Parvalbumin Content Research Articles

Immunoreactive parvalbumin concentrations in parahippocampal gyrus decrease in patients with Alzheimer's disease

By using a sensitive enzyme immunoassay system for rat parvalbumin, we determined parvalbumin contents in the 4 cerebrocortical regions (superior frontal gyrus of frontal lobe, parahippocampal gyrus of temporal lobe, superior parietal lobule of parietal lobe, and calcarine area of occipital lobe) of patients with Alzheimer's disease and age-matched controls. Among the 4 regions, concentrations of parvalbumin were the highest in calcarine area (68.6 ± 6.7 ng/mg protein, rat parvalbumin equivalents, mean ± SE) and the lowest in the parahippocampal gyrus (11.0 ± 1.7 ng/mg protein) in the controls. A similar regional difference of the concentration was observed also in the patients with Alzheimer's disease. When compared with the controls, however, concentrations of parvalbumin in parahippocampal gyrus of patients with Alzheimer's disease (4.0 ± 0.9 ng/mg protein) were significantly low ( P<0.01), showing less than a half of the control values. In contrast, the concentrations in the 3 other regions showed little difference between Alzheimer's disease and the controls.

Parvalbumin, labile heat and slowing of relaxation in mouse soleus and extensor digitorum longus muscles.

1. Parvalbumin content, heat rate and rate of relaxation were measured in two mouse muscles: the slow-twitch soleus and the fast-twitch extensor digitorum longus (EDL). 2. No trace of parvalbumin was found in the soleus; EDL contained a mean of 4.86 mg of this protein per gram of fresh muscle (S.D. = 1.25). 3. Heat rate during 7 s isometric tetani in isolated soleus muscle at 20 degrees C can be described by the sum of an exponentially decaying term and a constant term. The exponential term is reduced by 67% in a second tetanus performed 1 s after a first one; its repriming is complete after a resting period of about 1 min. The exponential term has therefore the properties of labile heat. 4. Relaxation rate measured during 15 s of isometric interrupted tetani at 20 degrees C is nearly constant in the soleus, but decreases continuously with increasing tetanus duration in the EDL. In the latter, isometric tension also decreases continuously. 5. Therefore, parvalbumin can account neither for the labile heat production in mouse soleus nor for the slowing of relaxation associated with muscle fatigue observed after a few seconds of tetanus in EDL. The role of parvalbumin in striated muscles is thus reassessed, and other possible causes of labile heat production and slowing of relaxation are discussed.

Fiber type-specific distribution of parvalbumin in rabbit skeletal muscle

A highly sensitive sandwich ELISA for parvalbumin (PA), based on a fluorometric detection system, was developed. This assay detected PA concentrations as low as 20 pg/ml (2 pg per assay) and was used for measuring PA contents in fragments of single muscle fibers isolated from freeze-dried 100-150 microns thick cross sections. The fibers were typed according to their histochemically assessed mATPase in parallel cross sections. Type I fibers from rabbit tibialis anterior (TA) and vastus lateralis (VL) muscles contained extremely low PA concentrations (2-5 micrograms/g w.wt.). Type IIA fibers displayed slightly higher values with mean values of 17 and 29 micrograms/g w.wt. (range 5-65) in TA and VL, respectively. Much higher PA concentrations were found in type IIB fibers with wide ranges from 75-1150 micrograms/g w.wt. in TA and 440-1370 micrograms/g w.wt. in VL. Whereas the IIB fibers of the TA displayed a continuum, two subgroups were distinguishable according to their PA contents (means of 590 and 1230 micrograms/g w.wt.) in VL. Possibly, the population with the lower PA content which was histochemically defined as type IIB in the present study, corresponds to fiber type IID. The finding that PA is predominantly present in type IIB fibers was also confirmed by the parallel decay of PA and type IIB fibers during chronic low-frequency stimulation. The use of freeze substitution, or alternatively, of freeze-drying, made it possible to demonstrate PA immunohistochemically without artifacts and to evaluate the staining intensity by microphotometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscle inactivity reduces the content of parvalbumin in rat thymic myoid cells in vitro

We investigated the influence of muscle inactivity by the addition of 1 μM tetrodotoxin in culture medium (complete abolishment of spontaneous firings); feeding with 50 mM KCl in the culture medium (inactivation of action potentials by depolarization) and the addition of 50 μM dantrolene sodium in culture medium (excitation-contraction uncoupling) on the content of parvalbumin in a cultured rat thymic myoid cell line (R615B2: M cells) using a highly sensitive enzyme immunoassay for rat parvalbumin. This is the first report of the content of parvalbumin in thymic myoid cells. Our studies clearly show that the content of parvalbumin is significantly decreased in paralyzed M cells compared with controls which had continuous muscle contractions. It is strongly suggested that muscle contractile activity plays an important role on the expression of parvalbumin in thymic myoid cells.

