Estudio comparativo de la técnica del ganglio centinela entre los casos de carcinoma de mama multifocal y unifocal / Comparative study of sentinel node biopsy in patients with multifocal breast carcinoma versus in those with unifocal breast carcinoma

Objetivo valorar los resultados de la biopsia del ganglio centinela (BGC) en pacientes con cáncer de mama multifocal (CMMF) en comparación con el unifocal (CMUF). Pacientes y métodos se han realizado e incluido en una base de datos de manera prospectiva 1.535 BGC a pacientes de 9 centros hospitalarios. De ellos 174 presentaban CMMF. Para la BGC se utilizaron coloides de Tc-99m y la vía de administración fue mayoritariamente la profunda, repartiendo el trazador en los diferentes focos. Resultados el índice de detección global fue del 93,8%, sin encontrar diferencias entre ambos grupos (el 94,8% en CMMF frente al 93,4%). La media de GC detectados fue de 1,46, siendo mayor en el grupo CMMF (1,58 frente a 1,45; p=0,036). La localización fue extraaxilar en el 19,6%, más frecuente en el grupo CMMF (el 23,4 frente al 18,9%, no significativo) y más en el territorio de la cadena mamaria interna y en el nivel III axilar. La incidencia de metástasis en los GC biopsiados fue del 27,3%, mayor en el grupo CMMF (el 29,1 frente al 26,7%, no significativo), con una media de GC afectados mayor (0,42 frente a 0,32, no significativo). En la linfadenectomía axilar se identificó afectación de ganglios adicionales en una proporción igual en ambos grupos (29,7%). Conclusionesla BGC parece tener un rendimiento similar en tumores unifocales y multifocales. En tumores multifocales, parece haber un patrón de drenaje linfático específico, con mayor número de GC detectados y probablemente con mayor número de localizaciones de GC extraaxilares (AU)

Objective To evaluate the results for sentinel node biopsy (SNB) in patients with multifocal breast cancer (MBC) in comparison to in those with unifocal breast cancer (UBC). Patients and methods A total of 1535 prospective SNB (174 on patients with MBC) were performed at 9 hospitals. In most patients, Tc-99m album in colloids were injected intraparenchymally into each tumoral focus for SNB. Results The overall identification rate was 93.8%; no differences between groups were observed (94.8% in MBC vs 93.4% in UBC). The mean number of sentinel nodes detected was 1.46, being higher in the MBC group than in the UBC group (1.58 vs 1.45; p=0.036). Extra-axillary sentinel nodes were found in 19.6%; extra-axillary sentinel nodes were more common in the MBC group (23.4% vs 18.9%, ns) and in the internal mammary chain and in level III axillary lymph nodes. The incidence of sentinel node metastasis was 27.3% (29.1% MBC vs 26.7% UBC, ns), and the mean number of positive sentinel nodes was 0.42 in the MBC group vs 0.32 in the UBC group (p=ns). Axillary dissection identified the same rate of positive additional nodes (29.7%) in both groups. Conclusions The diagnostic yield of SNB seems similar in MBC and UBC. In MBC, there appears to be a specific pattern of lymphatic drainage, with a higher number of sentinel nodes detected and probably a higher number of extra-axillary sentinel nodes (AU)

[Comparative study of sentinel node biopsy in patients with multifocal breast carcinoma versus in those with unifocal breast carcinoma.]. / Estudio comparativo de la técnica del ganglio centinela entre los casos de carcinoma de mama multifocal y unifocal.

