Interaction of bepridil with the cardiac troponin C/troponin I complex.

We have investigated the binding of bepridil to calcium-saturated cardiac troponin C in a cardiac troponin C/troponin I complex. Nuclear magnetic resonance spectroscopy and [(15)N,(2)H]cardiac troponin C permitted the mapping of bepridil-induced amide proton chemical shifts. A single bepridil-binding site in the regulatory domain was found with an affinity constant of approximately 140 microM(-1). In the presence of cardiac troponin I, bepridil binding to the C domain of cardiac troponin C was not detected. The pattern of bepridil-induced chemical shifts is consistent with stabilization of more open regulatory domain conformational states. A similar pattern of chemical shift perturbations was observed for interaction of the troponin I cardiac-specific amino-terminus with the cardiac troponin C regulatory domain. These results suggest that both bepridil and the cardiac-specific amino-terminus may mediate an increase in calcium affinity by interacting with and stabilizing open regulatory domain conformations. Chemical shift mapping suggests a possible role for inactive calcium-binding site I in the modulation of calcium affinity.

Magnesium-calcium exchange in cardiac troponin C bound to cardiac troponin I.

Understanding the process of Ca(2+)/Mg(2+)exchange during muscle excitation and relaxation is fundamental to elucidating the mechanism of Ca(2+)-regulated muscle contraction. During the resting phase, the C-domain of cardiac troponin C may be occupied by either Ca(2+)or Mg(2+). Here, complexes of recombinant cardiac troponin C(81-161) and the N terminus of cardiac troponin I, representing residues 33-80, were generated in the presence of saturating Mg(2+). Heteronuclear multi-dimensional nuclear magnetic resonance experiments were used to obtain backbone assignments of the Mg(2+)-loaded complex. In the presence of cardiac troponin I, the affinity of site IV for Mg(2+)is increased. Comparison of Mg(2+)and Ca(2+)-loaded complexes reveals that chemical shift differences are primarily localized to metal-binding sites III and IV, defining positions within these sites that have distinct Ca(2+)/Mg(2+)conformations. The observed transition from the Mg(2+)-loaded to Ca(2+)-loaded form demonstrates that sites III and IV fill simultaneously with Ca(2+)displacing Mg(2+). However, even in the absence of excess Ca(2+), Mg(2+)does not readily displace Ca(2+)in the isolated binary complex. Thus, the Mg(2+)-loaded conformer may only represent a small fraction of the total cardiac troponin C found in the sarcomere.

Regulatory domain conformational exchange and linker region flexibility in cardiac troponin C bound to cardiac troponin I.

Previously, we utilized (15)N transverse relaxation rates to demonstrate significant mobility in the linker region and conformational exchange in the regulatory domain of Ca(2+)-saturated cardiac troponin C bound to the isolated N-domain of cardiac troponin I (Gaponenko, V., Abusamhadneh, E., Abbott, M. B., Finley, N., Gasmi-Seabrook, G., Solaro, R.J., Rance, M., and Rosevear, P.R. (1999) J. Biol. Chem. 274, 16681-16684). Here we show a large decrease in cardiac troponin C linker flexibility, corresponding to residues 85-93, when bound to intact cardiac troponin I. The addition of 2 m urea to the intact cardiac troponin I-troponin C complex significantly increased linker flexibility. Conformational changes in the regulatory domain of cardiac troponin C were monitored in complexes with troponin I-(1-211), troponin I-(33-211), troponin I-(1-80) and bisphosphorylated troponin I-(1-80). The cardiac specific N terminus, residues 1-32, and the C-domain, residues 81-211, of troponin I are both capable of inducing conformational changes in the troponin C regulatory domain. Phosphorylation of the cardiac specific N terminus reversed its effects on the regulatory domain. These studies provide the first evidence that the cardiac specific N terminus can modulate the function of troponin C by altering the conformational equilibrium of the regulatory domain.

Polychlorinated biphenyl- and mercury-associated alterations on benthic invertebrate community structure in a contaminated salt marsh in southeast Georgia.

