Longitudinal impact of the Women's Health Initiative study on hormone therapy use in Australia.

Purpose: This study aimed to examine the longitudinal impact of evidence changes on menopausal hormone therapy (MHT) use in Australia. Methods: We analyzed two datasets of subsidized and total MHT use (2000-2016) using segmented regression analysis to explore the impact of the Women's Health Initiative (WHI) 2002 and 2007 studies. Analyses were stratified by class, route, and strength. Use was measured in defined daily dose/1000 women/day (DDD/1000/day) or packs/1000 women/month (packs/1000/month). Results: The drop in total MHT use after the WHI 2002 was substantial. The biggest decreases in class, route, and strength were estrogens (28.99 DDD/1000/day, 95% confidence interval [CI] 23.97, 34.01), oral (46.07 DDD/1000/day, 95% CI 41.13, 51.01), and medium strength (34.95 packs/1000/month, 95% CI 30.17, 39.73), respectively. However, vaginal use remained stable (-1.83 DDD/1000/day, 95% CI -3.83, 0.17). Profiles of total and subsidized use were similar over time. Utilization levels were relatively unchanged after 2007. Decreased utilization contributed to product discontinuation, with a lag of up to 4 years. Product discontinuation in 2009 further decreased utilization. Discussion and conclusions: MHT use remained low after 2002 despite evidence favoring its use in women younger than 60 years or within 10 years postmenopause. Continued low use could relate to the WHI 2002 media coverage, therapy objectives, key stakeholder uncertainty, health policies, and medicine availability.

The impact of clozapine on hospital use: a systematic review and meta-analysis.

OBJECTIVE: The objective of this study was to perform a systematic review and meta-analysis of studies reporting the impact of clozapine on hospital use in people with a psychotic illness. METHOD: PubMed, EMBASE, PsycINFO and the Cochrane Schizophrenia Group Trials Register were systematically searched from inception to 12 October 2016. We included all trials and observational studies, except case reports. RESULTS: Thirty-seven studies were included. Clozapine significantly reduced the proportion of people hospitalised compared to control medicines (RR = 0.74; 95% CI: 0.69-0.80, P < 0.001, 22 studies, n = 44 718). There were significantly fewer bed days after clozapine treatment compared to before clozapine treatment in both controlled (MD = -34.41 days; 95% CI: -68.22 to -0.60 days, P = 0.046, n = 162) and uncontrolled studies (MD = -52.86 days; 95% CI: -79.86 days to -25.86 days, P < 0.001, n = 2917). Clozapine and control medicines had a similar time to rehospitalisation (-19.90 days; 95% CI: -62.42 to 22.63 days, P = 0.36). CONCLUSION: Clozapine treatment reduced the number of people hospitalised and the number of bed days after treatment compared with before treatment. Clozapine has the potential to reduce acute hospital use among people with treatment refractory schizophrenia.

International trends in clozapine use: a study in 17 countries.

OBJECTIVE: There is some evidence that clozapine is significantly underutilised. Also, clozapine use is thought to vary by country, but so far no international study has assessed trends in clozapine prescribing. Therefore, this study aimed to assess clozapine use trends on an international scale, using standardised criteria for data analysis. METHOD: A repeated cross-sectional design was applied to data extracts (2005-2014) from 17 countries worldwide. RESULTS: In 2014, overall clozapine use prevalence was greatest in Finland (189.2/100 000 persons) and in New Zealand (116.3/100 000), and lowest in the Japanese cohort (0.6/100 000), and in the privately insured US cohort (14.0/100 000). From 2005 to 2014, clozapine use increased in almost all studied countries (relative increase: 7.8-197.2%). In most countries, clozapine use was highest in 40-59-year-olds (range: 0.6/100 000 (Japan) to 344.8/100 000 (Finland)). In youths (10-19 years), clozapine use was highest in Finland (24.7/100 000) and in the publicly insured US cohort (15.5/100 000). CONCLUSION: While clozapine use has increased in most studied countries over recent years, clozapine is still underutilised in many countries, with clozapine utilisation patterns differing significantly between countries. Future research should address the implementation of interventions designed to facilitate increased clozapine utilisation.

