Effects of potential detoxifying agents on growth performance and deoxynivalenol (DON) urinary balance characteristics of nursery pigs fed DON-contaminated wheat.

Two experiments were conducted to evaluate potential detoxifying agents on growth of nursery pigs fed deoxynivalenol (DON)-contaminated diets. Naturally DON-contaminated wheat (6 mg/kg) was used to achieve desired DON levels. In a 21-d study, 238 pigs (13.4 ± 1.8 kg BW) were used in a completely randomized design with a 2 × 2 + 1 factorial arrangement. Diets were: 1) Positive control (PC; < 0.5 mg/kg DON), 2) PC + 1.0% Product V (Nutriquest LLC, Mason City, IA), 3) Negative control (NC; 4.0 mg/kg DON), 4) NC + 1.0% Product V, and 5) NC + 1.0% sodium metabisulfite (SMB; Samirian Chemicals, Campbell, CA). There were 6 or 7 replicate pens/treatment and 7 pigs/pen. Analyzed DON was decreased by 92% when pelleted with SMB, but otherwise matched formulated levels. Overall, a DON × Product V interaction was observed for ADG ( 0.05) with a tendency for an interaction for ADFI ( 0.10). As anticipated, DON reduced ( 0.001) ADG and ADFI, but the interaction was driven by even poorer growth when Product V was added to NC diets. Pigs fed NC diets had 10% poorer G:F ( 0.001) than PC-fed pigs. Reductions in ADG due to DON were most distinct (50%) during the initial period. Adding SMB to NC diets improved ( 0.01) ADG, ADFI, and G:F, and improved ( 0.02) ADG and G:F compared to the PC diet. A urinary balance study was conducted using diets 3 to 5 from Exp. 1 to evaluate Product V and SMB on DON urinary metabolism. A 10 d adaptation was followed by a 7 d collection using 24 barrows in a randomized complete block design. Pigs fed NC + SMB diet had greater urinary DON output ( 0.05) than pigs fed NC + Product V, with NC pigs intermediate. Daily DON excretion was lowest ( 0.05) in the NC + SMB pigs. However, degradation of DON-sulfonate back to the parent DON molecule was observed as pigs fed NC + SMB excreted more DON than they consumed (164% of daily DON intake), greater ( 0.001) than pigs fed the NC (59%) or NC + Product V (48%). Overall, Product V did not alleviate DON effects on growth nor did it reduce DON absorption and excretion. However, hydrothermally processing DON-contaminated diets with 1.0% SMB restored ADFI and improved G:F. Even so, the urinary balance experiment revealed that some of the converted DON-sulfonate can degrade back to DON under physiological conditions. While further research is needed to discern the stability of the DON-sulfonate, SMB appears promising to restore performance in pelleted DON-contaminated diets.

Determining the impact of increasing standardized ileal digestible lysine for primiparous and multiparous sows during lactation.

Two experiments were conducted to evaluate the effects of increasing dietary SID Lys in lactation on sow and litter performance. In Exp. 1, a total of 111 primiparous sows (Line 241; DNA Genetics, Columbus, NE) were allotted to 1 of 4 dietary treatments on d 110 of gestation. Dietary treatments included increasing dietary standardized ileal digestible (SID) Lys (0.80, 0.95, 1.10, and 1.25%). During lactation, there were no differences in ADFI or sow BW at weaning (d 21), resulting in no differences in BW loss. However, backfat loss during lactation decreased (linear, P = 0.046) as SID Lys increased. There were no differences in litter weaning weight, litter gain from d 2 to weaning, percentage of females bred by d 7 after weaning, d 30 conception rate, farrowing rate or subsequent litter characteristics. In Exp. 2, a total of 710 mixed parity sows (Line 241; DNA Genetics) were allotted to 1 of 4 dietary treatments at d 112 of gestation. Dietary treatments included increasing SID Lys (0.75, 0.90, 1.05, and 1.20%). Sow BW at weaning increased (quadratic, P = 0.046), and sow BW loss from post-farrow to weaning or d 112 to weaning decreased (quadratic, P ≤ 0.01) as SID Lys increased. Sow backfat loss increased (linear, P = 0.028) as SID Lys increased. Conversely, longissimus muscle depth loss decreased (linear, P = 0.002) as SID Lys increased. Percentage of females bred by d 7 after weaning increased (linear, P = 0.047) as SID Lys increased in parity 1 sows, with no difference in parity 2 or 3+ sows. Litter weight at d 17 and litter gain from d 2 to 17 increased (quadratic, P = 0.01) up to 1.05% SID Lys with no improvement thereafter. For subsequent litter characteristics, there were no differences in total born, percentage born alive, stillborn, or mummies. In conclusion, our results suggest that increasing dietary SID Lys can reduce sow protein loss in lactation. The optimal level of dietary SID Lys required by the sow may vary based on response criteria and parity.

