Adrenal hemorrhage in newborn: how, when and why- from case report to literature review.

BACKGROUND: Neonatal adrenal hemorrhage is a relatively uncommon condition (0.2-0.55%). Various risk factors have been reported in addition to birth asphyxia, such as sepsis, coagulation disorders, traumatic delivery, and perinatal injuries. Adrenal hemorrhage usually affects the right adrenal gland (about 70% of cases) while it involves the bilateral adrenal gland only in 10% of cases. In most cases, the event is asymptomatic but, in others, it may be so devastating to determine death by bleeding or adrenal insufficiency. CASE PRESENTATION: A case of bilateral neonatal adrenal hemorrhage, with adrenal insufficiency, but with no important risk factors and favorable evolution in a male infant. CONCLUSIONS: This case emphasizes the importance of keeping a non-interventional attitude, avoiding early surgery but carrying out a serial sonographic follow-up. Serial ultrasound monitoring is the most reliable approach during conservative management.

Presynaptic calcium stores underlie large-amplitude miniature IPSCs and spontaneous calcium transients.

The cellular mechanisms responsible for large miniature currents in some brain synapses remain undefined. In Purkinje cells, we found that large-amplitude miniature inhibitory postsynaptic currents (mIPSCs) were inhibited by ryanodine or by long-term removal of extracellular Ca2+. Two-photon Ca2+ imaging revealed random, ryanodine-sensitive intracellular Ca2+ transients, spatially constrained at putative presynaptic terminals. At high concentration, ryanodine decreased action-potential-evoked rises in intracellular Ca2+. Immuno-localization showed ryanodine receptors in these terminals. Our data suggest that large mIPSCs are multivesicular events regulated by Ca2+ release from ryanodine-sensitive presynaptic Ca2+ stores.

Suprasellar keratinous cyst: A case report and review on its radiological features and treatment outcome.

BACKGROUND: Keratinous or epidermoid cysts (ECs) are encapsulated lesions lined by squamous cell epithelium. They comprise approximately 1% of intracranial lesions. Contrary to dermoid cysts, they lack dermal elements such as sebaceous or apocrine glands and hair follicles. The sellar region is the second most common intracranial site following the cerebellopontine angle. Here, we report a case of EC in a patient who complained of endocrine disturbances. We also performed a systematic review on previously published cases to analyze clinical and radiological characteristics and report the treatment outcomes of suprasellar ECs. CASE DESCRIPTION: A 42-year-old woman presented with a one-year history of amenorrhea, weight gain, severe headache, and visual disturbances for 6 months. Work-up identified an elevated prolactin level and a temporal field defect of the right eye. Magnetic resonance imaging (MRI) showed a cystic suprasellar lesion pushing on the optic chiasm. She underwent endoscopic trans-sphenoidal surgery, which confirmed a keratinous cyst on histology. Postoperatively, complete resection was confirmed on imaging. She did well although her hospital stay was prolonged due to diabetes insipidus and hypocortisolism. CONCLUSION: Chronic endocrine disturbances can be the presenting complaints of a suprasellar EC, whose T1-weighted MRI appearance can be non-specific, mimicking other differential diagnoses, such as a Rathke's cleft cyst. However, the T2-weighted MRI appearances of ECs are generally hyper-intense and lesions show diffusion restriction. Treatment is surgical and yields good outcomes in most cases reported.

Release of calcium into the myofibrillar space in response to active shortening of striated muscle.

