Copper-Ligand Interactions and Physiological free Radical Processes. Part 2. Influence of Cu2+ Ions on Cu+-Driven Oh Generation and Comparison with Their Effects on Fe2+-Driven Oh Production

In our search to establish a reference ·OH production system with respect to which the reactivity of copper(II) complexes could then be tested, the influence of free Cu²⁺ ions on the Cu⁺/H₂O₂ reaction has been investigated.

This influence depends on the C_Cu²⁺/C_Cu⁺ ratio. At low Cu²⁺ concentrations, ·OH damage to various detector molecules decreases with increasing Cu²⁺ concentrations until C_Cu²⁺/C_Cu⁺ reaches unity. Above this value, ·OH damage increases sharply until C_Cu²⁺/C_Cu⁺ becomes equal to 5 with salicylate and 2 with deoxyribose, ratios for which the protective effect of Cu²⁺ cancels. Finally, at higher concentrations, Cu²⁺ ions logically add their own ·OH production to that normally expected from Cu⁺ ions. The possible origin of this unprecedented alternate effect has been discussed. The possible influence of Cu⁺ ions on the generation of ·OH radicals by water gamma radiolysis has also been tested and, as already established for Cu²⁺ in a previous work, shown to be nonexistent. This definitely confirms that either form of ionised copper cannot scavenge ·OH radicals in the absence of a Iigand.     

A label-free DNAzyme sensor for lead(II) detection by quantitative polymerase chain reaction

A label-free sensor was developed for sensitive detection of lead(II), combining high selectivity of a Pb²⁺-dependent DNAzyme with enormous signal amplification of quantitative polymerase chain reaction (QPCR). Specifically, a substrate strand was designed to have two primer-hybridization sequences at either terminus. The presence of lead ion (Pb²⁺) catalyzed cleavage of the substrate strands. This resulted in a concentration decrease of the substrate strand that could be detected by QPCR. Compared with existing DNAzyme-based protocols for Pb²⁺ assay, this strategy circumvented the use of various optical or electrical labels that might be difficult to be synthesized. Also, the incorporation of QPCR furnished our approach with high sensitivity and superb reproducibility. In addition, QPCR allowed an immediate quantification of the cleavage efficiency that could be useful for evaluation of the DNAzyme activity. The results obtained revealed that our approach exhibited a dynamic response toward Pb²⁺ within a three-decade concentration range from 10 nM to 5 μM with a detection limit of 1 nM. This approach also demonstrated good selectivity against other metal ions that commonly coexisted with Pb²⁺.     

Biosorption Performance of Multimetal Resistant Fungus Penicillium chrysogenum XJ-1 for Removal of Cu2+ and Cr6+ from Aqueous Solutions

Adsorption for heavy metals via biomaterials such as fungal biomass presents a practical remediation technique for polluted water. Among all known filamentous fungi, Penicillium chrysogenum is widespread in nature and can serve as a biosorbent for heavy metals. In the current study, the ability of P. chrysogenum XJ-1 to remove copper (Cu²⁺) and chromium (Cr⁶⁺) from water was evaluated. The maximum biosorption capacity of XJ-1 for Cu²⁺ reached 42.83 ± 0.57 mg g^?1 dry biomass at pH 5.0 after the equilibrium time of 1.5 h. The maximum biosorption capacity for Cr⁶⁺ at pH 3.0 reached 52.69 ± 1.68 mg g^?1 dry biomass after the equilibrium time of 1.5 h. The biosorption data of XJ-1 biomass were well fitted to the Freundlich isotherm model and the pseudo-second-order Lagergren kinetic model. Laundry powder-treated and HCl-treated XJ-1 biomass significantly enhanced its adsorption capacity to Cu²⁺ and Cr⁶⁺, respectively. HCl and NaOH were suitable desorbents for Cu²⁺/Cr⁶⁺ loading biomass, respectively. Fourier transform infrared spectroscopy analyses revealed that hydroxyl, amine, and sulfonyl groups on the biosorbent contributed to binding Cu²⁺ and Cr⁶⁺ and that carbonyl and carboxyl groups were also vital binding sites of Cu²⁺. Scanning electron microscopy and energy-dispersive x-ray (SEM-EDX) analyses confirmed that considerable amounts of metals were precipitated on the cell surface of XJ-1. Our results suggested that XJ-1 might be used to purify multimetal-contaminated water. This low-cost and eco-friendly biomass of XJ-1 seems to have a broad use in the restoration of metal-contaminated water.     