Influence of dietary fat source and feeding duration on finishing pig growth performance, carcass composition, and fat quality.

A total of 160 finishing pigs (PIC 327 × 1050; initially 45.6 kg) were used in an 84-d experiment to evaluate the effects of dietary fat source and feeding duration on growth performance, carcass characteristics, and carcass fat quality. There were 2 pigs per pen with 8 pens per treatment. The 10 dietary treatments were a corn-soybean meal control diet with no added fat and a 3 × 3 factorial with main effects of fat source (4% tallow, 4% soybean oil, or a blend of 2% tallow and 2% soybean oil) and feeding duration (d 0 to 42, 42 to 84, or 0 to 84). The control corn-soybean meal diet was fed in place of added fat diets when needed for duration treatment purposes. On d 0, 1 pig was identified in each pen and fat biopsy samples of the back, belly, and jowl were collected on d 0, 41, and 81 for fatty acid analysis. At the conclusion of the study, all pigs were harvested, carcass characteristics were determined, and back, belly, and jowl fat samples were collected for analysis. Overall (d 0 to 84), there were no differences among pigs fed the different fat sources for growth and carcass characteristics; however, pigs fed diets with added fat for the entire study had improved ( = 0.036) G:F compared with pigs fed the control diet without added fat. Pigs fed supplemental fat throughout the entire study also had improved ( < 0.05) ADG and G:F as well as heavier d-84 BW ( = 0.006) compared with pigs fed additional fat during only 1 period. Adding fat for the entire study increased ( = 0.032) backfat and tended to reduce ( = 0.079) the fat free lean index compared with pigs fed the control diet without added fat. Added fat also increased ( < 0.05) the iodine value (IV) when compared with pigs fed the control diet. Increasing the feeding duration of soybean oil lowered MUFA and increased PUFA concentrations for all fat depots, whereas these values remained relatively unchanged by the addition of tallow (duration × fat source interactions, < 0.05). Our study failed to show any feeding period × fat source interactions ( < 0.05) in fatty acid composition or IV for jowl fat, whereas this interaction occurred for belly fat and backfat, which would indicate a longer turnover rate for jowl fat. In conclusion, feeding additional fat improved ADG and G:F; however, feeding soybean oil for an increased duration, either alone or in combination with tallow, negatively affected the fatty acid composition and IV of different fat depots.

Equations generated to predict iodine value of pork carcass back, belly, and jowl fat.

Data from existing literature were used to generate equations to predict finishing pig back, belly, and jowl fat iodine values (IV) and an experiment was conducted to evaluate these equations. The final database included 24, 21, and 29 papers for back, belly, and jowl fat IV, respectively. For experiments that changed dietary fatty acid composition, initial (INT) diets were defined as those fed before the change in diet composition and final (FIN) diets were those fed after. The predictor variables tested were divided into 5 groups: 1) diet fat composition (dietary percent C16:1, C18:1, C18:2, C18:3, EFA, unsaturated fatty acids, and IV product) for both INT and FIN diets, 2) day feeding the INT and FIN diets, 3) ME or NE of the INT and FIN diet, 4) live performance criteria (initial BW, final BW, ADG, ADFI, and G:F), and 5) carcass criteria (HCW and backfat thickness). The PROC MIXED procedure of SAS (SAS Inst., Inc., Cary, NC) was used to develop regression equations. Evaluation of models with significant terms was then conducted based on the Bayesian information criterion. The optimum equations to predict back, belly, and jowl fat IV were backfat IV = 84.83 + (6.87 × INT EFA) - (3.90 × FIN EFA) - (0.12 × INT days) - (1.30 × FIN days) - (0.11 × INT EFA × FIN days) + (0.048 × FIN EFA × INT days) + (0.12 × FIN EFA × FIN days) - (0.0060 × FIN NE) + (0.0005 × FIN NE × FIN days) - (0.26 × backfat depth); belly fat IV = 106.16 + (6.21 × INT EFA) - (1.50 × FIN days) - (0.11 × INT EFA × FIN days) - (0.012 × INT NE) + (0.00069 × INT NE × FIN days) - (0.18 × HCW) - (0.25 × backfat depth); and jowl fat IV = 85.50 + (1.08 × INT EFA) + (0.87 × FIN EFA) - (0.014 × INT days) - (0.050 × FIN days) + (0.038 × INT EFA × INT days) + (0.054 × FIN EFA × FIN days) - (0.0066 × INT NE) + (0.071 × INT BW) - (2.19 × ADFI) - (0.29 × backfat depth). Dietary treatments from the evaluation experiment consisted of a corn-soybean meal control diet with no added fat or a 3 × 3 factorial arrangement with main effects of fat source (4% tallow, 4% soybean oil, or a blend of 2% tallow and 2% soybean oil) and feeding duration (d 0 to 42, 42 to 84, or 0 to 84). The back, belly, and jowl fat IV equations tended to overestimate IV when observed IV were less than approximately 65 g/100 g and underestimate belly fat IV when actual IV are greater than approximately 74 g/100 g or when the fat blend was fed from d 0 to 84 or 42 to 84. Overall, with the exceptions noted, the regression equations were an accurate tool for predicting carcass fat quality based on dietary and pig performance factors.

