Troponin C isoform composition determines differences in Sr(2+)-activation characteristics between rat diaphragm fibers

Single fibers of rat diaphragm containing different naturally occurring combinations of myofibrillar protein isoforms were used to evaluate the contribution of troponin C (TnC) isoforms to fiber type-related differences with respect to sensitivity to Sr²⁺ of the contractile system. Mechanically skinned fibers were studied for their isometric force vs. Sr²⁺ concentration ([Sr²⁺]) relationships and then analyzed electrophoretically for myofibrillar protein isoform composition. Our data demonstrate that fiber-type differences in Sr²⁺ dependence of contractile activation processes are primarily determined by the TnC isoform composition, with the slow isoform conferring on average a sevenfold greater sensitivity to Sr²⁺ than the fast isoform. Moreover, the ratio of TnC isoforms determined functionally from the force-pSr (–log₁₀ [Sr²⁺]) curves is tightly (r² = 0.97) positively correlated with that estimated electrophoretically. Together, these results validate the use of Sr²⁺ activation characteristics to distinguish fibers containing different proportions of fast and slow TnC isoforms and to study the mechanisms by which divalent cations activate the contractile apparatus. We also found that the functionally and electrophoretically determined ratios of TnC isoforms present in a fiber display similar sigmoidal relationships with the ratio of myosin heavy chain (MHC) isoform types expressed. These relationships 1) offer further insight in the functional and molecular expression of TnC in relation to the molecular expression of MHC isoform types and 2) may provide the basis for predicting sensitivity to Sr²⁺, TnC, and MHC isoforms in pure and hybrid skeletal muscle fibers. muscle contraction; skeletal muscle; myofibrillar proteins; single fiber; sensitivity to strontium; sensitivity to calcium     

Electrophoretic and functional identification of two troponin C isoforms in toad skeletal muscle fibers

The differential sensitivity of frog twitch and slow-tonic fibers to Ca²⁺ and Sr²⁺ suggests that these two fiber types express different troponin C (TnC) isoforms. To date, only one TnC isoform from anurans (resembling the mammalian fast-twitch isoform) has been isolated and characterized. In this study, we examined the possibility that anuran striated muscle contains more than one TnC isoform. Toward this end, we determined the TnC isoform composition of 198 single fibers from the rectus abdominis of the cane toad (a mixed slow-tonic and twitch muscle) and of toad cardiac muscle using a method that enables the identification of TnC isoforms on the basis of the effect of Ca²⁺ on their electrophoretic mobility. The fibers were typed according to their myosin heavy chain (MHC) isoform composition. The data indicate that striated muscle of the cane toad contains two TnC isoforms, one of which (TnC-t) is present in all fibers displaying only twitch MHC isoforms and the other of which (TnC-T/c) is present in fibers displaying the tonic MHC isoform and in cardiac muscle. For a subpopulation of 15 fibers, the TnC isoform composition was also compared with Ca²⁺ and Sr²⁺ activation characteristics. Fibers containing the TnC-T/c isoform were 3-fold more sensitive to Ca²⁺, 40-fold more sensitive to Sr²⁺, and responded to a 4.6-fold broader range of [Ca²⁺] than did fibers containing the TnC-t isoform. The Ca²⁺ activation properties of toad fibers containing the TnC-T/c isoform appear to be consistent with the previously reported physiological characteristics of amphibian slow-tonic muscle fibers. myofibrillar proteins; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; alanine SDS-PAGE; hybrid fibers; Ca²⁺-binding proteins; single fiber; muscle protein polymorphism; fiber type     

Sustained norepinephrine contraction in the rat portal vein is lost when Ca(2+) is replaced with Sr(2+)

