Distribution of sarcoplasmic reticulum Ca-ATPase and of calsequestrin at the polar regions of rat, rabbit and cat intrafusal fibers

Sarcoplasmic reticulum (SR) Ca2+-pumping ATPase (Ca-ATPase) and calsequestrin (CaS) were visualized by indirect immunofluorescence at the polar regions of adult rat, rabbit and cat intrafusal fibers. The immunohistochemical reaction products were regarded as histochemical markers of the SR and as valid indicators of the distribution of the two Ca2+-sequestering proteins. Static nuclear bag2 fibers displayed lower levels of both Ca-ATPase and CaS than the other two intrafusal fiber types. Nuclear chain fibers presented the highest Ca-ATPase levels and, together with dynamic nuclear bag1 fibers, they also exhibited relatively high amounts of CaS. The level of Ca-ATPase was lower in bag 1 fibers than in nuclear chain fibers, but not as low as in bag2 fibers. The comparatively high levels of Ca-ATPase and CaS seen in nuclear chain fibers coincided with their reported faster contractile speeds compared to nuclear bag fibers.     

Distribution of SERCA isoforms in human intrafusal fibers

The sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) is a membrane protein that plays a crucial role in muscle relaxation by transporting cytosolic Ca²⁺ into the lumen of the sarco/endoplasmic reticulum. In this study, the presence of SERCA1 and SERCA2 was investigated in human intrafusal fibers by immunocytochemistry. Nuclear bag₁ fibers contained both SERCA1 and SERCA2 isoforms, with predominant staining seen with SERCA2 in the A and B regions. Most nuclear bag₂ fibers also contained SERCA1 and SERCA2 isoforms and their coexistence frequently occurred in the A region. SERCA1 was present whereas SERCA2 was generally absent in the nuclear chain fibers. The staining intensity seen with the SERCA1 monoclonal antibody varied in the order of chain>bag₁>bag₂. The expression of SERCA1 isoform was found to correlate with the presence of fast myosin heavy chain (MyHC) isoform in nuclear chain fibers, whereas for nuclear bag fibers there was no such apparent correlation between patterns of expression of SERCA and MyHC isoforms. The phenotype revealed for the human bag fibers was very sophisticated and adapted to attain a very wide range of contraction and relaxation velocities.     

Histochemical profiles of cat intrafusal muscle fibers and their motor innervation

Summary Muscle spindles were examined histochemically in serial transverse sections of cat tenuissimus muscles. The myofibrillar adenosine triphosphatase (ATPase) staining reaction was used to identify nuclear bag₁, bag₂ and nuclear chain intrafusal muscle fibers. Regional differences in ATPase staining occurred along the bag₁ and bag₂ fibers but not along the chain fibers. All intrafusal fiber types displayed regional variability in staining for nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR). Motor nerve terminals were demonstrated along the poles of bag₁, bag₂ and chain fibers by staining for cholinesterase (ChE). There was no consistent spatial correlation between the intensity of regional ATPase staining along the bag fibers and location, number or type of motor endings. However, most ChE deposits occurred in intrafusal fiber regions that displayed the greatest NADH-TR variability. Some fiber poles or whole intrafusal fibers were devoid of any ChE deposits but their ATPase and NADH-TR content was comparable to that of fibers bearing ChE deposits. The observations suggested that motor nerve fibers per se may not play a major role in determining the histoenzymatic content of intrafusal fibers.     

Aggregation of myonuclei and the spread of slow-tonic myosin immunoreactivity in developing muscle spindles