Triptan use in Australia 1997-2015: A pharmacoepidemiological study.

OBJECTIVE: This study examined the use of triptan derivatives in Australia between 1997 and 2015, based on a national drug reimbursement database, and compared patterns of use with available international data. METHODS: We obtained publically available data on the number of prescriptions for triptans marketed in Australia (sumatriptan, eletriptan, rizatriptan, zolmitriptan, naratriptan). Dispensed use was measured as defined daily dose (DDD per 1000 population per day) for Australia's concessional beneficiaries (low-income earners, people with disabilities, and seniors). RESULTS: Total triptan use increased at an average annual rate of 112% over the 18-year period. Sumatriptan was the preferred triptan throughout (average annual increase 45%). Zolmitriptan and naratriptan use peaked in 2004, then decreased. Rizatriptan and eletriptan became available in 2010. There were 3.2-fold and 5.9-fold annual increases in their use from 2011 to 2105. There was some evidence suggesting that pattern of triptan use in concessional beneficiaries probably reflected pattern of overall triptan use in Australia. CONCLUSIONS: The use of triptan derivatives in Australia per head of population for treating migraine attacks continued to increase over the 18-year period studied, with use of recently introduced derivatives more than substituting for decreased use of older triptans. This suggests that the available treatments of migraine attacks had achieved what were considered less than adequate therapeutic outcomes.

Off-label use of tumour necrosis factor-alpha inhibitors and anakinra at an Australian tertiary hospital.

BACKGROUND: Tumour necrosis factor-alpha inhibitors (anti-TNFα) and anakinra are monoclonal antibodies against pro-inflammatory cytokines overexpressed in many systemic inflammatory diseases. In Australia, they are registered for the treatment of several rheumatological, gastroenterological and dermatological indications. Despite increasing observational evidence for their use in off-label indications, there is a paucity of outcome research from the Australian hospital sector. AIMS: To describe the off-label use of anti-TNFα and anakinra at a tertiary referral hospital in Queensland, Australia and consideration of a drug register to inform future clinical decision-making. METHODS: We performed an in-depth retrospective chart audit of off-label treatment with anti-TNFα or anakinra at the Royal Brisbane and Women's Hospital from mid-2010 to mid-2014, linking demographic, phenotypic, pathology and outcome data with these drugs. RESULTS: Off-label use was identified in 10 patients. The most frequent indications were sarcoidosis and dermatological conditions. Three patients required sequential therapy with a second anti-TNFα (total responses = 13). Complete response occurred in 46%, partial response in 38% and primary non-response in 8%. Response was unable to be determined in 8%. We recorded 14 adverse events (infections most common). CONCLUSION: This study suggests that anti-TNFα may be beneficial for some off-label indications (e.g. sarcoidosis). However, the observational design of this study (and pre-existing research) limits the ability to infer causality and generalise results. We propose the creation of a mandatory drug register to monitor off-label use. Whilst comparative efficacy cannot be established without a matched placebo arm, a register would enable some reporting on effectiveness in rare diseases and identify infrequent but serious adverse events.

Increasing Clozapine Dispensing Trends in Queensland, Australia 2004-2013.

INTRODUCTION: Clozapine is the most effective treatment for treatment-resistant schizophrenia but its use is suboptimal. METHODS: Clozapine dispensing data from Queensland, Australia were extracted (2004-2013). The number of people dispensed clozapine each year and mean maintenance doses were calculated. The 18-week and 5-year cessation and treatment interruption rates were calculated using Kaplan-Meier analysis. RESULTS: Clozapine dispensings increased 36.4% (p<0.001) from 44 to 60 people per 100,000. This was estimated as 8.3% of people with schizophrenia and 33.3% of people with treatment resistant schizophrenia dispensed clozapine in 2013. Mean maintenance dose did not significantly change (364-399 mg) over 5 years of treatment. One in 7 (14.2%) people ceased within the first 3 weeks. 3-quarters (72.7%) reached maintenance therapy. The 5-year actuarial estimate of the proportion of people a) dispensed clozapine was 0.610 (S.E. 0.011) and b) with an interruption to treatment was 0.422 (S.E. 0.013). DISCUSSION: The number of patients being dispensed clozapine increased between 2004 and 2013 but clozapine is still underused. Increased use combined with continued monitoring for adverse effects will improve quality use of clozapine.