Agonist-induced activation of smoothmuscle involves a rise in intracellular Ca²⁺ concentrationand sensitization of myosin light chain phosphorylation toCa²⁺. Sr²⁺ can enter through Ca²⁺channels, be sequestered and released from sarcoplasmic reticulum, andreplace Ca²⁺ in activation of myosin light chainphosphorylation. Sr²⁺ cannot replace Ca²⁺ infacilitation of agonist-activated Ca²⁺-dependentnonselective cation channels. It is not known whether Sr²⁺can replace Ca²⁺ in small G protein-mediated sensitizationof phosphorylation. To explore mechanisms involved in-receptor-activated contractions in smooth muscle, effects ofreplacing Ca²⁺ with Sr²⁺ were examined in ratportal vein. Norepinephrine (NE) at >3.0 × 10⁷ Min the presence of Ca²⁺ resulted in a strong sustainedcontraction, whereas this sustained component was absent in thepresence of Sr²⁺; only the amplitude of phasic contractionsincreased. Pretreatment with low (~0.05 mM) free Ca²⁺followed by 2.5 mM Sr²⁺ resulted in a sustained componentof the NE response. In -escin-permeabilized preparations,phenylephrine in the presence of GTP or guanosine 5'-O-(3-thiotriphosphate) alone induced sensitization toSr²⁺. In conclusion, a Ca²⁺-regulatedmembrane/channel process is required for the sustained component of NEresponses in rat portal vein. Sensitization alone is not responsiblefor the sustained phase of the NE contraction.

     

Fiber type populations and Ca2+-activation properties of single fibers in soleus muscles from SHR and WKY rats

Electrophoretic analyses of muscle proteins in whole musclehomogenates and single muscle fiber segments were used to examine myosin heavy chain (MHC) and myosin light chain 2 (MLC2) isoform composition and fiber type populations in soleus muscles from spontaneously hypertensive rats (SHRs) and their age-matchednormotensive controls [Wistar-Kyoto (WKY) rats], at threestages in the development of high blood pressure (4 wk, 16 wk, and 24 wk of age). Demembranated (chemically skinned with 2% Triton X-100),single fiber preparations were used to determine the maximumCa²⁺-activated force percross-sectional area, calcium sensitivity, and degree of cooperativityof the contractile apparatus andCa²⁺-regulatory system withrespect to Ca²⁺. The results showthat, at all ages examined, 1) SHRsoleus contained a lower proportion of MHCI and MLC2 slow (MLC2s) and ahigher proportion of MHCIIa, MHCIId/x, and MLC2 fast (MLC2f )isoforms than the age-matched controls;2) random dissection of single fibers from SHR and WKY soleus produced four populations of fibers: type I (expressing MHCI), type IIA (expressing MHCIIa), hybrid typeI+IIA (coexpressing MHCI and MHCIIa), and hybrid type IIA+IID (coexpressing MHCIIa and MHCIId/x); and3) single fiber dissection from SHRsoleus yielded a lower proportion of type I fibers, a higher proportionof fast-twitch fibers (types IIA and IIA+IID), and a higher proportionof hybrid fibers (types I+IIA and IIA+IID) than the homologous musclesfrom the age-matched WKY rats. Because the presence of hybrid fibers isviewed as a marker of muscle transformation, these data suggest thatSHR soleus undergoes transformation well into adulthood. Our data showalso that, for a given fiber type, there are no significant differencesbetween SHR and WKY soleus muscles with respect to any of theCa²⁺-activation propertiesexamined. This finding indicates that the lower specific tensionsreported in the literature for SHR soleus muscles are not due tostrain- or hypertension-related differences in the function of thecontractile apparatus or regulatory system.     

