The inhibition of the conversion of testosterone into 5α-dihydrotestosterone in the reproductive organs of the male rat

The inhibition of testosterone 5α-reductase activity by 3-oxo-4-androstene-17β-carboxylic acid in the male reproductive organs of the rat was demonstrated $\text{in vitro}$. The medium for incubation of caput epididymis showed the highest concentration of 5α-dihydrotestosterone (5α-DHT) whereas the highest concentration of testosterone (T) was recorded in medium for incubation of decapsulated testis after two hours of incubation. The 3-oxo-4-androstene-17β-carboxylic acid (1.58 × 10^?5M) inhibited the conversion of T to 5α-DHT in all the organs tested (testis, caput and cauda epididymis and ventral prostate) under identical incubation conditions.     

Formation of 5α-dihydrotestosterone from 5α-androstane-3α,17β-diol in prostate cancer LAPC-4 cells – Identifying inhibitors of non-classical pathways producing the most potent androgen

5α-Dihydrotestosterone (5α-DHT) possesses a great affinity for the androgen receptor (AR), and its binding to AR promotes the proliferation of prostate cancer (PC) cells in androgen-dependent PC. Primarily synthesized from testosterone (T) in testis, 5α-DHT could also be produced from 5α-androstane-3α,17β-diol (3α-diol), an almost inactive androgen, following non-classical pathways. We reported the chemical synthesis of non-commercially available [4-¹⁴C]-3α-diol from [4-¹⁴C]-T, and the development of a biological assay to identify inhibitors of the 5α-DHT formation from radiolabeled 3α-diol in LAPC-4 cell PC model. We measured the inhibitory potency of 5α-androstane derivatives against the formation of 5α-DHT, and inhibition curves were obtained for the most potent compounds (IC₅₀ = 1.2–14.1 μM). The most potent inhibitor 25 (IC₅₀ = 1.2 μM) possesses a 4-(4-CF₃-3-CH₃O-benzyl)piperazinyl methyl side chain at C3β and 17β-OH/17α-CCH functionalities at C17 of a 5α-androstane core.     

Stimulation of Ca2+ uptake in the human liver fluke Opisthorchis viverrini by praziquantel

⁴⁵Ca²⁺ uptake by the human liver fluke Opisthorchis viverrini is enhanced by praziquantel. The drug-induced ⁴⁵Ca²⁺ uptake was dependent on the presence of Ca²⁺ and was attenuated in the presence of 10 mM Mg²⁺. La³⁺ and vanadate at concentration of 1mM partially reduced the amount of ⁴⁵Ca²⁺ uptake into the liver fluke in response to praziquantel treatment. The stimulating effect of praziquantel was eliminated in the presence of 10 μM verapamil. These findings suggest that praziquantel increases the permeability of the liver fluke tegument to Ca²⁺ probably by interfering with the mechanism that regulates Ca²⁺ binding or trnasport across the tegumental membrane.     

Simultaneous loss and reappearance of α1-adrenergic responses and [3H]prazosin binding sites in rat liver after irreversible blockade by phenoxybenzamine

The relative influences of the in vivo administration of phenoxybenzamine on in vitro binding to α₁-adrenergic receptors and α₁-receptor-mediated responses were studied. Phenoxybenzamine treatment reduced maximal specific binding of the α₁-selective antagonist [³H]prazosin to liver cell membranes. This response was rapid (< 90 min) and half-maximal following a phenoxybenzamine dose of approx. 10 mg/kg. A similar decrease in the ability of phenylephrine to stimulate glucose release and ⁴⁵Ca²⁺ efflux from liver slices was also noted after phenoxybenzamine treatment. During the recovery period following administration of 30 mg/kg phenoxybenzamine, [³H]prazosin specific binding and phenylephrine-stimulated glucose release and ⁴⁵Ca²⁺ efflux returned to their respective control levels with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values of 42, 49 and 38 h, respectively. At all times studied during the recovery period, α₁-binding and both of the α₁-responses were similar fractions of their respective control values. These observations indicate that a close relationship exists between the density of [³H]prazosin binding sites and the ability of rat liver to respond to α₁-stimulation. We suggest that the binding sites identified in studies using the antagonist [³H]prazosin and those through which the agonist phenylephrine stimulates glucose release and ⁴⁵Ca²⁺ efflux are either identical or in equilibrium with each other.     