OBJECTIVE: To evaluate the results for sentinel node biopsy (SNB) in patients with multifocal breast cancer (MBC) in comparison to in those with unifocal breast cancer (UBC). PATIENTS AND METHODS: A total of 1535 prospective SNB (174 on patients with MBC) were performed at 9 hospitals. In most patients, Tc-99m albumin colloids were injected intraparenchymally into each tumoral focus for SNB. RESULTS: The overall identification rate was 93.8%; no differences between groups were observed (94.8% in MBC vs 93.4% in UBC). The mean number of sentinel nodes detected was 1.46, being higher in the MBC group than in the UBC group (1.58 vs 1.45; p=0.036). Extra-axillary sentinel nodes were found in 19.6%; extra-axillary sentinel nodes were more common in the MBC group (23.4% vs 18.9%, ns) and in the internal mammary chain and in level III axillary lymph nodes. The incidence of sentinel node metastasis was 27.3% (29.1% MBC vs 26.7% UBC, ns), and the mean number of positive sentinel nodes was 0.42 in the MBC group vs 0.32 in the UBC group (p=ns). Axillary dissection identified the same rate of positive additional nodes (29.7%) in both groups. CONCLUSIONS: The diagnostic yield of SNB seems similar in MBC and UBC. In MBC, there appears to be a specific pattern of lymphatic drainage, with a higher number of sentinel nodes detected and probably a higher number of extra-axillary sentinel nodes.

Sentinel node biopsy as a practical alternative to axillary lymph node dissection in breast cancer patients: an approach to its validity.

BACKGROUND: Sentinel node biopsy (SNB) has been proposed as an alternative to axillary lymph-node dissection (ALND) in breast cancer. Before implementing SNB in our practice, we wished to test its validity by comparing it to the standard ALND, both in our hands and with other reported series. PATIENTS AND METHODS: One hundred thirty-two patients were included prospectively. SNB and immediate ALND were performed. For SNB, a technetium-colloid was used to produce preoperative lymphoscintigraphy and intraoperative gamma-probe search for the SN. Serial sectioning and immunostains were used on the SN. A comprehensive review of the literature was done in order to run a meta-analysis of diagnostic tests using a summary receiver operating characteristic curve (SROC) to calculate the pooled parameters of sensitivity and associated 95% confidence interval (95% CI), including our own data. RESULTS: Our technical success rate was 96%. Local sensitivity was 96%, with a 95% CI from 85%-99%. Seven patients were upstaged by the SNB. A literature search identified 18 studies published from 1996-1999. Estimates of sensitivity ranged from 83%-100%. The pooled data meta-analysis gave a global sensitivity of 91%, with a 95% CI from 89%-93%. The area under the global SROC curve was 0.9967. CONCLUSIONS: The minimally invasive SNB was shown to be a practical alternative to ALND. We propose to use local as well as global sensitivity and associated 95% CI to test the validity of SNB in the clinical setting. Due to limitations of ALND as the golden standard, SNB can in fact be considered a more accurate method for nodal staging.

Resultados de la biopsia del ganglio centinela en 100 pacientes con cáncer de mama / Results of sentinel node biopsy in 100 patients with breast cancer

Comunicamos los resultados obtenidos con la biopsia del ganglio linfático (BGC) en 100 pacientes consecutivas con cáncer de mama en las que además se realizó vaciado axilar. Se utilizaron exclusivamente trazadores isotópicos y sonda gamma para la linfogammagrafía y el rastreo intraoperatorio. El análisis histopatológico incluyó cortes seriados e inmunohistoquímica para citoqueratinas en los GC. Los resultados mostraron una eficacia técnica del 97 por ciento. El número de GC por paciente fue de 2,0 ñ 1,2. En el 23 por ciento de las pacientes se encontraron GC fuera de la localización habitual (mamaria interna, intramamarios, etc.). En total, 37 pacientes mostraron metástasis ganglionares. La sensibilidad de la BGC fue del 94,6 por ciento, el valor predictivo del 96,8 por ciento y la tasa de falsos negativos del 5,4 por ciento. Se reestadiaron al alza seis pacientes (9,1 por ciento de las consideradas N0 por el vaciado axilar). Nuestros resultados confirman el valor de la BGC, que en el futuro tenderá a sustituir a la linfadenectomía axilar convencional. (AU)

Effects of ramp shortening during linear phase of relaxation on [Ca2+]i in intact skeletal muscle fibers.

