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R J Solaro

Publications and source records attributed to R J Solaro.

At least 91 records · Page 5Linked to original sources
PubMed1992

Alterations in myofibrillar function and protein profiles after complete global ischemia in rat hearts.

We studied changes in myofibrillar function and protein profiles after complete global ischemia with anoxia in rat hearts. Hearts were exposed to global ischemia and anoxia (CGI) for 30 or 60 minutes at 37 degrees C, and myofibrils were prepared for measurement of Ca(2+)-dependent Mg(2+)-ATPase activity at pH 7.0 and 6.5. Hearts incubated in cold saline (1 +/- 1 degrees C) and nonincubated hearts served as controls. Maximum ATPase activity was unchanged at pH 7.0 and pH 6.5 in myofibrils from hearts treated with 30 or 60 minutes of CGI. At pH 7.0, the Hill coefficient, which is an index of cooperative interactions among thin-filament proteins, was unchanged after 30 minutes of CGI but was significantly increased after 60 minutes of CGI. A similar trend for increased cooperativity was observed when myofibrillar ATPase activity was measured at pH 6.5 in myofibrils from rat hearts made ischemic for 30 or 60 minutes. Both 30 and 60 minutes of CGI resulted in increased pCa50 values (half-maximally activating free [Ca2+]) at pH 7.0 and pH 6.5. Densitometric analysis of myofibrillar proteins separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that troponin I and troponin T were degraded during 60 minutes of CGI. Two new protein bands appearing in ischemia-treated myofibrils were identified as partially degraded troponin I and troponin T with Western blots. The troponin I fragment could be phosphorylated by cAMP-dependent protein kinase. In addition, we observed phosphorylation of a protein band that corresponded to myosin light chain-2 in myofibrils from CGI-treated hearts. These results suggest that degradation of thin-filament proteins may contribute to the changes in cooperativity of Ca2+ regulation of ATPase activity observed in the myofibrils from rat hearts exposed to CGI.

Animals↗
PubMed1992

Electrophysiological actions of the pimobendan metabolite, UD-CG 212 Cl, in guinea pig myocardium.

Pimobendan (UD-CG 115 BS), an inotropic agent and inhibitor of type III phosphodiesterase activity, is demethylated in vivo to form UD-CG 212 Cl, which is a more potent type III phosphodiesterase inhibitor. This study examined cyclic AMP (cAMP)-mediated actions of UD-CG 212 Cl. In guinea pig papillary muscles, UD-CG 212 Cl increased cAMP and stimulated Ca(++)-dependent slow action potentials (APs) in a dose-dependent manner. When compared to previous studies using pimobendan, UD-CG 212 Cl was approximately 100-fold more potent. UD-CG 212 Cl had no additional effects on slow APs in the presence of a maximal dose of isoproterenol (1 microM). Propranolol had little effect on UD-CG 212 Cl-induced slow APs. These results, along with previous studies, indicate that slow AP induction by UD-CG 212 Cl was cAMP-dependent, and the increase in cAMP levels was most likely due to phosphodiesterase inhibition and not beta receptor stimulation. Experiments with tetraethylammonium.Cl suggested that UD-CG 212 Cl probably did not induce slow APs by blocking K+ channels. In voltage-clamped ventricular myocytes UD-CG 212 Cl (100 microM) could stimulate Ca++ current (+21 +/- 5%) when basal cAMP levels were enhanced with a submaximal dose of isoproterenol (10(-9)-10(-8) M). Isoproterenol was not required to observe the stimulating effect of UD-CG 212 Cl on Ca++ current in intact, nondialyzed cells prepared using the nystatin-perforated patch method. Studies with the stereoisomers of UD-CG 212 Cl showed that the D-isomer was more potent than the L-isomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1991

Identification and functional significance of troponin I isoforms in neonatal rat heart myofibrils.