Summary The pattern of regional expression of a slow-tonic myosin heavy chain (MHC) isoform was studied in developing rat soleus intrafusal muscle fibers. Binding of the slow-tonic antibody (ATO) began at the equator of prenatal intrafusal fibers where sensory nerve endings are located, and spread into the polar regions of nuclear bag₂ and bag₁ fibers but not nuclear chain fibers during ontogeny. The onset of the ATO reactivity coincided with the appearance of equatorial clusters of myonuclei (nuclear bag formations) in bag₁ and bag₂ fibers. Moreover, the intensity of the ATO reaction was strongest in the region of equatorial myonuclei and decreased with increasing distance from the equator of bag₁ and bag₂ fibers at all stages of prenatal and postnatal development. The polar expansion of ATO reactivity continued throughout the postnatal development of bag₁ fibers, but ceased shortly after birth in bag₂ fiber coincident with innervation by motor axons. Thus, afferents that innervate the equator might induce the slow-tonic MHC isoform in bag₂ and bag₁ fibers by regulating the myosin gene expression by equatorial myonuclei, and efferents or twitch contractile activity might inhibit the spread of the slow-tonic MHC isoform into the poles of bag₂ but not bag₁ fibers. Absence of ATO binding in chain fibers suggests that chain myotubes may not be as susceptible to the effect of afferents as are myotubes that develop into bag₂ and bag₁ fibers. The different patterns of slow-tonic MHC expression in the three types of intrafusal fiber may therefore result from the interaction of three elements: sensory neurons, motor neurons, and intrafusal myotubes.     

The topography of long nuclear chain intrafusal fibers in the cat muscle spindle

Summary Muscle spindles were studied histochemically in serial transverse sections of specimens of the cat tenuissimus muscle. The nuclear chain intrafusal muscles fibers were separated into three subtypes, called long, intermediate and typical. The long chain and intermediate chain fibers tended to assume a particular position within the axial bundle of intrafusal fibers. The fibers were usually located in that layer of chain fibers that was positioned farthest away from the bag₂ fiber. Furthermore, they were usually situated adjacent to the bag₁ fiber throughout much of the extent of the spindle pole. Some long chain and intermediate chain fibers had several fiber nuclei abreast at the equator rather than a single row of central nuclei, as in most nuclear chain fibers. The relative position of intrafusal fibers within the cat spindle may reflect their order of formation during development, with the fibers retaining, to a variable degree, their association with the bag₂ fiber which acted as template. Thus, the axial position of long chain and intermediate chain fibers suggests that they are among the first nuclear chain fibers to form. This may play a role in the known preferential innervation of these chain fibers by skeleto-fusimotor axons.     

Comparison of motor endings of the cat's muscle spindle stained for NADH-tetrazolium reductase and cholinesterase

Summary Cat muscle spindles were examined histochemically in serial transverse sections of tenuissimus muscles stained for ATPase, NADH-TR and ChE alternating sequentially. Motor nerve terminals on nuclear bag₁, bag₂ and nuclear chain intrafusal muscle fibers were identified in periodic sections stained for ChE. Intrafusal fiber regions that carried ChE-active areas were then examined in staining for NADH-TR. The motor endings on the three types of intrafusal fiber differed in their apparent histochemical content of both ChE and NADH-TR. The observations suggest that functional differences may exist among motor nerve terminals on the various intrafusal fiber types.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

Summary The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations.Nuclear bag₁ fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag₂ fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with antineonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag₁ fibers lacked staining for M-protein, whereas bag₂ fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag₁ fibers were less reactive and clear striations were not observed, in contrast to bag₂ and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition, (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested.Taken together, the data show that in adult rat solcus, slow tonic and neonatal myosin heavy, chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Origin of intrafusal muscle fibers in the rat

Summary The expression of several isoforms of myosin heavy chain (MHC) by intrafusal and extrafusal fibers of the rat soleus muscle at different stages of development was compared by immunocytochemistry. The first intrafusal myotube to form, the bag₂ fiber, expressed a slow-twitch MHC isoform identical to that expressed by the primary extrafusal myotubes. The second intrafusal myotube to form, the bag₁ fiber, expressed a fast-twitch MHC similar to that initially expressed by the secondary extrafusal myotubes. At subsequent stages of development, the equatorial and juxtaequatorial regions of bag₂ and bag₁ intrafusal myofibers began to express a slow-tonic myosin isoform not expressed by extrafusal fibers, and ceased to express some of the MHC isoforms present initially. Myotubes which eventually matured into chain fibers expressed initially both the slow-twitch and fast-twitch MHC isoforms similar to some secondary extrafusal myotubes. In contrast, adult chain fibers expressed the fast-twitch MHC isoform only. Hence intrafusal myotubes initially expressed no unique MHCs, but rather expressed MHCs similar to those expressed by extrafusal myotubes at the same chronological stage of muscle development. These observations suggest that both intrafusal and extrafusal fibers develop from common pools of bipotential myotubes. Differences in MHC expression observed between intrafusal and extrafusal fibers of rat muscle might then result from a morphogenetic effect of afferent innervation on intrafusal myotubes.     