The serum response element can mediate induction of c-fos by growth hormone.

The c-fos protooncogene is transcriptionally activated by a wide variety of agents including serum, growth factors, and phorbol esters. This induction is rapid and transient and is mediated through a number of identified promoter elements. Growth hormone (GH) is also known to induce transcription of c-fos in a variety of cell types including NIH 3T3 fibroblasts and 3T3-F442A preadipocytes. To identify DNA sequences in the c-fos gene regulated by GH, this study sought to determine whether induction of c-fos by GH involves previously identified c-fos promoter elements. A plasmid containing a growth factor-sensitive fragment of the upstream region of the c-fos promoter from -361 to -264 bp was tested for GH sensitivity. The fragment was cloned upstream of a human c-fos reporter [designated FOS by Human Gene Mapping 11 (1991)], which included basal promoter elements. In transiently transfected mouse NIH 3T3 fibroblasts, the promoter fragment conferred GH sensitivity on the human c-fos reporter. To identify a specific GH-sensitive DNA sequence in this promoter, a serum response element (SRE)-reporter plasmid was tested and found to be stimulated by GH. GH was effective in inducing expression through the SRE over a range of physiological GH concentrations. Since GH was recently found to synergize with serum factors in inducing c-fos transcription, the effect of GH and serum on SRE function was examined for insight into the mechanism for such synergism. The combined effect of GH and serum to induce reporter expression through the SRE was greater than the added effects of GH and serum, indicating that the synergism between GH and serum in inducing c-fos involves the SRE sequence. These studies identify the SRE as one specific DNA sequence in the c-fos promoter functionally regulated by GH. It is notable that GH is effective at physiological concentrations. Furthermore, synergism in c-fos induction between GH and serum factors is evident through the SRE.

Excitation of skinned muscle fibers by imposed ion gradients. IV. Effects of stretch and perchlorate ion.

Depolarizing ion gradients stimulate 45Ca release in skeletal muscle fibers skinned by microdissection. Several lines of indirect evidence suggest that sealed transverse (T) tubules rather than sarcoplasmic reticulum (SR) are the locus of such stimulatory depolarization. Two implications of this hypothesis were tested. (a) A requirement for signal transmission was evaluated from the stimulation of 45Ca efflux in fibers that had been highly stretched, an intervention that can impair the electrical stimulation of intact fibers. Length was increased over approximately 95-115 s, after loading with 45Ca and rinsing at normal length; prestimulus 45Ca loss due to stretch itself was very small. In the first study, stimulation of 45Ca release by KCl replacement of K propionate was inhibited completely in fibers stretched to twice slack length, compared with fibers at 1.05-1.1 times slack length. Identical protocols did not alter 45Ca release stimulated by caffeine or Mg2+ reduction, implying that SR Ca release per se was fully functional and inhibition was selective for a preceding step in ionic stimulation. In a second study, stimulation by choline Cl replacement of K methanesulfonate, at constant [K+] [Cl-] product, was inhibited strongly; total 45Ca release decreased 69%, and stimulation above control loss decreased 78%, in segments stretched to twice the length at which sarcomere spacing had been 2.2 micron, compared with paired controls from the same fibers kept at 2.3 micron. (b) Perchlorate potentiation of T tubule activation was evaluated in fibers stimulated at constant [K+] [Cl-] at normal length (2.3 micron); this anion shifts the voltage dependence of intramembrane charge movement and contractile activation in intact fibers. Perchlorate (8 mM) potentiated both submaximal stimulation of Ca2+-dependent 45Ca release by partial choline Cl replacement of K methanesulfonate and the small Ca2+-insensitive 45Ca efflux component stimulated by nearly full replacement in the presence of 5 mM EGTA. These results provide independent support for the hypothesis that the T tubules are the locus of stimulation by depolarizing ion gradients, with junctional transmission of this signal causing SR 45Ca release.

