[Membrane testosterone receptors in cultured vascular smooth muscle cells].

OBJECTIVE: To determine the presence of membrane testosterone receptors in cultured vascular smooth muscle cells (VSMC), and investigate their relationship with classical intracellular androgen receptors (iAR). METHODS: VSMCs were cultured from the thoracic aorta of male Sprague-Dawley rats by the explant method. Subconfluent VSMCs were incubated with serum-free medium for 24 h to obtain quiescent non-dividing cells, and then treated with the indicated agents. The aliquots of VSMCs were labeled with testosterone-BSA-FITC (T-BSA-FITC) and analyzed by flow cytometry. Classical iARs in intact- and permeabilized-cells were detected with anti-iAR antibodies and FITC-labeled secondary antibodies by immunofluorescence, followed by flow cytometry analysis. RESULTS: Incubation of VSMCs with T-BSA-FITC obviously increased their relative fluorescence intensity at 10 sec as compared with the untreated controls (P < 0.01), and so did it at 10 min in comparison with the treatment with BSA-FITC alone or together with free testosterone (P < 0.01). Pretreatment with iAR antagonist flutamide exhibited no significant influence on the relative fluorescence intensity of VSMCs (P = 0.318). Traditional iARs were not detectable on the surface of intact VSMCs, although permeabilized cells contained iARs. CONCLUSION: VSMCs contain testosterone receptors in the plasma membrane, and these membrane receptors are not identical to classical iARs.

[Testosterone at physiological level inhibits PGF2alpha-induced increase in intracellular Ca2+ in cultured vascular smooth muscle cells].

OBJECTIVE: To explore the acute effects of testosterone at the physiological level on PGF2alpha-induced increase in intracellular Ca2+ in cultured vascular smooth muscle cells (VSMCs). METHODS: VSMCs from the thoracic aorta of male Sprague-Dawley rats were cultured using the explant method. The subconfluent VSMCs were incubated with serum-free medium for 24 hours to obtain quiescent non-dividing cells and then treated with the indicated agents. For the measurement of [Ca2+]i, the VSMCs were loaded with fura-2. Changes of [Ca2+]i were determined ratiometrically with a Nikon TE-2000E system. RESULTS: The resting level of [Ca2+]i was around 100 nmol/L in the VSMCs. Exposing cells to perfusate containing 10 micromol/L PGF2alpha triggered an immediate and transient peak in [Ca2+]i, which gradually decreased afterwards. Interference at the peak with the physiological concentration (40 nmol/L) of testosterone significantly decreased the peak-to-baseline time of [Ca2+]i, compared with ethanol vehicle (104.9 +/- 27.0 s vs 153.5 +/- 40.4 s, P < 0.01). Pretreatment with testosterone at 40 nmol/L for 2 minutes also reduced the peak-to-baseline time of [Ca2+]i significantly in comparison with the ethanol control (120.6 +/- 32.0 s vs 151.4 +/- 27.4 s, P < 0.01), but it had no significant effect on the peak level of PGF2alpha-induced intracellular Ca2+ (390.0 +/- 126.0 nmol/L vs 403.4 +/- 160.7 nmol/L, P > 0.05). CONCLUSION: Testosterone at physiological concentration inhibits PGF2alpha-induced Ca2+ fluxes, probably via receptor-operated calcium channels by a non-genomic mechanism in VSMCs, which may be involved in the vasodilatory effect of testosterone.

[Regulation of androgen receptor mRNA expression by testosterone in cultured vascular smooth muscle cells].

OBJECTIVE: To investigate the effects of testosterone exposure on androgen receptor (AR) mRNA expression in cultured vascular smooth muscle cells (VSMCs). METHODS: VSMCs were cultured from the thoracic aorta of male SD rats using explant method. The total RNA was extracted by one-step guanidine isothiocyanate method and subjected to Northern blotting analysis for determining AR mRNA level. The effect of testosterone on the viability and growth of VSMCs were studied by means of cell counting and tritiated thymidine incorporation assay. RESULTS: Testosterone treatment of the synchronized VSMCs for 24 h increased intracellular AR mRNA expression in a dose-dependent manner, with relative mRNA level of 97.67+/-7.22, 98.00+/-13.58, 143.33+/-10.99, 177.67+/-14.62 and 185.67+/-19.97 corresponding to testosterone doses of 0, 4 nmol/L, 40 nmol/L, 400 nmol/L and 4 micromol/L, respectively. Incubation of synchronized VSMCs with testosterone at a physiological level of 40 nmol/L for 24 h resulted in a mean of 30% up-regulation of AR mRNA level, compared with that of untreated cells. During AR up-regulation, testosterone had no significant effects on the cell number and DNA synthesis of VSMCs as measured by cell counting and tritiated thymidine incorporation assay. CONCLUSION: Self-initiated up-regulation of AR mRNA expression occurs in synchronized VSMCs, which is independent of testosterone that influences apoptosis or growth rate of the cells, suggesting the involvement of AR in androgen regulational at the transcription level in VSMCs.