The effects of shortening distance at Vu, the unloaded shortening speed, and filament overlap on the amount of extra Ca2+ released during relaxation in muscle, as indicated by the bump area, were studied. Single, intact frog skeletal muscle fibers at 3 degreesC were used. The myoplasmic free Ca2+ concentration ([Ca2+]i) was estimated by using fura 2 salt injected into the myoplasm. Ramps were applied, either at full overlap with different sizes or at varying overlaps with a fixed size, in the linear phase of relaxation. At full overlap, a plot of bump area vs. ramp size was fit by using a sigmoidal curve with one-half of the bump area equal to 25.9 nm. With a fixed ramp size of 100 nm/half-sarcomere, the plot of bump area vs. mean sarcomere length (SLm) was fit by a straight line intersecting the SLm axis at approximately 3.5 micrometers, close to just no overlap. The results suggest that the transition in the distribution of attached cross bridges from the isometric case to one appropriate for unloaded shortening at Vu is completed within 50 nm/half-sarcomere and support the view that attached cross bridges in the overlap zone influence the affinity of Ca2+ for troponin C in the thin filament.

Effects of isoflurane on [Ca2+]i, SR Ca2+ content, and twitch force in intact trabeculae.

The goal was to test whether isoflurane exerts its depressant effect on the heart by mainly affecting the intracellular Ca2+ transient [Ca2+]i. Intact rat ventricular trabeculae, paced at 0.5 Hz and 30 degreesC with extracellular [Ca2+] ([Ca2+]o) of 2 mM, were used. The [Ca2+]i was monitored using fura 2 injected into the myoplasm. The sarcoplasmic reticulum (SR) Ca2+ content was estimated using rapid cooling with or without caffeine to induce Ca2+ release and contracture. A plot of peak twitch force versus peak [Ca2+]i transient with increasing isoflurane concentration declines linearly so that a 56% reduction in the peak [Ca2+]i transient would abolish twitch force. This relationship is intermediate between those obtained with lowering [Ca2+]o, which depresses twitch force through a reduction of the [Ca2+]i transient, and adding 2,3-butanedione monoxime, which reduces the responsiveness of the contractile system to [Ca2+]i. The isoflurane effect is different from that of halothane with respect to both the above relationship and the rapid-cooling response. Isoflurane abolishes the ability of rapid cooling to liberate Ca2+ from the SR.

Effects of rapid shortening on rate of force regeneration and myoplasmic [Ca2+] in intact frog skeletal muscle fibres.

1. The effect of rapid shortening on rate of force regeneration (dF/dtR) was examined in single, intact frog (Rana temporaria) skeletal muscle fibres (3.0 C). Step releases leading to unloaded shortening were applied after 500 ms of stimulation, during the plateau of an isometric tetanus. Initial mean sarcomere length ranged from 2.05 to 2.35 micrometer; force regeneration after shortening was at 2.00 micrometer. 2. Values for dF/dtR following a 25 nm half-sarcomere-1 release were 3.17 +/- 0.17 (mean +/- s.e.m., n = 8) times greater than the initial rate of rise of force before release (dF/dtI). As release size was increased from 25 to 175 nm half-sarcomere-1, the relationship between release size and dF/dtR decreased sharply before attaining a plateau value that was 1.34 +/- 0.09 times greater than dF/dtI. Despite wide variations in dF/dtR, the velocity of unloaded shortening remained constant (2.92 +/- 0.08 micrometer half-sarcomere-1 s-1; n = 8) for the different release amplitudes used in this study. 3. To investigate its role in the attenuation of dF/dtR with increased shortening, the effects of rapid ramp (constant velocity) shortening on intracellular free Ca2+ concentration ([Ca2+]i) were monitored using the Ca2+-sensitive fluorescent dye furaptra. Compared with an isometric contraction, rapid fibre shortening was associated with a transient increase in [Ca2+]i while force regeneration after shortening was associated with a transient reduction in [Ca2+]i. The greatest reductions in [Ca2+]i were associated with the largest amplitude ramps. 4. Cross-bridge-mediated modifications of the Ca2+ affinity of troponin C (TnC) may explain the fluctuations in [Ca2+]i observed during and after ramps. Associated fluctuations in TnC Ca2+ occupancy could play a role in the reduction of dF/dtR with increasing release size.