Properties of a Zn2+-glycerophosphocholine cholinephosphodiesterase from bovine brain membranes

A Zn²⁺-glycerophosphocholine cholinephosphodiesterase was purified with a specific activity of 4.6 μmole/min·mg protein from bovine brain membranes by procedures involving PI-PLC solubilization, concanavalin A affinity chromatography, CM-sephadex chromatography and Sephadex G-150 chromatography. Based on molecular weight determination gel chromatography and SDS polyacrylamide gel electrophoresis, the phosphodiesterase activity appears to be a dimeric protein (110 kDa) composed of two subunits with a molecular weight of approximately 54 kDa. The Km value for p-nitrophenylphosphocholine and the optimum pH were found to be 16 μM and pH 10.5, respectively. The phosphodiesterase was inhibited by Cu²⁺, but not the other divalent metal ions. The activity of the apoenzyme was remarkably activated by Co²⁺ or Zn²⁺, but not Mn²⁺ or Mg²⁺. In addition, the inactivation of the enzyme in glycine buffer was prevented by Mn²⁺ or Zn²⁺, but not Co²⁺ or Mg². In a separate experiment, comparing properties of the purified and membrane-bound phosphodiesterases, the forms of two enzymes were quite similar except in stability. Both enzymes were more stable at pH 7.4 than pH 5 or 10. However, the membrane-bound enzyme was more stable than the soluble enzyme at all three pHs. These data suggest that the activity of the phosphodiesterase may be stabilized in-vivo.     

Model of active transport of ions in cardiac cell

A model of the active transport of ions in a cardiac muscle cell, which takes into account the active transport of Na⁺, K⁺, Ca²⁺, Mg²⁺, HCO₃⁻ and Cl⁻ ions, has been constructed. The model allows independent calculations of the resting potential at the biomembrane and concentrations of basic ions (sodium, potassium, chlorine, magnesium and calcium) in a cell. For the analysis of transport processes in cardiac cell hierarchical algorithm “one ion-one transport system” was offered. The dependence of the resting potential on concentrations of the ions outside a cell has been established. It was shown, that ions of calcium and magnesium, despite their rather small concentration, play an essential role in maintenance of resting potential in cardiac cell. The calculated internal concentrations of ions are in good agreement with the corresponding experimental values.     

Effect of lanthanum ions (La3+) on the reactive oxygen species scavenging enzymes in wheat leaves

Physiological effects of lanthanum ions on the activities of the enzymes in the reactive oxygen species (ROS) scavenging system in leaves of wheat (Triticum aestivum L.) seedlings were studied. Wheat leaves treated in Hogland solution with 0.1 mM LaCl₃ for 48 h showed increased levels of superoxide dismutase (SOD), catalase (CAT), ascorbate-specific peroxidase (AsA-POD), and dehydroascorbate reductase (DHAR). However, a minor effect was observed on the levels of monodehydroascorbate reductase (MDAR) and glutathione reductase (GR), which regulate the release of energy required by the ROS scavenging system. The whole system was linked up by H⁺ transmission. Our results indicated that the activities of the enzymes that function directly to remove ROS were elevated by La³⁺ treatment, which is consistent with the observations that La³⁺-treated plants had increased tolerance to environmental stresses. The remaining levels of MDAR and GR suggested that these two enzymes might be regulated differently from that of the other four enzymes studied.     

Mono- and bis-N-functionalised cyclen with benzimidazolylmethyl pendant arms: Sensitive and selective fluorescent probes for zinc and copper ions

The Zn²⁺ and Cu²⁺ complexes of L¹ and L² ligands (L¹: 1-(benzimidazol-2-ylmethyl)-1,4,7,10-tetraazacyclododecane, L²: 1,7-bis(benzimidazol-2-ylmethyl)-1,4,7,10-tetraazacyclododecane) were synthesised and characterised by means of NMR, EPR, and UV-Vis spectroscopies, X-ray determination and molecular modelisation (HF-DFT(B3LYP)/LANL2DZ). These studies showed that the 1:1 complexes were formed in which the benzimidazole arm(s) are coordinated to the metal ion. On addition of successive amounts of Zn²⁺ in CH₃CN, the fluorescence emission of L¹ increased linearly by a factor of 50 and the one of L² by a factor of 5 while on addition of successive amounts of Cu²⁺ in CH₃CN, the fluorescence emission of L² decreased linearly to 80% of its initial value.     

