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     

Properties and Possible Physiological Significance of Cell Wall Calcium Binding in Etiolated Pea Epicotyls

Baydoun, E. A-H. and Brett, C. T. 1988. Properties and possiblephysiological significance of cell wall calcium binding in etiolatedpea epicotyls.—J. exp. Bot. 39: 199–208. The binding of ⁴⁵Ca²⁺ ions to cell walls prepared from pea epicotylswas examined in young and old parts of the epicotyl, and wasfound to be considerably greater, on a carbohydrate basis, inthe older, non-growing cells. A similar comparison between light-and dark-grown stems showed greater binding in the dark-grownstems. The polygalacturonase-insensitive component of the bindingcontained at least three types of binding with different affinities,and had an apparent pK of 4.3. The specificity of the bindingfor calcium ions was examined and a considerable degree of specificitywas observed. The specificity of inhibition by calcium of epicotylelongation was similar to the specificity of calcium binding.A specific calcium chelator, EGTA, when present at a concentrationof above 10 mol m^–3, promoted the extension of matureregions of the epicotyl, while inhibiting extension of youngertissue. Key words: Cell wall, calcium, pea epicotyl     

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.     

Structure and Function of the Transverse Tubular System in Crustacean Muscle Fibers

The structure and function of the transverse tubular system(TTS) in two types of crustacean muscle fibers are examined.Giant fibers from the barnacle,Balanus nubilus, which are gradedlycontracting, are compared with allor-none twitch fibers fromthe crab, Carcinus maenas. Both fiber types were found to havedeep sarcolemmal invaginations which serve both to increasethe fiber surface area and to kfeep the length of the tubulesshort enough for electrotonic propagation.The ultrastructureof the tubular system in both types of fiber is compared.Thesystem is better developed in Carcinus than in Balanus, butthe slow Balanus fibers do have a relatively well developedTTS and sarcoplasmic reticulum in contrast to slow vertebratefibers. The apparent high, membrane-capacitance values of crustaceanfibers are the result of investigators not taking into considerationthe large increase in surface area due to the sarcolemmal infoldings.Thetubular membranes in Carcinus fibers were found to be permselectiveto chloride ions, and could be made to swell (as confirmed byelectron microscopy) by establishing an outward gradient forchloride across them. The capacitance of the tubular membranerelative to the plasma membrane was found to increase when thetubuleswere swollen. The implication of a fiber having two spatiallyseparated, differentially permeable membranes on excitation-contractioncoupling is discussed.     

Effects of BAPTA on force and Ca2+ transient during isometric contraction of frog muscle fibers

The effects of1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid (BAPTA) on force and intracellularCa²⁺ transient were studied duringisometric twitches and tetanuses in single frog muscle fibers. BAPTAwas added to the bathing solution in its permeant AM form (50 and 100 µM). There was no clear correlation between the changes in force andthe changes in Ca²⁺ transient.Thus during twitch stimulation BAPTA did not suppress theCa²⁺ transient until the force hadbeen reduced to <50% of its control value. At the same time, thepeak myoplasmic free Ca²⁺concentration reached during tetanic stimulation was markedly increased, whereas the force was slightlyreduced by BAPTA. The effects of BAPTA were not duplicated by usinganother Ca²⁺ chelator, EGTA,indicating that BAPTA may act differently as aCa²⁺ chelator. Stiffnessmeasurements suggest that the decrease in mechanical performance in thepresence of BAPTA is attributable to a reduced number of active crossbridges. The results could mean that BAPTA, under the conditions used,inhibits the binding of Ca²⁺ totroponin C resulting in a reduced state of activation of the contractile system.

     

Velocity, force, power, and Ca2+ sensitivity of fast and slow monkey skeletal muscle fibers