Parvalbumin content and Ca2+ and Mg2+ dissociation rates correlated with changes in relaxation rate of frog muscle fibres.

1. Experiments were done to test the hypothesis that parvalbumin (PA) promotes relaxation in frog skeletal muscle. Single fibres and purified PA from Rana temporaria skeletal muscle were used to determine the relationship between PA concentration ( [PA] ), Ca2+ and Mg2+ dissociation rates from PA and changes in rate of relaxation as a function of tetanus duration at 0 degrees C. 2. Total [PA] in fibres from tibialis anterior muscles is 0.76 +/- 0.20 mmol PA l-1 myoplasmic water (mean +/- S.D., n = 25) with 65% PA IVa and 35% PA IVb, where PA IVa and PA IVb are PA isoforms. 3. Relaxation rate from an isometric tetanus shows progressively as a function of tetanus duration with an exponential time course and a rate constant of 1.18 +/- 0.35 s-1 (n = 17). Time course of recovery of relaxation rate after a prolonged tetanus is exponential with a rate constant of 0.12 +/- 0.02 s-1 (n = 14). 4. The extent of recovery of relaxation rate after a prolonged tetanus was correlated with total [PA] in fibres (correlation coefficient (r) = 0.80, n = 7; P less than 0.05). 5. Dissociation rate constants for Mg2+ and Ca2+ from purified PA are 0.93 +/- 0.02 s-1 (n = 5) and 0.19 +/- 0.01 s-1 (n = 5), respectively. Dissociation rate constants were not significantly different for PA isoforms IVa and IVb. These rate constants are similar to the rate constants determined for the time courses of slowing and recovery of relaxation rate, respectively. 6. Results suggest that the time courses of slowing and recovery of relaxation rate may be controlled, to a large extent, by Mg2+ and Ca2+ dissociation from PA, respectively. This evidence supports a role for PA in facilitating relaxation during a tetanus in frog skeletal muscle at 0 degrees C.

Sensitive immunoassay for rat parvalbumin: tissue distribution and developmental changes

A sensitive enzyme immunoassay for measurements of rat parvalbumin was established using antibodies raised in rabbits with parvalbumin purified from skeletal muscles. Antibodies in the antiserum were purified with a parvalbumin-coupled Sepharose column. The sandwich-type immunoassay system for parvalbumin was composed of polystyrene balls with immobilized purified antibodies and the same antibodies labeled with β- d-galactosidase from Escherichia coli. The assay was highly sensitive and the minimum detection limit was 1 pg parvalbumin/tube. The assay did not cross-react with other calcium binding proteins, including human S-100a 0 and S-100b proteins, rat 28-kDa calbindin-D, and bovine calmodulin. High concentrations of parvalbumin were observed in the skeletal muscles, especially in those composed of fast-twitch fibers, and in the diaphragm and tongue, but not in heart muscle. A relatively high concentration was estimated in the central nervous tissue. Parvalbumin was detected in the cerebral cortex and cerebellum of gestational 15-day fetuses. However, the levels of parvalbumin in the muscle tissues and central nervous tissue were very low in rats before 1 week of age. Thereafter, they increased sharply, reaching the adult levels by 5 weeks in most of the tissues. Parvalbumin concentrations in adult rat soleus muscle increased <20-fold within 10 days after transection of the ipsilateral sciatic nerve, while the concentrations in the extensor digitorum longus muscle did not change in the same period.

