Changes In Calcium Sensitivity Research Articles

Role of the amino-terminal domain in regulating interactions of annexin I with membranes: Effects of amino-terminal truncation and mutagenesis of the phosphorylation sites

Phosphorylation of the N-terminal tail by protein kinase C strongly inhibits the ability of bovine or human annexin I to aggregate chromaffin granules by increasing the calcium requirement 4-fold (Wang, W., & Creutz, C. E. (1992) Biochemistry 31, 9934-9936). In the present study three forms of human annexin I truncated in the amino terminus at residue Trp-12, Lys-26, or Lys-29 exhibit dramatic differences in their sensitivities to calcium in a chromaffin granule aggregation assay, while the [Ca2+](1/2)max values for binding of the truncated proteins to granule membranes are similar. Cleavage at Trp-12 causes a 3-fold decrease in calcium sensitivity in the membrane aggregation assay, while cleavage at Lys-26 causes a 4-fold enhancement of calcium sensitivity. In contrast, cleavage at Lys-29 results in virtually no change in calcium sensitivity. Mutagenic substitution with negatively charged amino acids of Ser-27, a site for phosphorylation by protein kinase C, or Tyr-21, a site for phosphorylation by the epidermal growth factor receptor kinase, mimics the inhibition of granule-aggregating activity seen with phosphorylation by protein kinase C. When bovine chromaffin cells are stimulated to secrete by nicotine, annexin I is phosphorylated in the amino terminus. Thr-24 and Ser-28, which are sites for phosphorylation by protein kinase C in vitro, are two of the sites phosphorylated in vivo in stimulated chromaffin cells. These data demonstrate that the ability of annexin I to promote membrane aggregation is highly sensitive to changes in the structure of the N-terminal domain of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in force and calcium sensitivity in the developing avian heart

The aim of this study was to characterize the development of the contractile properties of intact and chemically skinned muscle from chicken heart and to compare these characteristics with those of developing mammalian heart reported by others. Small trabeculae were dissected from left ventricles of Arbor Acre chickens between embryonic day 7 and young adulthood (7 weeks post-hatching). At all ages, increasing extracellular calcium (0.45-3.6 mM) progressively increased twitch force of electrically stimulated trabeculae. Twitch force at 1.8 mM extracellular calcium, normalized to cross-sectional area, increased to a maximum at 1 day post-hatching, remained constant through 3 weeks post-hatching, but then decreased at 7 weeks post-hatching. The maximal calcium-activated force of trabeculae chemically skinned with Triton X-100 detergent increased to a maximum 2 days before the time of hatching and was not significantly changed up to 7 weeks post-hatching. Over the ages studied, average twitch force in 1.8 mM calcium was between 26 and 66% of maximal calcium-activated force after skinning, suggesting that the contractile apparatus is not fully activated during the twitch in normal Ringer. In skinned trabeculae, the calcium sensitivity of the contractile apparatus was higher in the embryo than in the young adult. These age-dependent changes in calcium sensitivity are correlated with isoform switching in troponin T. A decrease in pH from 7.0 to 6.5 decreased the calcium sensitivity of the contractile apparatus to a greater degree in skinned trabeculae from young adult hearts than in those from embryonic hearts. This change in susceptibility to acidosis is temporally associated with isoform switching in troponin I.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental Changes in Effects of Halothane and Isoflurane on Contractile Properties of Rabbit Cardiac Skinned Fibers

Immature hearts of various animal species and humans have been demonstrated to be more sensitive than adult hearts to the myocardial depressant effects of volatile anesthetics. To further investigate the mechanisms involved, the calcium sensitivity and maximal activated tension of detergent-treated left ventricular fibres of fetuses (30 days), newborn (1-day-old), immature (3-, 8-, and 17-day-old), and adult rabbits were determined by stepwise exposure to increasing Ca2+ concentrations. Responses were measured prior to and after exposure to equianesthetic concentrations of halothane (1%) or isoflurane (1.5%) applied in a random order. In control conditions maximal developed tension was the lowest in fetuses (11.1 +/- 0.6 mN.mm-2), intermediate in newborn and immature rabbits, and highest in adults (25.6 +/- 2.9 mN.mm-2). There were also age-related changes in calcium sensitivity; pCa (= -log10[Ca2+]) for half-activation (pCa50) was significantly less in 1-, 3-, and 8-day-old rabbits (5.444 +/- 0.036, 5.425 +/- 0.017, and 5.385 +/- 0.019, respectively) than in adults (5.517 +/- 0.010), whereas it was not different in fetuses (5.521 +/- 0.017). During anesthetic exposure both calcium sensitivity and maximal developed tension decreased significantly in all age groups of animals, with both anesthetics having a similar effect in animals of identical age. However, calcium sensitivity decreased significantly more in newborn animals (0.192 and 0.196 pCa unit for halothane and isoflurane, respectively) compared with adults (0.122 and 0.137 pCa units, respectively). By contrast, fetuses were less sensitive to the myocardial depressant effects of anesthetics than were newborn animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Length and myofilament spacing-dependent changes in calcium sensitivity of skeletal fibres: effects of pH and ionic strength.