Expression levels of RyR1 and RyR3 control resting free Ca2+ in skeletal muscle

To better understand the role of the transient expression of ryanodine receptor (RyR) type 3 (RyR3) on Ca²⁺ homeostasis during the development of skeletal muscle, we have analyzed the effect of expression levels of RyR3 and RyR1 on the overall physiology of cultured myotubes and muscle fibers. Dyspedic myotubes were infected with RyR1 or RyR3 containing virions at 0.2, 0.4, 1.0, and 4.0 moieties of infection (MOI), and analysis of their pattern of expression, caffeine sensitivity, and resting free Ca²⁺ concentration ([Ca²⁺]_r) was performed. Although increased MOI resulted in increased expression of each receptor isoform, it did not significantly affect the immunopattern of RyRs or the expression levels of calsequestrin, triadin, or FKBP-12. Interestingly, myotubes expressing RyR3 always had significantly higher [Ca²⁺]_r and lower caffeine EC₅₀ than did cells expressing RyR1. Although some of the increased sensitivity of RyR3 to caffeine could be attributed to the higher [Ca²⁺]_r in RyR3-expressing cells, studies of [³H]ryanodine binding demonstrated intrinsic differences in caffeine sensitivity between RyR1 and RyR3. Tibialis anterior (TA) muscle fibers at different stages of postnatal development exhibited a transient increase in [Ca²⁺]_r coordinately with their level of RyR3 expression. Similarly, adult soleus fibers, which also express RyR3, had higher [Ca²⁺]_r than did adult TA fibers, which exclusively express RyR1. These data show that in skeletal muscle, RyR3 increases [Ca²⁺]_r more than RyR1 does at any expression level. These data suggest that the coexpression of RyR1 and RyR3 at different levels may constitute a novel mechanism by which to regulate [Ca²⁺]_r in skeletal muscle. ryanodine receptor; calcium release; ryanodine binding; muscle fibers     

Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

Osteoblasts subjected to fluid shearincrease the expression of the early response gene, c-fos, andthe inducible isoform of cyclooxygenase, COX-2, two proteins linked tothe anabolic response of bone to mechanical stimulation, in vivo. Theseincreases in gene expression are dependent on shear-induced actinstress fiber formation. Here, we demonstrate that MC3T3-E1osteoblast-like cells respond to shear with a rapid increase inintracellular Ca²⁺ concentration([Ca²⁺]_i) that wepostulate is important to subsequent cellular responses to shear. Totest this hypothesis, MC3T3-E1 cells were grown on glass slides coatedwith fibronectin and subjected to laminar fluid flow (12 dyn/cm²). Before application of shear, cells were treatedwith two Ca²⁺ channel inhibitors or various blockers ofintracellular Ca²⁺ release for 0.5-1 h. Althoughgadolinium, a mechanosensitive channel blocker, significantly reducedthe [Ca²⁺]_i response, neithergadolinium nor nifedipine, an L-type channel Ca²⁺ channelblocker, were able to block shear-induced stress fiber formation andincrease in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid-AM, an intracellular Ca²⁺ chelator, or thapsigargin,which empties intracellular Ca²⁺ stores, completelyinhibited stress fiber formation and c-fos/COX-2 production in shearedosteoblasts. Neomycin or U-73122 inhibition of phospholipase C, whichmediates D-myo-inositol 1,4,5-trisphosphate (IP₃)-induced intracellular Ca²⁺ release, alsocompletely suppressed actin reorganization and c-fos/COX-2 production.Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform ofU-73122, did not inhibit these shear-induced responses. These resultssuggest that IP₃-mediated intracellular Ca²⁺release is required for modulating flow-induced responses in MC3T3-E1 cells.

     

Altered Ca(2+) handling by ryanodine receptor and Na(+)-Ca(2+) exchange in the heart from ovariectomized rats: role of protein kinase A

Our previous study has demonstrated that ovariectomy (Ovx) significantly increased the left ventricular developed pressure (LVDP) and the maximal rate of developed pressure over time (±dP/dt_max) in the isolated perfused rat heart and the effects were reversed by female sex hormone replacement. In the present investigation, we studied the effects of Ovx for 6 wk on Ca²⁺ homeostasis that determines the contractile function. Particular emphasis was given to Ca²⁺ handling by ryanodine receptor (RyR) and Na⁺-Ca²⁺ exchange (NCX). ⁴⁵Ca²⁺ fluxes via the RyR, NCX, and Ca²⁺-ATPase (SERCA) were compared with their expression in myocytes from Ovx rats with and without estrogen replacement. Furthermore, we correlated the handling of Ca²⁺ by these Ca²⁺ handling proteins with the overall Ca²⁺ homeostasis by determining the Ca²⁺ transients induced by electrical stimulation and caffeine, which reveals the dynamic changes of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in the heart. In addition, we determined the expression and contribution of protein kinase A (PKA) to the regulation of the aforementioned Ca²⁺ handling proteins in Ovx rats. It was found that after Ovx there were 1) increased Ca²⁺ fluxes via RyR and NCX, which were reversed not only by estrogen replacement, but more importantly by blockade of PKA; 2) an increased expression of PKA; and 3) no increase in expression of NCX and SERCA. We suggest that hyperactivities of RyR and NCX are a result of upregulation of PKA. The increased release of Ca²⁺ through RyR and removal of Ca²⁺ by NCX are believed to be responsible for the greater contractility and faster relaxation after Ovx. ovariectomy     