Effect of Cu2+-ascorbic acid on lipid peroxidation,Mg2+-ATPase activity and spectrin of RBC membrane and reversal by erythropoietin

The effect of erythropoietin (Ep), a glycoprotein hormone, has been studied on lipid peroxidation induced by Cu²⁺ and ascorbate in vitro, Mg²⁺ ATPase activity and spectrin of RBC membrane. Our present investigation reveals that Cu²⁺ and ascorbic acid increases lipid peroxidation of RBC membrane significantly. It has further been observed that under the same experimental condition spectrin, a major cytoskeleton membrane protein, and Mg²⁺-ATPase activity of RBC membrane decrease significantly. However, exogenous administration of Ep completely restores lipid peroxidation and Mg²⁺-ATPase activity and partially recovers spectrin of RBC membrane.     

Preconcentration with Bacillus subtilis–Immobilized Amberlite XAD-16: Determination of Cu2+ and Ni2+ in River,Soil, and Vegetable Samples

Solid-phase extraction (SPE) method was developed for the preconcentration of Cu²⁺ and Ni²⁺ before their determination by inductively coupled plasma optical emission spectrometry (ICP-OES). Bacillus subtilis–immobilized Amberlite XAD-16 was used as biosorbent. Effects of critical parameters such as pH, flow rate of samples, amount of Amberlite XAD-16 and biosorbent, sample volume, eluent type, and volume and concentration of eluent on column preconcentration of Cu²⁺ and Ni²⁺ were optimized. Applicability of the method was validated through the analysis of the certified reference tea sample (NCS ZC73014). Sensitivity of ICP-OES was improved by 36.4-fold for Cu²⁺ and 38.0-fold for Ni²⁺ by SPE-ICP-OES method. Limit of quantitation (LOQ) was found to be 0.7 and 1.1 ng/ml for Cu²⁺ and Ni²⁺, respectively. Concentrations of Cu²⁺ and Ni²⁺ were determined by ICP-OES after application of developed method. Relative standard deviations (RSDs) were lower than 4.9% for Cu²⁺ and 7.9% for Ni²⁺. The Tigris River that irrigates a large agricultural part of Southeast Turkey is polluted by domestic and industrial wastes. Concentrations of Cu²⁺ and Ni²⁺ were determined in water, soil, and some edible vegetables as a biomonitor for heavy metal pollution.     

Synthesis, structure and properties of unsymmetrical μ-alkoxo-dicopper(II) complexes: biological relevance to phosphodiester and DNA cleavage and cytotoxic activity

The unsymmetric dinucleating ligand N-(2-hydroxybenzyl)-N,N′,N′-tris(2-pyridylmethyl)-1,3-diaminopropan-2-ol (L = H₂btppnol) and the corresponding copper(II) complex [Cu₂(Hbtppnol)(μ-CH₃COO)](ClO₄)₂ (1) have been recently reported in part in a short communication [Inorg. Chem. Commum. 8 (1999) 334]. In this study, we investigated the ability of complex 1 to promote the hydrolysis of P-O phosphate diester bonds in bis(2,4-dinitrophenyl) phosphate (2,4-BDNPP) and the cleavage of genomic and plasmid DNA molecules. Reaction of 1 with excess of the diester 2,4-BDNPP, at pH 7.0, results in the formation of the monoester phosphate coordinated [Cu₂(Hbtppnol)(μ-((NO₂)₂-C₆H₃)PO₄)]ClO₄ (3) complex, which was also characterized by X-ray crystallography. In addition, the stable μ-phosphate complex [Cu₂(Hbtppnol)(μ-(NO₂-C₆H₄)PO₄)](ClO₄) (2) obtained from the reaction of 4-nitrophenyl phosphate with complex 1 was also characterized by X-ray crystallography, indicating that 1 is unable to cleave monoester-phosphate bonds. The kinetics for the promotion of bis(2,4-dinitrophenyl) phosphate (2,4-BDNPP) hydrolysis by complex 1 was investigated as a function of pH, catalyst concentration and substrate concentration. On the basis of kinetic and potentiometric studies, the deuterium isotope effect (k^H/k^D ∼ 1) and the X-ray structure of the monoester phosphate coordinated [Cu₂(Hbtppnol)(μ-((NO₂)₂-C₆H₃)PO₄)]ClO₄ (3) complex as the product of the reaction, we demonstrated that the aquo/hydroxo complex is the active species and the reaction occurs through the formation of a ternary complex in which one Cu^II binds the substrate and the second copper center has a terminal bound hydroxide to attack the phosphorus atom, at physiological pH. A rate enhancement factor of ∼100 was calculated relative to that measured for the uncatalyzed reaction under identical conditions. Complex 1 effectively promotes the cleavage of double-stranded genomic and plasmid DNA, at physiological pH, probably through a hydrolytic mechanism in agreement with that proposed for the reaction of 1 with 2,4-BDNPP. Finally, cytotoxic activity of 1 in a human small cell lung carcinoma cell line (GLC4) and its cisplatin resistant subline (GLC4/CDDP) was studied and the IC₅₀ values were determined.     