We investigated the mechanism(s) responsible for differences in the effects of acidic pH on Ca2+ activation of the activity of adult and neonatal rat heart myofilaments. Studies on preparations of myofilaments reconstituted with adult troponin-tropomyosin (Tn-Tm) and either adult or neonatal thick filaments indicated that the difference in effect of acidic pH is related to differences in Tn-Tm and not other myofilament proteins. Immunoblotting analysis showed that development of the rat heart myofibrils is associated with isoform switching from slow skeletal TnI to cardiac TnI and from a slow mobility isoform of TnT (TnT1) to a faster Mr isoform (TnT2. Expression of slow skeletal TnI was associated with a relative insensitivity of myofilament Ca2+ activation to deactivation by acidic pH. Moreover, the effect of acidic pH on Ca2+ activation of ATPase activity of soleus myofibrils, which contain cardiac TnC and slow skeletal TnI, was essentially the same as the effect of acidic pH on rat cardiac myofibrils in the early neonatal period. Neonatal myofilaments also contained a relative abundance of a set of polypeptides copurifying with the thin filaments. We have identified these proteins as histones. The relative amount of histones among a variety of preparations from different species was not correlated with the pH sensitivity of myofibrillar Ca2+ activation. Shifts in TnT isoforms among these species were also not correlated with an altered response to acidic pH. Our data provide evidence in support of the hypothesis that the relative insensitivity of neonatal myofilament activity to acidic pH is due to the presence of slow skeletal TnI in the thin-filament regulatory complex.

Acids↗
PubMed1991

Contractile deactivation and uncoupling of crossbridges. Effects of 2,3-butanedione monoxime on mammalian myocardium.

We investigated the effects of 1 and 3 mM 2,3-butanedione monoxime (BDM, diacetyl monoxime) on excitation and contraction of cardiac muscle in several types of preparations at various levels of organization. We selected a concentration of BDM that was not expected to affect sarcolemmal calcium flux and action potential duration in cardiac tissue. Two indicators were used to record intracellular calcium. Aequorin, a bioluminescent calcium indicator, was used in studies with ferret papillary muscle preparations, and fura-2, a fluorescent calcium indicator, was used in studies with guinea pig cardiac myocytes. In both cases, addition of BDM resulted in a reduction of peak intracellular calcium released from the sarcoplasmic reticulum and a reduction of peak twitch force. The duration of the action potential of isolated myocytes was slightly abbreviated in the presence of BDM. In studies on the calcium current in the myocytes, addition of BDM was associated with reduced calcium current at any potential. Peak calcium current was reduced by 7.9 +/- 1% in the presence of BDM. In tetanized ferret papillary muscles, BDM reduced maximal calcium-activated force by 30 +/- 5% and increased the calcium ion concentration required for half-maximal force by 0.1 +/- 0.01 microM. The Hill coefficient was reduced from 5.00 +/- 0.11 to 3.40 +/- 0.20. Maximal shortening velocity of ferret papillary muscles was increased in the presence of BDM from 1.55 +/- 0.24 to 2.04 +/- 0.33 mm/sec. Ca2+ binding to troponin C in skinned fiber preparations from guinea pig, bovine, and canine hearts was unaffected by addition of up to 10 mM BDM. Our results indicate that BDM affects both calcium availability and responsiveness of the myofilaments to Ca2+. Uncoupling of contractile activation from excitation may also result from altered crossbridge kinetics.

Animals↗
PubMed1991

Regulation of Ca(2+)-signaling in cardiac myofilaments.

The contraction and relaxation of heart muscle cells is associated with a transient change in the intracellular level of free Ca2+. This transient reflects a release from the sarcoplasmic reticulum of Ca2+ ions that turn on the reaction of myosin cross-bridges of the thick filaments with actin of the thin filaments. Without this transient no contraction occurs. The first part of this paper is concerned with the molecular processes by which Ca2+ signals the actin-myosin reaction. It will show that the activation in reality involves release of the thin filaments from a prevailing inhibited state. The inhibited state depends on the activity of the troponin (TN) complex and tropomyosin (TM); disinhibition requires Ca(2+)-binding to TN. The second part of the paper describes evidence that the signaling process is itself modulated by the mechanical and chemical state of the myofilaments in the short term and by altered gene expression in the long term.

Actin Cytoskeleton↗
PubMed1990

Slowly exchanging calcium binding sites unique to cardiac/slow muscle troponin C.

Evidence is presented for the existence of slowly exchanging Ca2(+)-binding sites in troponin C (CTNC) of cardiac and slow twitch skeletal muscles. These sites were revealed in the course of experiments aimed at measuring the Ca2(+)-binding properties of TNC in the myofilament lattice. 45Ca bound to chemically skinned muscle fibers or myofibrils of cardiac and soleus muscles was eluted by EGTA in a two-exponential timecourse with a slow phase of a rate constant of about 2 x 10(-4)/s. The slow phase was not found in skinned fiber or myofibrils of psoas, a fast skeletal muscle. However, skinned psoas fibers in which the native TNC was replaced by CTNC exhibited the slow 45Ca elution characteristic of soleus and cardiac preparations, indicating that the slowly-exchanging sites are located in CTNC. These sites are tentatively identified as the Ca2(+)-Mg2+ sites of CTNC on the basis that the slow phase was observed under conditions known to restrict Ca2+ binding to the Ca2(+)-Mg2+ sites.