Excitation of skinned muscle fibers by imposed ion gradients. III. Distribution of permeant ions in unstimulated and stimulated fibers.

Ion gradients imposed across an internal membrane system stimulate skinned muscle fibers; to evaluate the sarcoplasmic reticulum (SR) as the primary target site, SR polarization under resting and stimulatory conditions was assessed from fiber uptake of permeant probe ions. Solvent spaces were estimated from simultaneous [14C]urea (U) or [3H]deoxyglucose (DOG) uptake in segments of fibers from bullfrog semitendinosus muscle, skinned by microdissection. The distribution spaces, i.e., virtual solvent volumes at bath concentrations (Vu and VDOG), of these uncharged probes correlated well with the protein content of the same segments, which validated the tracer methodology for volume normalization. The membrane-bounded volume fraction (Vm), derived from the difference between total solvent volume (Vs) and the non-membrane-bounded solvent volume (Vc), was sufficient to detect appreciable SR ion accumulation. The Vm estimated from the difference between VU and VDOG assayed simultaneously with 2 or 5-6 min exposures was 10-11%, which is consistent with the morphometric volume fraction (mostly SR) in frog fibers; however, the change in this difference after membrane permeabilization corresponded to Vm only 5%. The change in permeant ion distribution space caused by member permeabilization was used to assess SR membrane polarization, assuming the free ions distribute across the intact membrane according to the Nernst ratio. Resting polarization (SR lumen positive) was assessed from [14C]SCN- or [14C]propionate- distribution spaces in unstimulated fibers, expressed relative to VDOG (assayed simultaneously). The ratios for (a) [14C]SCN- space (carrier 2 mM) and (b) [14C]propionate- space (carrier 120 mM) were not decreased by membrane permeabilization. This indicated that anion distribution was independent of membrane integrity and did not reflect an SR transmembrane potential, although a was more and b was less than 1. Polarization under stimulatory conditions (lumen negative) was assessed from 86Rb+ distribution, before and after an imposed ion gradient (choline Cl replacement of K methanesulfonate (KMes) at constant [K+] [Cl-]) that theoretically could generate a 48-fold transmembrane cation ratio; Ca release was minimized by EGTA. The ratio of 86Rb+ space to VU, greater than 1 in KMes (120 mM K, the effective carrier), was higher in choline Cl (2.5 mM K) but not decreased by membrane permeabilization; this indicated that 86Rb+ distribution did not reflect an SR transmembrane potential. Similar results in the presence of valinomycin ruled out the possibility of inadequate 86Rb+ equilibration.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms of stimulated 45Ca efflux in skinned skeletal muscle fibers.