AIM: The study was undertaken to explore whether shortening of striated muscle during activity is associated with release of bound Ca2+ into the myofibrillar space as has previously been proposed in order to explain the depressant effect of active shortening. METHODS: The experiments were carried out on single muscle fibres isolated from the anterior tibialis muscle of Rana temporaria. The fibres were loaded with the calcium sensitive indicator Fluo-3. The fibres, stimulated to produce a partially fused isometric tetanus, were subjected to a shortening ramp or, alternatively, to a stretch ramp during activity while force, fibre length, sarcomere length and the Fluo-3 signal were recorded. RESULTS: A shortening ramp performed during a partially fused tetanus caused an increase in the myofibrillar free calcium concentration and produced, simultaneously, a decrease in active force. The isometric force recovered gradually after the shortening ramp, while the intracellular Ca2+ concentration stayed above the control level during the remainder of the stimulation period. A stretch ramp applied during a partially fused tetanus caused a considerably smaller change in the myofibrillar Ca2+ concentration. CONCLUSION: The results provide evidence that the myosin cross-bridges interact with the calcium binding sites on the thin filaments during active shortening, causing sustained release of calcium and reduced contractile strength.

The effect of ATP on calcium efflux in dialyzed barnacle muscle fibres.

Calcium efflux has been studied in barnacle muscle fibres under internal dialysis conditions. Prolonged dialysis of these fibres, with a medium free of ATP and containing 2 mM cyanide and 1 mM iodoacetate, causes the ATP in the perfusion effluent to fall to less than 20 micrometer. The mean calcium efflux from fibres dialyzed with EGTA buffered solution containing 0.3 micrometer ionized Ca and and no ATP is 0.6 pmol-cm-2-s-1. A two-fold stimulation of the calcium efflux is observed when ATP is added to fibres previously dialyzed with an ATP-free medium. Withdrawal of Na+ and Ca2+ from the external medium causes a marked drop in the Ca2+ efflux in the presence of internal ATP.

Currents related to the sodium-calcium exchange in squid giant axon.

We report the measurement of a Cai-activated membrane current in dialyzed squid axon under membrane potential control with a low-noise voltage clamp. Two additional voltage clamp systems were used to clamp the external guard plates to a value that prevented the establishment of potential differences between the central and lateral compartments of the experimental chamber. This reduced to a minimum the contribution of membrane currents generated at the axon ends to the current measured in the central pool. This latter current was reduced by using internal and external solutions designed to diminish at a maximum membrane currents, while maintaining the conditions for optimal operation of the Na+-Ca2+ exchange. Thus TTX was used to block Na+ channels and prolonged exposure to K+-free media was used to eliminate K+ conductance. The maximum concentration of external sodium was 200 mM. The addition of fixed amounts of free ionic calcium to the internal solution, activated a current whose direction and magnitude depended on the thermodynamic driving forces for calcium and sodium. When the experimental conditions determined an inwardly directed current, this depended on the presence of external sodium, and lithium could not substitute for it. The Cai-activated current, was blocked by external lanthanum and showed a high temperature dependence. In experiments in which the reversal potential was measured for the Cai-activated current, it was found to be strikingly similar to the value calculated according to Er = 3ENa - 2ECa, suggesting that the current is the electrical manifestation of the Na+-Ca2+ exchange operating with an stoichiometry of 3Na+:1Ca2+.

Direct measurement of intracellular free magnesium in frog skeletal muscle using magnesium-selective microelectrodes.

Mg2+-selective microelectrodes have been used to measure the intracellular free Mg2+ concentration in frog skeletal muscle fibers. Glass capillaries with a tip diameter of less than 0.4 micron were backfilled with the Mg2+ sensor, ETH 1117. In the absence of interfering ions, they gave Nernstian responses between 1 and 10 mM free Mg2+. In the presence of an ionic environment resembling the myoplasm, the microelectrode response was sub Nernstian (18-24 mV) but still useful. The electrodes were calibrated before and after muscle-fiber impalements . In quiescent fibers from sartorius muscle (Rana pipiens), with resting membrane potentials not less than -82 mV, the intracellular free Mg2+ concentration was 3.8 +/- 0.41 (S.E.) mM (n = 58) at 22 degrees C. No significant change in the intracellular free Mg2+ was observed following extensive (approx. 6 h) incubation in Mg2+-free media. Increasing the external concentration of magnesium from 4 to 20 mM (approx. 15 min) produced a slow and small enhancement (1.8 mM) of [Mg2+]i, which was fully reverted when the divalent cation was removed from the bathing solution. No change in ionic magnesium resting concentration was observed when the muscle fibers were treated either with caffeine 3 mM or with Na+-free solutions. In depolarized muscle fibers (-23 +/- 2.7 mV) treated with 100 mM K+, the myoplasmic [Mg2+] was 3.7 +/- 0.45 (S.E.) mM, n = 6, immediately after the spontaneous relaxation of the contracture. Similar determinations in muscle fibers during stimulation at low frequency (5 Hz), and after fatigue development, showed no changes in the concentration of free cytosolic Mg2+. These results point out that [Mg2+]i is not modified under these three different experimental conditions.