Immunohistochemical demonstration of embryonic myosin heavy chains in adult mammalian intrafusal fibers

Summary Serial cross sections of rat, rabbit and cat intrafusal fibers from muscle spindles of normal adult hindlimb muscles were incubated with a monoclonal antibody against embryonic myosin heavy chains. Intrafusal fiber types were identified by noting their staining patterns in adjacent sections incubated for myofibrillar ATPase after acid or alkaline preincubation. In rat and rabbit muscle spindles dynamic nuclear bag₁ fibers reacted strongly at the polar and juxtaequatorial regions. Static nuclear bag₂ fibers reacted weakly or not at all at the polar region, but showed a moderate amount of activity at the juxtaequator. At the equatorial region both types of nuclear bag fibers displayed a rim of fluorescence surrounding the nuclear bags, while the areas occupied by the nuclear bags themselves were negative. Nuclear chain fibers in rat and rabbit muscle spindles were unreactive with the specific antibody over their entire length. In cat muscle spindles both types of nuclear bag fibers presented profiles which resembled those of the nuclear bag fibers in the other two species, but unlike in rat and rabbit spindles, cat nuclear chain fibers reacted as strongly as dynamic nuclear bag₁ fibers.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Immunohistochemical differences in myosin composition among intrafusal muscle fibres

Summary Mammalian intrafusal fibre types (nuclear chain, nuclear bag₁ and nuclear bag₂ fibres) are known to differ in their ultrastructure, intensity of the myofibrillar histochemical ATP-ase reaction, type of innervation and time course of contraction. The present study concerns the myosin composition of these intrafusal fibre types in the soleus muscle (mouse) and the extensor digitorum longus muscle (rat). We used an immunohistochemical method with three myosin antisera raised in rabbits: anti chicken pectoral myosin, anti chicken heart myosin (1) and anti chicken heart myosin (2) (=anti chicken heart myosin (1) adsorbed with muscle powder from soleus muscle of guinea pig). The results showed that three intrafusal fibre types differed in their myosin composition. A comparison of intrafusal fibre types with extrafusal fibre types for the histochemical myofibrillar ATP-ase reactivity and the reactivity with myosin antisera showed a resemblance of nuclear chain fibres with extrafusal type II fibres and a difference between nuclear bag₁ and nuclear bag₂ fibres and all other fibre types.     

Ultrastructure of Type 2A Extrafusal Muscle Fibers and Muscle Spindle Intrafusal Fibers of Adult Rats after Prolonged Swimming

We compared the ultrastructure of type 2A extrafusal muscle fibers, the nuclear chain, and other intrafusal fibers of muscle spindle (muscle stretch mechanoreceptor) in adult rats after prolonged swimming (5–10 h/day, 10 days). The Golgi apparatus was expressed moderately in type 2A extrafusal fibers and hypertrophied in the motor B zone of nuclear chain intrafusal fibers. Intense development of the Golgi apparatus in the nuclear chain intrafusal fiber appears to be related to glycogenolysis in the autophagous vacuoles, involvement in the lysosome activity, and plasma membrane renewal. Recapitulation of the mechanism of glycogen autophagy, which is observed in newborn rats during mobilization of glycogen from the liver and muscle, was demonstrated in adult rats under the influence of physical load in the nuclear chain and nuclear bag₂ intrafusal fibers and in type 2A extrafusal fibers. This is accounted for by a weak differentiation of the intrafusal muscle fibers: structurally, they are similar to myotubes and have specific features of blood supply and innervation. Individual features of experimental adult rats may also play a certain role.     

