Quantitative aspects of drug-receptor interactions. II. The role of Ca2+men in desensitization and spasmolytic activity

The implications of the basic model of agonist-receptor interactions outlined in paper I² are further examined with specific reference to the role of Ca_mem²⁺ in desensitization and the action of spasmolytic agents (papaverine). Using the same series of 1, 3-dioxolane agonists it has been found that only the full agonists produce desensitization and that this process is enhanced by high agonist and low Ca_ext²⁺ concentrations. It is proposed that one form of desensitization is derived from a critical reduction in [Ca_mem²⁺] leading to a membrane conformational change to an inactive state. The selectivity of papaverine, chosen as a typical spasmolytic agent, for the tonic component of response is only seen with full agonists at high concentrations. It is suggested that the same factor controlling desensitization (Ca_mem²⁺) also determines the selectivity of papaverine action: papaverine is proposed to bind preferentially to Ca²⁺-depleted membrane states. The basic conclusion from both papers is that receptor activation consists of a transition between Ca²⁺-associated and Ca²⁺-dissociated membrane states and that the extent of this activation determines the properties of many agonist and antagonist drugs.     

Longitudinal smooth muscle of the mammalian intestine

Ca²⁺ mobilization in muscle cells from the circular muscle layer of the mammalian intestine is mediated by IP₃-dependent Ca²⁺ release. Ca²⁺ mobilization in muscle from the adjacent longitudinal muscle layer involves a distinct, phosphoinositide-independent pathway. Receptors for contractile agonists in longitudinal muscle cells are coupled via a pertussis toxinsensitive G protein to activation of PLA₂ and formation of arachidonic acid (AA). The latter activates Cl⁻ channels resulting in depolarization of the plasma membrane and opening of voltage-sensitive Ca²⁺ channels. Ca²⁺ influx via these channels induces Ca²⁺ release by activating sarcoplasmic ryanodine receptor/Ca²⁺ channels. The increase in [Ca²⁺]_i activates membrane-bound ADP ribosyl cyclase, and the resultant formation of cADPR enhances Ca²⁺-induced Ca²⁺ release.     

Purification and Characterization of a Cation-stimulated Adenosine Triphosphatase from Corn Roots

A membrane-bound, monovalent cation-stimulated ATPase from Zea mays roots has been purified to a single band on sodium dodecyl sulfate gel electrophoresis. Microsomal preparations with K⁺ -stimulated ATPase activity were extracted with 1 m NaClO₄, and the solubilized enzyme was purified by chromatography on columns of n-hexyl-Sepharose, DEAE-cellulose, and Sephadex G-100 Superfine. A 500-fold purification over the activity present in the microsomes was obtained. The K⁺ -stimulated activity shows positive cooperativity with increasing KCl concentrations. The purified enzyme shows K⁺ -stimulated activity with ATP, GTP, UTP, CTP, ADP, α + β-glycerophosphate, p-nitrophenyl phosphate, and pyrophosphate as substrates. Under most conditions ATP is the best substrate. Although dicyclohexyl carbodiimide and Ca²⁺ inhibit and alkylguanidines stimulate the K⁺ -ATPase while bound to microsomes, they have no effect on the purified enzyme.     

Extracellular calcium is involved in stomatal movement through the regulation of water channels in broad bean

Involvement of extracellular Ca²⁺ in stomatal movement through the regulation of water channels was investigated in broad bean (Vicia faba L.). Leaf peels were first incubated to open stomata, and then transferred to buffers in the presence of different CaCl₂ concentrations. Stomatal status was observed under magnification and stomatal aperture (pore width/length) was measured. Stomatal closure was significantly induced and aperture oscillation occurred at lower extracellular concentrations of calcium ([Ca²⁺]_ext), while at higher concentrations, no significant change in stomatal aperture was observed, which was similar to the response recorded with HgCl₂. Lower [Ca²⁺]_ext-induced stomatal closure could be reversed using depolarizing buffer. It is suggested that lower [Ca²⁺]_ext regulates water channels through an indirect way and at higher concentrations, extracellular Ca²⁺ is involved in regulating stomatal aperture by directly influencing water channels to retard aperture change.     

