including non-specific adsorption to bulk tissue and plastic surfaces, and indirect competition by calcium [36,37,41,42]. Our finding that the large amount of membranes assayed contributed to the low efficacy of thapsigargin was supported by recent investigations (I. K. Franklin and M. J. Hubbard, unpublished work) of permeabilized enamel cells, where thapsigargin was effective at 20-fold lower concentrations than in this study.…”

Alterations in the force‐frequency relationship by tert‐ butylbenzohydroquinone, a putative SR Ca2+ pump inhibitor, in rabbit and rat ventricular muscle

“…As such a reversal is currently explained by abolition of trans-sarcolemmal Ca2" buffering by the SR, it is likely that a fraction of pumps unblocked by 30 uM TBQ is still able to buffer incoming Ca2". Unfortunately, solubility problems and possible non-specific effects related to membrane partition of hydrophobic compounds (Baudet et al, 1993;Du et al, 1994) precludes complete SR Ca2" pump inhibition and investigation of the reversal phenomenon in multicellular preparations.…”

“…In multicellular preparations, the effects of these putative Ca21 pump inhibitors have yielded more disappointing results as concentrations of TG or CPA at least 10 times higher (>50 MM) than in isolated cells have to be used to record significant, and sometimes modest, changes in inotropism and variable effects on SR Ca2+ content (Baudet et al, 1993;Pery-Man et al, 1993;Yard et al, 1994). The use of high concentrations of hydrophobic molecules could provoke membrane partitioning, non-specific binding and possible non-selective effects (Baudet et al, 1993;Du et al, 1994;Yard et al, 1994). Regarding the staircase phenomenon, Yard et al (1994) have reported that application of 30 gM CPA attenuated the positive force-frequency relationship of the guinea-pig atrium, but these data may not be extrapolated to the ventricle, nor did this study investigate the Ca21 load of the SR. Our own experience with TG and CPA in rabbit ventricle (Baudet et al, 1993) (Bers, 1991 for rabbits or with ether followed by exsanguination for rats.…”

Malignant hyperthermia: Effects of sarcoplasmic reticulum Ca2+ pump inhibition

“…Other studies support our choice of 10 µmol/L CPA, which inhibits SR Ca 2+ uptake in skeletal muscle by approximately 70% in frog skinned fibers 4 and by as much as 100% in rat skinned fibers. 14 Higher concentrations have additional effects, such as increasing the Ca 2+ sensitivity and decreasing peak tension of skinned ferret slow twitch fibers, 12 and increasing the Ca 2+ sensitivity of the contractile apparatus of skinned rat fast twitch fibers.…”