In this study,we determined the contractile properties of single chemically skinnedfibers prepared from the medial gastrocnemius (MG) and soleus (Sol)muscles of adult male rhesus monkeys and assessed the effects of thespaceflight living facility known as the experiment support primatefacility (ESOP). Muscle biopsies were obtained 4 wk before andimmediately after an 18-day ESOP sit, and fiber type was determined byimmunohistochemical techniques. The MG slow type I fiber wassignificantly smaller than the MG type II, Sol type I, and Sol type IIfibers. The ESOP sit caused a significant reduction in the diameter oftype I and type I/II (hybrid) fibers of Sol and MG type II and hybridfibers but no shift in fiber type distribution. Single-fiber peak force(mN and kN/m²) was similarbetween fiber types and was not significantly different from valuespreviously reported for other species. The ESOP sit significantlyreduced the force (mN) of Sol type I and MG type II fibers. Thisdecline was entirely explained by the atrophy of these fiber typesbecause the force per cross-sectional area (kN/m²) was not altered. Peakpower of Sol and MG fast type II fiber was 5 and 8.5 times that of slowtype I fiber, respectively. The ESOP sit reduced peak power by 25 and18% in Sol type I and MG type II fibers, respectively, and, for theformer fiber type, shifted the force-pCa relationship to the right,increasing the Ca²⁺ activationthreshold and the free Ca²⁺concentration, eliciting half-maximal activation. The ESOP sit had noeffect on the maximal shortening velocity(V_o) of anyfiber type. V_o ofthe hybrid fibers was only slightly higher than that of slow type Ifibers. This result supports the hypothesis that in hybrid fibers theslow myosin heavy chain would be expected to have a disproportionatelygreater influence onV_o.

     

Image-analysis-based assessment of the effects of the "Ca2+-jump" technique on sarcomere uniformity

A new imageanalysis-based technique was used to quantitatively examine the effectsof the "Ca²⁺-jump"activation protocol on the maintenance of fiber quality in skinnedrabbit psoas muscle fiber segments. Specifically, contractions in pCa4.6 were preceded by short-duration "preactivation" soaks in asolution in which EGTA was replaced with thelow-Ca²⁺ buffering capacity analoghexamethylenediamine-N, N, N', N'-tetraacetate, which facilitated rapid Ca²⁺equilibration within the fiber segments. Fiber quality was assessed byexamining the Fourier spectra of the muscle fiber images before, during, and after activation. Segment lengths were typically below 500 µm, thus allowing the majority of the sarcomeres to be visualized inthe field of view (×200 and ×400 magnification). Thepreactivation protocol resulted in less deterioration of fiber qualitywith repetitive activation. In addition, there was also a significant reduction in the time required to reach the 50% level of maximum tension, with no significant change in the maximum tension level.

     

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     

Ca2+-Activated Force Production and Calcium Handling by the Sarcoplasmic Reticulum of Crustacean Muscles During Molt-Induced Atrophy

Growth in crustaceans is an intermittent process centered aroundthe principal event of ecdysis. A major problem facing decapodcrustaceans at the time of ecdysis is the withdrawal of thelarge muscle mass of the chelae through the narrow basi-ischialjoints. To overcome this problem the muscle undergoes an atrophytriggered by the molt, which reduces the muscle mass. Once theanimal is freed from the old exoskeleton, the muscle fibers,must elongate to accommodate the new larger exoskeleton. Despitethis major myofibrillar remodification, the muscles are thoughtto remain functional over the molt cycle. Studies using skinnedmuscle fibers have shown that long-sarcomere fibers maintaintheir function over the molt cycle while the contractile propertiesof the short-sarcomere fibers are modified, as fibers couldnot withstand maximal activation with Ca²⁺ during the premoltstage. In this study the maximum Ca²⁺-activated force productionand the ability of the sarcoplasmic reticulum (SR) to releaseaccumulated Ca²⁺ has been investigated in the two major fibertypes in the claw muscle of Cherax destructor, in the stagesjust prior to ecdysis and during inter molt. In both long- andshort-sarcomere fibers, the amount of Ca²⁺ released by the SRwas not different in premolt and intermolt stages. However,the maximum releasing capacity of the SR was reached in a shortertime during the premolt suggesting that Ca²⁺ is being accumulatedat a faster rate. The force production was greatly reduced andwas graded during the premolt in both fiber types. This modulationof force appears to be the most likely candidate regulatingthe magnitude of the force development in the periods when fibersare undergoing myofibrillar remodification and thus may serveto prevent fiber damage.     

Ca2+-sensitizing effects of the mutations at Ile-79 and Arg-92 of troponin T in hypertrophic cardiomyopathy

Several mutationsin human cardiac troponin T (TnT) gene have been reported to causehypertrophic cardiomyopathy (HCM). To explore the effects of themutations on cardiac muscle contractile function under physiologicalconditions, human cardiac TnT mutants, Ile79Asn and Arg92Gln, as wellas wild type, were expressed in Escherichiacoli and exchanged into permeabilized rabbit cardiac muscle fibers, and Ca²⁺-activatedforce was determined. The freeCa²⁺ concentrations required fortension generation were found to be significantly lower in the mutantTnT-exchanged fibers than in the wild-type TnT-exchanged fibers,whereas no significant differences were found in tension-generatingcapability under maximal activating conditions and in cooperativity.These results suggest that a heightenedCa²⁺ sensitivity of cardiac musclecontraction is one of the factors to cause HCM associated with theseTnT mutations.