Animals↗
PubMed1990

Changes in myofibrillar activation and troponin C Ca2+ binding associated with troponin T isoform switching in developing rabbit heart.

Postnatal development of the mammalian heart is associated with changes in the population of isoforms of the thin filament proteins. We correlated the change in thin filament proteins, which occur in rabbit hearts between 5 days and 22 days of age, with changes in Ca2+ dependence of myofibrillar ATPase activity, force generation, and troponin C Ca2+ binding. The preparations derived from the 5-day-old animals exhibited a high molecular weight isoform of troponin T not found in the hearts of the 22-day-old animals. Other troponin T isoforms were also found to be present in different relative amounts. No other major differences in thin filament protein composition could be identified. Compared with the 5-day-old rabbit heart preparations, the ATPase activity of myofibrils from 22-day-old rabbit hearts exhibited a reduced Ca2+ sensitivity. The pCa50 (negative log of the half-maximal-activity free Ca2+) of the MgATPase activity was shifted by 0.15 pCa units with maturation. Maturation of the myofibrils was also associated with an increased effect of Mg2+ on pCa50. On increasing the Mg2+ from 2 to 10 mM at constant MgATP2-, the pCa50 of 5-day myofibrils was increased (shifted to the right) by 0.39 pCa units for 5-day-old rabbit hearts and 0.45 pCa units for 22-day-old rabbit hearts. Although similar changes in pCa50 of force developed by myofibrils were marginally significant, fibers from hearts of 5-day-old rabbits exhibited a greater Hill coefficient than hearts from 22-day-old rabbits (3.0 vs. 2.1). Despite the increased sensitivity of 5-day-old rabbit hearts to Ca2+, these hearts exhibited significantly less Ca2+ bound to myofibrillar troponin C than did the 22-day-old rabbit hearts. Moreover, the models that best described the Ca2+ binding data are different for the two age groups. Our data indicate that the Ca2+ activation and Ca2+ binding properties of myofibrillar troponin C are altered in developing cardiac myofibrils and that the changes in these properties may be influenced by changes in the troponin T isoforms present in the myofibril.

Animals↗
PubMed1990

Effect of thyroid status on thin-filament Ca2+ regulation and expression of troponin I in perinatal and adult rat hearts.

There is evidence for the existence of developmental changes in expression of troponin I (TNI) in cardiac thin filaments; however, regulation of TNI expression has not been described. We tested whether thyroid state affects expression of TNI using neonatal and adult rats made hypothyroid by treatment with 6-n-propyl-2-thiouracil. Polyacrylamide gels of myofibrils from hearts of 7-, 14-, 21-, and 28-day-old animals indicated that both euthyroid and hypothyroid rats display a developmental shift toward the adult form of TNI. However, hypothyroid rats displayed a lower percentage of adult TNI at each age studied. When adult rats were made hypothyroid, the proportion of adult TNI decreased slightly. Thin-filament activity was determined from measurements of the effect of acidic pH on calcium activation of myofibrillar ATPase activity. Sensitivity to acidic pH was measured by the magnitude of shift in pCa50 (-log of half-maximally activating molar Ca2+) between pH 7.0 and 6.5. Euthyroid rats displayed developmental increases in pH sensitivity. At 7, 14, and 28 days of development, shifts in pCa50 were 0.11, 0.38, and 0.43 units, respectively. Hypothyroid rats displayed less pH sensitivity with pCa50 shifts of 0.07, 0.21, and 0.15 units at 7, 14, and 28 days of development. Adult hypothyroid rats displayed a 0.38-unit shift in pCa50, whereas euthyroid adults displayed a 0.44-unit shift. Our results indicate that pH sensitivity and expression of cardiac TNI are influenced by developmental stage and hormonal status.

Adenosine Triphosphatases↗
PubMed1990

Developmental difference in the stimulation of cardiac myofibrillar Mg2(+)-ATPase activity by calmidazolium.