A small-molecule inhibitor of skeletal muscle myosin II.

We screened a small-molecule library for inhibitors of rabbit muscle myosin II subfragment 1 (S1) actin-stimulated ATPase activity. The best inhibitor, N-benzyl-p-toluene sulphonamide (BTS), an aryl sulphonamide, inhibited the Ca2+-stimulated S1 ATPase, and reversibly blocked gliding motility. Although BTS does not compete for the nucleotide-binding site of myosin, it weakens myosin's interaction with F-actin. BTS reversibly suppressed force production in skinned skeletal muscle fibres from rabbit and frog skin at micromolar concentrations. BTS suppressed twitch production of intact frog fibres with minimum alteration of Ca2+ metabolism. BTS is remarkably specific, as it was much less effective in suppressing contraction in rat myocardial or rabbit slow-twitch muscle, and did not inhibit platelet myosin II. The isolation of BTS and the recently discovered Eg5 kinesin inhibitor, monastrol, suggests that motor proteins may be potential targets for therapeutic applications.

Trends in Australian dental prescribing of antibiotics: 2005-2016.

BACKGROUND: Prescribing of antibiotics by dentists for surgical prophylaxis or as an adjunct to managing dental infections is a substantial part of the overall landscape for prescribed antibiotics in health care settings. METHODS: We explored trends in the antibiotic prescribing patterns of Australian dentists over the 12-year period, 2005-2016. We obtained data on dispensed prescriptions of antibiotics from registered dentists subsidized on the Pharmaceutical Benefits Scheme. RESULTS: Australian dentists were responsible for almost 7 million dispensed prescriptions of antibiotics over 12 years; an average of 24 prescriptions per year per dentist. The most commonly prescribed antibiotic was amoxicillin, followed by amoxicillin + clavulanic acid and metronidazole. These top three antibiotics constituted more than 80% of all antibiotics prescribed and their use increased dramatically over time. There was a large increase in the prescribing of broad-spectrum antibiotics over time, most of which occurred from 2011 to 2016. CONCLUSIONS: Excessive prescribing of broad-spectrum antibiotics runs contrary to national antimicrobial stewardship (AMS) initiatives and guidelines. Multifaceted educational strategies are essential to align prescribing with current best practice. High-level evidence to inform clear guidelines on antibiotic prescribing in dental infections, with audit and feedback, should reduce the inappropriate use of antibiotics in dentistry.

Changing patterns of antiepileptic drug use in pregnant Australian women.

OBJECTIVE: To trace the pattern of antiepileptic drug (AED) use in pregnant Australian women annually from 1999 to 2007, and correlate it with the pattern of AED use in the wider community. METHODS: Analysis of data from the Australian Register of AEDs in Pregnancy, related to Australian population data for AED prescriptions. RESULTS: Over the study period, prescribing of carbamazepine, phenytoin and valproate for pregnant women decreased, and prescribing of lamotrigine, topiramate and levetiracetam increased. These changes tended to parallel prescribing trends in the wider community, except for valproate, whose prescribing in the overall community increased as its prescribing, and its dosage prescribed, decreased in pregnancy. Concomitant with this, there was a trend towards fewer births of foetuses with abnormalities. CONCLUSIONS: While otherwise following national AED prescribing trends, Australian prescribers are reducing the use and dose of valproate in pregnant women, likely in recognition of the teratogenic hazards of this drug.

Dental opioid prescribing rates after the up-scheduling of codeine in Australia.