Excitation-contraction (E-C) coupling in skeletal muscle can be studied in skinned fibers by direct assay of 45Ca efflux and simultaneous isometric force, under controlled conditions. Recent work provides evidence that such studies can address major current questions about the mechanisms of signal transmission between transverse tubules and sarcoplasmic reticulum and sarcoplasmic reticulum calcium release, as well as operation of the sarcoplasmic reticulum active Ca transport system in situ. Stimulation by imposed ion gradients at constant [K+][Cl-] product results in 45Ca release with two components: a large Ca2+-dependent efflux, responsible for contractile activation, and a small Ca2+-insensitive efflux. The Ca2+-insensitive stimulation is sustained, consistent with sustained depolarization, and appears to gradate the Ca2+-dependent stimulation; this component is likely to reflect intermediate steps in E-C coupling. Several lines of evidence suggest that the depolarizing stimulus acts on the transverse tubules. It is inhibited by the impermeant glycoside ouabain applied before skinning, which should specifically inhibit polarization of subsequently sealed transverse tubules. Sealed polarized transverse tubules also are the only plausible target for stimulation of 45Ca release by monensin and gramicidin D, which can rapidly dissipate Na+ and K+ gradients; a protonophore and the K+-specific ionophore valinomycin are ineffective. Ionophore stimulation is prevented by the permeant glycoside digitoxin; it is also highly Ca2+ dependent. Stimulation of 45Ca release by imposed ion gradients is potentiated by perchlorate, which potentiates charge movements and activation in intact fibers, and is inhibited selectively in highly stretched fibers, presumably by transverse tubule-sarcoplasmic reticulum uncoupling. These results relate the Ca2+-dependent sarcoplasmic reticulum efflux channel to the physiological transverse tubule-sarcoplasmic reticulum coupling pathway, which also could involve Ca2+.

Ca2+ dependence of transverse tubule-mediated calcium release in skinned skeletal muscle fibers.

Isometric force and 45Ca efflux from the sarcoplasmic reticulum were measured at 19 degrees C in frog skeletal muscle fibers skinned by microdissection. After Ca2+ loading, application of the ionophores monensin, an Na+(K+)/H+ exchanger, or gramicidin D, an H+ greater than K+ greater than Na+ channel-former, evoked rapid force development and stimulated release of approximately 30% of the accumulated 45Ca within 1 min, whereas CCCP (carbonyl cyanide pyruvate p-trichloromethoxyphenylhydrazone), a protonophore, and valinomycin, a neutral, K+-specific ionophore, did not. When monensin was present in all bathing solutions, i.e., before and during Ca2+ loading, subsequent application failed to elicit force development and to stimulate 45Ca efflux. 5 min pretreatment of the skinned fibers with 50 microM digitoxin, a permeant glycoside that specifically inhibits the Na+,K+ pump, inhibited monensin and gramicidin D stimulation of 45Ca efflux; similar pretreatment with 100 microM ouabain, an impermeant glycoside, was ineffective. Monensin stimulation of 45Ca efflux was abolished by brief pretreatment with 5 mM EGTA, which chelates myofilament-space calcium. These results suggest that: monensin and gramicidin D stimulate Ca2+ release from the sarcoplasmic reticulum that is mediated by depolarization of the transverse tubules, which seal off after sarcolemma removal and form closed compartments; a transverse tubule membrane potential (myofilament space-negative) is maintained and/or established by the operation of the Na+,K+ pump in the transverse tubule membranes and is sensitive to the permeant inhibitor digitoxin; the transverse tubule-mediated stimulation of 45Ca efflux appears to be entirely Ca2+ dependent.

Excitation of skinned muscle fibers by imposed ion gradients. II. Influence of quercetin and ATP removal on the Ca2+-insensitive component of stimulated 45Ca efflux.

Ionic gradients imposed by choline Cl replacement of K methanesulfonate (Mes) at constant [K][Cl] product stimulate 45Ca efflux from skinned muscle fibers; a small, sustained Ca2+-insensitive efflux component, observed in EGTA, appears to grade a much larger Ca2+-dependent component responsible for contractile activation and is likely to reflect intermediate steps in excitation-contraction coupling. The present studies examined ATP-related effects on the Ca2+-insensitive stimulation. 45Ca efflux was measured on segments of frog semitendinosus muscle skinned by microdissection, with isometric force monitored continuously. The Ca2+-insensitive component was potentiated by quercetin, a flavonoid thought to inhibit the sarcoplasmic reticulum (SR) Ca pump by stabilizing a phosphorylated intermediate. Quercetin increased the stimulated net 45Ca release in the absence of EGTA, as expected from inhibition of reaccumulation, but its effectiveness in EGTA indicated potentiation of unidirectional efflux as such. Quercetin also increased unstimulated (control) 45Ca efflux in EGTA, to a smaller extent; potentiation appeared to be a function of efflux, with stimulation above control loss increased approximately 2.6-fold. ATP removal before stimulation, which led to rigor force and increased stiffness, prevented all quercetin effects in EGTA. ATP removal by itself inhibited ionic stimulation of the Ca2+-insensitive component, with little residual increase above the parallel control loss. Addition of the nonhydrolyzable ATP analogue AMP-PCP ([adenylyl-beta,gamma-methylene]diphosphate) (0.8 mM) after ATP removal gave similar results to ATP-free solution, which suggests that adenine nucleotide binding alone does not support stimulation by choline Cl. These results imply a fundamental role for ATP in the excitation of skinned fibers by imposed diffusion potentials; they also suggest that ATP regulates the SR Ca efflux channel, in a manner that could provide the positive feedback in Ca2+-dependent Ca release.