Regucalcin increases Ca2+-ATPase activity and ATP-dependent calcium uptake in the microsomes of rat kidney cortex

The effect of regucalcin, a Ca²⁺-binding protein, on Ca²⁺ transport system in rat renal cortex microsomes was investigated. The presence of regucalcin (10^-8 to 10^-6 M) in the reaction mixture caused a significant increase in Ca²⁺-ATPase activity and ATP-dependent⁴⁵ Ca²⁺ uptake in the microsomes. Regucalcin (10^-7 M) increased Ca²⁺-ATPase activity independently of increasing concentrations of CaCl_{_2}. The microsomal Ca²⁺-ATPase activity and⁴⁵ Ca²⁺ uptake were markedly decreased by the presence of vanadate (0.1 mM) or N-ethylmaleimide (NEM; 5 mM) in the absence or presence of regucalcin. Dithiothreitol (DTT; 5 mM) markedly elevated Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake in the microsomes. The DTT effects were not further enhanced by regucalcin (10^-7 M). Meanwhile, the microsomal Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake were significantly decreased by the presence of dibutyryl cyclic AMP (DcAMP; 10^-5 and 10^-3 M) or inositol 1,4, 5-trisphosphate (IP3; 10^-7 and 10^-5 M). The effect of regucalcin (10^-7 M) on Ca²⁺ ATPase activity and ⁴⁵Ca²⁺ uptake was weakened in the presence of DcAMP or IP₃. The present results demonstrate that regucalcin has a stimulatory effect on ATP-dependent Ca²⁺ uptake in the microsomes of rat renal cortex due to acting on the thiol groups of Ca²⁺-ATPase.     

Structural Basis for Difference in Heat Capacity Increments for Ca Binding to Two α-Lactalbumins

Thermodynamic parameters for the unfolding of as well as for the binding of Ca²⁺ to goat α-lactalbumin (GLA) and bovine α-lactalbumin (BLA) are deduced from isothermal titration calorimetry in a buffer containing 10 mM Tris-HCl, pH 7.5 near 25°C. Among the different parameters available, the heat capacity increments (ΔC_p) offer the most direct information for the associated conformational changes of the protein variants. The ΔC_p values for the transition from the native to the molten globule state are rather similar for both proteins, indicating that the extent of the corresponding conformational change is nearly identical. However, the respective ΔC_p values for the binding of Ca²⁺ are clearly different. The data suggest that a distinct protein region is more sensitive to a Ca²⁺-dependent conformational change in BLA than is the case in GLA. By analysis of the tertiary structure we observed an extensive accumulation of negatively charged amino acids near the Ca²⁺-binding site of BLA. In GLA, the cluster of negative charges is reduced by the substitution of Glu-11 by Lys. The observed difference in ΔC_p values for the binding of Ca²⁺ is presumably in part related to this difference in charge distribution.     

Calcium pump activity of the renal plasma membrane and renal microsomes

ATP-dependent Ca²⁺ uptake distinct from that of the mitochondria is found in both plasma membrane and microsomal membranes of rat kidney. Activity attributed to these fractions is enhanced by ammonium oxalate and is apparently insensitive to NaN₃. In contrast, rat kidney mitochondrial Ca²⁺ uptake is blocked by NaN₃. The pH of optimal activity is significantly higher for the mitochondrial fraction. Microsomal membrane Ca²⁺ uptake differs from that of the plasma membrane. Microsomal membranes are four times as active as the plasma membrane at high (5 mM) ATP levels. Apparent K_m values for Mg²⁺-ATP differ in the two preparations with a higher affinity for Mg²⁺-ATP found in the plasma membrane Ca²⁺ uptake activity of the plasma membrane preparation is readily inhibited by Na⁺. Sucrose gradient density fractionation indicates that the observed microsomal membrane Ca²⁺ pump activity is associated with membrane vesicles derived from the endoplasmic reticulum. Ca²⁺ pump activity of both plasma membrane and microsomal fraction is depressed din the adrenalectomized rat. This activity is not restored by a single natriuretic dose of aldosterone.     