The calcium sensitivity of force was measured in glycerinated rabbit psoas fibres at sarcomere lengths (SL) from 2.3 to 3.4 micron. Increased SL caused calcium sensitivity to increase and the slope of force-calcium relations to decrease. We have hypothesized that length-dependent changes in myofilament lattice spacing and the presence of fixed charge on the myofilaments are important in determining calcium sensitivity. Lattice spacing changes were monitored by measuring fibre diameter (D). D was decreased by increasing SL, decreasing bathing solution pH and by osmotic compression with 3% PVP. 3% PVP caused D to decrease by about 15% at all SLs and pH values tested. Force-calcium relations were measured at different SLs and pH values, with and without 3% PVP in the bathing solutions. At all pH values D at SL 2.3 micron with 3% PVP was comparable to the value at 3.4 micron, without PVP. At pH 7.5 and 7.0 calcium sensitivity was about the same at both SL, although the slope of the force-calcium relation was less at longer SL. The similarity of the calcium sensitivity at the same D, but much different SL, indicates that lattice spacing is important in determining calcium sensitivity, while SL and the degree of myofilament overlap are important in determining the slope of force-calcium relations. In order to test for the role of myofilament charge in determining calcium sensitivity, pH and ionic strength were varied. Decreasing pH caused decreased maximum force and calcium sensitivity. In addition, the influence of SL on calcium sensitivity decreased as pH was lowered, with minimal SL dependence at pH 5.5; even though lattice spacing still decreased with increasing SL. When D was decreased with PVP, calcium sensitivity increased at all SLs in pH 7.5 and 7.0 while the same lattice spacing changes at pH 6.0 and 5.5 resulted in greatly reduced shifts in calcium sensitivity. These results indicate that the effect of lattice spacing on calcium sensitivity depends on myofilament charge. At pH 6.0, even though osmotic compression of the lattice has no effect, increasing SL causes about half the shift in calcium sensitivity seen at pH 7.0. Lowering ionic strength from 200 to 110 mM caused an increase in both the magnitude and length dependence of calcium sensitivity at pH 7.0, while at pH 5.5 both decreased.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscle force and stiffness during activation and relaxation. Implications for the actomyosin ATPase.

Isolated skinned frog skeletal muscle fibers were activated (increasing [Ca2+]) and then relaxed (decreasing [Ca2+]) with solution changes, and muscle force and stiffness were recorded during the steady state. To investigate the actomyosin cycle, the biochemical species were changed (lowering [MgATP] and elevating [H2PO4-]) to populate different states in the actomyosin ATPase cycle. In solutions with 200 microM [MgATP], compared with physiological [MgATP], the slope of the plot of relative steady state muscle force vs. stiffness was decreased. At low [MgATP], cross-bridge dissociation from actin should be reduced, increasing the population of the last cross-bridge state before dissociation. These data imply that the last cross-bridge state before dissociation could be an attached low-force-producing or non-force-producing state. In solutions with 10 mM total Pi, compared to normal levels of MgATP, the maximally activated muscle force was reduced more than muscle stiffness, and the slope of the plot of relative steady state muscle force vs. stiffness was reduced. Assuming that in elevated Pi, Pi release from the cross-bridge is reversed, the state(s) before Pi release would be populated. These data are consistent with the conclusion that the cross-bridges are strongly bound to actin before Pi release. In addition, if Ca2+ activates the ATPase by allowing for the strong attachment of the myosin to actin in an A.M.ADP.Pi state, it could do so before Pi release. The calcium sensitivity of muscle force and stiffness in solutions with 4 mM [MgATP] was bracketed by that measured in solutions with 200 microM [MgATP], where muscle force and stiffness were more sensitive to calcium, and 10 mM total Pi, where muscle force and stiffness were less sensitive to calcium. The changes in calcium sensitivity were explained using a model in which force-producing and rigor cross-bridges can affect Ca2+ binding or promote the attachment of other cross-bridges to alter calcium sensitivity.