Isolation and characterization of parvalbumins from skeletal muscles of a tropical amphibian, Leptodactylus insularis

Parvalbumins were isolated from skeletal muscles of a tropical amphibian, Leptodactylus insularis, and three new isotypes were identified. The total concentration of parvalbumins in L. insularis was the same as the total amounts found in an amphibian from the temperate or variable zone (Rana temporaria). Muscles of the thigh and foreleg had the maximum parvalbumin concentration (0.35 mmol.kg wet weight-1). Samples from pectoralis and rectus abdominis muscles had significantly less (0.29 mmol.kg-1). Three previously unknown parvalbumin isotypes (IV, IIIa, and IIIb) were isolated from the tropical amphibian. They were different from the isotypes (IVa and IVb) predominant in R. temporaria skeletal muscle. Parvalbumins are thought to have a role in the short-term removal of myoplasmic Ca2+ during muscle relaxation. Hence, the unique isotypes in L. insularis may reflect optimal molecular adaptations retained during the animal's evolution in a constantly warm environment.

Age‐Related changes and tissue distribution of parvalbumin in normal and dystrophic mice of strain 129 ReJ

In murine muscular dystrophy, hindlimb muscle contains a functionally defective thiol protease inhibitor (TPI) which has been implicated in the onset and progression of the disease in mice. More recently, this protease inhibitor has been identified as parvalbumin, a calcium binding protein. In this study, a polyclonal antibody against mouse muscle parvalbumin was used to study the concentration and distribution of this protein in normal and dystrophic male mice at various ages. Immunodetection assays were used to screen extracts of hindlimb, forelimb, brain, heart, lung, liver, and kidney in 60-day-old normal and dystrophic male mice for parvalbumin content. Parvalbumin was detected in relatively high amounts in both hindlimb and forelimb muscle extracts, while much lower concentrations were detected in brains of normal and dystrophic animals. No parvalbumin was detected in the lung, liver, heart, or kidney extracts using the immunoassay. With aging, the parvalbumin concentration in hindlimb muscle of normal mice remained fairly constant for 90 days, whereupon the level increased at 120 days. In contrast, the parvalbumin concentration in hindlimb muscle of dystrophic mice decreased steadily with age to about 22%% of normal animals at 120 days. The parvalbumin content was also reduced in dystrophic brain.

Calcium-binding proteins are concentrated in the CA2 field of the monkey hippocampus: a possible key to this region's resistance to epileptic damage

Previous immunocytochemical studies have shown a heterogeneous distribution of parvalbumin (PA) and calbindin (CB) in the rat hippocampal formation. The results of the present study showed a heterogeneous distribution of PA and CB in primate Ammon's horn. The density and intensity of immunoreactivity for both of these calcium-binding proteins was greatest in CA2 as compared to CA1 and CA3. CB-immunoreactivity was localized to the cell bodies, dendrites, and axon initial segments of pyramidal cells whereas PA-immunostaining was found in the axon terminals, dendrites and cell bodies of interneurons that have features similar to GABAergic inhibitory neurons. Based on previous studies that have shown a protective role of calcium-binding proteins in neurons exposed to hyperstimulation, these results suggest that the resistance of CA2 pyramidal cells in temporal lobe epilepsy is due to the high concentration of CB and PA in this region of Ammon's horn.

Effects of temperature acclimation on muscle relaxation in the carp: A mechanical, biochemical, and ultrastructural study

AbstractCommon carp (Cyprinus carpio L.) were acclimated to either 5–8°C or 20–25°C for a minimum of 1 month, and the twitch contraction kinetics of a myotomal nerve‐muscle preparation were investigated. A significant compensation for the acute effects of temperature was achieved in twitch kinetics by acclimation to 8°C. An acute drop in temperature from 20 to 8°C in preparations from warm‐acclimated fish led to approximately two‐ to three‐fold increases in the half‐times for activation and relaxation. At 8°C, values were 50% faster in 8°C‐ than in 20°C‐acclimated fish, indicating a partial capacity adaptation in rates of both twitch activation and relaxation.The mechanisms underlying temperature compensation of twitch contraction kinetics were investigated. A quantitative ultrastructural study was performed on the sarcoplasmic reticulum (S.R.) of fast and slow myotomal muscles. No differences were found in the surface and volume densities of the S.R. in fast‐twitch fibres between cold‐ and warm‐acclimated fish. The surface densities of terminal cisternae and S.R. were higher in slow‐twitch fibres from the warm‐acclimated fish. Parvalbumin concentration in fast fibres was found to be independent of acclimation temperature (0.61–0.68 mmol/kg wet weight). Although the relative proportions of parvalbumin isoforms (I–IV) showed considerable individual variation this was not correlated with environmental temperature. The Ca2+‐ATPase activity of S.R.‐enriched microsomes prepared from fast myotomal muscle was 60% higher at 8°C in cold‐ than warm‐acclimated carp. This suggests that changes in the kinetics and/or density of Ca2+ pumps contribute to the observed capacity adaptation in relaxation rate with temperature acclimation. Other possible mechanisms underlying the plasticity of twitch contraction kinetics in carp are briefly discussed.