Measurements of intracellular ionized calcium in squid giant axons using calcium-selective electrodes.

Ca2+-selective electrodes have been used to measure free intracellular Ca2+ concentrations in squid giant axons. Electrodes made of glass cannulas of about 20 microns in diameter, plugged with a poly(vinyl chloride) gelled sensor were used to impale the axons axially. They showed a Nernstian response to Ca2+ down to about 3 microM in solutions containing 0.3 M K+ and 0.025 M Na+. Sub-Nernstian but useful responses were obtained up to pCa 8. The electrodes showed adequate selectivity to Ca2+ over Mg2+, H+, K+ and Na+. To calibrate them properly, a set of standard solutions were prepared using different Ca2+ buffers (EGTA, HEEDTA, nitrilotriacetic acid) after carefully characterizing their apparent Ca2+ association constants under conditions resembling the axoplasmic environment. In fresh axons incubated in artificial seawater containing 4 mM Ca2+, the mean resting intracellular ionized calcium concentration was 0.106 microM (n = 15). The Ca2+-electrodes were used to investigate effects of different experimental procedures on the [Ca2+]i. The main conclusions are: (i) intact axons can extrude calcium ions at low [Ca2+]i levels by a process independent of external Na+; (ii) poisoned axons can extrude calcium ions at high levels of [Ca2+]i by an external Na+-dependent process. The level of free intracellular Ca attained at these latter conditions is about an order to magnitude greater than the resting physiological value.

Properties of several protein kinases that copurify with rat spinal cord neurofilaments.

Several protein kinases that copurify with neurofilaments (NF) were identified and each kinase was assessed for its ability to phosphorylate NF proteins. NFs were isolated using an axonal flotation procedure and the kinases were extracted from NFs with 0.8 M KCl. NF kinases were incubated with peptide substrates for selected protein kinases, [32P]ATP and protein kinase cofactors and inhibitors to characterize the kinases. Using peptide substrates, three types of kinase were identified, and a fourth was identified using NF protein as substrate. The first three kinases were the catalytic subunit of cAMP-dependent protein kinase, calcium-calmodulin dependent protein kinase II and a cofactor-independent kinase that phosphorylated prepro VIP sequence 156-170 and was inhibited by heparin. Using NF proteins as substrate, a fourth kinase was identified which was cofactor-independent and was not inhibited by heparin. Neither cofactor-independent kinase was casein kinase II. NF proteins were phosphorylated in vitro on serine and threonine, primarily by the two cofactor-independent kinases. Using [alpha-32P]8-N3ATP for affinity labeling, one kinase of 43,800 Da was identified. Thus, in addition to cAMP-dependent protein kinase and calcium-calmodulin dependent protein kinase II, two kinases have been found which are primarily responsible for NF phosphorylation in vitro and are cofactor-independent.

Telephone counseling improves adherence to colposcopy among lower-income minority women.