Reversible shift between two states of Ca2+-ATPase in human erythrocytes mediated by Ca2+ and a membrane-bound activator

The (Ca²⁺ + Mg²⁺)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) from human erythrocytes occurred in two different states, A-state and B-state, depending on the membrane preparation.The A-state showed low maximum activity (V) and the Ca²⁺ activation was characterized by a Hill coefficient, n_H, of about 1 and a Michaelis constant, K_Ca, about 30 μM.The B-state showed high V, a n_H above 1, which indicates positive cooper-activity of Ca²⁺ activation, and a K_Ca of about 1 μM.With varying ATP concentrations, both the A-state and the B-state showed negative cooperativity and slightly different values of K_m.The B-state was shifted to the A-state when the membranes were exposed to low Ca²⁺ concentrations. The shift reached 50% at approx. 0.5 μM Ca²⁺. At the low Ca²⁺ concentrations an activator was released from the membranes.The A-state was shifted to the B-state when the membranes were exposed to Ca²⁺ in the presence of the activator. The shift reached 50% at about 30 μM Ca²⁺. The recovery of high V was time dependent and lasted several minutes. Increasing concentrations of Ca²⁺ and activator accelerated the recovery.It is suggested that the A-state and the B-state correspond to enzyme free of activator and enzyme associated with activator, respectively. Furthermore, the two states may represent a resting and an active state, respectively, of the calcium pump.     

Interaction between the M2- and M3-Receptor Subtypes in Muscarinic Electrical and Mechanical Responses of Intestinal Smooth Muscles

In various gastrointestinal smooth muscles, two different muscarinic receptor subtypes, M₂ and M₃, are expressed; these receptors are the target for the parasympathetic neurotransmitter acetylcholine. Although the number of M₂ receptors is much greater than that of M₃ receptors, the functional role of the former receptor subtype has yet to be fully defined, since pharmacological analyses of the contractile responses to acetylcholine and other muscarinic agonists have revealed that such responses are mediated extensively by the minor M₃ subtype. The M₃ receptor links to Ca²⁺ store release, and the released Ca²⁺ ions may contribute to the contraction. However, many studies indicated the importance of Ca²⁺ influx through voltage-gated Ca²⁺ channels, rather than Ca²⁺ release, in muscarinic contractions, since the contractile responses are markedly inhibited by Ca²⁺ channel blockers. The major M₂ receptors link to the opening of cationic channels leading to the membrane depolarization, which in turn activates voltage-gated Ca²⁺ channels. Thus, there should be somewhere a point of contact between the M₃- and M₂-mediated signal transductions, as if M₃ receptor stimulation is connected with membrane depolarization. Our electrophysiological and pharmacological findings suggest that the M₂-mediated cationic channel opening and a resulting increase in the membrane electrical activity are the primary mechanism for mediating the contractile response to muscarinic agonists. An allosteric interaction between M₂ and M₃ receptors such that M₃ activation intensifies the M₂/cation channel pathway may account at least in part for the failure of many previous analyses to detect M₂ participation in the contractile responses to full agonists.     

Characterization of Ca and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor

The interactions between Ca²⁺ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca²⁺ or phosphocholine were injected. Binding of Ca²⁺ induced a 10-fold higher signal than expected from the molecular weight of Ca²⁺. It was interpreted to result from the conformational change that occurs on binding of Ca²⁺. Two sites with different characteristics were distinguished: a high-affinity site with K_D = 0.03 mM and a low-affinity site with K_D = 5.45 mM. The pH dependencies of the two Ca²⁺ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K_D = 5 μM at 10 mM Ca²⁺. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and suggest that Ca²⁺ is an efficient regulator under physiological conditions.     