Denervation produces different single fiber phenotypes in fast- and slow-twitch hindlimb muscles of the rat

Using a single, mechanically skinned fiber approach, we tested the hypothesis that denervation (0 to 50 days) of skeletal muscles that do not overlap in fiber type composition [extensor digitorum longus (EDL) and soleus (SOL) muscles of Long-Evans hooded rats] leads to development of different fiber phenotypes. Denervation (50 day) was accompanied by 1) a marked increase in the proportion of hybrid IIB/D fibers (EDL) and I/IIA fibers (SOL) from 30% to >75% in both muscles, and a corresponding decrease in the proportion of pure fibers expressing only one myosin heavy chain (MHC) isoform; 2) complex muscle- and fiber-type specific changes in sarcoplasmic reticulum Ca²⁺-loading level at physiological pCa 7.1, with EDL fibers displaying more consistent changes than SOL fibers; 3) decrease by 50% in specific force of all fiber types; 4) decrease in sensitivity to Ca²⁺, particularly for SOL fibers (by 40%); 5) decrease in the maximum steepness of the force-pCa curves, particularly for the hybrid I/IIA SOL fibers (by 35%); and 6) increased occurrence of biphasic behavior with respect to Sr²⁺ activation in SOL fibers, indicating the presence of both slow and fast troponin C isoforms. No fiber types common to the two muscles were detected at any time points (day 7, 21, and 50) after denervation. The results provide strong evidence that not only neural factors, but also the intrinsic properties of a muscle fiber, influence the structural and functional properties of a particular muscle cell and explain important functional changes induced by denervation at both whole muscle and single cell levels. mechanically skinned fibers; myosin heavy chain isoforms; lineage; sarcoplasmic reticulum; Ca²⁺; Sr²⁺ sensitivity; Long-Evans hooded rat     

Effect of lactate on depolarization-induced Ca(2+) release in mechanically skinned skeletal muscle fibers

Itis unclear whether accumulation of lactate in skeletal muscle fibersduring intense activity contributes to muscle fatigue. Usingmechanically skinned fibers from rat and toad muscle, we were able toexamine the effect of L(+)-lactate onexcitation-contraction coupling independently of other metabolicchanges. We investigated the effects of lactate on the contractileapparatus, caffeine-induced Ca²⁺ release from thesarcoplasmic reticulum, and depolarization-induced Ca²⁺release. Lactate (15 or 30 mM) had only a small inhibitory effect directly on the contractile apparatus and caused appreciable(20-35%) inhibition of caffeine-induced Ca²⁺ release,seemingly by a direct effect on the Ca²⁺ release channels.However, 15 mM lactate had no detectable effect on Ca²⁺release when it was triggered by the normal voltage sensor mechanism, and 30 mM lactate reduced such release by only <10%. These results indicate that lactate has only a relatively small inhibitory effect onnormal excitation-contraction coupling, indicating that lactate accumulation per se is not a major factor in muscle fatigue.

     

Effects of dihydropyridine receptor II-III loop peptides on Ca(2+) release in skinned skeletal muscle fibers