A developmental change in the content of parvalbumin in normal and dystrophic mouse ( mdx) muscle

A highly sensitive enzyme immunoassay for mouse parvalbumin was developed in this study. The amount of parvalbumin was determined by a sandwich enzyme immunoassay method using anti-parvalbumin IgG-coated polystyrene balls and an anti-parvalbumin Fab'-horseradish peroxidase conjugate. Parvalbumin could not be detected in normal and dystrophic skeletal muscles of newborn mice. In normal mice, it appeared in the first postnatal week and increased linearly thereafter until the 12th week in fast twitch muscle. Rapid increase in parvalbumin was seen during 3rd and 8th week. On the other hand, parvalbumin detected in the first postnatal week increased gradually, but did not yet reach the adult level at the 16th postnatal week in slow twitch muscle. In mdx mice, fast twitch muscles such as the gastrocnemius and tibialis anterior were found to contain significantly decreased amounts of parvalbumin, compared with those in control mice. In fast twitch muscle parvalbumin in mdx mice could not be detected in the newborn, increased until 4th week and thereafter did not increase as that in normal mice. In slow twitch muscle the postnatal increase in parvalbumin content was not different from that in control mice. These results suggest that the decrease in the content of parvalbumin in dystrophic muscle may contribute to the elevation of the level of sarcoplasmic free Ca 2+ and the activated Ca 2+-dependent proteolysis.

Differential effect of development on parvalbumin content of chandelier and basket neurons in primate neocortex : Mayada Akil, M.D., David A. Lewis, M.D. Departments of Psychiatry and Behavioral Neuroscience, Western Psychiatric Institute and Clinic, University of Pittsburgh, Pittsburgh, PA 15213

Differential effect of development on parvalbumin content of chandelier and basket neurons in primate neocortex : Mayada Akil, M.D., David A. Lewis, M.D. Departments of Psychiatry and Behavioral Neuroscience, Western Psychiatric Institute and Clinic, University of Pittsburgh, Pittsburgh, PA 15213

Purification and characterization of the parvalbumin from monkey skeletal muscle

1. 1. A high affinity Ca 2+ binding and low mol. wt protein, parvalbumin, was purified from monkey skeletal muscle. 2. 2. As compared with other animals, only one component and a lower content of monkey parvalbumin were found. 3. 3. This may suggest that both the component and the content of parvalbumin decreases with biological evolution. 4. 4. The parvalbumin was found to have a mol. wt of 11,400, a pI of 5.1, a high aspartic acid and lysine content, maximum absorption at around 260nm, a blocked amino-terminal, an immunological distinction, 2 mol Ca 2+ binding/mol, and a conformational change by Ca 2+ binding. 5. 5. Parvalbumin was shown to have alpha type properties.

Soluble calcium-binding proteins: Parvalbumins and calmodulin from eel skeletal muscle

1. 1. Eel skeletal muscle contains three parvalbumin isoforms. The overall parvalbumin concentration in the muscle is 0.5 mmol kg −1 wet weight. 2. 2. Calmodulin (0.1 μmol kg −1 wet weight) was purified by extraction with ethylenediamine tetraacetate-containing buffer, fractionation with trichloroacetic acid and separation by ion exchange chromatography on DEAE-cellulose and by molecular sieving on Ultrogel AcA 54. 3. 3. Troponin-C-free calmodulin was obtained by fast protein liquid chromatography on a Mono Q column.