Excitation of skinned muscle fibers by imposed ion gradients. I. Stimulation of 45Ca efflux at constant [K][Cl] product.

45Ca efflux from skinned muscle fibers is stimulated transiently, by a highly Ca2+-dependent mechanism, by KCl replacement of K propionate. In the present studies, Cl replaced the much less permeant anion methanesulfonate (Mes) either (a) at constant [K], in which increased [K][Cl] permits net KCl and water flux across internal membranes, or (b) at constant [K][Cl] (choline substitution), in which the imposed gradients and diffusion potentials should dissipate slowly. 45Ca efflux and isometric force were measured simultaneously on segments of frog semitendinosus fibers skinned by microdissection. EGTA was applied to chelate released 45Ca either (a) shortly after high [Cl] (interrupted response), to minimize reaccumulation, (b) before high [Cl] (pretreated response), to evaluate Ca2+ dependence, or (c) under control conditions in KMes. KCl replacement of KMes stimulated release of 65% fiber 45Ca within 1 min in interrupted responses; EGTA pretreatment was only moderately inhibitory with substantial residual stimulation. In contrast, choline Cl replacement of KMes induced release of 26-35% fiber 45Ca in interrupted responses; EGTA pretreatment was strongly inhibitory, but release significantly exceeded control with a small, sustained increase in Ca2+-insensitive efflux. These differences in 45Ca release and EGTA inhibition suggest that Cl replacement of Mes at constant [K] stimulates efflux by osmotic effects as well as imposed diffusion potentials; at least half the stimulated 45Ca loss (above control) in interrupted KCl responses is attributable to an osmotic component with low Ca2+ sensitivity. In the highly Ca2+-sensitive stimulation at constant [K][Cl], 45Ca release (above control) in interrupted responses correlated well with that in the pretreated responses of segments from the same fiber, with a slope of 8.4. This relationship suggests that imposed diffusion potentials stimulate a small Ca2+-insensitive component that gradates a much larger Ca2+-dependent efflux. The Ca2+-insensitive component apparently reflects intermediate steps in the excitation-contraction coupling that require positive feedback to result in sufficient Ca release for contraction.

The role of free calcium ion in calcium release in skinned muscle fibers.

Major questions in excitation--contraction coupling of fast skeletal muscle concern the mechanism of signal transmission between sarcolemma and sarcoplasmic reticulum (SR), the mechanism of SR Ca release, and operation of the SR active transport system during excitation. Intracellular Ca movement can be studied in skinned muscle fibers with more direct control, analysis of 45Ca flux, and simultaneous isometric force measurements. Ca release can be stimulated by bath Ca2+ itself, ionic "depolarization," Mg2+ reduction, or caffeine. The effectiveness of bath Ca2+ has suggested a possible role for Ca2+ in physiological release, but this response is difficult to analyze and evaluate. Related evidence emerged from analysis of other responses: with all agents studied, stimulation of 45Ca efflux is highly Ca2+-dependent. The presence of a Ca chelator prevents detectable stimulation by ionic "depolarization" or Mg2+ reduction and inhibits the potent caffeine stimulus; inhibition is graded with chelator concentration and caffeine concentration, and is synergistic with inhibition by increased Mg2+. The results indicate that a Ca2+-dependent pathway mediates most or all of stimulated 45Ca efflux in skinned fibers, and has properties compatible with a function in physiological Ca release.

Magnesium effects on activation of skinned fibers from striated muscle.