Inskeletal muscle fibers, the intracellular loop between domains II andIII of the ₁-subunit of the dihydropyridine receptor (DHPR) may directly activate the adjacent Ca²⁺ releasechannel in the sarcoplasmic reticulum. We examined the effects ofsynthetic peptide segments of this loop on Ca²⁺ release inmechanically skinned skeletal muscle fibers with functional excitation-contraction coupling. In rat fibers at physiological Mg²⁺ concentration ([Mg²⁺]; 1 mM), a20-residue skeletal muscle DHPR peptide[A_S(20);Thr⁶⁷¹-Leu⁶⁹⁰; 30 µM], shown previously toinduce Ca²⁺ release in a triad preparation, caused onlysmall spontaneous force responses in ~40% of fibers, although itpotentiated responses to depolarization and caffeine in all fibers. TheCOOH-terminal half of A_S(20)[A_S(10)] induced much larger spontaneousresponses but also caused substantial inhibition of Ca²⁺release to both depolarization and caffeine. Both peptides induced orpotentiated Ca²⁺ release even when the voltage sensors wereinactivated, indicating direct action on the Ca²⁺ releasechannels. The corresponding 20-residue cardiac DHPR peptide [A_C(20);Thr⁷⁹³-Ala⁸¹²] was ineffective, but itsCOOH-terminal half [A_C(10)] had effects similar to A_S(20). In the presence of lower[Mg²⁺] (0.2 mM), exposure to eitherA_S(20) or A_C(10) (30 µM) induced substantial Ca²⁺ release. PeptideC_S (100 µM), a loop segment reported to inhibit Ca²⁺ release in triads, caused partial inhibition ofdepolarization-induced Ca²⁺ release. In toad fibers, eachof the A peptides had effects similar to or greater than those in ratfibers. These findings suggest that the A and C regions of the skeletalDHPR II-III loop may have important roles in vivo.

     

Muscarinic stimulation increases basal Ca(2+) and inhibits spontaneous Ca(2+) transients in murine colonic myocytes

Localized Ca²⁺ transients inisolated murine colonic myocytes depend on Ca²⁺ releasefrom inositol 1,4,5-trisphosphate (IP₃) receptors.Localized Ca²⁺ transients couple to spontaneous transientoutward currents (STOCs) and mediate hyperpolarization responses inthese cells. We used confocal microscopy and whole cell patch-clamprecording to investigate how muscarinic stimulation, which causesformation of IP₃, can suppress Ca²⁺ transientsand STOCs that might override the excitatory nature of cholinergicresponses. ACh (10 µM) reduced localized Ca²⁺ transientsand STOCs, and these effects were associated with a rise in basalcytosolic Ca²⁺. These effects of ACh were mimicked bygeneralized rises in basal Ca²⁺ caused by ionomycin(250-500 nM) or elevated external Ca²⁺ (6 mM).Atropine (10 µM) abolished the effects of ACh. Pretreatment of cellswith nicardipine (1 µM), or Cd²⁺ (200 µM) had no effecton responses to ACh. An inhibitor of phospholipase C, U-73122, blockedCa²⁺ transients and STOCs but did not affect the increasein basal Ca²⁺ after ACh stimulation. Xestospongin C (Xe-C;5 µM), a membrane-permeable antagonist of IP₃ receptors,blocked spontaneous Ca²⁺ transients but did not prevent theincrease of basal Ca²⁺ in response to ACh. Gd³⁺(10 µM), a nonselective cation channel inhibitor, prevented the increase in basal Ca²⁺ after ACh and increased thefrequency and amplitude of Ca²⁺ transients and waves.Another inhibitor of receptor-mediated Ca²⁺ influxchannels, SKF-96365, also prevented the rise in basal Ca²⁺after ACh and increased Ca²⁺ transients and development ofCa²⁺ waves. FK-506, an inhibitor ofFKBP12/IP₃ receptor interactions, had no effect onthe rise in basal Ca²⁺ but blocked the inhibitory effectsof increased basal Ca²⁺ and ACh on Ca²⁺transients. These results suggest that the rise in basalCa²⁺ that accompanies muscarinic stimulation of colonicmuscles inhibits localized Ca²⁺ transients that couldcouple to activation of Ca²⁺-activated K⁺channels and reduce the excitatory effects of ACh.