The misuse of pharmaceutical opioids is a major public health issue. In Australia, codeine was re-scheduled on 1 February 2018 to restrict access; it is now only available on prescription. The aim of this study was to measure the change in dental opioid prescriptions, one year before and after the codeine re-scheduling in Australia and to assess dental prescribing rates of opioids for 2018 by population and by clinician. Data was extracted for dental opioids for the year immediately prior and after the codeine up-schedule (1 February 2017-31 January 2019) from the publicly-available national prescription database (Pharmaceutical Benefits Scheme). Descriptive statistics, T-tests and odds ratios were used to identify significant prescribing differences. Codeine, codeine/paracetamol, oxycodone and tramadol use increased significantly the year after the codeine restriction than the previous year (13.8-101.1%). Australian dentists prescribed 8.6 prescriptions/1,000 population in 2018, with codeine/paracetamol accounting for most prescriptions (96%). The significant increase in opioid prescribing highlights that Australian dentists may be contributing to the misuse of pharmaceutical opioids. Educational efforts should be targeted at the appropriate use of opioids and patient selection. Dentists should be added to the prescription monitoring system SafeScript so they can make informed decisions for patients who are potentially misusing opioids.

Prescribing patterns of dental practitioners in Australia from 2001 to 2012. Antimicrobials.

BACKGROUND: The development of antibiotic resistance by bacteria is of global concern. Inappropriate prescribing has the potential to exacerbate this issue. We aimed to examine the patterns of prescribing of antimicrobial medicines by dental practitioners in Australia from 2001 to 2012. METHODS: Data were collected from Medicare Australia on prescriptions from dental practitioners dispensed to concessional beneficiaries between 2001 and 2012. We examined patterns of use over time. RESULTS: There was an overall increase in number of prescriptions and in dispensed use (standardized by dose and population) of antibiotics and antifungals for the concessional population over the 12-year period. The use of dentally prescribed antibiotics increased 50%. Amoxicillin was the most commonly prescribed antibiotic accounting for 66% of all prescriptions in 2012. Generally, there was preferential prescribing of the highest dose formulations. The use of the two antifungals increased 30% over the study period with a preference for amphotericin B (74%) rather than nystatin. CONCLUSIONS: These data show a concerning increase in prescribing of antibiotics and antifungals by dentists in Australia. It would appear that Australian dentists may not be prescribing these medicines appropriately; however, further research is needed to understand prescribing behaviours and decision-making by dentists.

Model of sarcomeric Ca2+ movements, including ATP Ca2+ binding and diffusion, during activation of frog skeletal muscle.

Cannell and Allen (1984. Biophys. J. 45:913-925) introduced the use of a multi-compartment model to estimate the time course of spread of calcium ions (Ca2+) within a half sarcomere of a frog skeletal muscle fiber activated by an action potential. Under the assumption that the sites of sarcoplasmic reticulum (SR) Ca2+ release are located radially around each myofibril at the Z line, their model calculated the spread of released Ca2+ both along and into the half sarcomere. During diffusion, Ca2+ was assumed to react with metal-binding sites on parvalbumin (a diffusible Ca2+- and Mg2+-binding protein) as well as with fixed sites on troponin. We have developed a similar model, but with several modifications that reflect current knowledge of the myoplasmic environment and SR Ca2+ release. We use a myoplasmic diffusion constant for free Ca2+ that is twofold smaller and an SR Ca2+ release function in response to an action potential that is threefold briefer than used previously. Additionally, our model includes the effects of Ca2+ and Mg2+ binding by adenosine 5'-triphosphate (ATP) and the diffusion of Ca2+-bound ATP (CaATP). Under the assumption that the total myoplasmic concentration of ATP is 8 mM and that the amplitude of SR Ca2+ release is sufficient to drive the peak change in free [Ca2+] (Delta[Ca2+]) to 18 microM (the approximate spatially averaged value that is observed experimentally), our model calculates that (a) the spatially averaged peak increase in [CaATP] is 64 microM; (b) the peak saturation of troponin with Ca2+ is high along the entire thin filament; and (c) the half-width of Delta[Ca2+] is consistent with that observed experimentally. Without ATP, the calculated half-width of spatially averaged Delta[Ca2+] is abnormally brief, and troponin saturation away from the release sites is markedly reduced. We conclude that Ca2+ binding by ATP and diffusion of CaATP make important contributions to the determination of the amplitude and the time course of Delta[Ca2+].