A role for Ca(v)1.3 in rat intestinal calcium absorption

Active Ca²⁺ absorption through epithelial Ca²⁺ channels TRPV5/6 in duodenum is activated by hyperpolarisation. However, when diet and Ca²⁺ are plentiful, digestion products cause depolarisation. We therefore used homology-based PCR from a rat jejunal mucosal cDNA preparation to reveal the presence of the neuroendocrine L-type isoform Ca_v1.3α₁. Immunocytochemical labelling and immunoblotting localised Ca_v1.3α₁ protein in apical membrane from proximal jejunum to mid ileum. Perfusion studies in vivo with 1.25 mM luminal Ca²⁺ revealed L-type channel activity. Inhibition of glucose absorption with phloridzin strongly inhibited ⁴⁵Ca²⁺ absorption; absorption was inhibited by nifedipine and Mg²⁺ and activated by Bay K 8644, none of which affect TRPV5/6. At 10 mM Ca²⁺, nifedipine inhibited ⁴⁵Ca²⁺ absorption with a time course similar to that at 1.25 mM Ca²⁺: absorption was therefore channel-mediated rather than paracellular. We suggest that in times of dietary sufficiency, Ca_v1.3 may mediate a significant route of Ca²⁺ absorption into the body.     

Ca binding to sarcoplasmic reticulum ATPase phosphorylated by Pi reveals four thapsigargin-sensitive Ca sites in the presence of ADP

Sarcoplasmic reticulum (SR) Ca²⁺-ATPase was phosphorylated by P_i at pH 8.0 in the presence of dimethyl sulfoxide (Me₂SO). Under these conditions, it was possible to measure transient ⁴⁵Ca²⁺ binding to the phosphoenzyme. Binding reached 1.2 Ca²⁺ per phosphoenzyme (E-PCa_x) within 10 min in 30% Me₂SO, 20 mM MgCl₂ and 0.1 mM P_i and the phosphoenzyme only decreased by 23% during this period. This Ca²⁺ binding was abolished by thapsigargin, showing that it is associated with functional sites of the Ca²⁺-ATPase. At 40% Me₂SO, simultaneous addition of Ca²⁺ and ADP increased Ca²⁺ binding up to almost four Ca²⁺ per phosphoenzyme (ADPE-PCa_y), revealing a species bearing simultaneously four Ca²⁺ sites. Both E-PCa_x and ADPE-PCa_y were further identified as distinct species by (2′,3′-O-2(2,4,6-trinitrophenyl)adenosine 5′-triphosphate) fluorescence, which revealed long-range modifications in the Ca²⁺-transport sites induced by ADP binding to E-P. In addition, E-PCa_x was shown to be a functional intermediate of the cycle leading to ATP synthesis provided that Me₂SO was diluted. These findings indicate that more than two functional Ca²⁺-sites exist on the functional Ca²⁺-ATPase unit, and that the additional sites become accessible upon ADP addition. This is compatible with a four-site model of the SR Ca²⁺-ATPase allowing simultaneous binding of Ca²⁺ at lumenal and cytosolic sites. The stoichiometries for Ca²⁺ binding found here could either be interpreted as binding of four Ca²⁺ on a Ca²⁺-ATPase monomer considered as the functional unit or as binding of two Ca²⁺ per monomer of a functional dimer.     