PURPOSE: A randomized trial was conducted to evaluate the impact of a telephone counseling intervention to improve patient adherence to colposcopic examination for suspected cervical intraepithelial neoplasia (CIN). METHODS: Subjects were lower-income, minority women who missed a scheduled initial appointment for colposcopy at an urban medical clinic. Patients were randomly assigned to either a control condition (n = 42) or a telephone counseling condition (n = 48). The 15-minute, structured telephone counseling intervention protocol addressed educational, psychosocial, and practical barriers to colposcopy adherence. RESULTS: The most common patient-reported barriers to colposcopy adherence included a lack of understanding of the purpose of colposcopy (50%), worry about or fear of cancer (25%), and forgetting (23%). Telephone counseling was found to be highly effective in addressing these barriers and improving adherence to diagnostic follow-up and treatment. Of patients in the control condition, 43% complied with a rescheduled colposcopy appointment, compared with 67% in the telephone counseling condition. Logistic regression analysis indicated that the effect of telephone counseling was independent of sociodemographic confounder variables (odds ratio = 2.6; P less than .003). Additionally, 74% of patients who received the initial telephone counseling adhered to recommended treatment, compared with 53% of patients in the control condition. CONCLUSION: Brief, structured telephone contact may be a cost-effective mechanism for improving adherence to diagnostic follow-up and treatment for a variety of cancer screening tests.

Volume and twitch tension changes in single muscle fibers in hypertonic solutions.

Single muscle fibers were exposed to solutions made hypertonic (approximately 460 milliosmols/kg water) by addition of either NaCl, glycerol, urea, acetamide, ethylene glycol, or propylene glycol. The changes in either the fiber twitch tension or the volume were measured. In the case of NaCl both fiber volume and twitch tension fall rapidly to 64 and 27% of the respective initial value. These two values were maintained for the duration of the exposure. In the case of the other substances, the fiber volume and twitch tension also decreased but in these cases the effect was transient and the fibers recovered their initial volume and twitch tension. The rate of recovery in the different hypertonic media increased in the order: glycerol < urea < ethylene glycol < propylene glycol < acetamide. In the cases of the last three substances, the initial twitch value was recovered in less than 5 min and even surpassed. However, on returning to normal Ringer the fibers' ability to twitch or to develop potassium contractures was lost. The return of the fibers to normal Ringer after exposure to these hypertonic solutions causes a transient swelling of the fibers. However, when fibers were swelled by exposure to hypotonic media, they did not lose their ability to twitch on return to the normal Ringer.

Caffeine- and potassium-induced contractures of frog striated muscle fibers in hypertonic solutions.

The effect of hypertonic solutions on the caffeine- and KCl-induced contractures of isolated fibers of frog skeletal muscle was tested. Hypertonic solutions, twice the normal osmotic strength, prepared by adding NaCl or sucrose, potentiate the caffeine-induced contractures. The fibers may develop tensions of 3.6 kg/cm(2) of fiber transverse section. The same hypertonic medium reduced the peak tension of KCl-induced contractures. Thus the hypertonic condition does not affect the contractile mechanism itself. These findings give further support to the view that the differential effect of hypertonic solution is on the excitation-contraction coupling mechanism. Extracellular calcium is not essentially required for the first few of a series of caffeine-induced contractures either in hypertonic or in isotonic solutions.

Contractile activation phenomena in voltage-clamped barnacle muscle fiber.

Tension development in voltage-clamped barnacle muscle fibers occurs with depolarizing pulses so small as not to activate the potassium and calcium conductance systems. Peak tension and the tension time integral appear to be graded by both amplitude and duration of the depolarizing pulses. Subthreshold depolarizing conditioning pulses shorter than 500 ms potentiate the response to a given test pulse. This effect diminishes and reverts when the duration of the conditioning pulse is increasingly prolonged. The relationship between fiber membrane potential and tension developed in response to depolarizing pulses is described by an S-shaped curve. The tension saturates at a membrane potential of about +10 mV (inside positive). For a given pulse duration the saturation value remains constant even when the fiber interior reaches a value of +230 mV, which is well above what may be estimated to be the equilibrium potential of calcium ions (Eca = +120). In the presence of 5 mM external procaine, the shape of the tension-potential curve changes; the maximum value tension besides being diminished is not sustained by falls when the potential approaches the estimated value for Eca. These results suggest that under physiological conditions the contractile activator is probably released from an internal store, and that the calcium entering the fiber as inward current does not play a direct major role in contractile activation.