We probed possible developmentally related changes in thin filament activity in rat hearts with the aid of calmidazolium (CDZ). CDZ is a calmodulin antagonist that also binds to troponin C and stimulates Ca2+ troponin C-dependent activation of cardiac myofibrillar contractile activity. In paired experiments, we compared the effects of 10, 30, 50, 70, and 100 microM CDZ on Mg2(+)-dependent ATPase activity of myofibrillar preparations from adult and neonatal rat hearts. Over the dose-response curve, the ATPase activity of neonatal myofibrils was significantly less stimulated than was the ATPase activity of the adult preparations. To know whether this difference in response to CDZ was related to differences in the thin or thick filaments, we studied hybrid adult and neonatal myofibrillar preparations. These hybrid myofibrils had native thin filaments, but the thick filaments were displaced with rabbit skeletal myosin. The relative insensitivity of the neonatal preparations to the effect of CDZ was retained in the hybrid myofibrils. This suggested that developmental transitions in the population of thin filament proteins are responsible for the difference between adult and neonatal myofibrils in their response to CDZ. Recently, we and others have reported developmental switching of troponin I isoforms in the rat heart. Since troponin I reacts strongly with troponin C in a Ca2(+)-dependent manner, we speculate that developmentally related changes in troponin I isoforms may contribute to the differential effect of CDZ in neonatal cardiac myofibrils.

Age Factors↗
PubMed1989

Ca2+, pH and the regulation of cardiac myofilament force and ATPase activity.

When the pH surrounding myofilaments of striated muscle is reduced there is an inhibition of both the actin-myosin reaction as well as the Ca2+-sensitivity of the myofilaments. Although the mechanism for the effect of acidic pH on Ca2+-sensitivity has been controversial, we have evidence for the hypothesis that acidic pH reduces the affinity of troponin C (TNC) for Ca2+. This effect of acidic pH depends not only on a direct effect of protons on Ca2+-binding to TNC, but also upon neighboring thin filament proteins, especially TNI, the inhibitory component of the TN complex. Using fluorescent probes that report Ca2+-binding to the regulatory sites of skeletal and cardiac TNC, we have shown, for example, that acidic pH directly decreases the Ca2+-affinity of TNC, but only by a relatively small amount. However, with TNC in whole TN or in the TNI-TNC complex, there is about a 2-fold enhancement of the effects of acidic pH on Ca2+-binding to TNC. Acidic pH decreases the affinity of skeletal TNI for skeletal TNC, and also influences the micro-environment of a probe positioned at Cys-133 of TNI, a region of interaction with TNC. Other evidence that the effects of acidic pH on Ca2+-TNC activation of myofilaments are influenced by TNI comes from studies with developing hearts. In contrast, to the case with the adult preparations, Ca2+-activation of detergent extracted fibers prepared from dog or rat hearts in the peri-natal period are weakly affected by a drop in pH from 7.0 to 6.5. This difference in the effect of acidic pH appears to be due to a difference in the isoform population of TNI, and not to differences in isotype population or amount of TNC.

Actin Cytoskeleton↗
PubMed1989

Myofibrillar Ca++ activation and heart failure--Ca++ sensitization by the cardiotonic agent APP 201-533.

Certain forms of cardiac failure appear to be associated with a decrease in the Ca++ sensitivity of the contractile structures, possibly due to troponin I phosphorylation. Interference of cardiotonic drugs with myofibrillar Ca++ activation instead of enhancement of Ca++ influx may therefore provide a more causal therapeutic concept in the treatment of cardiac insufficiency. APP 201-533 (3-Amino-6-methyl-5-phenyl-2(1H)-pyridinone) (the structure of which is shown below) is a novel cardiotonic agent acting neither via beta adrenoceptor stimulation nor inhibition of Na+/K+ ATPase. In the 100 microM concentration range, it increases the Ca++ sensitivity and the Ca++ affinity of functionally isolated cardiac contractile structures. This coincides with an inhibitory effect on the cAMP-dependent protein kinase from rat liver. A possible relation with the regulation of troponin I phosphorylation is discussed.

Animals↗
PubMed1989

Inotropic responses to isoproterenol and phosphodiesterase inhibitors in intact guinea pig hearts: comparison of cyclic AMP levels and phosphorylation of sarcoplasmic reticulum and myofibrillar proteins.