The effect of length on the relationship between tension and intracellular [Ca2+] in intact frog skeletal muscle fibres.

1. The relationship between tension and intracellular calcium concentration ([Ca2+]i) in intact frog skeletal muscle fibres was determined at two fibre lengths, corresponding to mean sarcomere lengths (SL) of 2.2 and 2.9 micron. Tension and [Ca2+]i were recorded during the slow decline of tension following stimulation in the presence of cyclopiazonic acid (CPA), a sarcoplasmic reticulum Ca2+-uptake pump inhibitor. [Ca2+]i was estimated by injecting the K+ salt form of the fluorescent dye fura-2 into the fibres. Experimental temperature was 3.0 C. 2. At a SL of 2.2 micron, where thick and thin filaments fully overlap, the [Ca2+]i corresponding to 50 % tension generation ([Ca2+]50) was 1.09 +/- 0.02 microM (mean +/- S.E.M., n = 61 contractions). At a SL of 2.9 micron, where overlap is approximately 50 %, the [Ca2+]50 was significantly lower, 0.69 +/- 0. 02 microM (n = 22 contractions). This is in agreement with previous results from skinned fibres. 3. The relationship between tension and [Ca2+]i was very steep, as reported previously from experiments at a SL of 2.2 micron in which the membrane permeant acetoxymethyl ester form of fura-2 was used. The fall in tension from 90 to 10 % occurred in 0.12 +/- 0.01 pCa units (mean +/- S.E.M., n = 61) for a SL of 2.2 micron and 0.17 +/- 0.01 pCa units (n = 22) for a SL of 2.9 micron, corresponding to Hill coefficients of 15.4 and 10.9, respectively. 4. We conclude that the increase in sensitivity of tension to [Ca2+] that occurs in skinned skeletal muscle fibres upon stretch also occurs in intact fibres, that the steepness of the relation between tension and [Ca2+]i in intact fibres reported previously cannot be attributed to the use of the acetoxymethyl ester form of fura-2 to report [Ca2+]i, and that the steepness decreases as myofilament overlap decreases.

Basis for late rise in fura 2 R signal reporting [Ca2+]i during relaxation in intact rat ventricular trabeculae.

Intact rat ventricular trabeculae were injected with the salt form of fura 2, and the fura 2 ratio signal (R) was used to report intracellular Ca2+ concentration ([Ca2+]i). The fixed end relaxation phase of a twitch is associated with a slowing of the decay of the R signal, or even a reversal, to form a distinct bump, indicating a transient rise in [Ca2+]i. The bump is most prominent at 30 degrees C, and motion artifact is not its cause. Increasing doses of 2,3-butanedione monoxime caused progressive attenuation of the twitch and bump. Increasing the bathing Ca2+ concentration potentiated the twitch and enhanced the bump. Imposed muscle shortening during relaxation caused a much quicker force decline, and this led to the appearance of a much more prominent associated bump. The amplitude of the bump depends on the amplitude of twitch force and the rate of relaxation. These findings can be explained, as in skeletal muscle, by making cross-bridge attachment and Ca2+ binding to troponin C strongly cooperative; therefore, the bump during fast relaxation is produced by a reversal of this cooperatively, leading to rapid dissociation of Ca2+ from troponin C into the myoplasm.

Effects of halothane on [Ca2+]i transient, SR Ca2+ content, and force in intact rat heart trabeculae.