Effects of external calcium deprivation on single muscle fibers.

Deprivation of external calcium causes sudden potentiation of the twitch response of single muscle fibers. The potentiation was 64 +/- 8%. Potentiation is simultaneous with membrane depolarization occurring after Ca(++) removal. This depolarization amounted to 9 +/- 2 mv. Ca(++) removal also alters the action potential. 3 min after calcium withdrawal, action potential amplitude fell by 36 +/- 3 mv; maximum rates of rise and fall of the spike decreased by 55 +/- 5 and 63 +/- 5% respectively. Changes in shape of the A. P. differ from those seen with other potentiators of the twitch response, such as Zn(++). After short exposure to calcium-free media, potassium-induced contractures show potentiation of peak tension. The S-shaped curve relating potassium contracture tension to log [K](o) shifts to the left after such treatment. Calcium deprivation also increased the rate of relaxation of the contractures. This effect depends on the duration of calcium deprivation, and is probably related to the effect of calcium lack on the membrane. The change in relaxation occurred immediately after calcium deprivation, and was reversed by sudden readmission of calcium. Relaxation of twitch and tetanus responses also were affected by Ca lack, but not as rapidly as potassium contractures. The results suggest that external calcium is not directly involved in the process responsible for tension development, supporting the view that this process is mediated by translocation of intracellular calcium. The relaxation process, however, appears to be rapidly affected by deprivation of external calcium.

Contractile inactivation in frog skeletal muscle fibers. The effects of low calcium, tetracaine, dantrolene, D-600, and nifedipine.

Short muscle fibers (approximately 1.5 mm) of Rana pipiens were voltage-clamped with a two-microelectrode technique at a holding potential of -100 mV. Using conditioning depolarizing ramps, with slopes greater than 0.2 mV/s, partially inactivated responses are obtained at threshold values between -55 and -35 mV. With slopes equal to or slower than 0.1 mV/s, one inactivates contraction without ever activating it. When the membrane potential is brought slowly to values more positive than about -40 mV, test pulses, applied on top of the ramps, bringing the membrane potential to values up to +100 mV, are ineffective in eliciting contractile responses, which indicates complete inactivation. After inactivation, contractile threshold is shifted by perhaps 10 mV, to about -40 mV. The sensitivity of fibers to depolarizing ramps is increased by D-600 (50 microM), dantrolene (50 microM), tetracaine (100 microM), and low calcium (10(-8) M). In the presence of these agents, complete inactivation was obtained using ramp slopes of 1, 0.8, 0.4, and 0.2 mV/s, respectively. Nifedipine was less effective. With D-600, once inactivation had been induced, no repriming occurred after repolarization to -100 mV, and partial recovery occurred after washing out the drug. With low calcium, tetracaine, and nifedipine, the tension-voltage relationship was not affected, whereas the steady state inactivation curve (obtained in repriming experiments) was shifted by 10-25 mV toward more negative potentials. With D-600, the activation curve was not modified, whereas the inactivation curve could not be obtained, because of repriming failure. With dantrolene, the inactivation curve was not affected, whereas the activation curve was shifted toward less negative potentials and peak tension diminished, depending on the pulse duration. The results indicate that it is possible to induce complete inactivation without activation, and to differentiate activation and inactivation parameters pharmacologically, which suggests that the two are separate processes.

Ultraslow contractile inactivation in frog skeletal muscle fibers.