The influence of selective (milrinone: 10, 50, 100 microM) and nonselective phosphodiesterase (isobutylmethylxanthine: 0.1, 10, 100 microM) inhibitors and beta-adrenergic stimulation (isoproterenol: 0.01, 0.1 microM) on phospholamban and myofibrillar protein phosphorylation was studied in guinea pig hearts perfused with [32P]orthophosphate. Changes in protein phosphorylation were compared to alterations in tissue cyclic AMP (cAMP) levels and positive inotropic effects induced by these agents. Isoproterenol (0.01 microM), milrinone (50 microM), and isobutylmethylxanthine (100 microM) all produced similar, twofold increases in dP/dt and -dP/dt but only stimulation with isobutylmethylxanthine and isoproterenol was associated with significant increases in phospholamban phosphorylation. At these equipotent doses, the effects of isobutylmethylxanthine were associated with higher increases (3.1-fold) in cAMP than those observed with isoproterenol (twofold). Milrinone (50 microM) produced a 2.5-fold increase in cAMP levels but failed to change phospholamban phosphorylation. Higher doses of milrinone (100 microM) resulted in relatively high (4.1-fold) cAMP levels, and this was associated with increased (1.5-fold) phosphorylation of phospholamban. Phosphorylation of troponin I was significantly increased at 0.01 microM and 0.1 microM isoproterenol, while phosphorylation of C protein was observed only at 0.1 microM isoproterenol. Isobutylmethylxanthine and milrinone did not significantly increase phosphorylation of either troponin I or C protein at any of the doses studied. These findings indicate that cardiotonic agents acting via the cAMP pathway may produce similar inotropic responses at different levels of cAMP and phosphorylation of sarcoplasmic reticulum and myofibrillar proteins.

Animals↗
PubMed1989

Changes in phosphoinositide turnover in isolated guinea pig hearts stimulated with isoproterenol.

The incorporation of 32Pi into phospholamban, troponin I, phosphatidylinositols, and inositol trisphosphates was studied in Langendorff-perfused guinea pig hearts stimulated with isoproterenol. Hearts were perfused with Krebs-Henseleit buffer containing [32P]Pk and freeze-clamped at different times during the positive inotropic response. Exposure of the hearts to 0.1 microM isoproterenol for up to 1 minute was associated with significant (up to threefold) increases in phospholamban and troponin I phosphorylation, but there was no significant increase in 32P incorporation into phospholipids. However, longer exposure (2 minutes or more) to isoproterenol was associated with increases in the degree of 32P labeling of phosphatidylinositols and phosphatidic acid. Examination of 32P labeling of inositol trisphosphates in the same hearts revealed that the radioactivity associated with these compounds decreased with time. The decreases were significant at times of exposure of 2 minutes or longer to beta-adrenergic stimulation. The tissue levels of the inositol 1,4,5-trisphosphate isoform were also measured in hearts perfused with isoproterenol for 3 minutes, and they were found to be significantly lower compared with values obtained in control hearts. The effects of isoproterenol on 32P incorporation into phospholipids and proteins were observed in the presence of prazosin, and they were completely abolished by the beta-receptor blocker propranolol. Examination of the phosphoinositide-specific phospholipase C activity in the perfused hearts revealed that isoproterenol stimulation was associated with a decrease in the membrane-associated enzymatic activity at physiological calcium concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

The positive inotropic effect of pimobendan involves stereospecific increases in the calcium sensitivity of cardiac myofilaments.

We have studied the effect of pimobendan (UD-CG 115 BS) on the electrical, mechanical, and biochemical activity of intact and detergent-skinned preparations of cardiac muscle. Racemic pimobendan increased the contractile force of guinea pig papillary muscle preparations and this positive inotropic action was associated with potentiation of the Ca2+-dependent slow action potentials (APs). However, in the presence of 25 mM [K]0 and maximally activating concentrations of isoproterenol, isometric force was increased further by addition of 50 microM pimobendan with no effect on the slow action potential. Experiments with chemically skinned heart muscle fibers showed that pimobendan, in a dose-dependent manner, increased active tension developed at submaximally activating concentrations of Ca2+. The tension-cost (unit increase in ATPase rate/unit increase in force) was unchanged in the presence of pimobendan. Force-pCa and ATPase-pCa relations of skinned fiber preparations contracting isometrically were shifted to the left by 0.15-0.20 pCa units in the presence of 50 microM pimobendan. The mechanism for this effect was shown to be an increase in the Ca affinity of the regulatory binding sites of troponin C (TNC). These effects are due mainly to the l optical isomer of pimobendan. Addition of either the d or l isomer of pimobendan to preparations. maximally stimulated by 1 microM isoproterenol, did not affect the slow AP parameters, but did increase contractile force to 124% of control by the d isomer and to 184% of control by the l isomer. The Ca2+-sensitizing effect of l-pimobendan on skinned fiber preparations was substantially greater than that of the d isomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗
PubMed1988

Phosphorylation of phospholipids in isolated guinea pig hearts stimulated with isoprenaline.