Absorbance signals from resting frog skeletal muscle fibers injected with the pH indicator dye, phenol red.

Singly dissected twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with the pH indicator dye, phenol red. Dye-related absorbances in myoplasm, denoted by A0(lambda) and A90(lambda), were estimated as a function of wavelength lambda (450 nm less than or equal to lambda less than or equal to 640 nm) with light polarized parallel (0 degrees) and perpendicular (90 degrees) to the fiber axis respectively. At all lambda, A0(lambda) was slightly greater than A90(lambda), indicating that some of the phenol red molecules were bound to oriented structures accessible to myoplasm. The phenol red "isotropic" signal, [A0(lambda) + 2A90(lambda)]/3, a quantity equal to the average absorbance of all the dye molecules independent of their orientation, had a spectral shape that was red-shifted by approximately 10 nm in comparison with in vitro dye calibration curves measured in 140 mM KCl. The red-shifted spectrum also indicates that some phenol red molecules were bound in myoplasm. A quantitative estimate of indicator binding was obtained from measurements of the dye's apparent diffusion constant in myoplasm, denoted by Dapp. The small value of Dapp, 0.37 x 10(-6) cm2 s-1 (at 16 degrees C), can be explained if approximately 80% of the dye was bound to myoplasmic sites of low mobility. To estimate the apparent myoplasmic pH, denoted by pHapp, the isotropic absorbance of phenol red was fitted by in vitro calibration spectra. pHapp was found to be independent of dye concentration (0.2-2 mM), but varied widely (range, 6.8-7.5; mean value, 7.17) among fibers judged from functional characteristics to be normal. When fibers were subjected to acid or alkaline loads by exposure to Ringer's solution containing, respectively, dissolved CO2 or NH3, the changes in pHapp were in agreement with those expected from pH micro-electrode studies. It is concluded that in spite of the several indications for the presence of bound phenol red inside muscle cells, the pHapp signal from the indicator is useful for monitoring changes in myoplasmic pH in response to physiological and pharmacological manipulations.

Changes in phenol red absorbance in response to electrical stimulation of frog skeletal muscle fibers.

Intact single twitch fibers from frog muscle were stretched to long sarcomere length, micro-injected with the pH indicator dye phenol red, and activated by action potential stimulation. Indicator-related absorbance changes (denoted by delta A0 and delta A90) were measured with 0 degree and 90 degrees polarized light (oriented, respectively, parallel and perpendicular to the fiber axis). Two components of delta A were detected that had generally similar time courses. The "isotropic" component, calculated as the weighted average (delta A0 + 2 delta A90)/3, had the wavelength dependence expected for a change in myoplasmic pH. If calibrated in pH units, this signal's peak amplitude, which occurred 15-20 ms after stimulation, corresponded to a myoplasmic alkalization of average value 0.0025 +/- 0.0002 (+/- SEM; n = 9). The time course of this change, as judged from a comparison with that of the fibers' intrinsic birefringence signal, was delayed slightly with respect to that of the myoplasmic free [Ca2+] transient. On average, the times to half-peak and peak of the phenol red isotropic signal lagged those of the birefringence signal by 2.4 +/- 0.2 ms (+/- SEM; n = 8) and 8.4 +/- 0.5 ms (+/- SEM; n = 4), respectively. The other component of the phenol red signal was "dichroic," i.e., detected as a difference (delta A0-delta A90 greater than 0) between the two polarized absorbance changes. The wavelength dependence of this signal was similar to that of the phenol red resting dichroic signal (Baylor and Hollingworth. 1990. J. Gen. Physiol. 96:449-471). Because of the presence of the active dichroic signal, and because approximately 80% of the phenol red molecules appear to be bound in the resting state to either soluble or structural sites, the possibility exists that myoplasmic events other than a change in pH underlie the phenol red isotropic signal.

