Effects of serum proteins on estrogen action in the perfused rat liver

To determine the effects of serum proteins on the biologic activity of estrogens, we perfused isolated livers from ovariectomized female rats with oxygenated Krebs-Henseleit-bicarbonate buffer (KHBB), with and without 4% human serum albumin (4% HSA), with and without added estrogens, or with charcoal-stripped human serum (CSHS) with and without added estradiol. At the end of the perfusions, the cytosolic and nuclear estrogen receptors were measured by an exchange assay. When added to KHBB, estradiol 10(-9) or 10(-8) M or estrone 10(-8) M did not cause any significant increase in the percent of receptors measured in the nucleus. When the livers were perfused with KHBB containing 4% HSA and estradiol 10(-9) to 10(-7) M or estrone 10(-8) M, there was an increase in nuclear receptors. Perfusion with estradiol 10(-8) M in CSHS resulted in significantly less receptor in the nucleus than after estradiol in KHBB plus 4% HSA. We conclude that the presence of 4% HSA in the perfusion medium increases the biologic activity of estradiol and estrone on the isolated rat liver, and this increase is inhibited in the presence of sex hormone-binding globulin. The exact mechanism by which HSA increases the biologic activity is uncertain, but may be due in part to better diffusion of estrogen through the liver.     

Induction of porcine uterine estrogen sulfotransferase activity by progesterone

Studies have been carried out which were designed to examine the hormonal requirement for the appearance of estrogen sulfotransferase activity in porcine uteri. Mature, ovariectomized (OVX) gilts were housed for 3 weeks before being treated with various regimens of estradiol-17 beta (E2) and progesterone (P). Uteri were then removed, minced, incubated for 2 h with [3H] E2 (10(-8) M) and Na2 35SO4 (10(-4) M) and the labeled metabolic products were extracted and analyzed. Endometrial samples were also taken for the determination of E2 and P cytoplasmic and nuclear receptors (R). It was found that 4 daily injections of 250 micrograms of E2 was sufficient to bring plasma E2 concentrations to that representative of a normal estrous cycle (approx. 30 pg/ml) and to induce cytoplasmic PR to high levels (7000--19000 fmol/mg DNA). Estrogen sulfotransferase activity, which was negligible in OVX and E2-treated pigs, increased to near normal secretory levels (4 pmol product/h per 0.4 g tissue) only in pigs primed with E2 and subsequently treated with E2 and P (25--250 mg/day, 3 days). This treatment also brought about the translocation of PR to the nuclear compartment. The steroid alcohol sulfotransferase activity in these tissues decreased upon ovariectomy and remained unaffected by the hormone treatments. Endometria from treated and untreated pigs were cultured for a period up to 7 days. During this time E2 (10(-8) M) induced and/or maintained PR and P (10(-6) M) was shown to stimulate estrogen sulfurylation concomitant with the translocation of PR to the nucleus. These studies have demonstrated that, in OVX pigs and endometrial cultures, P stimulated uterine estrogen sulfotransferase activity to a level normally found in secretory uteri. In order for P to bring about elevated levels of estrogen sulfurylation it was necessary that the endometrium contain adequate concentrations of cytoplasmic PR (which required E2 priming of the system) and the P receptor complex must display nuclear translocation.     

Prostaglandin F2 alpha output by human endometrium under superfusion and organ culture conditions

Specimens of proliferative and secretory human endometrium were incubated under organ culture or superfusion conditions and the levels of PGF2 alpha in the medium were measured by radioimmunoassay. Basal rates of PGF2 alpha output during short-term superfusions and long-term (1-2 day) batch incubations, performed on the same tissue specimens, were similar. Basal output of PGF2 alpha by proliferative endometrium (230-280 ng/mg protein X d) was significantly higher than that of secretory tissue under both experimental conditions. Estradiol (10(-8) M) increased PGF2 alpha output significantly (4-fold) only in secretory endometrium under organ culture conditions; Progesterone (10(-7) M) decreased it significantly (to 1/2-1/4 of the basal level) in both types of endometria during long-term incubations and in proliferative endometrium during superfusion. Glands isolated from proliferative and secretory endometrium produced PGF2 alpha during superfusion at a rate comparable to that of endometrial tissue under similar conditions. PGF2 alpha output by glands isolated from secretory endometrium increased significantly (3-fold) when estradiol was added to the superfusion medium.     