The goal of this study was to test whether the well-known cardiodepressant effect of halothane is caused mainly by depression of the transient increase in intracellular Ca2+ concentration ([Ca2+]i) during a twitch. Intact rat ventricular trabeculae, paced at 0.5 Hz and 30 degrees C with a bathing extracellular Ca2+ concentration ([Ca2+]o) of 2 mM, were used. The [Ca2+]i was monitored with the use of fura 2 injected into the myoplasm. The sarcoplasmic reticulum (SR) Ca2+ content was estimated with the use of rapid cooling (RC)-induced contracture force and Ca2+ release. The relationship of the peak [Ca2+]i transient versus peak twitch force obtained with halothane is intermediate between those obtained with lowered [Ca2+]o and varying doses of 2,3-butanedione monoxime. The data indicate that the negative inotropic action of halothane at low (0.18 mM) dose is mainly achieved by reduction in the Ca2+ sensitivity of the contractile apparatus, whereas, at high dose (0.55 mM), halothane acts both by reducing the [Ca2+]i transient and the Ca2+ sensitivity of the contractile system. At moderate (0.37 mM) dose, the effects were midway between low and high doses. With the use of RC contracture force alone, the reduction of SR Ca2+ content caused by halothane is overestimated.

The relationship between tension and slowly varying intracellular calcium concentration in intact frog skeletal muscle.

1. The relationship between intracellular calcium concentration, [Ca2+]i, and fixed-end tension was investigated in intact single muscle fibres from frogs. A slow decline of tension was produced by cyclopiazonic acid (CPA), a sarcoplasmic reticulum Ca2+ uptake pump inhibitor. The fluorescent dyes fura-2 and furaptra (mag-fura-2) were used to estimate [Ca2+]i. 2. Neither the steepness nor the position of the curve changed consistently over a wide range of tension decay times from a few seconds to over 100 s. For these near-steady-state curves, the 10-90% tension change occurred, on average, in 0.07 pCa units, corresponding to a Hill coefficient > 25, much steeper than previously reported. Possible artifacts could reduce that to 15. 3. Methoxyverapamil (D600) reduces the calcium released in response to an action potential. Contractions with D600 and CPA had a slow rise composed of many small steps, and a slow fall. Comparing rise and fall showed little or no hysteresis in the tension-[Ca2+]i relationship. 4. A model involving co-operativity between the binding of Ca2+ and myosin to thin filaments is shown to produce a tension-pCa relationship that is substantially altered by the mean rate constant for detachment of myosin cross-bridges, which in turn is likely to be affected by sarcomere movements. 5. Such a model is shown to be capable of reproducing the small rise in [Ca2+]i previously reported during the late phase of fixed-end relaxation of intact fibres.

Pacing rate, halothane, and BDM affect fura 2 reporting of [Ca2+]i in intact rat trabeculae.

Experiments were done on intact trabeculae from rats. Fura 2 in the salt form was microinjected directly into the myoplasm. The experiments were conducted at 30 degrees C, with 2 mM extracellular Ca2+ concentration and pacing at either 0.5 or 5 Hz. The aims were to establish a new method for in vivo calibration of fura 2 and to determine the effect of autofluorescence changes on intracellular Ca2+ concentration ([Ca2+]i) reported by fura 2. Autofluorescence was recorded under optimal conditions for fura 2 fluorescence (emission at 510 nm). By alteration of the oxidation-reduction state, it was shown that NADH is the main component of autofluorescence in heart. An increase in pacing frequency caused a decrease in autofluorescence. Both halothane and 2,3-butanedione monoxime (BDM) at 5-Hz pacing produced a substantial rise in autofluorescence, approaching the levels observed at 0.5-Hz pacing. The values for the dissociation constant (678 nM) and maximum fluorescence ratio of fura 2 for Ca2+ for the in vivo calibration are 3.4 times larger and 2.6 times smaller, respectively, than those found in vitro. Using the parameters obtained in vivo, we found that the diastolic and systolic [Ca2+]i of a twitch at 30 degrees C were 0.2 and 2.4 microM, respectively. Proper correction of the autofluorescence change unmasks the [Ca2+]i elevation caused by 5-Hz pacing. It was concluded that autofluorescence is not constant and that interventions affecting autofluorescence need correction if fura 2 is used to report [Ca2+]i.