A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels

The two human CLC Cl⁻ channels, ClC-Ka and ClC-Kb, are almost exclusively expressed in kidney and inner ear epithelia. Mutations in the genes coding for ClC-Kb and barttin, an essential CLC-K channel β subunit, lead to Bartter syndrome. We performed a biophysical analysis of the modulatory effect of extracellular Ca²⁺ and H⁺ on ClC-Ka and ClC-Kb in Xenopus oocytes. Currents increased with increasing [Ca²⁺]_ext without full saturation up to 50 mM. However, in the absence of Ca²⁺, ClC-Ka currents were still 20% of currents in 10 mM [Ca²⁺]_ext, demonstrating that Ca²⁺ is not strictly essential for opening. Vice versa, ClC-Ka and ClC-Kb were blocked by increasing [H⁺]_ext with a practically complete block at pH 6. Ca²⁺ and H⁺ act as gating modifiers without changing the single-channel conductance. Dose–response analysis suggested that two protons are necessary to induce block with an apparent pK of ∼7.1. A simple four-state allosteric model described the modulation by Ca²⁺ assuming a 13-fold higher Ca²⁺ affinity of the open state compared with the closed state. The quantitative analysis suggested separate binding sites for Ca²⁺ and H⁺.A mutagenic screen of a large number of extracellularly accessible amino acids identified a pair of acidic residues (E261 and D278 on the loop connecting helices I and J), which are close to each other but positioned on different subunits of the channel, as a likely candidate for forming an intersubunit Ca²⁺-binding site. Single mutants E261Q and D278N greatly diminished and the double mutant E261Q/D278N completely abolished modulation by Ca²⁺. Several mutations of a histidine residue (H497) that is homologous to a histidine that is responsible for H⁺ block in ClC-2 did not yield functional channels. However, the triple mutant E261Q/D278N/H497M completely eliminated H⁺ -induced current block. We have thus identified a protein region that is involved in binding these physiologically important ligands and that is likely undergoing conformational changes underlying the complex gating of CLC-K channels.     

Ca2+ release and contraction induced by IP3 and contractile agonists in mammalian gastric smooth muscle

The source, time course and stoichiometry of cytosolic free Ca²⁺ ([Ca²⁺]_i) during contraction were examined in smooth muscle cells isolated from the guinea pig and human stomach. Contraction by receptor-linked agonists (eg, acetylcholine, cholecystokinin octapeptide and Met-enkephalin) was preceded by stoichiometric increases in [Ca²⁺]_i and net ⁴⁵Ca²⁺ efflux that were maintained in the absence of extracellular Ca²⁺ or in the presence of a Ca²⁺ channel blocker (13600). The intracellular Ca²⁺ store could be depleted by repeated stimulation with all agonists in Ca²⁺-free medium or in the presence of 13600 resulting in loss of contractile response; response was restored by re-exposure of the cells to Ca²⁺.The source of intracellular Ca²⁺ an the signal for its release were examined in saponin-permeabilized muscle cells. The cells retained their ability to contract in response to receptor-linked agonists and developed an ability to contract in response to inositol trisphosphate (IP₃). The cells accumulated Ca²⁺ to the same extent as intact muscle cells, but only in the presence of ATP. IP₃ caused a prompt, concentration-dependent increase in contraction, [Ca²⁺]_i and net ⁴⁵Ca²⁺ efflux. These effects were maximally similar to those produced by CCK-8 alone or in combination with IP₃: Depletion of the Ca²⁺ store by repeated stimulation of single muscle cells in Ca²⁺-free medium with IP₃, acetylcholine or CCK-8 separately resulted in loss of contractile response to all three agents; the response was restored by re-exposure of the muscle cell to a cytosol-like perfusate (Ca²⁺ 180 nM).The studies demonstrate that a product of membrane phosphoinositide hydrolysis is capable of mobilizing Ca²⁺ from a depletable, non-mitochondrial intracellular store that is utilized by receptor-linked agonists. The magnitude of IP₃-induced Ca²⁺ release is correlated with contraction.     

Identification of an l-Phenylalanine Binding Site Enhancing the Cooperative Responses of the Calcium-sensing Receptor to Calcium

Functional positive cooperative activation of the extracellular calcium ([Ca²⁺]_o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca²⁺]_o or amino acids elicits intracellular Ca²⁺ ([Ca²⁺]_i) oscillations. Here, we report the central role of predicted Ca²⁺-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca²⁺-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca²⁺]_o. Next, we identify an adjacent l-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca²⁺]_o and l-Phe in eliciting CaSR-mediated [Ca²⁺]_i oscillations. The heterocommunication between Ca²⁺ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca²⁺]_o signaling by positively impacting multiple [Ca²⁺]_o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca²⁺]_o and amino acids into intracellular signaling events.     