Identification and quantification in single muscle fibers of four isoforms of parvalbumin in the iliofibularis muscle of Xenopus laevis

The major parvalbumins present in the iliofibularis muscle of Xenopus laevis were identified and the total parvalbumin content of different types of single fibers of this muscle was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS). The criteria used in the identification of proteins as parvalbumins were: a relative molecular mass (Mr) between 10,000 and 14,000, an isoelectric point (pI) between 4.0 and 5.0, and a Ca2+-dependent mobility when run on a polyacrylamide gel in the absence of SDS. Four proteins were thus identified as parvalbumins: PA1, Mr 14,000, pI 4.90; PA2, Mr 11,000, pI 4.90; PA3, Mr 11,000, pI 4.95; and PA4, Mr 11,000, pI 4.25. An ultraviolet absorbance spectrum characteristic of parvalbumins was recorded for a purified preparation of these four proteins. Because the apparent Mr of rabbit parvalbumin in the gel system used was 14,000, whereas the true value is 12,100, it is not excluded that the Mr of component PA1 of 14,000 is an overestimation. The total parvalbumin content of muscles and single muscle fibers was determined using the supernatant obtained after centrifugation of tissue homogenates. Analysis of the protein pattern after electrophoresis in the presence of SDS of this fraction indicated that the Mr 14,000 and 11,000 protein bands contained virtually only parvalbumin. Quantification of the total parvalbumin content of relatively fast (type 1) and slow (type 2) contracting and relaxing single muscle fibers, using laser densitometric analysis of minigels, yielded mean values (mg protein/g wet wt., +/- S.D.) of 5.2 +/- 0.8 for nine type 1 fibers, and 1.9 +/- 1.0 for five type 2 fibers. Both fiber types contained about 2.5-times as much of the Mr 14,000 isoform relative to the combined Mr 11,000 isoforms.

Parvalbumin increases in the caudate putamen of rats with vitamin D hypervitaminosis.

The influence of chronic vitamin D3 application on the concentration of the four calcium-binding proteins parvalbumin, the 28-kDa calbindin-D, calmodulin, and S-100 was studied in various brain regions and in the kidney. Young rats were administered daily 20,000 international units of vitamin D3 per kg (body weight) over a period of 4 months. This chronic treatment resulted in a clinically mild hypervitaminosis that did not affect the content of calmodulin, the 28-kDa calbindin-D, and S-100. Also the concentration of parvalbumin in the cerebral cortex, hippocampus, and kidney remained unchanged. On the other hand, parvalbumin was increased about 50% in the caudate putamen of hypervitaminotic animals as compared to controls. Our results indicate that the metabolism of parvalbumin in the caudate putamen can be influenced by variations of the blood level of this steroid hormone.

Functional regeneration in the hindlimb skeletal muscle of the mdx mouse.

The pattern of spontaneous skeletal muscle degeneration and clinical recovery hindlimb muscles of the mdx mutant mouse was examined for functional and metabolic confirmation of apparent structural regeneration. The contractile properties, histochemical staining and myosin light chain and parvalbumin contents of extensor digitorum longus (EDL) and soleus (Sol) muscles of mdx and age-matched control mice were studied at 3-4 and 32 weeks. Histochemical staining (myofibrillar ATPase and NADH-tetrazolium reductase) revealed no significant change in slow-twitch-oxidative (SO) or fast-twitch-oxidative-glycolytic (FOG) fibre type proportions in mdx Sol apart from the normal age-related increase in SO fibres. At 32 weeks mdx EDL, however, showed significantly smaller fast-twitch-glycolytic (FG) and larger FOG proportions than those in control EDL. These fibre type distributions were confirmed by differential staining with antibodies to myosin slow-twitch and fast-twitch heavy chain isozymes. Frequency distribution of cross-sectional area for each fibre type showed a wider than normal range of areas especially in FOG fibres of mdx Sol, and FG fibres of mdx EDL, supporting previous observations using autoradiography of myofibre regeneration. Isometric twitch and tetanic tensions in Sol were significantly less than in controls at 4 weeks, but by 32 weeks, values were not different from age-matched controls. In mdx EDL at 3 weeks, twitch and tetanus tensions were significantly less, and time-to-peak twitch tensions were significantly faster than in control EDL. By 32 weeks, mdx EDL twitch and tetanus tensions expressed relative to muscle weight continued to be significantly lower than in age-matched controls, despite normal absolute tensions. The maximum velocity of shortening in 32-week mdx EDL was significantly lower than in control EDL. Myosin light chain distribution in mdx Sol exhibited significantly less light chain 2-slow (LC2s) and more light chain 1b-slow(LC1bs) at 32 weeks than age-matched control Sol. Gels of EDL from 32-week-old mdx mice showed significantly less light chain 2-fast-phosphorylated (LC2f-P) and light chain 3-fast (LC3f) and significantly more light chain 1-fast (LC1f) and light chain 2-fast (LC2f), but normal parvalbumin content compared to age-matched controls. These observations suggest that mdx hindlimb muscles are differentially affected by the disease process as it occurs in murine models of dystrophy. However, the uniqueness of mdx Sol and to a lesser extent EDL is that they also undergo an important degree of functional regeneration which is able to compensate spontaneously for degenerative influences of genetic origin.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental control of the excitability of muscle: transplantation experiments on a myotonic mouse mutant.