Simulation of calcium sparks in cut skeletal muscle fibers of the frog.

Spark mass, the volume integral of Delta F/F, was investigated theoretically and with simulations. These studies show that the amount of Ca2+ bound to fluo-3 is proportional to mass times the total concentration of fluo-3 ([fluo-3T]); the proportionality constant depends on resting Ca2+ concentration ([Ca2+]R). In the simulation of a Ca2+ spark in an intact frog fiber with [fluo-3T] = 100 microM, fluo-3 captures approximately one-fourth of the Ca2+ released from the sarcoplasmic reticulum (SR). Since mass in cut fibers is several times that in intact fibers, both with similar values of [fluo-3T] and [Ca2+]R, it seems likely that SR Ca2+ release is larger in cut fiber sparks or that fluo-3 is able to capture a larger fraction of the released Ca2+ in cut fibers, perhaps because of reduced intrinsic Ca2+ buffering. Computer simulations were used to identify these and other factors that may underlie the differences in mass and other properties of sparks in intact and cut fibers. Our spark model, which successfully simulates calcium sparks in intact fibers, was modified to reflect the conditions of cut fiber measurements. The results show that, if the protein Ca2+-buffering power of myoplasm is the same as that in intact fibers, the Ca2+ source flux underlying a spark in cut fibers is 5-10 times that in intact fibers. Smaller source fluxes are required for less buffer. In the extreme case in which Ca2+ binding to troponin is zero, the source flux needs to be 3-5 times that in intact fibers. An increased Ca2+ source flux could arise from an increase in Ca2+ flux through one ryanodine receptor (RYR) or an increase in the number of active RYRs per spark, or both. These results indicate that the gating of RYRs, or their apparent single channel Ca2+ flux, is different in frog cut fibers--and, perhaps, in other disrupted preparations--than in intact fibers.

The amplitude and time course of the myoplasmic free [Ca2+] transient in fast-twitch fibers of mouse muscle.

Bundles of 10-100 fibers were dissected from the extensor digitorum longus muscle of mouse, mounted in an apparatus for optical recording, and stretched to long sarcomere length (> or = 3.6 microns). One fiber within the bundle was microinjected with furaptra, a fluorescent indicator that responds rapidly to changes in myoplasmic free [Ca2+] (delta [Ca2+]). Twitches and brief tetani were initiated by external stimulation. At myoplasmic furaptra concentrations of approximately 0.1 mM, the indicator's fluorescence signal during fiber activity (delta F/F) was well resolved. delta F/F was converted to delta [Ca2+] under the assumption that furaptra's myoplasmic dissociation constant for Ca2+ is 98 microM at 16 degrees C and 109 microM at 28 degrees C. At 16 degrees C, the peak amplitude of delta [Ca2+] during a twitch was 17.8 +/- 0.4 microM (+/-SEM; n = 8) and the half-width of delta [Ca2+] was 4.6 +/- 0.3 ms. At 28 degrees C, the peak and half-width values were 22.1 +/- 1.8 microM and 2.0 +/- 0.1 ms, respectively (n = 4). During a brief high-frequency tetanus, individual peaks of delta [Ca2+] were also well resolved and reached approximately the same amplitude that resulted from a single shock; the initial decays of delta [Ca2+] from peak slowed substantially during the tetanus. For a single twitch at 16 degrees C, the amplitude of delta [Ca2+] in fast-twitch fibers of mouse is not significantly different from that recently measured in fast-twitch fibers of frog (16.5 +/- 0.9 microM; Zhao, M., S. Hollingworth, and S.M. Baylor. 1996. Biophys. J. 70:896-916); in contrast, the half-width of delta [Ca2+] is surprisingly brief in mouse fibers, only about half that measured in frog (9.6 +/- 0.6 ms). The estimated peak rate at which Ca2+ is released from the sarcoplasmic reticulum in response to an action potential is also similar in mouse and frog, 140-150 microM/ms (16 degrees C).