The effect of calcium antagonists on contractions of the sensitized and normal guinea pig ileum

The purpose of this study was to learn wether a number of Ca²⁺ antagonists were effective in reducing contractile response of the isolated ileum of the sensitized and normal guinea pig. Contractions of the normal ileum in response to LTD₄, acetylcholine, histamine, and potassium chloride were obtained before and after verapamil, diltiazen and papaverine. Ovalbumin-induced contractions of the ovalbumin-sensitized ileum were obtained in the presence of the three Ca²⁺ antagonists. In the normal ileum, all the Ca²⁺ antagonists were highly effective in diminishing the contractile responses to LTD₄, acetylcholine, histamine and potassium chloride. In the sensitized ileum, ovalbumin-evoked contractions, with subsequent release of a potent contractile mediator (presumably SRS-A), were Ca²⁺-dependent since verapamil, diltiazem and papaverine caused a concentration-related reduction of contractions. Thus, the influx of extracellular Ca²⁺ plays a key role in the contractile responses of the normal and sensitized guinea pig ileum when stimulated by various potent agonists acting on specific receptors or on the cell membrane.     

Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1^−/−) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca²⁺ cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1^−/− compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca²⁺ transient (Δ[Ca²⁺]_i) responses in NOS1^−/− cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca²⁺]_i across the range of pacing frequencies and increased myofilament Ca²⁺ sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca²⁺ reuptake, and restored SR Ca²⁺ content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca²⁺ leak, HYD improves Ca²⁺ cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.     

Oleic acid-induced Ca2+ mobilization in human platelets: is oleic acid an intracellular messenger?

The purpose of this study was to explore the effect of oleic acid (OA) on intracellular Ca²⁺ mobilization in human platelets. When applied extracellularly, OA produced a concentration dependent rise in cytosolic [Ca²⁺] [Ca²⁺]_cyt) when extracellular [Ca²⁺] ([Ca²⁺]_ext was zero (presence of EGTA), suggesting that OA caused an intracellular release of Ca²⁺. Intracellular Ca²⁺ release was directly proportional to entry of OA into platelets and OA entry was indirectly proportional to [Ca²⁺]_ext. In permeabilized platelets, OA caused the release of ⁴⁵Ca²⁺ from ATP dependent intracellular stores. Finally, our results show that thrombin stimulated the release of [³H]OA from platelet phospholipids. The saturated fatty acids stearic and palmitic acid did not stimulate an increase in [Ca²⁺]_cyt under these conditions, but the unsaturated fatty acid, linolenic acid produced effects similar to those of OA, suggesting specificity among fatty acids for effects on [Ca²⁺]_cyt. Taken together, our experiments suggest that OA which has been incorporated into platelet phospholipids was released intothe cytosol by thrombinstimulation. Our experiments also show that OA stimulates Ca²⁺ release from intracellular stores. These results support the hypothesis that OA may serve as an intracellular messenger in human platelets.     

Kinetic parameters of calcium binding to tissue structures in human acute hypercalcemia

The kinetics of calcium binding by tissue structures was analyzed in 19 healthy volunteers (13 men and 6 women) from the age group of 33 ± 6.5 years under the conditions of acute hypercalcemia followed by drip intravenous infusion of calcium gluconate for 2.5 h. At the end of each 30-min period, the calcium amount retained by the tissue structures was recorded and the kinetic parameters of calcium binding were determined according to Langmuir and Scatchard. In all volunteers, there was a region of binding isotherm with positive cooperativity (linear regression in Scatchard’s coordinates) with the same buffer capacity (β_tis) for calcium in Langmuir’s coordinates (0.58 ± 0.24 L kg). Half of the volunteers exhibited cooperativity at [Ca²⁺] = 1.3–1.5 mmol/L, while others, at [Ca²⁺] = 1.0–1.3 mmol/L, which corresponded to the differences in the association constant (K_a) and the number of interactive sites (n) with [Ca²⁺] = 1 mmol/L. Additionally, two segments of the binding isotherm were detected with the successive binding of calcium to one set of non-interactive centers with similar kinetic parameters of calcium binding (β_tis, K_a, n). Four different types of calcium binding in healthy volunteers were found. The results of this study may serve as the basis for a functional diagnostic test of disorders of the tissue calcium-binding properties in different pathological conditions.     