The community structure of a benthic macroinvertebrate assemblage in a contaminated salt marsh was evaluated as part of an ecological characterization of a former chloralkali production facility in Georgia. Sample locations were chosen based on a gradient of the primary contaminants of concern, total mercury and polychlorinated biphenyls (PCBs), primarily Aroclor 1268. Sediment concentrations of Aroclor 1268 ranged from 2.3 to 150 mg/kg dry weight, while mercury concentrations ranged from 15 to 170 mg/kg dry weight in the study area. Mercury and PCBs were determined to be co-located in the sediments. Total organic carbon composition of the sediments was negatively associated with PCB and mercury concentrations. A total of 29 benthic taxa was identified in 49 samples; replicate samples were taken at each of five sampling locations. Mean infaunal density across all sampling locations was estimated at approximately 61,000 to 234,000 organisms m(-2). Overall, polychaetes comprised 57% of the infaunal community with Manayunkia aestuarina as the dominant species. Oligochaetes, nematodes, crustacea, insects, and gastropods comprised 23.0, 18.0, 1.0, 0.7, and 0.2% of the overall benthic community, respectively. Density estimates of individual species between sampling locations showed no consistent patterns in response to pollutants. However, an analysis of higher taxonomic levels revealed some general trends. In uncontaminated areas, the benthic community was dominated by nematodes and oligochaetes, whereas moderate to highly contaminated areas were dominated by polychaetes and a smaller percentage of oligochaetes and nematodes. A trophic analysis of the same data set revealed that the community shifted from an evenly distributed percentage of surface and subsurface feeders in the uncontaminated areas to a community dominated by surface feeders in the more contaminated locations. Carnivores comprised from 0.13 to 0.90% of the trophic structure, with the percentage of carnivores generally decreasing with increasing contamination. Mercury and PCBs were bioaccumulating in representative marsh benthic invertebrates, presenting a potential source of contaminants to marsh consumers. Tissue PCB and tissue mercury concentrations were positively related to sediment PCB and mercury concentrations, respectively. A standard 14-day toxicity test using the amphipod Leptocheirus plumulosus showed no acute toxicity across the sampling locations.

NMR analysis of cardiac troponin C-troponin I complexes: effects of phosphorylation.

Phosphorylation of the cardiac specific amino-terminus of troponin I has been demonstrated to reduce the Ca2+ affinity of the cardiac troponin C regulatory site. Recombinant N-terminal cardiac troponin I proteins, cardiac troponin I(33-80), cardiac troponin I(1-80), cardiac troponin I(1-80)DD and cardiac troponin I(1-80)pp, phosphorylated by protein kinase A, were used to form stable binary complexes with recombinant cardiac troponin C. Cardiac troponin I(1-80)DD, having phosphorylated Ser residues mutated to Asp, provided a stable mimetic of the phosphorylated state. In all complexes, the N-terminal domain of cardiac troponin I primarily makes contact with the C-terminal domain of cardiac troponin C. The nonphosphorylated cardiac specific amino-terminus, cardiac troponin I(1-80), was found to make additional interactions with the N-terminal domain of cardiac troponin C.

Solution structures of the C-terminal domain of cardiac troponin C free and bound to the N-terminal domain of cardiac troponin I.

The N-terminal domain of cardiac troponin I (cTnI) comprising residues 33-80 and lacking the cardiac-specific amino terminus forms a stable binary complex with the C-terminal domain of cardiac troponin C (cTnC) comprising residues 81-161. We have utilized heteronuclear multidimensional NMR to assign the backbone and side-chain resonances of Ca2+-saturated cTnC(81-161) both free and bound to cTnI(33-80). No significant differences were observed between secondary structural elements determined for free and cTnI(33-80)-bound cTnC(81-161). We have determined solution structures of Ca2+-saturated cTnC(81-161) free and bound to cTnI(33-80). While the tertiary structure of cTnC(81-161) is qualitatively similar to that observed free in solution, the binding of cTnI(33-80) results mainly in an opening of the structure and movement of the loop region between helices F and G. Together, these movements provide the binding site for the N-terminal domain of cTnI. The putative binding site for cTnI(33-80) was determined by mapping amide proton and nitrogen chemical shift changes, induced by the binding of cTnI(33-80), onto the C-terminal cTnC structure. The binding interface for cTnI(33-80), as suggested from chemical shift changes, involves predominantly hydrophobic interactions located in the expanded hydrophobic pocket. The largest chemical shift changes were observed in the loop region connecting helices F and G. Inspection of available TnC sequences reveals that these residues are highly conserved, suggesting a common binding motif for the Ca2+/Mg2+-dependent interaction site in the TnC/TnI complex.