Comparison of simulated and measured calcium sparks in intact skeletal muscle fibers of the frog.

Calcium sparks in frog intact skeletal muscle fibers were modeled as stereotypical events that arise from a constant efflux of Ca(2+) from a point source for a fixed period of time (e.g., 2.5 pA of Ca(2+) current for 4.6 ms; 18 degrees C). The model calculates the local changes in the concentrations of free Ca(2+) and of Ca(2+) bound to the major intrinsic myoplasmic Ca(2+) buffers (troponin, ATP, parvalbumin, and the SR Ca(2+) pump) and to the Ca(2+) indicator (fluo-3). A distinctive feature of the model is the inclusion of a binding reaction between fluo-3 and myoplasmic proteins, a process that strongly affects fluo-3's Ca(2+)-reaction kinetics, its apparent diffusion constant, and hence the morphology of sparks. DeltaF/F (the change in fluo-3's fluorescence divided by its resting fluorescence) was estimated from the calculated changes in fluo-3 convolved with the microscope point-spread function. To facilitate comparisons with measured sparks, noise and other sources of variability were included in a random repetitive fashion to generate a large number of simulated sparks that could be analyzed in the same way as the measured sparks. In the initial simulations, the binding of Ca(2+) to the two regulatory sites on troponin was assumed to follow identical and independent binding reactions. These simulations failed to accurately predict the falling phase of the measured sparks. A second set of simulations, which incorporated the idea of positive cooperativity in the binding of Ca(2+) to troponin, produced reasonable agreement with the measurements. Under the assumption that the single channel Ca(2+) current of a ryanodine receptor (RYR) is 0.5-2 pA, the results suggest that 1-5 active RYRs generate an average Ca(2+) spark in a frog intact muscle fiber.

Myoplasmic calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra.

Furaptra (Raju, B., E. Murphy, L. A. Levy, R. D. Hall, and R. E. London. 1989. Am. J. Physiol. 256:C540-C548) is a "tri-carboxylate" fluorescent indicator with a chromophore group similar to that of fura-2 (Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. J. Biol. Chem. 260:3440-3450). In vitro calibrations indicate that furaptra reacts with Ca2+ and Mg2+ with 1:1 stoichiometry, with dissociation constants of 44 microM and 5.3 mM, respectively (16-17 degrees C; ionic strength, 0.15 M; pH, 7.0). Thus, in a frog skeletal muscle fiber stimulated electrically, the indicator is expected to respond to the change in myoplasmic free [Ca2+] (delta[Ca2+]) with little interference from changes in myoplasmic free [Mg2+]. The apparent longitudinal diffusion constant of furaptra in myoplasm was found to be 0.68 (+/- 0.02, SEM) x 10(-6) cm2 s-1 (16-16.5 degrees C), a value which suggests that about half of the indicator was bound to myoplasmic constituents of large molecular weight. Muscle membranes (surface and/or transverse-tubular) appear to have some permeability to furaptra, as the total quantity of indicator contained within a fiber decreased after injection; the average time constant of the loss was 302 (+/- 145, SEM) min. In fibers containing less than 0.5 mM furaptra and stimulated by a single action potential, the calibrated peak value of delta[Ca2+] averaged 5.1 (+/- 0.3, SEM) microM. This value is about half that reported in the preceding paper (9.4 microM; Konishi, M., and S. M. Baylor. 1991. J. Gen. Physiol. 97:245-270) for fibers injected with purpurate-diacetic acid (PDAA). The latter difference may be explained, at least in part, by the likelihood that the effective dissociation constant of furaptra for Ca2+ is larger in vivo than in vitro, owing to the binding of the indicator to myoplasmic constituents. The time course of furaptra's delta[Ca2+], with average values (+/- SEM) for time to peak and half-width of 6.3 (+/- 0.1) and 9.5 (+/- 0.4) ms, respectively, is very similar to that of delta[Ca2+] recorded with PDAA. Since furaptra's delta[Ca2+] can be recorded at a single excitation wavelength (e.g., 420 nm) with little interference from fiber intrinsic changes, movement artifacts, or delta[Mg2+], furaptra represents a useful myoplasmic Ca2+ indicator, with properties complementary to those of other available indicators.