Localization, solubilization and characterization of plant membrane-associated calcium-dependent protein kinases

Membrane fractions from mature silver beet (Beta vulgaris) deveined leaf and leaf stem homogenates have associated Ca²⁺ -dependent protein kinase. The Ca²⁺ -dependent protein kinase activity is associated with plasma membranes (density 1.14-1.18 grams per cubic centimeter) as determined from copurification on isopycnic centrifugation with plasma membrane markers such as β-glucan synthetase, eosin-5-maleimidelabeling, and specific naphthylphthalamic acid-binding. The Ca²⁺ -dependent protein kinase is not specifically associated with chloroplasts or mitochondria. The membrane-bound Ca²⁺ -dependent protein kinases were solubilized with 0.8% (volume/volume) Nonidet P40. The solubilized enzymes were extensively purified by a protocol involving binding to diethylaminoethyl-cellulose (Whatman DE-52), Ca²⁺ -dependent binding to phenyl-Sepharose CL-4B, gradient elution from diethylaminoethyl-Sephacel (resolving two distinct Ca²⁺ -dependent protein kinases), and gel filtration on Ultrogel AcA 44. These two membrane-derived enzymes have similar molecular weights but differ in protein substrate specificity, in K_m values for ATP, and in Ca²⁺ -independent activation by unsaturated fatty acids. The membrane-bound enzymes correspond closely in these properties to two Ca²⁺ -dependent protein kinases present in the soluble phase.     

The oppositely directed Ca2+ and Na+ transmembrane transport in algal cells

Summary The countertransport of Ca²⁺ and Na⁺ across the membranes of the unicellular fresh-water algaChlamydomonas reinhardtii CW-15 and twoDunaliella species differing in salt tolerance was studied. All algae used are devoid of cell walls. The calcium uptake by twoDunaliella species depended markedly on the intracellular sodium concentration. This calcium uptake was accompanied by Na⁺ release. For 15 and 30 s after artificial gradient formation (Na_int ⁺ greater than Na_ext ⁺) the ratio of released Na⁺ to absorbed Ca²⁺ was 31 and 41, respectively. For the extremely halotolerantD. salina, the apparent Michaelis constant of the Ca²⁺ uptake was 33 M, and for the marine halotolerant algaD. maritima, it was equal to 400 M, presuming more efficient Na⁺-for-Ca²⁺ exchange inD. salina cells. Ouabain, an inhibitor of Na⁺/K⁺-ATPase, suppressed Na⁺ transfer by 25%, whereas the agents blocking Ca²⁺-channels did not affect the transport of Ca²⁺ and Na⁺. The oppositely directed transmembrane Ca²⁺ and Na⁺ transfer was shown to depend on the external concentrations of Na⁺ and H⁺. In the fresh-water algaC. reinhardtii CW-15 (Na_ext ⁺ greater than Na_int ⁺), the direction of Ca²⁺ and Na⁺ fluxes across the plasma membrane was opposite to those described for Dunaliella cells. The results obtained point to the ability of the Na⁺-Ca²⁺ exchanger function in plasma membranes of algal cells.     

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and α-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n ? 6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n ? 3 fatty acids (α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n ? 6 fatty acids (linoleic acid and arachidonic acid), the total n ? 3 fatty acyl content was reduced in all the phospholipids examined. In n ? 3 and n ? 6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n ? 9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appear to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca²⁺]_i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n ? 3 and n ? 6 PUFA but not in n ? 9 monounsaturated fatty acid modified cells. In Ca²⁺ free medium, OKT3-induced transient increase in [Ca²⁺]_i, representing Ca²⁺ release from the inositol 1,4,5-triphosphate-sensitive Ca²⁺ stores, were similar in control and UFA modified cells. Using Mn²⁺ entry as an index of plasma membrane Ca²⁺ permeability, the rate of fura-2 fluorescence quenching as a result of Mn²⁺ influx stimulated by OKT3 in n ? 9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n ? 3 and n ? 6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca²⁺ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and n ? 9 monounsaturated fatty acids appears to be important for the maintenance of a functional Ca²⁺ influx mechanism.     