Developmental aspects of an animal model of myotonia, the mouse mutant called "arrested development of righting response" (ADR phenotype), were studied. Adult ADR muscle is characterized by a low chloride conductance of the membrane, leading to hyperexcitability, and by a low parvalbumin content. The myotonic hyperexcitability (as measured by the extent of "aftercontractions") of ADR muscle increased steeply between postnatal days 9 and 18, by which time it had approached the adult level. To study the tissue autonomy of the myotonic phenotype, muscle grafts were performed in all four combinations between ADR and wildtype (WT phenotype) donors and hosts. In most experiments, the relative contributions of donor and host to the regenerated muscles were determined by an allelic marker (glucose phosphate isomerase). In WT and ADR hosts, ADR grafts showed myotonic responses that in WT nude mouse hosts were incomplete and similar to those of juvenile ADR muscle. In no case did grafts from WT donors show any myotonia. This shows that the myotonic ADR phenotype is based on an intrinsic muscle property most likely related to the plasma membrane. The parvalbumin contents of grafted muscles, when compared with those of untransplanted muscles, indicated graft-host interaction in the expression of this secondary phenotypic property.

Fibre types, calcium-sequestering proteins and metabolic enzymes in denervated and chronically stimulated muscles of the rat.

1. Fibre types, Ca2+-sequestering proteins (parvalbumin, sarcoplasmic reticulum Ca2+-ATPase), enzyme activities of energy metabolism, and lactate dehydrogenase isozymes were studied in extensor digitorum longus (EDL) and soleus muscles of adult rats after denervation and direct stimulation for 45-61 days. The stimulation resembled the firing patterns of normal motor units (Hennig & Lømo, 1985) in either soleus (20 Hz, high amount) or EDL (150 Hz, low and high amount). 2. Low- and high-amount 150 Hz stimulation maintained essentially normal properties in the denervated EDL and induced many EDL-like properties in the denervated soleus, e.g. pronounced increases in Ca2+-ATPase and parvalbumin contents, a rise in the glycolytic enzyme activities, as well as a reversal of the lactate dehydrogenase H/M subunit ratio. The 150 Hz high-amount stimulation was not as efficient as the 150 Hz low amount in maintaining the fast properties of denervated EDL with the exception of the enzymes of aerobic oxidative metabolism. High-amount 20 Hz stimulation maintained essentially normal properties in the soleus but induced only moderate slow muscle characteristics in the EDL. 3. The maintenance of essentially normal properties by 'native' and the changes induced by 'foreign' stimulus patterns in the absence of the nerve indicate that evoked muscle activity plays a major role in controlling the phenotypic expression of muscle properties. However, the different responses of the denervated fast- and slow-twitch muscles to identical stimulus patterns further suggest that rat EDL and soleus muscles contain intrinsically different muscle fibres.

Relationship between parvalbumin content and the speed of relaxation in chronically stimulated rabbit fast-twitch muscle.

The time courses of changes in parvalbumin (PA) content, isometric twitch tension, and half-relaxation time (1/2 RT) were studied in rabbit tibialis anterior muscle following chronic 10 Hz nerve stimulation of 1-21 days. Up to 5 days stimulation had no effect on PA content, but it induced a slight (10-15%) increase in the 1/2 RT. This change occurred together with the previously observed 50% decrease in Ca2+-uptake by the SR (Leberer et al. 1987). While prolonged stimulation produced no further decrease in the Ca2+-uptake by the SR, PA content declined after 5 days of stimulation. The reduction in PA content was accompanied by a progressive lengthening of the 1/2 RT. However, the increase in 1/2 RT was particularly pronounced after PA had fallen below 50% of its normal value. A 90% reduction in PA coincided with a 60% increase in the 1/2 RT. By this time the staircase phenomenon, normally observed in fast-twitch muscle, was completely abolished. Although the changes in PA content and 1/2 RT were not linearly related, these results suggest that PA plays an important role in the relaxation process of mammalian fast-twitch muscle.