The effects of repeated active stretches on tension generation and myoplasmic calcium in frog single muscle fibres.

1. A series of contractions with stretches (eccentric contractions) beyond the optimal length for tension generation (optimum) were shown to induce a shift in that optimum in single muscle fibres of frog, as has been previously reported for whole muscles. Shifts averaging 0.129 micron (sarcomere)-1 or 6% were found, without apparent damage to the fibre. 2. The stiffness of fibres was found to fall during a stretch, even though tension was rising. In addition, the isometric stiffness fell as a result of a series of eccentric contractions. 3. Calcium-sensitive fluorescent dyes indicated that such contractions did not reduce the amplitude of the intracellular calcium transient, but did increase its duration. A rise in resting [Ca2+] was found to accompany damage, but not necessarily the shift in optimum. 4. The twitch potentiator nitrate was shown to increase myoplasmic [Ca2+] during twitch and tetani, but not to reverse the shift in optimum length due to eccentric contractions. Both eccentric contractions and twitch potentiation reduced the maximum stimulation rate to which a fibre could respond with propagated action potentials. 5. These results exclude reduced myoplasmic [Ca2+] as the cause of the shift in optimum length in this preparation.

Rapid cooling contractures in rat skinned myocardium: effects of isoflurane and halothane.

Rapid cooling contractures (RCCs) have been elicited in rat ventricular cardiac muscle skinned by saponin (50 micrograms/ml). The size and shape of the RCC in skinned cardiac muscle were similar to those observed in intact cardiac muscle. The ATP-dependent Ca2-uptake pump in the sarcoplasmic reticulum (SR) was active at low temperature and could efficiently load the SR with Ca2+ even at 3 degrees C. Halothane reduced both RCC and caffeine contracture in a dose-dependent way when halothane treatment was applied before either RCC or caffeine. This action is consistent with halothane-induced depletion of the SR Ca2+. Under similar conditions, isoflurane inhibited RCC but had little effect on the caffeine contracture. This may suggest that rapid cooling and caffeine have different modes of action on the SR Ca(2+)-release channel. Our results provide further strong supporting evidence for differential inhibitory actions on a key intracellular organelle in cardiac muscle by two vapor anesthetic agents.

The intracellular Ca2+ transient and tension in frog skeletal muscle fibres measured with high temporal resolution.

1. The purpose of this study was to determine, with high temporal resolution, the relationship between the intracellular Ca2+ transient (ICT) and the mechanical responses of intact, single skeletal muscle fibres of frogs following stimulation by a single, brief depolarization. 2. The time course of the ICT was monitored using the Ca(2+)-sensitive fluorescent dyes mag-fura-2 (furaptra) and mag-fura-5. The mag-fura dyes have a low affinity for Ca2+ and have been shown to track the ICT with no appreciable kinetic delay. Continuous records of mag-fura fluorescence, tension and stiffness responses were obtained simultaneously at high time resolution at a sarcomere length of 2.9 microns. Experimental temperature was 3 degrees C. 3. When a delay of 0.4 ms due to the low-pass filter associated with the photodetector was included, the onset of the fluorescence response preceded the onset of latency relaxation (the small fall in tension that precedes positive tension generation) by 3.1 +/- 0.2 ms (mean +/- S.E.M., n = 8). After its onset, the mag-fura fluorescence signal continued to change rapidly (indicating increasing intracellular [Ca2+]) to an extreme level that occurred 1.5 +/- 0.5 ms (mean +/- S.E.M., n = 7) before tension had recovered to its resting level following latency relaxation. The time delay from the extreme of the fluorescence signal to the peak of the tension signal was 239 +/- 27 ms (mean +/- S.E.M., n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of halothane, enflurane, and isoflurane on skinned rat myocardium activated by Ca2+.