Activatory effect of regucalcin on hepatic plasma membrane (Ca2+-Mg2+)-ATPase is impaired by liver injury with carbon tetrachloride administration in rats

The alteration of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver of rats administered orally carbon tetrachloride (CCl₄) solution was investigated. Rats received a single oral administration of CCl₄ (10, 25 and 50%, 1.0 ml/100 g body weight), and 3 or 24 h later they were sacrificed. CCl₄ administration caused a remarkable elevation of liver calcium content and a corresponding increase in liver plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity, indicating that the increased Ca²⁺ pump activity is partly involved in calcium accumulation in liver cells. Moreover, the participation in regucalcin, which is an intracellular activating factor on the enzyme, was examined by using anti-regucalcin IgG. The plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity increased by CCl₄ administration was not entirely inhibited by the presence of anti-regucalcin IgG (1.0 and 2.5 ug/ml) in the enzyme reaction mixture. However, the effect of regucalcin (0.25–1.0 uM) to activate (Ca²⁺-Mg²⁺)-ATPase in the liver plasma membranes of normal rats was not revealed in the liver plasma membranes obtained from CCl₄-administered rats. Also, the effect of regucalcin was not seen when the plasma membranes were washed with 1.0 mM EGTA, indicating that the disappearance of regucalcin effect is not dependent on calcium binding to the plasma membranes due to liver calcium accumulation. Now, the presence of dithiothreitol (5 mM) or heparin (20 ug/ml) caused a remarkable elevation of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver obtained from CCl₄-administered rats. Thus, the regucalcin effect differed from that of dithiothreitol or heparin. The present study suggests that the impairment of regucalcin effect on Ca²⁺ pump activity in liver plasma membranes is partly contribute to hepatic calcium accumulation induced by liver injury with CCl₄ administration.     

Calmodulin stimulation of calcium uptake and (Ca2+-Mg2+)-ATPase activities in microsomes from canine tracheal smooth muscle

The role of calmodulin in the regulation of microsomal ⁴⁵Ca²⁺ transport in canine tracheal smooth muscle was studied. Calmodulin stimulated ATP-dependent ⁴⁵Ca²⁺ uptake and (Ca²⁺Mg²⁺)-ATPase activities in microsomes treated with 0.5 mM EDTA and 0.5 mM EGTA. Oxalate also stimulated ATP-dependent ⁴⁵Ca²⁺ uptake and (Ca²⁺Mg²⁺)-ATPase activities and the stimulation was additive to the effects of calmodulin. The (Ca²⁺Mg²⁺)-ATPase and ATP-dependent ⁴⁵Ca²⁺ uptake activities are probably related as they exhibited similar [Ca²⁺]_free- and [calmodulin]-dependencies. These results indicate that calmodulin may play a role in the control of the cytosolic [Ca²⁺]_free in canine tracheal smooth muscle.     

Antiserum to 5alpha-dihydrotestosterone: production, characterization and use in radioimmunoassay

5α-Dihydrotestosterone (5α-DHT) was rendered antigenic by covalent attachment to bovine serum albumin (BSA) through position 1 of the steroid. Nucleophilic attack by β-mercaptopropionic acid on the 1,2-dehydro derivative of 5α-DHT yielded the corresponding 1α-thioether alkanoic acid which was coupled to bovine serum albumin by use of the carbodiimide reagent. The method should be generally applicable to 3-oxosteroids. Immunization of rabbits with 5α-DHT-1α-carboxyethyl-thioether-BSA gave rise to antisera of high affinity for 5α-DHT (K_a= 1.4 × 10⁹ 1/mol) that showed little cross reaction with 17β-hydroxy-5β-androstan-3-one (3%), and with a variety of 17-oxoandrostane compounds (≤0.5%). However the serum cross-reacted significantly with testosterone (10%) and with 5α-androstene-3α, 17β-diol (16%). A radioimmunoassay procedure for the determination of 5α-DHT in plasma is described. Chromatographic purification of the plasma extracts proved necessary for obtaining valid results. The plasma level of 5α-DHT(pg/ml; ean ± S.D.) was 364±79 (n = 7) in normal human adult males and 188 ± 62 (n = 5) in normal non-pregnant women.     