Chitosan signaling in guard cells requires endogenous salicylic acid

An elicitor chitosan (CHT) induces stomatal closure but the mechanism remains to be clarified. A phytohormone salicylic acid (SA) is crucial for elicitor-induced defense signaling in plants. Here we investigated whether endogenous SA is required for CHT signaling in guard cells. In the SA-deficient nahG mutant, treatment of CHT did not induce either apoplastic reactive oxygen species (ROS) production or stomatal closure but co-treatment of CHT and SA induced both apoplastic ROS production and stomatal closure, indicating the involvement of endogenous SA in CHT-induced apoplastic ROS production and CHT-induced stomatal closure. Furthermore, CHT induced transient cytosolic free calcium concentration increments in the nahG mutant in the presence of exogenous SA but not in the absence of exogenous SA. These results provide evidence that endogenous SA is a crucial element in CHT-induced stomatal closure.     

Calcium and copper transport ATPases: analogies and diversities in transduction and signaling mechanisms

The calcium transport ATPase and the copper transport ATPase are members of the P-ATPase family and retain an analogous catalytic mechanism for ATP utilization, including intermediate phosphoryl transfer to a conserved aspartyl residue, vectorial displacement of bound cation, and final hydrolytic cleavage of Pi. Both ATPases undergo protein conformational changes concomitant with catalytic events. Yet, the two ATPases are prototypes of different features with regard to transduction and signaling mechanisms. The calcium ATPase resides stably on membranes delimiting cellular compartments, acquires free Ca²⁺ with high affinity on one side of the membrane, and releases the bound Ca²⁺ on the other side of the membrane to yield a high free Ca²⁺ gradient. These features are a basic requirement for cellular Ca²⁺ signaling mechanisms. On the other hand, the copper ATPase acquires copper through exchange with donor proteins, and undergoes intracellular trafficking to deliver copper to acceptor proteins. In addition to the cation transport site and the conserved aspartate undergoing catalytic phosphorylation, the copper ATPase has copper binding regulatory sites on a unique N-terminal protein extension, and has also serine residues undergoing kinase assisted phosphorylation. These additional features are involved in the mechanism of copper ATPase intracellular trafficking which is required to deliver copper to plasma membranes for extrusion, and to the trans-Golgi network for incorporation into metalloproteins. Isoform specific glyocosylation contributes to stabilization of ATP7A copper ATPase in plasma membranes.     

Intracellular Ca can compensate for the lack of NADPH oxidase-derived ROS in endothelial cells

The aim of the present study is to determine the role of intracellular Ca²⁺ in VEGF signaling. We demonstrate that reduction in Ca²⁺ by chelating compound BAPTA-AM or by IP₃-endoplasmic reticulum blocker 2-APB selectively inhibited VEGF-induced activation of c-Src-PI3K-Akt but not ERK1/2 in human coronary artery endothelial cells (HCAEC). We also show that the selective inhibitory effects of NADPH oxidase knockdown on VEGF-mediated activation of c-Src-PI3K-Akt signaling and cell proliferation in HCAEC can be reversed by increase in intracellular Ca²⁺. These results suggest an essential role for Ca²⁺ in redox-dependent selective activation of c-Src-PI3K-Akt and endothelial cell proliferation.     