The effects of halothane (1.9 mM, 9.4 mM), enflurane (3.3 mM, 16.5 mM), and isoflurane (1.6 mM, 8.1 mM) on maximal Ca(2+)-activated force and Ca2+ sensitivity were studied in rat myocardial preparations rendered permeable by various methods. In preparations permeabilized either by mild homogenization or by saponin (50 micrograms/ml, 30 min), further disruption of the sarcolemma with 2% Triton X-100 resulted in increased maximal force and Ca2+ sensitivity. When membranes were mechanically disrupted or saponized, each vapor agent caused the myocardium to be more sensitive to Ca2+. In these preparations, maximal force was decreased by halothane but was not affected by enflurane or isoflurane. With cellular membranes completely disrupted by Triton, the previously observed increase in Ca2+ sensitivity caused by enflurane was reduced, whereas that caused by halothane and isoflurane was abolished and maximal force was decreased by halothane and isoflurane but was not affected by enflurane. These results indicate that 1) components associated with the cellular membrane systems normally modulate force in mammalian myocardium, and 2) halothane, enflurane, and isoflurane have complex effects on these components. These results therefore can explain some of the differences in inotropic effects that these agents exert on mammalian myocardium.

Tension as a function of sarcomere length and velocity of shortening in single skeletal muscle fibres of the frog.

1. Simple measurements of muscle tension at fixed fibre or segment length produce a range of length-tension relationships, depending primarily on the duration of the interval between stimulation onset and tension measurement, in contradiction with the simple predictions of current models. This has been explained by non-uniformity in sarcomere lengths, leading to internal motion and, in turn, to increasing tension because the force-velocity relationship has a much greater slope for slow lengthening than for slow shortening. 2. Previous attempts to reduce the effect of internal motion have been focused on decreasing the initial extent of non-uniformity and measuring tension early in a contraction, when non-uniformities are at a minimum. An alternative approach that has not been attempted previously is to reduce the non-linearity of the force-velocity relationship by avoiding the discontinuity in slope at zero velocity. This is accomplished by imposing overall fibre shortening at velocities sufficient to ensure that all sarcomeres are shortening. 3. When the tension maintained during shortening was measured and plotted against sarcomere length for each release velocity used, linear length-tension relationships resulted that extrapolated to a common sarcomere length intercept. This was true whether the release was applied early in the tetanus or near the end of the 'creep phase' of tension rise. These observations were duplicated by computer simulation using a multisarcomere model of a muscle fibre. 4. These results provide strong support for the view that cross-bridges function as independent force generators and for the explanation of the creep phase of fibre or segment isometric tension as being due to internal motion. The results also imply that the force-velocity relationship scales with sarcomere length without changing shape. 5. Using this novel method for obtaining length-tension relationships, the sarcomere length at which active tension fell to zero was found, by extrapolation, to be 3.65 microns in semitendinosus fibres and 3.53 microns in tibialis anterior fibres from the frog (Rana temporaria).

Unloaded shortening velocity of skinned rat myocardium: effects of volatile anesthetics.

The slack test method has been adapted for measurement of unloaded velocity of shortening in rat ventricular trabeculae that were skinned with saponin (50 micrograms/ml for 30 min). The method was sensitive enough to detect a 17% reversible change in the unloaded velocity of shortening produced by a 3 degrees C change in temperature. At pCa 5.30 (80-90% activation), halothane, enflurane, and isoflurane each slowed the shortening velocity by 25-30% at dose levels of 8 mM or greater but not at 4 mM or less. At pCa 5.48 (50-60% activation), halothane slowed the shortening velocity by 20-45% at dose levels of 4 mM or greater but not at 2 mM. The slowing effect of anesthetics on shortening velocity showed saturation at 8 mM for halothane, enflurane, and isoflurane when activation was at pCa 5.30. Saturation occurred at 4 mM for halothane when the pCa was 5.48. This result indicates that the dose-response relationship may be narrow, such that it can be demonstrated between 2 and 4 mM halothane for pCa 5.48 and between 4 and 8 mM halothane for pCa 5.30. The anesthetic dose dependence of isometric force and length axis intercept did not generally follow the same relationship as for the shortening velocity. Thus in several instances force did not significantly decrease when the velocity of shortening did. This may be interpreted as lack of simple inhibition by anesthetics on the number of interacting cross-bridges and as direct influence by anesthetics on the cross-bridge cycle.