In vivo calcium regulation in diabetic skeletal muscle

In skeletal muscle, dysfunctional contractile activity has been linked to impaired intracellular Ca²⁺ concentration ([Ca²⁺]_i) regulation. Muscle force production is impaired and fatigability and muscle fragility deteriorate with diabetes. Use of a novel in vivo model permits investigation of [Ca²⁺]_i homeostasis in diabetic skeletal muscle. Within this in vivo environment we have shown that diabetes perturbs the Ca²⁺ regulatory system such that resting [Ca²⁺]_i homeostasis following muscle contractions is compromised and elevations of [Ca²⁺]_i are exacerbated. This review considers the impact of diabetes on the capacity of skeletal muscle to regulate [Ca²⁺]_i, following muscle contractions and, in particular, the relationship between muscle fatigue and elevated [Ca²⁺]_i in a highly ecologically relevant circulation-intact environment. Importantly, the role of mitochondria in calcium sequestration and the possibility that diabetes impacts this process is explored. Given the profound microcirculatory dysfunction in diabetes this preparation offers the unique opportunity to study the interrelationships among microvascular function, blood-myocyte oxygen flux and [Ca²⁺]_i as they relate to enhanced muscle fatigability and exercise intolerance.     

Thermodynamics and molecular dynamics simulations of calcium binding to the regulatory site of human cardiac troponin C: evidence for communication with the structural calcium binding sites

Human cardiac troponin C (HcTnC), a member of the EF hand family of proteins, is a calcium sensor responsible for initiating contraction of the myocardium. Ca²⁺ binding to the regulatory domain induces a slight change in HcTnC conformation which modifies subsequent interactions in the troponin–tropomyosin–actin complex. Herein, we report a calorimetric study of Ca²⁺ binding to HcTnC. Isotherms obtained at 25 °C (10 mM 2-morpholinoethanesulfonic acid, 50 mM KCl, pH 7.0) provided thermodynamic parameters for Ca²⁺ binding to both the high-affinity and the low-affinity domain of HcTnC. Ca²⁺ binding to the N-domain was shown to be endothermic in 2-morpholinoethanesulfonic acid buffer and allowed us to extract the thermodynamics of Ca²⁺ binding to the regulatory domain. This pattern stems from changes that occur at the Ca²⁺ site rather than structural changes of the protein. Molecular dynamics simulations performed on apo and calcium-bound HcTnC_1–89 support this claim. The values of the Gibbs free energy for Ca²⁺ binding to the N-domain in the full-length protein and to the isolated domain (HcTnC_1–89) are similar; however, differences in the entropic and enthalpic contributions to the free energy provide supporting evidence for the cooperativity of the C-domain and the N-domain. Thermograms obtained at two additional temperatures (10 and 37 °C) revealed interesting trends in the enthalpies and entropies of binding for both thermodynamic events. This allowed the determination of the change in heat capacity (?C _p ) from a plot of ?H verses temperature and may provide evidence for positive cooperativity of Ca²⁺ binding to the C-domain.     

Biochemical characterization of the plasma membrane Ca2+ pumping ATPase activity present in the gill cells of Mytilus galloprovincialis LAM

• 1.1. In the plasma membrane of mussel gill cells an ouabain insensitive, Ca²⁺-activated ATPase activity is present. The ATPase has high Ca²⁺ affinity (K_ma = 0.3 μM).
• 2.2. The optimum assay conditions to evaluate the enzymatic activity of the Ca²⁺-stimulated ATPase at 19°C are: 120–300 mM KCl ionic strength, pH 7.0 and 2 mM ATP. As for mammalian enzymes, the Ca²⁺ ATPase activity is stimulated by DTT (0.5–1 mM) and it is inhibited by low concentrations of vanadate (10–50 μM) and -SH inhibitors such as PCMB and PCMBS (10 μM); the enzyme appears to be calmodulin insensitive.
• 3.3. Electrophoretic analyses of plasma membrane proteins demonstrate that: (a) Ca²⁺ at n-μM concentrations is necessary to activate ATP hydrolysis with consequent formation of the enzyme-phosphate complex; (b) the steady state concentration of the phosphorylated intermediate is increased in the presence of La³⁺; (c) the mol. wt of Ca²⁺ ATPase is about 140 kDa.
• 4.4. Low Ca²⁺ concentrations (n-μM) are sufficient to stimulate the ATP-dependent Ca²⁺ uptake by plasma membrane inside-out vesicles.
• 5.5. The results indicate that the Ca²⁺ pump present in the gill plasma membranes could be responsible for Ca²⁺ extrusion and therefore involved in maintaining the cytosolic Ca²⁺ concentration within physiological levels.