Perturbation of sarcoplasmic reticulum calcium release and phenol red absorbance transients by large concentrations of fura-2 injected into frog skeletal muscle fibers.

Intact single twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with two indicator dyes: phenol red, to monitor a previously described signal (denoted delta pHapp; Hollingworth and Baylor. 1990. J. Gen. Physiol. 96:473-491) possibly reflective of a myoplasmic pH change following action potential stimulation; and fura-2, to monitor the associated change in the myoplasmic free calcium concentration (delta[Ca2+]). Additionally, it was expected that large myoplasmic concentrations of fura-2 (0.5-1.5 mM) might alter delta pHapp, since it was previously found (Baylor and Hollingworth. 1988. J. Physiol. 403:151-192) that the Ca2(+)-buffering effects of large fura-2 concentrations: (a) increase the estimated total concentration of Ca2+ (denoted by delta[CaT]) released from the sarcoplasmic reticulum (SR), but (b) reduce and abbreviate delta[Ca2+]. The experiments show that delta pHapp was increased at the larger fura-2 concentrations; moreover, the increase in delta pHapp was approximately in proportion to the increase in delta[CaT]. At all fura-2 concentrations, the time course of delta pHapp, through time to peak, was closely similar to, although probably slightly slower than, that of delta[CaT]. These properties of delta pHapp are consistent with an hypothesis proposed by Meissner and Young (1980. J. Biol. Chem. 255:6814-6819) and Somlyo et al. (1981. J. Cell Biol. 90:577-594) that a proton flux from the myoplasm into the SR supplies a portion of the electrical charge balance required as Ca2+ is released from the SR into the myoplasm. A comparison of the amplitude of delta pHapp with that of delta[CaT] indicates that, in response to a single action potential, 10-15% of the charge balance required for Ca2+ release may be carried by protons.

Calcium sparks in intact skeletal muscle fibers of the frog.

Calcium sparks were studied in frog intact skeletal muscle fibers using a home-built confocal scanner whose point-spread function was estimated to be approximately 0.21 microm in x and y and approximately 0.51 microm in z. Observations were made at 17-20 degrees C on fibers from Rana pipiens and Rana temporaria. Fibers were studied in two external solutions: normal Ringer's ([K(+)] = 2.5 mM; estimated membrane potential, -80 to -90 mV) and elevated [K(+)] Ringer's (most frequently, [K(+)] = 13 mM; estimated membrane potential, -60 to -65 mV). The frequency of sparks was 0.04-0.05 sarcomere(-1) s(-1) in normal Ringer's; the frequency increased approximately tenfold in 13 mM [K(+)] Ringer's. Spark properties in each solution were similar for the two species; they were also similar when scanned in the x and the y directions. From fits of standard functional forms to the temporal and spatial profiles of the sparks, the following mean values were estimated for the morphological parameters: rise time, approximately 4 ms; peak amplitude, approximately 1 DeltaF/F (change in fluorescence divided by resting fluorescence); decay time constant, approximately 5 ms; full duration at half maximum (FDHM), approximately 6 ms; late offset, approximately 0.01 DeltaF/F; full width at half maximum (FWHM), approximately 1.0 microm; mass (calculated as amplitude x 1.206 x FWHM(3)), 1.3-1.9 microm(3). Although the rise time is similar to that measured previously in frog cut fibers (5-6 ms; 17-23 degrees C), cut fiber sparks have a longer duration (FDHM, 9-15 ms), a wider extent (FWHM, 1.3-2.3 microm), and a strikingly larger mass (by 3-10-fold). Possible explanations for the increase in mass in cut fibers are a reduction in the Ca(2+) buffering power of myoplasm in cut fibers and an increase in the flux of Ca(2+) during release.