重金属Cd2+和Cu2+胁迫下泥蚶消化酶活性的变化

为了探讨重金属Cd²⁺和Cu²⁺胁迫对泥蚶消化酶活性的影响,运用酶学分析的方法,按《渔业水质标准》（GB 11607）规定的Cd²⁺、Cu²⁺最高限量值的1、2、5、10倍设置重金属离子Cd²⁺、Cu²⁺浓度及其组合,研究了在重金属Cd²⁺、Cu²⁺胁迫下,30d内泥蚶3种消化酶活性的变化规律。结果表明：与空白对照组相比,在重金属Cd²⁺、Cu²⁺或其组合的胁迫下,较低浓度组泥蚶的淀粉酶活性实验前期增强（即被诱导）,实验后期减弱（即被抑制）,较高浓度组泥蚶的淀粉酶活性从实验一开始就减弱,并保持在较低水平,毒性比较,同一重金属高浓度 > 低浓度,不同重金属及其组合Cu²⁺ > （Cd²⁺+Cu²⁺）组合 > Cd²⁺;泥蚶脂肪酶的活性实验前期增强,实验后期转为微减弱或减弱,毒性比较,同一重金属高浓度 > 低浓度,不同重金属及其组合（Cd²⁺+Cu²⁺）组合 > Cu²⁺ > Cd²⁺;泥蚶胃蛋白酶的活性实验前期增强,且活性呈现升高-降低-再升高-再降低的变化,实验后期分别表现微增强、微减弱和减弱,毒性比较,同一重金属高浓度 > 低浓度,不同重金属及其组合（Cd²⁺+Cu²⁺）组合 > Cu²⁺ > Cd²⁺。可见：环境中的Cd²⁺和Cu²⁺对泥蚶的消化酶活性起着明显的影响作用。     

Use of EPR spectroscopy to check the changes in organism radioresistance. Clinical results

It has been shown that changes in Fe³⁺-transferrin and Cu²⁺-ceruloplasmin pools, which are reliably checked by the EPR technique in whole blood, blood plasma, and serum, as well as changes in the extracellular DNA content in blood plasma are markers of changes in organism radioresistance. This has been proved during the medical examination of the Chernobyl liquidators and civilian population, including children, exposed to low-intensity radiation, as well as during clinical investigation of new radioprotectors.     

活性氧清除剂和钙离子通道阻断剂对人淋巴细胞化学发光的效应

本文报道了活性氧(ROS)清除剂——苯甲酸钠、维生素C、甘露醇、L-组氨酸、过氧化氢酶和超氧化物歧化酶对Con A诱导的人外周血淋巴细胞化学发光(Ly-CL)均有抑制效应,提示人Ly-CL与ROS的生成有关,参与人Ly-CL的ROS类型有·OH、¹O₂、H₂O₂和O₂^-_·。钙通道阻断剂——Verapamil对人Ly-CL也有抑制效应,表明人Ly-CL依赖于人淋巴细胞内钙离子浓度的增加。     

Effect of hypercholesterolemia on myocardial function in New Zealand white rabbits

Although hypercholesterolemia is a well-known risk factor for atherosclerosis, little is known about the effect of hypercholesterolemia on cardiac contractile function. The objective of this study was to examine the effect of hypercholesterolemia on myocardial contractility. Fifteen New Zealand white rabbits were fed standard chow (control group) and another 15 were fed a cholesterolenriched diet (HC group) for 12 weeks. The contractile response of ventricular muscle strips was measured in various extracellular calcium concentrations and at different pacing rates. The whole-cell calcium current recording, and mRNA and protein levels of cellular calcium-handling proteins were also analyzed. With 2 mM Ca²⁺ and stimulation at 3 Hz, the contractile force of HC strips was less than that of the controls (3.63±0.20 vs. 4.61±0.50 mN, p<0.05). The time to peak tension was longer for HC strips (93.3±2.16 vs. 82.2±2.81 ms, p < 0.05). The peak L-type calcium inward current density was slightly higher in HC myocytes but did not reach statistical significance (–14.90±0.94 vs. –12.44±0.84 pA/pF, p=0.15). The mRNA level of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA), normalized to GAPDH, was significantly lower in the HC than that in the control group (2.85±0.14 vs. 7.67±0.67, p<0.05), as was the ryanodine receptor (RyR; 0.42±0.06 vs. 0.71±0.13, p<0.05). The mRNA of the Na⁺/Ca²⁺ exchanger (NCX) was statistically higher in the HC group (0.90±0.12 vs. 0.48±0.05, p<0.05). Western blot experiments revealed that protein expression of SERCA in the HC strips decreased, but that of the NCX increased. The protein expression of the dihydropyridine receptor was similar between these two groups. We concluded that hypercholesterolemia results in suppression of the maximal contractile function and in a longer systolic contractile time course. These changes may partially be mediated through a decrease in SERCA and RyR but an increase in NCX expression.     