Slow-tonic MHC expression in paralyzed hindlimbs of fetal rats

Summary Whether nerve activity and active contraction of myotubes are essential for the assembly and initial differentiation of muscle spindles was investigated by paralyzing fetal rats with tetrodotoxin (TTX) from embryonic day 16 (E16) to E21, prior to and during the period when spindles typically form. TTX-treated soleus muscles were examined by light and electron microscopy for the presence of spindles and expression of myosin heavy chain (MHC) isoforms by the intrafusal fibers. Treatment with TTX did not inhibit the formation of a spindle capsule or the expression of a slow-tonic MHC isoform characteristic of intrafusal fibers, but did retard development of spindles. Spindles of TTX-treated E21 muscles usually consisted of one intrafusal fiber (bag₂) only rather than two fibers (bag₁ and bag₂) typically present in untreated (control) E21 spindles. Intrafusal fibers of TTX-treated spindles also had only one sensory region supplied by multiple afferents, and were devoid of motor innervation. These features are characteristic of spindles in normal E18–E19 muscles. Thus, nerve and/or muscle activity is not essential for the assembly of muscle spindles, formation of a spindle capsule, and transformation of undifferentiated myotubes into the intrafusal fibers containing spindle-specific myosin isoforms. However, activity may promote the maturation of intrafusal bundles, as well as the maturation of afferent and efferent nerve supplies to intrafusal fibers.     

The occurrence of “mixed” nuclear bag intrafusal fibers in the cat muscle spindle

Summary A total of 147 muscle spindles was studied histochemically in serial transverse sections of 42 cat tenuissimus muscle specimens. Nuclear bag₁, nuclear bag₂ and nuclear chain intrafusal muscle fibers were distinguished by the differential staining resulting from the reactions for myosin adenosine 5-triphosphatase and nicotinamide adenine dinucleotide tetrazolium reductase. The majority of intrafusal fibers were of the same histochemical type at both fiber poles. However, seven muscle spindles contained one nuclear bag fiber each that presented as a bag₁ in one pole and as a bag₂ in the other pole. These mixed nuclear bag fibers were found in spindles that also contained at least one bag₁ and one bag₂ fiber of equivalent histochemical presentation in both fiber poles. The mixed bag fibers displayed differences of apparent fiber diameter and relative polar length between the two fiber poles. The motor innervation pattern, as revealed by staining for cholinesterase, was also dissimilar between the two poles of mixed bag fibers. The study indicates that the spindle equatorial region may in some instances serve as a boundary between two morphologically and histochemically different poles of the same intrafusal fiber.     

One-bag-fiber muscle spindles in tenuissimus muscles of the cat

Summary Over 150 complete and 139 incomplete single muscle spindles were examined in serial transverse sections of cat tenuissimus muscles in search for spindles lacking one of the two types of nuclear bag intrafusal fiber. Several histochemical reactions were used to type the intrafusal muscle fibers and assess the spindle motor and sensory innervation. One complete spindle lacked a bag₁ fiber, and another spindle lacked a bag₂ fiber. Several incomplete spindles also lacked bag₁ fibers. In addition, ten double tandem spindles contained one capsular unit each that lacked the bag₁ fiber, and one triple tandem spindle had two such capsules. All one-bag-fiber spindles had primary sensory innervation, but none had secondary sensory innervation. Their motor innervation was similar to that of the usual two-bag-fiber spindles in the number and disposition of intrafusal motor endings. It is unclear whether the one-bag fiber spindles, either single or tandem-linked, are products of an aberrant spindle development or represent a true anatomical and functional subcategory of the cat muscle spindle.     