The intracellular Ca movements that control contraction and relaxation of striated muscle are regulated by the membrane potential and influenced by Mg2+. In skinned fibers, the internal composition can be manipulated directly by Ca movements estimated from isometric force transients, net changes in sarcoplasmic reticulum (SR) Ca, and 45Ca flux between fiber and bath. Stimulated Ca release, unlike unstimulated 45Ca efflux at low external [Ca2+], is highly [Mg2+]-sensitive at 20 C. Force and tracer measurements indicate three major sites of Mg2+-Ca2+ interaction in situ: Mg2+ can stimulate the SR active Ca transport system, inhibit a Ca2+-dependent Ca efflux pathway of SR, and shift the force-[Ca2+] relation, presumably by reducing Ca2+ binding to myofilament regulatory sites. These mechanisms constrain the resting Ca flux and are adaptive during relaxation. However, analysis of CI-stimulated 45Ca release and reaccumulation suggests that the depolarization process may inhibit Mg2+-dependent Ca influx, the membrane potential controlling both efflux and influx; recent studies on voltage-clamped cut fibers support this hypothesis. The Ca2+ and Mg2+ dependence of caffeine-stimulated 45Ca efflux suggests that Mg2+ inhibition of the Ca2+-dependent efflux pathway is small during rapid Ca2+ efflux. Therefore, both Mg2+ mechanisms, which minimize net release, may be reversed during normal activation.

Ca2+ dependence of stimulated 45Ca efflux in skinned muscle fibers.

Stimulation of sarcoplasmic reticulum Ca release by Mg reduction of caffeine was studied in situ, to characterize further the Ca2+ dependence observed previously with stimulation by Cl ion. 45Ca efflux and isometric force were measured simultaneously at 19 degrees C in frog skeletal muscle fibers skinned by microdissection; EGTA was added to chelate myofilament space Ca either before or after the stimulus. Both Mg2+ reduction (20 or 110 microM to 4 microM) and caffeine (5 mM) induced large force responses and 45Ca release, which were inhibited by pretreatment with 5 mM EGTA. In the case of Mg reduction, residual efflux stimulation was undetectable, and 45Ca efflux in EGTA at 4 microM Mg2+ was not significantly increased. Residual caffeine stimulation at 20 microM Mg2+ was substantial and was reduced further in increased EGTA (10 mM); at 600 microM Mg2+, residual stimulation in 5 mM EGTA was undetectable. Caffeine appears to initiate a small Ca2+-insensitive efflux that produces a large Ca2+-dependent efflux. Additional experiments suggested that caffeine also inhibited influx. The results suggest that stimulated efflux is mediated mainly or entirely by a channel controlled by an intrinsic Ca2+ receptor, which responds to local [Ca2+] in or near the channel. Receptor affinity for Ca2+ probably is influenced by Mg2+, but inhibition is weak unless local [Ca2+] is very low.

Activation of fast skeletal muscle: contributions of studies on skinned fibers.

The membrane potential of vertebrate twitch fibers closely controls Ca fluxes between intracellular compartments, which in turn control contraction. Recent work on intracellular Ca movement is reviewed in the general context of current efforts to synthesize physiological, biochemical, and structural observations on the contractile mechanism and its regulation, emphasizing the increasing role of functionally skinned fibers in this synthesis. Skinned fiber preparations, with removed or disrupted sarcolemma, bridge the gap between properties of isolated subsystems and their constrained operation in the intact fiber. Recent studies indicate that the surface action potential propagates along the transverse tubules, but not the sarcoplasmic reticulum (SR), which appears to be a distinct intracellular compartment. Voltage-dependent charge movements in the transverse tubules probably control Ca flux across the SR membranes. Current questions concern the mechanism of the signal that bridges the junctional gap between the two membrane systems, the mechanism and properties of the activated Ca efflux to the myofilament space, and the operation of the Ca pump of the SR during activation. New methods applied to intact fibers, cut fibers, skinned fibers, and subcellular systems are yielding the kind of information needed for a complete description of these central steps in excitation-contraction coupling and of Ca regulation of the myofilaments.

Properties of chloride-stimulated 45Ca flux in skinned muscle fibers.