The whole hexapeptide repeats domain from avian PrP displays untypical hallmarks in aspect of the Cu2+ complexes formation

Prions, the infectious agents responsible for the transmissible spongiform encephalopathies (TSEs) have defied full characterization for decades. Although the interactions of Cu(2+) ions with PrP both in vivo and in vitro are well documented, there are still a lot of ambiguities concerning the biological and chemical nature of these effects. In this work, we have investigated the interactions of Cu(2+) ions with whole repeat region of the copper-binding domain (hexapeptide repeats) of chicken PrP. Our results provide explanations for the structural and chemical basis of the specific interactions of Cu(2+) ions with the hexapeptide repeat region. Furthermore, we show that SOD-like activity depends on Cu(2+) complexes.     

Purification and partial characterization of a novel phosphodiesterase from the venom of Trimeresurus stejnegeri: inhibition of platelet aggregation

The phosphodiesterases (PDEs) are a superfamily of enzymes that have multiple roles in extracellular nucleotide metabolism and in the regulation of nucleotide-based intercellular signaling. Here we describe for the first time the isolation and partial characterization of a novel phosphodiesterase from Trimeresurus stejnegeri venom, named TS-PDE, using ion exchange and gel filtration chromatography. The purified TS-PDE is shown to be homogeneous as judged by SDS-PAGE and capillary isoelectric focusing. TS-PDE is a glycoprotein which contains 2.48% carbohydrate. Unlike other PDEs which are usually single polypeptide chain proteins with isoelectric points between 7.5 and 10.5, TS-PDE is a disulfide-linked heterodimer with an isoelectric point of 5.1 and a molecular mass of 100 kDa. The N-terminal amino acids of two chains are valine and serine, respectively. Furthermore, among all identified PDEs, only TS-PDE contains both of endogenous Cu²⁺ and Zn²⁺ which are essential for its phosphodiesterase activity. The purified TS-PDE exhibits broad phosphodiesterase substrate range with the order of specificity: nicotinamide guanine dinucleotide > ATP > nicotinamide adenine dinucleotide > ADP. The purified TS-PDE shows an exonuclease activity and no contamination with either alkaline phosphatase or 5′-nucleotidase activity. TS-PDE strongly inhibits ADP-induced platelet aggregation in human platelet-rich plasma by hydrolyzing ADP. Altogether, these results indicate that the novel TS-PDE is a unique phosphodiesterase with different structure from the known PDEs.     

Hormone-stimulated Mg(2+) accumulation into rat hepatocytes: a pathway for rapid Mg(2+) and Ca(2+) redistribution

Many diseases such as cardiac arrhythmia, diabetes, and chronic alcoholism are associated with a marked decrease of plasma and parenchymal Mg(2+), and Mg(2+) administration is routinely used therapeutically. This study uses isolated rat hepatocytes to ascertain if and under which conditions increases in extracellular Mg(2+) result in an increase in intracellular Mg(2+). In the absence of stimulation, changing extracellular Mg(2+) had no effect on total cellular Mg(2+) content. By contrast, carbachol or vasopressin administration promoted an accumulation of Mg(2+) that increased cellular Mg(2+) content by 13.2 and 11.8%, respectively, and stimulated Mg(2+) uptake was unaffected by the absence of extracellular Ca(2+). Mg(2+) efflux resulting from stimulation of alpha- or beta-adrenergic receptors operated with a Mg(2+):Ca(2+) exchange ratio of 1. These data indicate that cellular Mg(2+) uptake can occur rapidly and in large amounts, through a process distinct from Mg(2+) release, but operating only upon specific hormonal stimulation.