     

Effect of EGTA on Nyctinastic Closure Mediated by Phytochrome in Albizzia lophantha

The role of extracellular calcium on nyctinastic closure ofAlbizzia lophantha leaflets has been studied by testing theeffect of ethyleneglycol-bis-(ß-aminoethylether)-N,N,N',N'-tetraaceticacid (EGTA) and its reversibility by calcium. EGTA (1 and 5mM) causes an inhibition of nyctinastic closure and at a concentrationof 1 mM EGTA it decreases the difference between the effectof red light (R) and far-red light (FR) irradiation on leafletclosure. A simultaneous or subsequent supply of CaCl₂ (5 or10 mM) reverses EGTA (5 mM) inhibition on closure as well ascausing an additional promotion of closure. We suggest that external calcium could play a dual role in nyctinasticclosure. Phytochrome control of leaflet closure probably needsexternal Ca²⁺ and, in addition, Ca²⁺ could regulate the closuremechanism by controlling ionic fluxes through the plasma membranein pulvinular motor cells. (Received June 9, 1989; Accepted November 27, 1989)     

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     

Injection of a Ca2+-Chelating Agent into the Cytoplasm Retards the Progress of Turgor Regulation upon Hypotonic Treatment in the Alga, Lamprothamnium

The turgor regulation induced by hypotonic treatment (hypotonicturgor regulation) in the brackish-water alga Lamprothamniumsuccinctum is accompanied by a transient increase in the electricalconductance of the membrane, membrane depolarization and a transientincrease in the cytoplasmic concentration of free Ca²⁺ ([Ca²⁺([Ca²⁺]_c) (Okazaki and Tazawa 1990). In the present study, weloaded a Ca²⁺-chelating agent, EGTA, into the cytoplasm by mechanicalinjection in order to suppress the increase in [Ca²⁺]_c thatoccurs during the hypotonic turgor regulation. The rate of thecytoplasmic streaming was taken as an indirect indicator of[Ca²⁺]_c, since cytoplasmic streaming has been shown to be inhibitedby high [Ca²⁺]_c in Lamprothamnium cells. The lag time for theinhibition of the cytoplasmic streaming upon hypotonic treatmentwas significantly prolonged in EGTA-loaded cells as comparedto that in intact cells. This result indicates that the loadedcytoplasmic EGTA functioned as a buffer of Ca²⁺ to retard theincrease in [Ca²⁺]_c. It took a longer time for the membraneconductance to reach the peak value in EGTA-loaded cells thanin intact cells. Membrane depolarization was affected to aninsignificant extent by the cytoplasmic EGTA. The regulationof turgor pressure itself was partially inhibited. These resultsstrongly support the idea that the net efflux of ions that occursduring the recovery from hy-potonically induced changes in turgorpressure is controlled by [Ca²⁺]_c. (Received August 22, 1990; Accepted December 6, 1990)     

Effects of Calcium and Abscisic Acid on Volume Changes of Guard Cell Protoplasts of Commelina

Calcium ions contracted guard cell protoplasts (GCP) of Commelinacommunis L., being particularly effective within the concentrationrange of 0 to 0.2 mol m^–3. Abscisic acid (ABA) in thepresence of EGTA, which chelates free Ca²⁺ in the medium, contractedGCP to a similar extent to Ca²⁺ alone or Ca²⁺ and ABA together.Similarly, ABA in the absence of free Ca²⁺ (i.e. an ABA/EGTAtreatment) inhibited K⁺-induced swelling of contracted GCP,as did Ca²⁺ alone or ABA and Ca²⁺ together. Lanthanum, a Ca²⁺channel blocker, prevented the contraction of GCP by Ca²⁺ buthad no effect if ABA was also present with Ca²⁺. The inhibitionof swelling of GCP by Ca²⁺ was also prevented by the presenceof lanthanum or verapamil (another Ca²⁺ channel blocker). These results indicate that Ca²⁺ and ABA can act independentlyof each other in contracting swollen GCP and in preventing K⁺-inducedswelling of contracted GCP of C. communis. If swelling and contractionof GCP are equivalent to stomatal opening and closure, respectively,the results do not support the hypothesis that ABA opens Ca²⁺channels in the plasma membrane of guard cells allowing Ca²⁺to enter the cells and, as a second messenger, to set in motionclosing processes. Key words: Abscisic acid, calcium, guard cell protoplasts, stomata     