Ultrastructure of dynamic and static skeletofusimotor endings in a cat muscle spindle

Summary Endings of four skeletofusimotor axons in a spindle of the cat tenuissimus muscle were examined in semithin (1-m thick) and ultrathin transverse serial sections. Two (dynamic) axons terminated on the nuclear bag₁ intrafusal muscle fiber and on extrafusal fibers of the dark type. Two (static) axons terminated on the nuclear chain intrafusal fibers and extrafusal fibers of the intermediate type. The degree of indentation of axon terminals into the muscle surface, thickness of the sole plate and extent of folding of subjunctional membranes differed among intrafusal and extrafusal terminations of the same axon. Endings of axons on the bag₁ and chain fibers were also morphologically dissimilar. Motor axons may not determine ending morphology. Rather the form and structure of a bag₁ or chain ending may be determined by the type of intrafusal fiber on which the ending lies and the ending's distance from the primary sensory axon.     

Mitochondrial Ca2+-Handling in Fast Skeletal Muscle Fibers from Wild Type and Calsequestrin-Null Mice

Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca²⁺-binding protein inside SR, calsequestrin. Mitochondrial free Ca²⁺-concentration ([Ca²⁺]_mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca²⁺ release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca²⁺]_mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca²⁺ concentration. The rise in [Ca²⁺]_mito during electrical stimulation occurred in 20−30 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s⁻¹. Accordingly, frequency-dependent increase in [Ca²⁺]_mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca²⁺]_mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca²⁺]_mito transients were increased by inhibition of mitochondrial Na⁺/Ca²⁺ exchanger (mNCX). These results provide direct evidence for fast Ca²⁺ accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca²⁺-handling and a dependence of mitochondrial Ca²⁺-handling on intracellular (SR) and external Ca²⁺ stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca²⁺-leakage.     

Expression of an alpha cardiac-like myosin heavy chain in muscle spindle fibres

Summary In the present study we have investigated the reactivity of rat muscle to a specific monoclonal antibody directed against alpha cardiac myosin heavy chain. Serial cross sections of rat hindlimb muscles from the 17th day in utero to adulthood, and after neonatal denervation and de-efferentation, were studied by light microscope immunohistochemistry. Staining with anti- myosin heavy chain was restricted to intrafusal bag fibres in all specimens studied. Nuclear bag₂ fibres were moderately to strongly stained in the intracapsular portion and gradually lost their reactivity towards the ends, whereas nuclear bag₁ fibres were stained for a short distance in each pole. Nuclear bag₂ fibres displayed reactivity to anti- myosin heavy chain from the 21st day of gestation, whereas nuclear bag₁ fibres only acquired reactivity to anti- myosin heavy chain three days after birth. After neonatal de-efferentation, the reactivity of nuclear bag₂ fibres to anti- myosin heavy chain was decreased and limited to a shorter portion of the fibre, whereas nuclear bag₁ fibres were unreactive. We showed that a myosin heavy chain isoform hitherto unknown for skeletal muscle is specifically expressed in rat nuclear bag fibres. These findings add further complexity to the intricate pattern of isomyosin expression in intrafusal fibres. Furthermore, we show that motor innervation influences the expression of this isomyosin along the length of the fibres.     

Store-operated Ca2+ Entry in Malignant Hyperthermia-susceptible Human Skeletal Muscle

In malignant hyperthermia (MH), mutations in RyR1 underlie direct activation of the channel by volatile anesthetics, leading to muscle contracture and a life-threatening increase in core body temperature. The aim of the present study was to establish whether the associated depletion of sarcoplasmic reticulum (SR) Ca²⁺ triggers sarcolemmal Ca²⁺ influx via store-operated Ca²⁺ entry (SOCE). Samples of vastus medialis muscle were obtained from patients undergoing assessment for MH susceptibility using the in vitro contracture test. Single fibers were mechanically skinned, and confocal microscopy was used to detect changes in [Ca²⁺] either within the resealed t-system ([Ca²⁺]_t-sys) or within the cytosol. In normal fibers, halothane (0.5 mm) failed to initiate SR Ca²⁺ release or Ca²⁺_t-sys depletion. However, in MH-susceptible (MHS) fibers, halothane induced both SR Ca²⁺ release and Ca²⁺_t-sys depletion, consistent with SOCE. In some MHS fibers, halothane-induced SR Ca²⁺ release took the form of a propagated wave, which was temporally coupled to a wave of Ca²⁺_t-sys depletion. SOCE was potently inhibited by “extracellular” application of a STIM1 antibody trapped within the t-system but not when the antibody was denatured by heating. In conclusion, (i) in human MHS muscle, SR Ca²⁺ depletion induced by a level of volatile anesthetic within the clinical range is sufficient to induce SOCE, which is tightly coupled to SR Ca²⁺ release; (ii) sarcolemmal STIM1 has an important role in regulating SOCE; and (iii) sustained SOCE from an effectively infinite extracellular Ca²⁺ pool may contribute to the maintained rise in cytosolic [Ca²⁺] that underlies MH.     