Effects of troponin I phosphorylation on conformational exchange in the regulatory domain of cardiac troponin C.

Conformational exchange has been demonstrated within the regulatory domain of calcium-saturated cardiac troponin C when bound to the NH2-terminal domain of cardiac troponin I-(1-80), and cardiac troponin I-(1-80)DD, having serine residues 23 and 24 mutated to aspartate to mimic the phosphorylated form of the protein. Binding of cardiac troponin I-(1-80) decreases conformational exchange for residues 29, 32, and 34. Comparison of average transverse cross correlation rates show that both the NH2- and COOH-terminal domains of cardiac troponin C tumble with similar correlation times when bound to cardiac troponin I-(1-80). In contrast, the NH2- and COOH-terminal domains in free cardiac troponin C and cardiac troponin C bound cardiac troponin I-(1-80)DD tumble independently. These results suggest that the nonphosphorylated cardiac specific NH2 terminus of cardiac troponin I interacts with the NH2-terminal domain of cardiac troponin C.

Alveolar epithelial fluid clearance is mediated by endogenous catecholamines at birth in guinea pigs.

Transition from placental to pulmonary oxygenation at birth depends on a rapid removal of fetal lung fluid from the developing alveoli. Alveolar fluid clearance was examined in ventilated, anesthetized developing guinea pigs of the ages newborn, 2-d-old, 5-d-old, 30-d-old, and 60-d-old (adult). An isosmolar 5% albumin solution was instilled into the lungs of the guinea pigs; the guinea pigs were then studied for 1 h. Alveolar fluid clearance was measured from the increase in alveolar protein concentration as water was reabsorbed. Newborn guinea pigs had a very high alveolar fluid clearance rate that declined rapidly within the first 5 postnatal days towards adult levels. The high alveolar fluid clearance at birth was apparently mediated by the beta-adrenergic system as demonstrated by the elevated plasma epinephrine levels and the increased sensitivity to inhibition by the beta-adrenergic antagonist propranolol immediately after birth. Surprisingly, exogenous addition of epinephrine was not able to stimulate alveolar fluid clearance in the newborn lung, but exogenous epinephrine stimulation increased over time to adult levels. The elevated alveolar fluid clearance at birth was associated with a significantly greater amiloride sensitivity in the newborn guinea pig lung. Northern blot analysis of distal lung tissue as well as isolated alveolar epithelial type II cells showed and confirmed higher levels of the alpha-subunit of the epithelial sodium channel mRNA in the newborn lung that rapidly tapered off toward adult levels. In conclusion, these data demonstrate the importance of the beta-adrenergic system and amiloride-sensitive sodium transporting pathways for clearance of fetal lung fluid at birth.

Alveolar liquid clearance in the anesthetized ventilated guinea pig.

Alveolar liquid clearance was examined in ventilated, anesthetized guinea pigs. An isosmolar 5% albumin solution was instilled into the lungs. Alveolar liquid clearance was studied over 1 h and was measured from the increase in alveolar protein concentration as water was reabsorbed. Basal alveolar liquid clearance was 38% of instilled volume. The high basal alveolar liquid clearance was not secondary to endogenous catecholamine release. Compared with control animals, epinephrine and the general beta-adrenergic agonist isoproterenol increased alveolar liquid clearance to approximately 50% of instilled volume (P < 0.05), whereas the beta 2-adrenergic agonist terbutaline was without effect. The stimulation of alveolar liquid clearance by epinephrine or isoproterenol was completely inhibited by the addition of the general beta-adrenergic inhibitor propranolol or the beta 1-adrenergic inhibitor atenolol. Alveolar liquid clearance was inhibited by the sodium-channel inhibitor amiloride by 30-40% in control animals and in animals treated with epinephrine or isoproterenol. Isoproterenol and epinephrine, but not terbutaline, increased adenosine 3',5'-cyclic monophosphate in in vitro incubated lung tissue. The results suggest that alveolar liquid clearance in guinea pigs is mediated partly through amiloride-sensitive sodium channels and that alveolar liquid clearance can be increased by stimulation of primarily beta 1-adrenergic receptors.