     

Differential regulation of Ca(2+) sparks and Ca(2+) waves by UTP in rat cerebral artery smooth muscle cells

Uridine 5'-triphosphate (UTP), a potent vasoconstrictor that activatesphospholipase C, shifted Ca²⁺ signaling from sparks towaves in the smooth muscle cells of rat cerebral arteries. UTPdecreased the frequency of Ca²⁺ sparks and transientCa²⁺-activated K⁺ (K_Ca) currentsand increased the frequency of Ca²⁺ waves. The UTP-inducedreduction in Ca²⁺ spark frequency did not reflect adecrease in global cytoplasmic Ca²⁺, Ca²⁺influx through voltage-dependent Ca²⁺ channels (VDCC), orCa²⁺ load of the sarcoplasmic reticulum (SR), since globalCa²⁺ was elevated, blocking VDCC did not prevent theeffect, and SR Ca²⁺ load did not decrease. However,blocking protein kinase C (PKC) with bisindolylmaleimide I did preventUTP reduction of Ca²⁺ sparks and transient K_Cacurrents. UTP decreased the effectiveness of caffeine, which increasesthe Ca²⁺ sensitivity of ryanodine-sensitiveCa²⁺ release (RyR) channels, to activate transientK_Ca currents. This work supports the concept thatvasoconstrictors shift Ca²⁺ signaling modalities fromCa²⁺ sparks to Ca²⁺ waves through the concertedactions of PKC on the Ca²⁺ sensitivity of RyR channels,which cause Ca²⁺ sparks, and of inositol trisphosphate(IP₃) on IP₃ receptors to generateCa²⁺ waves.

     

Fiber type conversion alters inactivation of voltage-dependent sodium currents in murine C2C12 skeletal muscle cells

Each skeletal muscle of the body contains a unique composition of "fast" and "slow" muscle fibers, each of which is specialized for certain challenges. This composition is not static, and the muscle fibers are capable of adapting their molecular composition by altered gene expression (i.e., fiber type conversion). Whereas changes in the expression of contractile proteins and metabolic enzymes in the course of fiber type conversion are well described, little is known about possible adaptations in the electrophysiological properties of skeletal muscle cells. Such adaptations may involve changes in the expression and/or function of ion channels. In this study, we investigated the effects of fast-to-slow fiber type conversion on currents via voltage-gated Na⁺ channels in the C₂C₁₂ murine skeletal muscle cell line. Prolonged treatment of cells with 25 nM of the Ca²⁺ ionophore A-23187 caused a significant shift in myosin heavy chain isoform expression from the fast toward the slow isoform, indicating fast-to-slow fiber type conversion. Moreover, Na⁺ current inactivation was significantly altered. Slow inactivation less strongly inhibited the Na⁺ currents of fast-to-slow fiber type-converted cells. Compared with control cells, the Na⁺ currents of converted cells were more resistant to block by tetrodotoxin, suggesting enhanced relative expression of the cardiac Na⁺ channel isoform Na_v1.5 compared with the skeletal muscle isoform Na_v1.4. These results imply that fast-to-slow fiber type conversion of skeletal muscle cells involves functional adaptation of their electrophysiological properties. muscle plasticity; myosin heavy chain expression; sodium channel expression     

Ca(2+) mobilization through dorsal root ganglion Ca(2+)-sensing receptor stably expressed in HEK293 cells

The rat dorsal root ganglion (DRG) Ca²⁺-sensing receptor (CaR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168–175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [Ca²⁺] ([Ca²⁺]_e) from 0.5 to 1 mM resulted in increases in [Ca²⁺]_i levels, which were blocked by 30 µM 2-aminoethyldiphenyl borate. [Ca²⁺]_e-response studies indicate a Ca²⁺ sensitivity with an EC₅₀ of 1.75 ± 0.10 mM. NPS R-467 and Gd³⁺ activated the CaR. When [Ca²⁺]_e was successively raised from 0.25 to 4 mM, peak [Ca²⁺]_i, attained with 0.5 mM, was reduced by 50%. Similar reductions were observed with repeated applications of 10 mM Ca²⁺, 1 and 10 µM NPS R-467, or 50 and 100 µM Gd³⁺, indicating desensitization of the response. Furthermore, Ca²⁺ mobilization increased phosphorylated protein kinase C (PKC) levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca²⁺ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Ca_e²⁺. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca²⁺]_i transients by 49.9 ± 5.2% (at 1 mM Ca²⁺) and 40.5 ± 6.5% (at 2 mM Ca²⁺), compared with controls. The findings suggest involvement of PKC in the pathway for Ca²⁺ mobilization following CaR activation. desensitization; protein kinase C     