[Testosterone regulation of androgen receptor protein expression in cultured vascular smooth muscle cells].

OBJECTIVE: To investigate the effects of testosterone exposure on androgen receptor (AR) protein expression in vascular smooth muscle cells (VSMC) and the possible mechanisms mediating the effects. METHODS: VSMC were cultured from thoracic aorta of male Sprague-Dawley rats by using the explant method. Cytoplasmic and nuclear extracts were prepared by means of cell lysis and high salt extraction respectively, and subjected to western blotting analysis for determination of AR protein level. RESULTS: Treatment of synchronized VSMC with testosterone increased both cytoplasmic and nuclear AR protein expression in a dose (0 - 4 micro mol/L) and time (1 - 48 h)-dependent fashion, whereas exposure of VSMC to testosterone at a physiologically relevant concentration of 40 nmol/L for 10 min induced a transient down-regulation of AR protein. Pretreatment with transcription inhibitor actinomycin D and translation inhibitor cycloheximide repressed cytoplasmic AR protein leves to 46% and 12% (means) of the androgen treatment control level respectively. Furthermore, androgen up-regulation of intracellular AR protein was partially inhibited (50%) by nonsteroidal androgen antagonist, flutamide. CONCLUSION: Homologous up-regulation of AR protein expression exist in synchronized VSMC, and the auto-regulation is time and testosterone dose dependent, accompanied by nuclear translocation of AR protein, and requires functional AR protein. In addition, our present data suggest that testosterone increases AR protein expression in VSMC at the level of both transcription and translation.

Dimerization A Key Mechanism of Receptor Tyrosine Kinase Activation.

Receptor tyrosine kinase superfamily includes a group of growth factor receptors with intrinsic tyrosine kinase activity which share similar molecular structure. Their ligand-induced activation is mediated primarily by the receptor dimerization. It was proposed that ligand-induced homo- or hetero- dimerization is essential for the activation of receptor kinase however, the mechanisms of receptor dimerization are different. This review introduces the ligand-induced dimerization of different receptor tyrosine kinase subclasses and concentrates on heterodimerization between members of EGF receptor family, resulting the various cellular signaling pathways.

Synthesis of Human Proinsulin C-peptide and Preparation of Specific Antibody to It.

A HPLC and CE pure human proinsulin C-peptide was synthesized by solid-phase method and TSK column purification. Its amino acid sequence and MS were consistent with theoretical values. In comparison with the formly reported chemical synthesis of C-peptide, this method has the advantage of simplicity and higher overall yield (41%). To improve the immunogenicity and specificity of oligopeptide antibody, the acrylyl-C-peptides were transformed into a polymer the product had a poly-propionyl-core matrix with C-peptide branches. This treatment gave a macromolecule with a M(r) about 25 kD. By using the polymer to immunize New Zealand rabbits for 30 days, specific antiserum was obtained with titer of 2.5x10(4) (by ELISA), which did not cross react with BSA. Thus, the poly-propionyl-peptide system provided a new approach for preparing synthetic peptide antibody and therefore is promising for the preparation of synthetic peptide-based vaccine.

Mitochondria and Apoptosis.

Apoptosis is an important biological process it plays critical role in cell growth differentiation as well as the response toward outside stimulus. Recently it was found that the mitochondrial transmembrane potential and mitochondrial permeability transition have a remarkable role in the process of apoptosis. Hypothesis of a permeability transition pore complex located in the mitochondrial membranes was postulated and had got extensive interest. This subject was introduced in the present minireview.

Interaction of Apolipoprotein Model Peptides with Liposomes: A High Performance Liquid Chromatography Study.

The apparent partition constants of two amphiphilic peptides, Amp1 and Amp2, for partitioning into phosphatidylglycerol/phosphatidylcholine bilayer were measured using size-exclusion high performance liquid chromatograph. The exposed amino groups of vesicle-bound peptides were studied by TNBS assay. It was proposed that their N-terminals were exposed to the aqueous phase, and that the main explanation for the stronger interaction of Amp1 with lipid bilayer compared with Amp2 was its stronger lipid binding ability, though Amp1 was also buried deeper in the lipid membrane. It was also found that the two peptides were polymerized in buffer, with their amino groups almost totally buried within the polymers.