After a contracture response, skeletal muscle fibers enter into a state of contractile refractoriness or inactivation. Contractile inactivation starts soon after membrane depolarization, and causes spontaneous relaxation from the contracture response. Here we demonstrate that contractile inactivation continues to develop for tens of seconds if the membrane remains in a depolarized state. We have studied this phenomenon using short (1.5 mm) frog muscle fibers dissected from the Lumbricalis brevis muscles of the frog, with a two-microelectrode voltage-clamp technique. After a contracture caused by membrane depolarization to 0 mV, from a holding potential of -100 mV, a second contracture can be developed only if the membrane is repolarized beyond a determined potential value for a certain period of time. We have used a repriming protocol of 1 or 2 s at -100 mV. After this repriming period a fiber, if depolarized again to 0 mV, may develop a second contracture, whose magnitude and time course will depend on the duration of the period during which the fiber was maintained at 0 mV before the repriming process. With this procedure it is possible to demonstrate that the inactivation process builds up with a very slow time course, with a half time of approximately 35 s and completion in greater than 100 s. After prolonged depolarizations (greater than 100 s), the repriming time course is slower and the inactivation curve (obtained by plotting the extent of repriming against the repriming membrane potential) is shifted toward more negative potentials by greater than 30 mV when compared with similar curves obtained after shorter depolarizing periods (10-30 s). These results indicate that important changes occur in the physical state of the molecular moiety that is responsible for the inactivation phenomenon. The shift of the inactivation curve can be partially reversed by a low concentration (50 microM) of lanthanum ions. In the presence of 0.5 mM caffeine, larger responses can be obtained even after prolonged depolarization periods, indicating that the fibers maintain their capacity to liberate calcium.

Effects of D-600 on intramembrane charge movement of polarized and depolarized frog muscle fibers.

Intramembrane charge movement has been measured in frog cut skeletal muscle fibers using the triple vaseline gap voltage-clamp technique. Ionic currents were reduced using an external solution prepared with tetraethylammonium to block potassium currents, and O sodium + tetrodotoxin to abolish sodium currents. The internal solution contained 10 mM EGTA to prevent contractions. Both the internal and external solutions were prepared with impermeant anions. Linear capacitive currents were subtracted using the P-P/4 procedure, with the control pulses being subtracted either at very negative potentials, for the case of polarized fibers, or at positive potentials, for the case of depolarized fibers. In 63 polarized fibers dissected from Rana pipiens or Leptodactylus insularis frogs the following values were obtained for charge movement parameters: Qmax = 39 nC/microF, V = 36 mV, k = 18.5 mV. After depolarization we found that the total amount of movable charge was not appreciably reduced, while the voltage sensitivity was much changed. For 10 fibers, in which charge movement was measured at -100 and at 0 mV, Qmax changed from 46 to 41 nC/microF, while V changed from -41 to -103 mV and k changed from 20.5 to 30 mV. Thus membrane depolarization to 0 mV produces a shift of greater than 50 mV in the Q-V relationship and a decrease of the slope. Membrane depolarization to -20 and -30 mV, caused a smaller shift of the Q-V relationship. In normally polarized fibers addition of D-600 at concentrations of 50-100 microM, does not cause important changes in charge movement parameters. However, the drug appears to have a use-dependent effect after depolarization. Thus in depolarized fibers, total charge is reduced by approximately 20%. D-600 causes no further changes in the voltage sensitivity of charge movement in fibers depolarized to 0 mV, while in fibers depolarized to -20 and -30 mV it causes the same effects as that obtained with depolarization to 0 mV. These results are compatible with the idea that after depolarization charge 1 is transformed into charge 2. D-600 appears to favor the conversion of charge 1 into charge 2. Since D-600 also favors contractile inactivation, charge 2 could represent the state of the voltage sensor for excitation-contraction coupling in the inactivated state.

Effects of sulfhydryl inhibitors on depolarizations-contraction coupling in frog skeletal muscle fibers.