Deletions and Mutations in the Acidic Lipid-binding Region of the Plasma Membrane Ca2+ Pump: A STUDY ON DIFFERENT SPLICING VARIANTS OF ISOFORM 2*

Acidic phospholipids increase the affinity of the plasma membrane Ca²⁺-ATPase pump for Ca²⁺. They interact with the C-terminal region of the pump and with a domain in the loop connecting transmembrane domains 2 and 3 (A_L region) next to site A of alternative splicing. The contribution of the two phospholipid-binding sites and the possible interference of splicing inserts at site A with the regulation of the ATPase activity of isoform 2 of the pump by phospholipids have been analyzed. The activity of the full-length z/b variant (no insert at site A), the w/b (with insert at site A), and the w/a variant, containing both the 45-amino acid A-site insert and a C-site insert that truncates the pump in the calmodulin binding domain, has been analyzed in microsomal membranes of overexpressing CHO cells. The A-site insertion did not modify the phospholipid sensitivity of the pump, but the doubly inserted w/a variant became insensitive to acidic phospholipids, even if containing the intact A_L phospholipid binding domain. Pump mutants in which 12 amino acids had been deleted, or single lysine mutations introduced, in the A_L region were studied by monitoring agonist-induced Ca²⁺ transients in overexpressing CHO cells. The 12-residue deletion completely abolished the ATPase activity of the w/a variant but only reduced that of the z/b variant, which was also affected by the single lysine substitutions in the same domain. A structural interpretation of the interplay of the pump with phospholipids, and of the mechanism of their activation, is proposed on the basis of molecular modeling studies.     

The uptake of calcium by isolated chromaffin granules of the adrenal medulla

Bovine chromaffin secretory granules were purified by isopycnic Metrizamide gradient centrifugation and their Ca²⁺ sequestration pathways were characterized. The rate of Ca²⁺ sequestration at 37°C was first order, with a maximal uptake of 26.9 ±0.46 (mean ± S.D., n = 3) nmol Ca²⁺/mg protein and a first order rate constant (k) of 0.046 ± 0.002 min^–1. At 4°C the rate of uptake was substantially attenuated, with only 2.47 ± 0.2 (mean ± S.D, n = 3) nmol Ca²⁺/mg protein sequestered in 60 min. Ca²⁺ sequestration was 93% inhibited by 180 mM NaCl [I_50% of 78.7 ± 9.3 mM NaCl (mean ± S.D., n = 11)] but only slightly inhibited by KCl or MgCl₂. Ca ²⁺ sequestration was not stimulated by incubation with MgATP but was inhibited by 57% after incubation with 30 M monensin. Ca ²⁺ sequestration was dependent on extravesicular Ca ²⁺ with half-maximal sequestration at pCa²⁺ 6.81 ± 0.028 (mean ± S.D., n = 3). Sequestered Ca²⁺ could be exchanged with external ⁴⁵Ca²⁺, the exchange rate was first order (k of 0.042 ± 0.004: mean ± S.D., n = 3) and saturated at 27.7 ± 1.1 nmol Ca²⁺/mg (mean ± S.D., n = 3). The Ca²⁺/Ca²⁺ exchange system was totally inhibited by NaCl or KCl but only slightly by MgCl₂. About 75% of sequestered ⁴⁵Ca²⁺ could be released by incubation with NaCl, but only 8% was released by incubation with KCI. Half-maximal release of sequestered ⁴⁵Ca²⁺ required 69.3 ± 12.2 mM NaCl (mean ± S.D., n = 3). The Na⁺-induced release of sequestered ⁴⁵Ca²⁺ was rapid, t_0.5 of 2.80 ± 0.63 min (mean ± S.D., n = 3) and inhibited at 4°C. The concurrent incubation of chromaffin granules with ⁴⁵Ca²⁺ and either annexin proteins V or VI resulted in attenuated uptake of ⁴⁵Ca²⁺. These results suggest that Ca²⁺ uptake in adrenal chromaffin granules is regulated by Na⁺ and Ca²⁺ gradients and also possibly by annexins V and VI.Abbreviations EGTA ethylene glycol bis (-aminoethyl ether)-N,-N,N,N-tetraacetic acid - SDS Sodium dodecyl sulphate - PAGE Polyacrylamide gel electrophoresis - BSA bovine serum albumin - AI Annexin I - AIIt Annexin II tetramer - AIII Annexin III - AIV Annexin IV - AV Annexin V - AVI Annexin VI - k first order rate constant - AT total extent of Ca²⁺ uptake (nmol) - BufferA 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 5 mM EGTA - Buffer B 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) and 1 mM EGTA - Buffer C 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) - Buffer D 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.5 mM EGTA and 0.65 MM CaCl₂ - Buffer E 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.25 mM EGTA and 0.325 mM CaCl₂     