Effect of intermittent weight bearing on soleus fiber force-velocity-power and force-pCa relationships

Bangart, J. J., J. J. Widrick, and R. H. Fitts. Effectof intermittent weight bearing on soleus fiber force-velocity-power andforce-pCa relationships. J. Appl.Physiol. 82(6): 1905-1910, 1997.Ratpermeabilized type I soleus fibers displayed a 33% reduction in peakpower output and a 36% increase in the freeCa²⁺ concentration required forone-half maximal activation after 14 days of hindlimb non-weightbearing (NWB). We examined the effectiveness of intermittent weightbearing (IWB; consisting of four 10-min periods of weight bearing/day)as a countermeasure to these functional changes. At peak power output,type I fibers from NWB animals produced 54% less force and shortenedat a 56% greater velocity than did type I fibers from controlweight-bearing animals while type I fibers from the IWB rats produced26% more absolute force than did fibers from the NWB group andshortened at a velocity that was only 80% of the NWB group mean. As aresult, no difference was observed in the average peak power of fibers from the IWB and NWB animals. Hill plot analysis of force-pCa relationships indicated that fibers from the IWB group required similarlevels of free Ca²⁺ to reachhalf-maximal activation in comparison to fibers from the weight-bearinggroup. However, at forces <50% of peak force, the force-pCa curvefor fibers from the IWB animals clearly fell between the relationshipsobserved for the other two groups. In summary, IWB treatments1) attenuated the NWB-inducedreduction in fiber Ca²⁺sensitivity but 2) failed to preventthe decline in peak power that occurs during NWB because of opposingeffects on fiber force (an increase vs. NWB) and shortening velocity (adecrease vs. NWB).

     

Effects of a domain peptide of the ryanodine receptor on Ca2+ release in skinned skeletal muscle fibers

Mutations in the central domain of the skeletal muscle ryanodinereceptor (RyR) cause malignant hyperthermia (MH). A synthetic peptide(DP4) in this domain (Leu-2442-Pro-2477) produces enhanced ryanodine binding and sensitized Ca²⁺ release in isolatedsarcoplasmic reticulum, similar to the properties in MH, possiblybecause the peptide disrupts the normal interdomain interactions thatstabilize the closed state of the RyR (Yamamoto T, El-Hayek R, andIkemoto N. J Biol Chem 275: 11618-11625, 2000). Here, DP4 was applied to mechanically skinned fibers of rat muscle thathad the normal excitation-contraction coupling mechanism stillfunctional to determine whether muscle fiber responsiveness wasenhanced. DP4 (100 µM) substantially potentiated the Ca²⁺release and force response to caffeine (8 mM) and to low[Mg²⁺] (0.2 mM) in every fiber examined, with nosignificant effect on the properties of the contractile apparatus. DP4also potentiated the response to submaximal depolarization of thetransverse tubular system by ionic substitution. Importantly, DP4 didnot significantly alter the size of the twitch response elicited byaction potential stimulation. These results support the proposal thatDP4 causes an MH-like aberration in RyR function and are consistentwith the voltage sensor triggering Ca²⁺ release bydestabilizing the closed state of the RyRs.

     

Calcium influx through If channels in rat ventricular myocytes

The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac (I_f)/neuronal (I_h) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and I_h channels in neurons. This raises the possibility of Ca²⁺ permeation in I_f, the I_h counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca²⁺ signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by I_f in rat ventricular myocytes. We observed Ca²⁺ influx when HCN/I_f channels were activated. Ca²⁺ influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an I_f channel blocker, inhibited I_f and Ca²⁺ influx at the same time. Quantitative analysis revealed that Ca²⁺ flux contributed to 0.5% of current produced by the HCN2 channel or I_f. The associated increase in Ca²⁺ influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which I_f current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a Ca²⁺ chelator), preactivation of I_f channels significantly reduced the action potential duration, and the effect was blocked by another selective I_f channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of I_f channels had no effects on action potential duration. Our data extend our previous discovery of Ca²⁺ influx in I_h channels in neurons to I_f channels in cardiac myocytes. calcium ion flux; hyperpolarization-activated, cyclic nucleotide-gated/cardiac time- and volume-dependent cation current channels     