Halothane increases Ca2+ efflux via Ca2+ channels of sarcoplasmic reticulum in chemically skinned rat myocardium.

1. A method has been developed to study Ca2+ fluxes across the sarcoplasmic reticulum (SR) of chemically (saponin) skinned myocardium without interference from the SR Ca2+ pump. 2. Exposure of rat cardiac trabeculae to a solution containing 50 micrograms/ml saponin for 10 min or longer caused an SR Ca2+ efflux which was not blocked by Ruthenium Red (RRed) and did not require the presence of nucleotides. 3. Exposure of the saponin-treated cardiac preparation to 11 mM-AMP, when the SR Ca2+ pump was not active, enhanced Ca2+ release from the SR by a mechanism which was blocked by 10 microM-RRed. 4. The amount of Ca2+ loaded by the 10 min saponin-treated trabeculae was maintained constant for at least 3 min when the preparations were transferred to low [Ca2+] solutions (0.1 mM-EGTA; pCa greater than 7.5) containing ATP. This indicated that the Ca2+ pump can efficiently recycle Ca2+ lost from the SR under these conditions. 5. Halothane (0.47 and 1.89 mM) reversibly increased the rate of Ca2+ release from the SR regardless of whether or not the SR Ca2+ pump was active. This effect was more marked at 1.89 mM than at 0.47 mM. RRed (10 microM) completely blocked the Ca2+ release induced by both concentrations of halothane. 6. The presence of nucleotide (11 mM-AMP) did not affect the halothane-induced Ca2+ release when the Ca2+ pump was inactive. 7. Exposure of cardiac preparations to solutions containing more than 5 mM-halothane irreversibly damaged the ability of the SR to load Ca2+. 8. The results suggest that at lower doses (0.47 and 1.89 mM) halothane specifically and reversibly stimulates Ca2+ efflux via the RRed-sensitive SR Ca2(+)-release channel by a mechanism which does not require the presence of nucleotides or relatively high [Ca2+]. The results also suggest that AMP and halothane act independently and non-synergistically to increase Ca2+ efflux through the same SR Ca2(+)-release channel. At higher doses (greater than 5 mM) halothane irreversibly damages the SR membrane, presumably by disrupting the lipid bilayer.

Tension in frog single muscle fibers while shortening actively and passively at velocities near Vu.

Experiments were undertaken to determine the contribution of passive tension to total tension during rapid shortening in a stimulated muscle fiber. Results were obtained by applying shortening movements at constant velocities slightly less than Vu (the velocity of unloaded shortening) to intact twitch fibers isolated from the frog (Rana temporaria). The tension maintained by unstimulated fibers during such shortening movements ("dynamic passive tension") from moderately long lengths was greater than zero but much less than the passive tension measured under static conditions ("static passive tension") at the same lengths. Fibers maximally activated by electrical stimulation and then shortened at the same velocity over the same range of average sarcomere lengths maintained tension that was greater than zero but less than the dynamic passive tension. For average sarcomere lengths up to approximately 3.1 microns, the dynamic passive tension appeared to be substantially abolished by activation. The onset of the apparent disappearance of dynamic passive tension was studied by initiating the stimulation and the shortening movement simultaneously. The resulting tension response exhibited a latency relaxation that was increased in amplitude compared with the isometric case, followed by a brief tension rise, giving way to a steady tension level equal to that expected if stimulation had been initiated well before the release. These changes are qualitatively explained in terms of the establishment of a steady state distribution of deformations of attached cross-bridges.