We have studied the effects of the sulfhydryl reagents on contractile responses, using either electrically stimulated single muscle fibers or short muscle fibers that were voltage-clamped with a two-microelectrode voltage-clamp technique that allows the fiber tension in response to membrane depolarization to be recorded. The sulfhydryl inhibitors para-chloromercuribenzoic acid (PCMB) and parahydroximercuriphenyl sulfonic acid (PHMPS), at concentrations from 0.5 to 2 mM, cause loss of the contractile ability; however, before this effect is completed, they change the fiber contractile behavior in a complex way. After relatively short exposure to the compounds, < 20 min, before the fibers lose their contractile capacity, secondary tension responses may appear after electrically elicited twitches or tetani. After losing their ability to contract in response to electrical stimulation, the fibers maintain their capacity to develop caffeine contractures, even after prolonged periods (120 min) of exposure to PHMPS. In fibers under voltage-clamp conditions, contractility is also lost; however, before this happens, long-lasting (i.e., minutes) episodes of spontaneous contractile activity may occur with the membrane polarized at -100 mV. After more prolonged exposure (> 30 min), the responses to membrane depolarization are reduced and eventually disappear. The agent DTT at a concentration of 2 mM appears to protect the fibers from the effects of PCMB and PHMPS. Furthermore, after loss of the contractile responses by the action of PCMB or PHMPS, addition of 2 mM DTT causes recovery of tension development capacity.

Effects of sulfhydryl inhibitors on nonlinear membrane currents in frog skeletal muscle fibers.

The effect of sulhydryl reagents on nonlinear membrane currents of frog skeletal muscle fibers has been studied using the triple Vaseline gap voltage-clamp technique. These compounds, which are known to interfere with depolarization contraction coupling, also appear to diminish intramembranous charge movement recorded with fibers polarized to -100 mV (charge 1). This effect, however, is accompanied by changes in the fiber membrane conductance and in most cases by the appearance of an inwardly directed current in the potential range between -60 and +20 mV. This current is reduced by both cadmium and nifedipine and does not occur in Ca-free solution, suggesting that it is carried by calcium ions flowing through regular calcium channels that are more easily activated in the presence of SH reagent. These changes in the membrane electrical active and passive properties decrease the quality and reliability of the P/n pulse subtracting procedure normally used for charge movement measurements. These effects can be substantially reduced by cadmium ions (0.1 mM), which has no effect on charge movement. When SH reagents are applied in the presence of cadmium, no effects are observed, indicating that this cation may protect the membrane from the reagent effects. The effects of -SH reagents can be observed by applying them in the absence of cadmium, followed by addition of the cation. Under these conditions the conductance changes are reversed and the effects of the SH reagents on charge movement can be measured with a higher degree of confidence. Maximum charge is reduced by 32% in the presence of 1.5 mM PCMB and by 31% in the presence of 2 mM PHMPS. These effects do not occur in the presence of DTT and in some cases they may be reversed by this agent. Charge 2, recorded in depolarized muscle fibers, is also reduced by these agents.

Depolarization-contraction coupling in short frog muscle fibers. A voltage clamp study.

Short muscle fibers (1.5 mm) were dissected from hindlimb muscles of frogs and voltage clamped with two microelectrodes to study phenomena related to depolarization-contraction coupling. Isometric myograms obtained in response to depolarizing pulses of durations between 10 and 500 ms and amplitudes up to 140 mV had the following properties. For suprathreshold pulses of fixed duration (in the range of 20-100 ms), the peak tension achieved, the time to peak tension, and contraction duration increased as the internal potential was made progressively more positive. Peak tension eventually saturates with increasing internal potentials. For pulse durations of greater than or equal to 50 ms, the rate of tension development becomes constant for increasing internal potentials when peak tensions become greater than one-third of the maximum tension possible. Both threshold and maximum steepness of the relation between internal potential and peak tension depend on pulse duration. The relation between the tension-time integral and the stimulus amplitude-duration product was examined. The utility of this relation for excitation-contraction studies is based on the observation that once a depolarizing pulse configuration has elicited maximum tension, further increases in either stimulus duration or amplitude only prolong the contractile response, while the major portion of the relaxation phase after the end of a pulse is exponential, with a time constant that is not significantly affected by either the amplitude or the duration of the pulse. Hence, the area under the tension-response curve provides a measure of the availability to troponin of the calcium released from the sarcoplasmic reticulum in response to membrane depolarization. The results from this work complement those obtained in experiments in which intramembrane charge movements related to contractile activation were studied and those in which intracellular Ca++ transients were measured.