Calcium‐force relationships as detected with aequorin in two different vascular smooth muscles of the ferret.

The bioluminescent calcium indicator aequorin was chemically loaded into isolated strips of ferret portal vein and ferret aorta. Aequorin light emission (a function of [Ca2+]i) was recorded simultaneously with tension. Assuming an [Mg2+]i of 0.5 mM, [Ca2+]i was 1.8 X 10(-7) M in the unstimulated portal vein at 22 degrees C where there was negligible resting tone. In contrast, in the unstimulated aorta at 22 degrees C where there was significant basal tone, the [Ca2+]i was 2.7 X 10(-7) M. In both portal vein and aorta, potassium depolarization caused a monophasic rise in intracellular Ca2+ in parallel with the rise in tension, whereas phenylephrine caused an initial spike of light during the period of the force development which then fell to a much lower plateau level during the period of force maintenance. Calcium-force curves were generated by plotting calibrated aequorin light against force while intracellular [Ca2+] was made to change either by increasing degrees of potassium depolarization or decreasing extracellular [Ca2+]. The steady-state calcium-force curve in the presence of phenylephrine was shifted to the left of the curve in the presence of potassium depolarization in both the portal vein and aorta. In the aorta there was a counter-clockwise hysteresis in the calcium-force relationship. In contrast, in the portal vein there was no demonstrable hysteresis, indicating that the apparent change in calcium sensitivity of the contractile apparatus in the presence of phenylephrine must be caused by a second messenger other than calcium.

Calcium sensitivity of isometric force in intact and chemically skinned aortas during the development of aldosterone-salt hypertension in the rat.

We investigated the role of altered vascular calcium handling in the development of aldosterone-salt hypertension in the rat. The calcium sensitivity of isometric force in response to 50 mM KCl was compared in aortic rings from control and aldosterone-hypertensive rats. Over the entire range of calcium concentrations studied, responses in aortas from the hypertensives were significantly depressed compared to controls [ED50: aldosterone-hypertensive rats (n = 6), 0.739 +/- 0.137; controls (n = 7), 0.141 +/- 0.021 mM; P less than 0.001]. However, calcium sensitivity in response to 1 microM norepinephrine was similar in aortas from both hypertensives and controls [ED50: aldosterone-hypertensive rats (n = 7), 0.196 +/- 0.022; controls (n = 7), 0.180 +/- 0.024 mM]. The calcium sensitivity of Triton X-100 skinned aortic rings from aldosterone-hypertensive rats was likewise not significantly different from sensitivity in controls [ED50: aldosterone-hypertensive rats (n = 9), 3.61 X 10(-7) +/- 0.57; controls (n = 8), 3.89 X 10(-7) +/- 0.64 M]. Therefore, the observed decrease in calcium sensitivity in response to membrane depolarization in aortas from aldosterone-hypertensive rats probably is not due to a change in calcium sensitivity of the contractile system itself. The time course for development of changes in calcium handling in vessels from the aldosterone-hypertensive rats was found to be quite different from the time course for changes in monovalent ion metabolism. Whereas increases in monovalent ion permeability reportedly appear as early as one week after the start of aldosterone-salt treatment, significant alterations in calcium handling were not apparent until after four weeks of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the activation and troponin calcium binding of dog cardiac myofibrils by acidic pH.

The aim of experiments described here was to test whether deactivation of cardiac myofibrils in acidic pH is associated with decreases in amounts of calcium bound to myofilament troponin. We determined the amounts of myofibrillar bound calcium attributable to troponin, from measurements of calcium binding to myofibrils and to myosin and from determination of the troponin C content of the myofibrillar preparations (0.40 nmol troponin C/mg protein). In measurements done at 2 mM free magnesium, 2 mM (magnesium-adenosine triphosphate, ionic strength 0.12, 22 degrees C, the pCa50 (-log of the half maximally activating molar free calcium) for myofibrillar magnesium-adenosine triphosphatase activity was 5.87 at pH 7.0, 5.49 at pH 6.5, and 5.04 at pH 6.2. This change in calcium sensitivity of myofibrillar magnesium-adenosine triphosphatase activity was present whether or not ethyleneglycol-bis(beta-aminoethyl ether)-N, N'-tetraacetic acid, was used to buffer the free calcium and whether or not myofibrillar troponin I had been phosphorylated by cyclic adenosine 3',5'-monophosphate-dependent protein kinase. However, the change in pCa50 of myofibrillar adenosine triphosphatase activity induced by acidic pH, was greater when free magnesium was reduced from 2.0 to 0.05 mM, and less when free magnesium was increased from 2.0 mM to 10 and 15 mM. The change in pCa50 with acidic pH was less if the ionic strength was reduced from 0.12 to 0.035 M. The magnesium-adenosine triphosphatase activity of troponin/tropomyosin-free myofibrils was independent of pCa and unaffected by a reduction of pH from 7.0 to 6.5. The affinity of myofibrillar troponin C for calcium decreased as pH was reduced from 7.0 to 6.5 and to 6.2 with and without ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid, and in a manner predicted from the effect of acidic pH on pCa50 for myofibrillar activation. Our results are consistent with the idea that at least part of the mechanism responsible for deactivation of the adenosine triphosphatase activity of cardiac myofilaments in acidic pH is a reduction in the affinity of myofibrillar troponin C for calcium.