The concentration of free Ca(2+) in the sarcoplasmic reticulum of frog cut twitch skeletal muscle fibers estimated with tetramethylmurexide

One aim of this article was to determine the resting concentration of free Ca²⁺ in the sarcoplasmic reticulum (SR) of frog cut skeletal muscle fibers ([Ca²⁺]_SR,R) using the calcium absorbance indicator dye tetramethylmurexide (TMX). Another was to determine the ratio of [Ca²⁺]_SR,R to TMX's apparent dissociation constant for Ca²⁺ (K_app) in order to establish the capability of monitoring [Ca²⁺]_SR(t) during SR Ca²⁺ release – a signal needed to determine the Ca²⁺ permeability of the SR. To reveal the properties of TMX in the SR, the surface membrane was rapidly permeabilized with saponin to rapidly dissipate myoplasmic TMX. Results indicated that the concentration of Ca-free TMX in the SR was 2.8-fold greater than that in the myoplasm apparently due to binding of TMX to sites in the SR. Taking into account that such binding might influence K_app as well as a dependence of K_app on TMX concentration, the results indicate an average [Ca²⁺]_SR,R ranging from 0.43 to 1.70 mM. The ratio [Ca²⁺]_SR,R/K_app averaged 0.256, a relatively low value which should not depend on factors influencing K_app. As a result, the time course of [Ca²⁺]_SR(t) in response to electrical stimulation is well determined by, and approximately linearly related to, the active TMX absorbance signal.     

Variable luminal sarcoplasmic reticulum Ca buffer capacity in smooth muscle cells

Sarcoplasmic reticulum contains the internal Ca²⁺ store in smooth muscle cells and its lumen appears to be a continuum that lacks diffusion barriers. Accordingly, the free luminal Ca²⁺ level is the same all throughout the SR; however, whether the Ca²⁺ buffer capacity is the same in all the SR is unknown. We have estimated indirectly the luminal Ca²⁺ buffer capacity of the SR by comparing the reduction in SR Ca²⁺ levels with the corresponding increase in [Ca²⁺]_i during activation of either IP₃Rs with carbachol or RyRs with caffeine, in smooth muscle cells from guinea pig urinary bladder. We have determined that carbachol-sensitive SR has a 2.4 times larger Ca²⁺ buffer capacity than caffeine-sensitive SR. Rapid inhibition of SERCA pumps with thapsigargin revealed that this pump activity accounts for 80% and 60% of the Ca²⁺ buffer capacities of carbachol- and caffeine-sensitive SR, respectively. Moreover, the Ca²⁺ buffer capacity of carbachol-sensitive SR was similar to caffeine-sensitive SR when SERCA pumps were inhibited. Similar rates of Ca²⁺ replenishments suggest similar levels of SERCA pump activities for either carbachol- or caffeine-sensitive SR. Paired pulses of caffeine, in conditions of low Ca²⁺ influx, indicate the relevance of luminal SR Ca²⁺ buffer capacity in the [Ca²⁺]_i response. To further study the importance of luminal SR Ca²⁺ buffer capacity in the release process we used low levels of heparin to partially inhibit IP₃Rs. This condition revealed carbachol-induced transient increase of luminal SR Ca²⁺ levels provided that SERCA pumps were active. It thus appears that SERCA pump activity keeps the luminal SR Ca²⁺-binding proteins in the high-capacity, low-affinity conformation, particularly for IP₃R-mediated Ca²⁺ release.