Effects of sex steroids on calling,locomotor activity,and sexual behavior in castrated male Japanese quail

Castrated male Japanese quail were implanted with Silastic capsules containing testosterone (T), estradiol-17β (E₂), 5β-dihydrotestosterone (5β-DHT), Δ₄-androstenedione (Δ₄) 5α-androstanedione (A), 5α-dihydrotestosterone (5α-DHT) or with empty capsules. Calling, monitored continuously and automatically, was induced significantly by T and Δ₄. Locomotor activity, also monitored continuously by floor deflection, was enhanced by T, Δ₄, and E₂. Additional data concerning heterosexual and homosexual behavior were obtained from castrated quails after implantation of T, Δ₄, E₂, or 5α-DHT. T and Δ₄ restored hetero- and homosexual behavior as did E₂ but to a lesser extent. 5α-DHT did not induce either sexual behavior. Growth of the cloacal protrusion was induced in birds implanted with T, Δ₄, A, and 5α-DHT but not with 5β-DHT and E₂. These results indicate that calling and locomotor activity enhancement (including sexual behavior) are two different components of reproductive behavior which require different androgens or their metabolites to be activated.     

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.     

Mechanism of Cu2+-induced elevation of [3H]cimetidine binding to membranes in rat brain

The mechanism of increasing effect of CuCl₂ on specific [³H]cimetidine binding was examined in brain membranes of rats. CuCl₂-Induced elevation of [³H]cimetidine binding was high in Krebs-Ringer solution (pH 7.4) compared to those in 50 mM Na, K-phosphate buffer (pH 7.4) and in 50 mM Tris-HCl buffer (pH 7.4). CaCl₂ (5–50 mM) inhibited effect of CuCl₂, but NaCl (25–200 mM), KCl (5–100 mM) or MgCl₂ (5–50 mM) did not. CuCl₂ (50 μM) elevated 9.3- and 2.5-fold the binding in phosphate- and Tris—HCl buffer, respectively. EDTA-2Na decreased the binding elevated by 50 μM CuCl₂ in phosphate buffer to the similar level in Tris-HCl buffer, whereas it did not affect those in Tris-HCl buffer. The absorption spectra of cimetidine and CuCl₂ mixture showed a peak at 317 nm in phosphate buffer that was not observed in Tris-HCl buffer. It is suggested that cimetidine-Cu²⁺ chelate complex could be formed in phosphate buffer, resulting in higher amount of binding in phosphate buffer than in Tris-HCl buffer. PdCl₂ also caused a marked elevation in [³H]cimetidine binding, seeming to be due to formation of cimetidine-Pd²⁺ chelate complex. There were two types of [³H]cimetidine binding in the presence of 20 nM PdCl₂: high affinity binding with K_d = 0.7 ± 0.1 nM and low affinity binding with K_d = 44.3 ± 3.0 nM. It is suggested that cimetidine-Cu²⁺ complex binds to cimetidine binding sites in brain with higher affinity than cimetidine alone.     