Species-specific difference in distribution of voltage-gated L-type Ca(2+) channels of cardiac myocytes

Ca²⁺influx via sarcolemmal voltage-dependent Ca²⁺ channels(L-type Ca²⁺ channels) is the fundamental step inexcitation-contraction (E-C) coupling in cardiac myocytes.Physiological and pharmacological studies reveal species-specificdifferences in E-C coupling resulting from a difference in thecontribution of Ca²⁺ influx and intracellularCa²⁺ release to activation of contraction. We investigatedthe distribution of L-type Ca²⁺ channels in isolatedcardiac myocytes from rabbit and rat ventricle by correlativeimmunoconfocal and immunogold electron microscopy. Immunofluorescence labeling revealed discrete spots in the surface plasma membrane and transverse (T) tubules in rabbit myocytes. In ratmyocytes, labeling appeared more intense in T tubules than in thesurface sarcolemma. Immunogold electron microscopy extended thesefindings, showing that the number of gold particles in the surfaceplasma membrane was significantly higher in rabbit than rat myocytes.In rabbit myocyte plasma membrane, the gold particles were distributedas clusters in both regions that were associated with junctionalsarcoplasmic reticulum and those that were not. The findings areconsistent with the idea that influx of Ca²⁺ via surfacesarcolemmal Ca²⁺ channels contributes to intracellularCa²⁺ to a greater degree in rabbit than in rat myocytes.

     

Stretch-induced Ca(2+) release via an IP(3)-insensitive Ca(2+) channel

Various mechanicalstimuli increase the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). A part of the increase in [Ca²⁺]_i isdue to the release of Ca²⁺ from intracellular stores. Wehave investigated the effect of mechanical stimulation produced bycyclical stretch on the release of Ca²⁺ from theintracellular stores. Permeabilized VSMC loaded with ⁴⁵Ca²⁺ were subjected to 7.5% average (15%maximal) cyclical stretch. This resulted in an increase in⁴⁵Ca²⁺ rate constant by 0.126 ± 0.0035. Inhibition of inositol 1,4,5-trisphosphate (IP₃),ryanodine, and nicotinic acid adenine dinucleotide phosphate channels(NAADP) with 50 µg/ml heparin, 50 µM ruthenium red, and 25 µMthio-NADP, respectively, did not block the increase in⁴⁵Ca²⁺ efflux in response to cyclical stretch.However, 10 µM lanthanum, 10 µM gadolinium, and 10 µMcytochalasin D but not 10 µM nocodazole inhibited the increase in⁴⁵Ca²⁺ efflux. This supports the existence of anovel stretch-sensitive intracellular Ca²⁺ store in VSMCthat is distinct from the IP₃-, ryanodine-, and NAADP-sensitive stores.

     

Endoplasmic reticulum Ca(2+)-ATPase inhibitors stimulate membrane guanylate cyclase in pancreatic acinar cells

In this study, we show thatparticulate guanylate cyclase (GC) is present in rat pancreatic acinarcells and is located both on plasma membrane and membranes ofendoplasmic reticulum (ER). Western blot analysis indicates that theenzyme isoform GC-A is present in the acinar cell membranes. Thespecific inhibitors of ERCa²⁺-ATPase thapsigargin,2,5-di-(t-butyl)-1,4-hydroquinone(BHQ), and cyclopiazonic acid all activated particulate GC inpancreatic acini, both in membrane fractions and intact cells. Theseinhibitors also induced dephosphorylation of GC. Dose dependencies ofCa²⁺-ATPase inhibition and GCactivation by BHQ are very similar, and those for thapsigarginpartially overlap. ER Ca²⁺-ATPaseand GC are coimmunoprecipitated both by antisera against membrane GCand by antisera against ERCa²⁺-ATPase, suggesting a physicalassociation between the two enzymes. The results suggest thatthapsigargin and the other inhibitors act through ERCa²⁺-ATPase to activate membraneGC in pancreatic acinar cells, although their direct effect on GCcannot be excluded.