Isometric force and 45Ca loss from fiber to bath were measured simultaneously in skinned fibers from frog muscle at 19 degrees C. In unstimulated fibers, 45Ca efflux from the sarcoplasmic reticulum (SR) was very slow, with little or no dependence on EGTA (0.1-5 mM) or Mg++ (20 micrometer-1.3 mM). Stimulation by high [Cl] at 0.11 mM Mg++ caused rapid force transients (duration approximately 10 s) and 45Ca release. This response was followed for 55 s, with 5 mM EGTA added to chelate myofilament space (MFS) Ca either (a) after relaxation, (b) near the peak of the force spike, or (c) before or with the stimulus. When EGTA was present during Cl application, stimulation of 45Ca release was undetectable. Analysis of the time-course of tracer loss during the three protocols showed that when EGTA was absent, 16% of the fiber tracer was released from the SR within approximately 3 s, and 70% of the tracer still in the MFS near the peak of the force spike was subsequently reaccumulated. The results suggest that (a) the Cl response is highly Ca-dependent; (b) stimulation increases 45Ca efflux from the SR at least 100-200-fold; and (c) the rate of reaccumulation is much slower than the influx predicted from published data on resting fibers, raising the possibility that depolarization inhibits active Ca transport by the SR.

Influence of magnesium on chloride-induced calcium release in skinned muscle fibers.

Chloride-induced Ca release in skinned muscle fibers was studied by measuring isometric force transients and 45Ca loss from fiber to washout solutions. Skinned fibers prepared from muscles soaked in normal Ringer solution made large force transients in 120 mM Cl solution with 5 mM ATP and 1 mM Mg, but 3 mM Mg was inhibitory. Mg inhibition was antagonized by low temperature and by Cd, agents which slow active Ca uptake by the sarcoplasmic reticulum (SR). In low Mg++, Cl stimulated rapid 45Ca release from the SR in sufficient amounts to account for the force response. The increased 45Ca release was inhibited by EGTA, suggesting that release requires free Ca under these conditions. The 45Ca initially released was partially reaccumulated later. Reaccumulation was increased in higher Mg++. These results provide additional evidence that the Ca uptake rate is an important determinant of net release, and suggest that Mg++ acts primarily on this mechanism. Skinned fibers prepared from muscles soaked in low Cl solutions could give force responses to Cl solutions with 3 mM and 6 mM Mg. This observation suggests that the Cl stimulus varies with the [Cl] gradient across the internal membranes, and supports the hypothesis that applied Cl causes membrane depolarization.

Regulation by magnesium of intracellular calcium movement in skinned muscle fibers.

The effect of Mg on Ca movement between the sarcoplasmic reticulum (SR) and myofilament space (MFS) was studied in skinned muscle fibers by using isometric force as an indicator of MFS Ca. In Ca-loaded fibers at 20 degrees C, the large force spike induced by Ca in 1 mM Mg (5 mM ATP) was strongly inhibited in 3 mM Mg, and force development was extremely slow. After a brief Ca stimulus in 1 mM Mg, relaxation in Ca-free solution was significantly faster in 3 mM Mg. These changes were due to altered Ca movements, since the effect of 3 mM Mg on steady force in CaEGTA solutions was small. Changes in Mg alone induced force transients apparently due to altered Ca movement. In relaxed fibers, decreasing the Mg to 0.25 mM caused phasic force development. In contracting fibers in Ca solutions, increasing the Mg caused a large transient relaxation. The effects of increased Mg were antagonized by 0.5 mM Cd, an inhibitor of the SR Ca transport system. The results indicate that active Ca uptake by the SR in situ is stimulated by Mg, and that it can affect local MFS [Ca++] in the presence of a substantial Ca source. These results provide evidence that an increased rate of Ca uptake in 3 mM Mg could account for inhibition of the large force spike associated with Ca-induced Ca release in skinned fibers.

Potassium flux in smooth muscle of frog stomach.

In frog stomach muscle fibers, normal steady-state K flux, estimated directly from 42K uptake, was 0.17 pmol/cm2 per s at 5 degrees C and 0.63 pmol/cm2 per s at 15 degrees C. Influx characteristics were studied at 5 degrees C, where backflux and diffusional delay effects are minimized. Steady-state K influx was a saturating function of external [K] over the range 0.25-11 mM [K]o; influx at normal and higher [K]o did not differ significantly. Na loading (in K-free or low K solution) strongly stimulated influx, which showed altered saturation kinetics; maximal K influx was a quasilinear function of internal [Na]. Ouabain (10(-4) M) reduced normal and stimulated K influx markedly. Ethacrynic acid (10(-3) M) caused net K loss and Na gain, but increased K influx fourfold; ouabain inhibited the stimulated influx by 50%. These results indicate that K influx depends mainly on cycling of the Na-K pump and is normally limited by Na efflux. Ethacrynic acid may stimulate another mode of pump operation, K-K exchange, and uncouple the normal operation.