Senescence of Rice Leaves XXIV. Involvement of Calcium and Calmodulin in the Regulation of Senescence

Effects of compounds that influenced calcium uptake and calmodulininhibitors on the senescence of detached rice leaves were examined.Chelators, ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraaceticacid (EGTA) and l,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraaceticacid (BAPTA), significantly promoted senescence of detachedrice leaves in the dark and light. The effect of EGTA can bereversed by treating detached rice leaves with calcium. Verapamil,a calcium channel blocker, and lanthanum chloride, a calciumantagonist, promoted dark-induced, and suppressed BA- and light-retardedsenescence of detached rice leaves. Calcium ionophore A23187 [GenBank] and ruthenium red, believed to raise cytosolic level of Ca²⁺,were quite effective in retarding dark-induced and ABA-promotedsenescence of detached rice leaves. Calmodulin inhibitors, W-7,compound 48/80, chlorpromazine and trifluoperazine, significantlypromoted dark-induced, and suppressed BA- and light-retardedsenescence of detached rice leaves. It is concluded that cytosoliclevel of Ca²⁺ may regulate senescence of detached rice leavesthrough a calmodulin-dependent mechanism. (Received June 13, 1990; Accepted August 3, 1990)     

Responses of the stomata of Aster tripolium to calcium and sodium ions in relation to salinity tolerance

In previous work, the stomata of the maritime halophyte Astertripolium L. were shown to close when NaCl concentrations risein the vicinity of the guard cells. Further studies have nowrevealed important effects of calcium on the ionic responsesof the stomata. When the guard cells were presented with KCl,Ca²⁺ suppressed opening in a manner similar to that which hasbecome familiar in other species such as Commelina communisL. However, in the presence of NaCl, Ca²⁺ had the opposite effect,reducing the closing response to NaCl. This pattern of behaviouris discussed in relation to known salt effects on membranes,but the underlying physiological basis remains obscure. A previous study led to the hypothesis that the closing responseof the stomata to Na⁺ ions may make an important contributionto the salinity tolerance of this species. Here we report thatincreasing supplies of Ca²⁺ ions reduce the effect of salinityon stomatal conductance in the whole plant as well as in theisolated epidermis. This finding is consistent with the wellestablished role of calcium in increasing resistance to salinity:in the presence of high calcium the plant can tolerate a greatersalt intake, and hence there is a reduced need for transpirationto be restricted by partial stomatal closure. Key words: Sodium, calcium, Aster tripolium, stomata, salinity tolerance     

Ca2+ sparks are initiated by Ca2+ entry in embryonic mouse skeletal muscle and decrease in frequency postnatally

"Spontaneous" Ca²⁺ sparks and ryanodine receptor type 3 (RyR3) expression are readily detected in embryonic mammalian skeletal muscle but not in adult mammalian muscle, which rarely exhibits Ca²⁺ sparks and expresses predominantly RyR1. We have used confocal fluorescence imaging and systematic sampling of enzymatically dissociated single striated muscle fibers containing the Ca²⁺ indicator dye fluo 4 to show that the frequency of spontaneous Ca²⁺ sparks decreases dramatically from embryonic day 18 (E18) to postnatal day 14 (P14) in mouse diaphragm and from P1 to P14 in mouse extensor digitorum longus fibers. In contrast, the relative levels of RyR3 to RyR1 protein remained constant in diaphragm muscles from E18 to P14, indicating that changes in relative levels of RyR isoform expression did not cause the decline in Ca²⁺ spark frequency. E18 diaphragm fibers were used to investigate possible mechanisms underlying spark initiation in embryonic fibers. Spark frequency increased or decreased, respectively, when E18 diaphragm fibers were exposed to 8 or 0 mM Ca²⁺ in the extracellular Ringer solution, with no change in either the average resting fiber fluo 4 fluorescence or the average properties of the sparks. Either CoCl₂ (5 mM) or nifedipine (30 µM) markedly decreased spark frequency in E18 diaphragm fibers. These results indicate that Ca²⁺ sparks may be triggered by locally elevated [Ca²⁺] due to Ca²⁺ influx via dihydropyridine receptor L-type Ca²⁺ channels in embryonic mammalian skeletal muscle. calcium; ryanodine receptor; dihydropyridine receptor; muscle development