Regulation of rat brainstem tryptophan 5-monooxygenase. Calcium-dependent reversible activation by ATP and magnesium.

Tryptophan 5-monooxygenase in rat brainstem cytosol was activated about twofold by incubation with 0.5 mm ATP and 5 mm MgCl₂. The activation required micromolar concentrations of Ca²⁺ but was not dependent on either cyclic AMP or cyclic GMP. Rat brain cytosol was shown to possess an endogenous protein kinase which was markedly stimulated by the addition of Ca²⁺ using endogenous protein substrates. Following activation by ATP and Mg²⁺ in the presence of Ca²⁺, tryptophan 5-monooxygenase was reversibly deactivated to the original level by incubation at 30 °C after removal of Ca²⁺ by adding ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid and was then reactivated by incubation at 30 °C after subsequent addition of Ca²⁺ and ATP. The deactivation was markedly inhibited by the omission of Mg²⁺ or by the addition of NaF.     

Effect of extracellular adenosine 5′-triphosphate on Ca2+ efflux from freshly isolated adult rat cardiomyocytes

The effect of extracellular adenosine 5′-triphosphate (ATP) on Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes was examined. ATP stimulated the efflux of ⁴⁵Ca²⁺ from the cells in a concentration-dependent manner (0.01–1 mM). The ⁴⁵Ca²⁺ efflux from the cells was also stimulated by adenosine-5′-O-(3-thiotriphosphate) (ATP-γs) and α,β-methylene-ATP and adenosine 5′-diphosphate, but not by adenosine 5′-monophosphate and adenosine. The ATP-stimulated ⁴⁵Ca²⁺ efflux was not affected by deprivation of the extracellular Ca²⁺, but was dependent on the presence of extracellular Na⁺. These results indicate that ATP stimulates extracellular Na⁺-dependent ⁴⁵Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on the plasma membrane P₂ purinoceptors which may couple to Na⁺/Ca²⁺ exchange.     

The calmodulin-activated form of the Ca2+-pumping ATPase of the cardiac sarcolemmal membrane produces Ca2+ gradients with a thermodynamic efficiency of 100%

The thermodynamic efficiency of the calmodulin-activated form of the Ca²⁺-pumping ATPase of the bovine cardiac sarcolemma (SL) was evaluated in sealed vesicles under reversible conditions. The free internal Ca²⁺ concentration ([Ca²⁺]_i) established in the SL vesicle lumen by action of the ATPase was determined as a function of the [ATP]/([ADP][P_i]) ratio for the following experimental conditions: 250mM sucrose, 100mM KCI, 0.1mM Mg²⁺, 25mM HEPES, 25mM Tris, pH 7.40, at 37°C, [Ca²⁺]_o=50nM (1mM Ca/EGTA buffer), 0.75mM Mg-ATP, 0.1mM P_i, variable [ADP]. Under these conditions, with the pump working near itsK _m of 64nM, the [Ca²⁺]_i achieved was 18mM, decreasing with increasing [ADP] for [ADP] 0.84mM. A plot of the square of the [Ca²⁺]_i/[Ca²⁺]_o ratio against [ATP]/([ADP][P_i]) gave a straight line with a slope of 1.5×10⁷M. This was in agreement, within the experimental error, with the equilibrium constant for ATP hydrolysis under these conditions (1.09×10⁷M). These results demonstrate (1) tight coupling between Ca²⁺ transport and ATP hydrolysis with a stoichiometry of 2 Ca²⁺ moved per ATP split and (2) a low degree of passive leakage. Analysis at low [ADP] (<0.83mM) showed the unexpected result that ADP increases the rate of theforward reaction of the pump. The maximal effect on the initial rate is a 96±5% increase, with an EC₅₀ of approximately 0.4mM (ADP). Similar but lesser stimulation was observed with CDP. The implications of the above results for the energetics of the pump and for its physiological function in the beating heart are discussed.