     

Imaging caffeine-induced Ca2+ transients in individual fast-twitch and slow-twitch rat skeletal muscle fibers

Fast-twitch and slow-twitch rat skeletal muscles producedissimilar contractures with caffeine. We used digital imagingmicroscopy to monitor Ca²⁺ (withfluo 3-acetoxymethyl ester) and sarcomere motion in intact, unrestrained rat muscle fibers to study this difference. Changes inCa²⁺ in individual fibers weremarkedly different from average responses of a population. All fibersshowed discrete, nonpropagated, local Ca²⁺ transients occurring randomlyin spots about one sarcomere apart. Caffeine increased localCa²⁺ transients and sarcomeremotion initially at 4 mM in soleus and 8 mM in extensor digitorumlongus (EDL; ~23°C). Ca²⁺release subsequently adapted or inactivated; this was surmounted byhigher doses. Motion also adapted but was not surmounted. Prolonged exposure to caffeine evidently suppressed myofilament interaction inboth types of fiber. In EDL fibers, 16 mM caffeine moderately increasedlocal Ca²⁺ transients. In soleusfibers, 16 mM caffeine greatly increased Ca²⁺ release and producedpropagated waves of Ca²⁺(~1.5-2.5 µm/s). Ca²⁺waves in slow-twitch fibers reflect the caffeine-sensitive mechanism ofCa²⁺-inducedCa²⁺ release. Fast-twitch fiberspossibly lack this mechanism, which could account for their lowersensitivity to caffeine.

     

Ca2+ sparks and T tubule reorganization in dedifferentiating adult mouse skeletal muscle fibers

Ca⁺ sparks are rare in healthy adult mammalian skeletal muscle but may appear when adult fiber integrity is compromised, and occur in embryonic muscle but decline as the animal develops. Here we used cultured adult mouse flexor digitorum brevis muscle fibers to monitor occurrence of Ca²⁺ sparks during maintenance of adult fiber morphology and during eventual fiber morphological dedifferentiation after various times in culture. Fibers cultured for up to 3 days retain normal morphology and striated appearance. Ca²⁺ sparks were rare in these fibers. At 5–7 days in culture, many of the original muscle fibers exhibit sprouting and loss of striations, as well as the occurrence of spontaneous Ca²⁺ sparks. The average rate of occurrence of Ca²⁺ sparks is >10-fold higher after 5–7 days in culture than in days 1–3. With the use of fibers cultured for 7 days, application of the Ca²⁺ channel blockers Co²⁺ or nifedipine almost completely suppressed the occurrence of Ca²⁺ sparks, as previously shown in embryonic fibers, suggesting that Ca²⁺ sparks may be generated by similar mechanisms in dedifferentiating cultured adult fibers and in embryonic fibers before final differentiation. The sarcomeric disruption observed under transmitted light microscopy in dedifferentiating fibers was accompanied by morphological changes in the transverse (T) tubular system, as observed by fluorescence confocal imaging of both an extracellular marker dye and membrane staining dyes. Changes in T tubule morphology coincided with the appearance of Ca²⁺ sparks, suggesting that Ca²⁺ sparks may either be a signal for, or the result of, disruption of DHPR-ryanodine receptor 1 coupling. calcium ion signaling; muscle remodeling; fluo 4; calcium ion imaging     

The exchange of Ca2+-receptive protein complex (troponin) in the myofibrils of fast and slow skeletal muscles

In order to compare the role of the Ca²⁺-receptive protein (troponin), in the characteristic myofibrillar contractile response of chicken fast and slow skeletal muscles, the troponin in both kinds of myofibrils were partially exchanged, under slightly acidic conditions. The Ca²⁺- or Sr²⁺-activation of the ATPase of fast (or slow) skeletal myofibrils hybridized with slow (or fast) skeletal troponin profiles were also investigated. The results indicated that the Ca²⁺- or Sr²⁺-affinity of the myofibrillar ATPase activity were related to the species of troponin. This procedure for replacing troponin in myofibrils under physiological conditions in thus considered to be useful for the study of the Ca²⁺-regulatory mechanism in myofibrillar contraction.