Kinetic Properties of Choline Dehydrogenase.

The kinetic behavior of purified CDH had been investigated by steady-state initial velocity studies and inhibition studies with products. Variations in the concentration of one substrate led to changes in the K(m) and V(max) for the other substrate. The product betaine aldehyde was a noncompetitive inhibitor with respect to choline, whereas it competed with PMS. The results were consistent with a Bi-Bi Ping-Pong mechanism. 1-PC (1-pyrenebutyrylcholine bromide) and 9-AC (9-anthrolcholine bromide) behaved as mixed inhibitors, with K(i) values of 0.3 mM and 3.67 mM respectively.

Studies on the Spectral Properties of Choline Dehydrogenase.

The addition of the substrate didn't show any influence on the intrinsic emission spectra of purified CDH, which had a maximum at 335 nm. On the other hand, the 520 nm fluorescence of CDH increased after the addition of substrate. The secondary structure of solubilized CDH was examined by Fourier-transform infrared spectroscopy. The percentage distribution of its secondary structure assignment had been obtained: 53.4% alpha-helix, 24.5% beta-sheet, 13.9% 3(10)-helix and 0.5% beta-turn. The predominant conformation of CDH was alpha-helix and beta-sheet when there was no substrate. After the addition of substrate, the percentage of 3(10)-helix structure increased to 42%, whereas that of alpha-helix structure decreased to 35%, indicating that the conformation of CDH changed significantly after the binding of substrate of substrate to the enzyme.

Interaction of the Apolipoprotein A-I Model Peptides with Liposomes.

Two amphiphilic peptides, Amp1 and Amp2, were synthesized according to the sequence of the lipid-binding domain in apolipoprotein. Amp2 has a Val residue substituted for the Lys at the 4th position of Amp1. Intrinsic fluorescence spectra, peptide-induced leakage of calcein-laoded liposomes, quenching of tryptophan fluorescene by iodide and acrylamide, circular dichroism spectra, and measurement of the membrane penetration depth of tryptophan residue with spin-labeled phospholipids indicate unexceptionally that Amp1 interacted more strongly with the lipid bilayer than Amp2. It is proposed that class A amphiphilic alpha-helix is buried in the membrane in such a way that its long axis is oriented parallel to the membrane plane, and the electrostatic interaction between the positively charged residues located at the polar-nonpolar interface of the amphiphilic helix with the negatively charged head groups of phospholipids is important to the lipid affinity of the amphiphilic peptide.

Studies on the Denaturation and Conformation of Choline Dehydrogenase.

The substrate choline was able to improve the pH stability of choline dehydrogenase (CDH). It was found that during the thermal denaturation there were an increase of beta-structure and a decrease of the alpha-helix content. Changes in beta-structure were attributed to beta-turn and 3(10)-helix. The substrate had a protective effect on CDH thermal denaturation. The proportion of 3(10)-helix content of the SDS-denatured protein and that in the presence of substrate was 33% and 31.2% respectively; while, the corresponding proportion of beta-sheet was 29% and 10.6%, respectively. Moreover, when the absorption proportion of alpha-helix, random coil and the side-chain of tyrosine residues were concerned, it was found that the spectral property of the CDH treated by denaturants in the presence of substrate were rather similar to that of the non-denatured enzyme.

Influence of Lipid Composition on the Interaction of Apolipoprotein Model Peptides with Liposomes.

Two amphiphilic peptides, Ampl and Amp2, were synthesized according to the sequence of the lipid-binding domain in apoliporotein. Amp2 has a Val residue substituted for the Lys at the 4th position of Ampl. Interaction between Ampl / Amp2 and liposomes with different lipid compositions were compared by studying the blue shifts of the intrinsic fluorescence emission maxima, the peptide-induced lipsome leakage and the quenching of tryptophan fluorescence by acrylamide. The influence of temperature on interactions was studied as well. Ampo1 interacted stronger with acidic lipids while Amp2 interacted stronger with zwitterionic lipids. The interaction was reinforced with the increasing extent of lipid unsaturation as well as with the increase of temperature. The lipid unsaturation had amore prominent effect.

The Protection of Choline Dehydrogenase by Its Substrate.

Choline dehydrogenase, an enzyme bound to the mitochondrial inner membrane, plays an important role in the mitochondrial respiratory chain. The purified enzyme is different from the membrane bound enzyme in some properties. The inactivation effects of temperature and SDS on choline dehydrogenase were studied. It was found that the substrate choline had significant protective effect. It was suggested that its substrate could induce the conformational changes of choline dehydrogenase.