Barium responsiveness of the rat aorta and femoral artery during pregnancy

The barium responses of isolated aortic strips and femoral arteries from non-pregnant and pregnant rats were investigated. Barium caused concentration-related increases in tension of vessels from both pregnant and non-pregnant rats. The concentration-response curves of femoral arteries from non-pregnant and 3 week pregnant rats were not different; however contractility and slopes of concentration-response lines for thracic aortas from 1, 2 and 3 week pregnant rats were significantly less than those of aortas from non-pregnant rats. In addition, barium caused rhythmic contractions to develop in both femoral arteries and aortas of 3 week pregnant rats more frequently than vessels from non-pregnant rats. Rhythmic contractions did not develop in aortas from 3 week pregnant rats rats in calcium-free Krebs. Since the effects of barium on the electrical and mechanical activity of various muscles have been postulated to be similar to and/or dependent on calcium, these results may indicate that changes in calcium sensitivity of vascular smooth muscle occur during pregnancy. Such changes may contribute to the blood flow redistribution and other cardiovascular adaptations of pregnancy.

Development of rat brain uptake and calcium-dependent release of GABA.

Summary High-affinity uptake of GABA in the developing rat brain is greater in 7-day-old rats than in adults, which is quite different from the developmental profile observed for biogenic amine uptake in brain, where the magnitude of uptake increases in a linear fashion. In contrast to uptake, the neuronal calcium-dependent release system for GABA develops in a progressive fashion, similar to that observed for the biogenic amines, suggesting that the enhanced GABA uptake observed in the younger animals is not stored in a functionally releaseable pool. This conclusion is based on the findings that the lack of correlation between the magnitude of GABA release and uptake in the developing brain is not due to the enhanced uptake itself, to the presence of some identifiable subcellular constituent, or to a change in calcium sensitivity for the release process at the earlier time. Furthermore, uptake of β-alanine and leucine is also greater in the 7-day-old rat forebrain than adult, whereas other biogenic amines such as norepinephrine and dopamine are similar to serotonin with regard to the development of their uptake and release processes. These results suggest that high-affinity GABA uptake is a questionable marker for studying neuronal development of this transmitter system, and that calcium-dependent release is probably a more valid indicator of the functional state of GABA terminals.

Properties of aseptically-packed UHT milk: casein modification during storage and studies with model systems

SummaryStorage of aseptically packed UHT milk produced changes in the electrophoretic pattern of the milk caseins when the milk was stored at ambient or higher temperatures. Lower temperature storage at 4°C did not give rise to these changes.The alterations in electrophoretic properties of the caseins appeared to be due to the action of carbonyl compounds, produced by a Maillard type of reaction, which led to changes in the charge of the protein together with some degree of polymerization. These conclusions have been drawn from results obtained on model systems of casein-lactose subjected to various heat treatments, and on casein and milk treated with acetaldehyde.Changes in the sensitivity to calcium ions of individual caseins, whole casein and milk that had been subjected to various heat treatments or to treatment with acetaldehyde showed that all these different treatments gave rise to modified casein which, in general, became less sensitive to calcium. κ-Casein when treated alone rapidly lost its ability to protect αs1-casein from precipitation by calcium ions, while αs1-casein treated alone only gradually became more soluble in the presence of calcium. Thus, on treating whole casein there was evidence for a stability minimum when the protective ability of the κ-casein has been destroyed without a compensating gain in the stability of the αs1-casein.The importance of these changes in relation to the stability of UHT milks has not yet been elucidated but the results indicate that cross linking between protein chains and changes in calcium sensitivity occur during long-term storage.