Phosphorylation of phospholipids was studied in Langendorff perfused guinea pig hearts subjected to beta-adrenergic stimulation. Hearts were perfused with Krebs-Henseleit buffer containing [32P]Pi and freeze-clamped in a control condition or at the peak of the inotropic response to isoprenaline. 32P incorporation into total phospholipids, individual phospholipids and polyphosphoinositides was analysed in whole tissue homogenates and membranes, enriched in sarcoplasmic reticulum, prepared from the same hearts. Isoprenaline stimulation of the hearts did not result in any significant changes in the levels of phosphate incorporation in the total phospholipid present in cardiac homogenates (11.6 +/- 0.4 nmol of 32P/g for control hearts and 12.4 +/- 0.5 nmol of 32P/g for isoprenaline-treated hearts; n = 6), although there was a significant increase in the degree of phospholipid phosphorylation in sarcoplasmic reticulum (3.5 +/- 0.3 nmol of 32P/mg for control hearts and 6.7 +/- 0.2 nmol of 32P/mg for isoprenaline-treated hearts; n = 6). Analysis of 32P incorporation into individual phospholipids and polyphosphoinositides revealed that isoprenaline stimulation of the hearts was associated with a 2-3-fold increase in the degree of phosphorylation of phosphatidylinositol monophosphate and bisphosphate as well as phosphatidic acid in both cardiac homogenates and sarcoplasmic reticulum membranes. In addition, there was increased phosphate incorporation into phosphatidylinositol in sarcoplasmic reticulum membranes. Thus, perfusion of guinea pig hearts with isoprenaline is associated with increased formation of polyphosphoinositides and these phospholipids may be involved, at least in part, in mediating the effects of beta-adrenergic agents in the mammalian heart.

Animals↗
PubMed1988

Troponin I enhances acidic pH-induced depression of Ca2+ binding to the regulatory sites in skeletal troponin C.

Inhibition of muscle force development by acidic pH is a well known phenomenon, yet the exact mechanism by which a decrease in pH inhibits the Ca2+-activated force in striated myofilaments remains poorly understood. Whether or not the deactivation by acidic pH involves direct competition between Ca2+ and protons for regulatory binding sites on fast skeletal troponin C (TnC) or whether other proteins in thin filament regulation are important remains unclear. We measured the effects of acidic pH on Ca2+-dependent fluorescent changes in TnC labeled with the probe danzylaziridine (Danz), which reports Ca2+ binding to the regulatory (Ca2+-specific) sites. Measurements were also made with TnCDanz complexed with the inhibitory Tn unit, TnI, and in the whole Tn complex. Our results show that a drop in pH from 7.0 to 6.5 is associated with a 1.6-fold increase in the midpoint for the relation between free Ca2+ and Ca2+ binding to the regulatory sites on TnCDanz. However, when TnCDanz was present in its complex with either TnI alone or with TnI-TnT, the increase in midpoint free Ca2+ was increased by 3.5-fold. We tested whether this potentiation in the effect of acidic pH on Ca2+ binding to TnC is due to a pH-induced alteration in the binding of TnI to TnC. A decrease in pH from 7.0 to 6.5 was associated with a halving of the affinity of TnI for TnC. We also probed the effect of acidic pH on TnI. This was done (i) by measuring the intrinsic fluorescence of tryptophan residues in TnI alone and (ii) by measuring fluorescence of TnI (in the Tn complex) labeled at Cys-133 with 5-iodoacetamidofluorescein. A drop in pH from 7.0 to 6.5 was associated with a 15% decrease in intrinsic fluorescence and with a 30% decrease in the fluorescence of the 5-iodoacetamidofluorescein probe. We conclude, therefore, that while protons and Ca2+ may directly affect Ca2+ binding to regulatory sites on fast skeletal TnC, the effect of acidic pH on TnC Ca2+ binding is amplified in the TnI-TnC and Tn complexes by a pH-related effect on the affinity of TnI for TnC.

Animals↗