Binding characteristics of [3H]17beta-estradiol in the hypothalamus of juvenile rainbow trout, Oncorhynchus mykiss

Gonadal steroids in the salmonid brain, acting through cellular receptors, may be responsible for the modulation of neuronal activity and organization of reproductive behaviors. We report our findings on the use of [3H]17beta-estradiol (E2) to identify intracellular estrogen receptors (ERs) in the hypothalamus of juvenile rainbow trout, Oncorhynchus mykiss. Specific binding (B(SP)) of [3H]E2 was tissue dependent between 0.5 and 2.25 hypothalamus equivalents for cytosol and nuclear extract preparations, respectively. B(SP) in cytosol fractions increased with time and reached maximum levels (4.18 nM) at 2.5 h incubation; by contrast, B(SP) in nuclear extract increased with time to achieve maximum levels (3.9 nM) by 2 h incubation. The association rate constants (k(+1)) for cytosol and nuclear extract preparations were 1.10 +/- 0.02 x 10(8) M(-1) min(-1) and 1.27 +/- 0.04 x 10(8) M(-1) min(-1), respectively. Equilibrium bound B(SP) dissociated from cytosol preparations with a half life (t1-2) of 42 min and a dissociation rate constant (k(-1)) of 1.01 +/- 0.03 min(-1). B(SP) dissociated from nuclear extract preparations with a t1-2 = 45 min and k(-1)= 0.92 +/- 0.01 min(-1) x B(SP) was saturable in both extract preparations with a calculated equilibrium dissociation constant (Kd) of 1.46 +/- 0.1 nM (cytosol) and 2.37 +/- 0.2 nM (nuclear), and a maximum number of binding sites (B(MAX)) of 50.85 +/- 3.2 fmol mg(-1) protein and 61.74 +/- 2.65 fmol mg(-1) protein, respectively. In both preparations, B(SP) was differentially displaced by structurally similar compounds with a rank order of potency of E2 > estrone > estriol > 17alpha-ethynyl estradiol > testosterone > progesterone = tamoxifen > cortisol > dexamethasone > > beta-sitosterol. These properties of specifically bound [3H]E2 suggest the presence of an ER in the hypothalamus of juvenile rainbow trout comparable with ERs identified in salmonid liver.     

Effects of copper on cytoplasmic and nuclear binding of 17 beta-estradiol in the rat uterus

Interference of Cu++ with the initial events in estrogen action was tested by determining Cu++ effects on estradiol-receptor interactions. When immature rat uteri were incubated in vitro with [3H] estradiol ([3H]E2), steroid was bound in cytoplasmic fractions and rapidly accumulated in the nuclear fraction in a manner which was dependent upon time and hormone concentration. Uteri which were preincubated with 2 X 10(-4) M CuCl2 for 40-60 min and then exposed to [3H]E2 were found to have a 30-50% decrease in the amount of steroid bound in the cytoplasmic and nuclear fractions. When copper-treated uteri were exposed to [3H]E2 for variable times, the quantity of steroid bound in the cytoplasmic fraction was markedly depressed and the rate of nuclear accumulation of [3H]E2 was significantly decreased. These results show that Cu++ can inhibit [3H]E2 binding to tissue cytoplasmic receptors in vitro and thereby interfere with hormone delivery to target cell nuclei.     

Endocrine aging in C57 BL mice--II. Dynamics of estrogen receptors in the hypothalamic-pituitary axis

Specific cytosolic and nuclear binding sites for estrogens were measured in the hypothalamic-pituitary axis (HPA) of young (4-8 months) and old (16-18 months) C57 BL mice in order to determine any age-related alteration in hormone-receptor interaction. Our results indicated no age differences in the affinity (KD = 0.89 +/- 0.03 (SEM) vs 1.09 +/- 0.2 X 10(-9) M), the specificity, the sedimentation profile (6 s) or in the number (98.9 +/- 4.9 vs 84.4 +/- 2.3 fmol/mg protein) of unoccupied estrogen binding sites in the cytosols. Estradiol administration to young mice induced a complete translocation of cytosolic estrogen receptors to the nucleus, and two types of nuclear binding sites were observed: Type I were specific for estrogens with high affinity (KD = 0.51 +/- 0.06 X 10(-9) M) and low binding capacity (115.1 +/- 22.7 fmol/mg DNA) and sedimented in the 4.0 s area, while Type II binding sites showed a much higher capacity and lower affinity for R2858. HPA nuclear suspensions of aged untreated mice showed undetectable (less than 50 fmol/mg DNA) levels of nuclear estrogen receptors and E2 pre-treatment resulted in a significant increase in both types of binding sites. While no significant changes in the physicochemical characteristics of these nuclear receptors were observed, when compared to young animals, aging was manifested by a translocation defect in the HPA of C57 BL mice. These results suggest aging changes in the endocrine regulating centers of the brain with defective activation of estrogen receptors.     

Effect of the progesterone antagonist RU486 on human myometrial spontaneous contractility and PGI2 release.

We studied the effect of antiprogesterone RU 486 on spontaneous uterine contractility and PGI2 release with human myometrial strips superfused "in vitro". A decrease of PGI2 release into the superfusion medium was observed after 20 min superfusion. The inhibition was dose-dependent and reversible. After 20 min washing with tyrode medium without RU 486, the uterine strips recovered their initial rate of release. R5020, a progesterone agonist, did not affect PGI2 release nor dexamethasone and testosterone. Parallel to the decrease of PGI2 observed during RU 486 superfusion, the uterine spontaneous contraction frequency decreased, while the amplitude and duration of contractions increased. The alteration of uterine contractility was also rapid, dose-dependent and reversible. Modification of uterine strip spontaneous contractility, similar to those induced by RU 486, were also observed with superfusions of R5020 at concentrations as low as 10(-9)M, dexamethasone (10(-8)M), but not with superfusions of testosterone. These observations are not in favour of a progesterone-receptor mediated effect of RU 486 in our model. The mechanism of action may be related to the antiprogesterone specific structure i.e. the bulky substituent at the C-11 position. The RU 486 effect on uterine strip contractility, mimicked by other steroids, could point to a non-specific lipid/membrane interaction. However, the fact that testosterone did not affect motility, may indicate a possible specificity of steroids having a 3 oxo pregnene structure.     

Ca2+-dependent nuclear contraction in the heliozoon Actinophrys sol

Ca2+-dependent contractility was found to exist in the nucleus of the heliozoon protozoan Actinophrys sol. Upon addition of Ca2+ ([Ca2+]free = 2.0 x 10(-3) M), diameters of isolated and detergent-extracted nuclei became reduced from 16.5+/-1.7 microm to 11.0+/-1.3 microm. The threshold level of [Ca2+]free for the nuclear contraction was 2.9 x 10(-7) M. The nuclear contraction was not induced by Mg2+, and was not inhibited by colchicine or cytochalasin B. Contracted nuclei became expanded when Ca2+ was removed by EGTA; thus cycles of contraction and expansion could be repeated many times by alternating addition of Ca2+ and EGTA. The Ca2+-dependent nuclear contractility remained even after high salt treatment, suggesting a possible involvement of nucleoskeletal components in the nuclear contraction. Electron microscopy showed that, in the relaxed state, filamentous structures were observed to spread in the nucleus to form a network. After addition of Ca2+, they became aggregated and constructed a mass of thicker filaments, followed by re-distribution of the filaments spread around inside of the nucleus when Ca2+ was removed. These results suggest that the nuclear contraction is induced by Ca2+-dependent transformation of the filamentous structures in the nucleus.     

Replenishment and nuclear retention of oestradiol-17 beta receptors in rat uteri during postnatal development.

1. Uteri of 6--10-day-old rats do not show a late growth response to oestrogen (increase in rate of DNA synthesis and cell division) exhibited by fully competent (20 days or older) uteri. We posed the question whether the lack of the late growth response is due to an inability to replenish the cytoplasmic pool of oestrogen receptors or to curtailed retention of oestrogen binding in the nucleus. Uterine nuclear and cytoplasmic receptors were measured by a [3H]oestradiol-17 beta exchange assay, at 1, 3, 6, 14 and 24 h after oestrogen injection. 2. The replenishment of cytoplasmic oestrogen receptors showed a similar pattern in the uteri of 6 and 10-day-old (partially responsive) and in 20-day-old (fully responsive) rats. 3. Oestrogen was retained longer in uterine nuclei obtained from 5 and 10-day-old rats than in uterine nuclei of 20 and 25-day-old rats. 4. Oestrogen receptors resistant to 0.4 M KCl extraction (residual receptors) were found in uterine nuclei of 6 and 25-day-old rats after oestrogen injection at all the times tested. The concentration of these residual receptors during the late period (6--24 h after injection) was not significantly different in uterine nuclei of 6-day-old and 25-day-old rats. 5. We conclude that neither lack of oestrogen receptor replenishment nor curtailed retention of oestrogen binding in the nucleus is the factor which limits the complete responsiveness to oestrogen in uteri of rats during postnatal development.     

Serum albumin-lipid membrane interaction influencing the uptake of porphyrins

It is frequently observed in pharmaceutical practice that entrapped substances are lost rapidly when liposomes are used as carriers to introduce substances into cells. The reason for the loss is the interaction of serum components with liposomes. To elucidate the mechanism of this phenomenon the partition of mesoporphyrin (MP) was systematically studied in model systems composed of various lipids and human serum albumin (HSA). As surface charge is an important factor in the interaction, neutral (1, 2-dimyristoyl-sn-glycero-3-phosphatidylcoline, DMPC) and negatively charged (1,2-dimyristoyl-sn-glycero-3-phosphatidylcoline/1, 2-dimyristoyl-sn-glycero-3-phosphatidylglycerol, DMPC/DMPG = 19/1 w/w) lipids were compared. The liposome/apomyoglobin system was the negative control. The size distribution of sonicated samples was carefully analyzed by dynamic light scattering. Constants of association of MP to the proteins and to the liposomes were determined: K(p,1) = (2.5 +/- 0.7) x 10(7) M(-1), K(p,2) = (1.0 +/- 0.7) x 10(8) M(-1), K(L,1) = (1.3 +/- 0.3) x 10(5) M(-1), and K(L,2) = (3.2 +/- 0.6) x 10(4) M(-1) for HSA, apomyoglobin, DMPC, and DMPC/DMPG liposomes, respectively. These data were used to evaluate the partition experiments. The transfer of MP from the liposomes to the proteins was followed by fluorescence spectroscopy. In the case of apomyoglobin, the experimental points could be interpreted by ruling out the protein-liposome interaction. In the case of HSA, the efflux of MP from the liposomes was strongly inhibited above a critical HSA concentration range for negatively charged vesicles. This effect was interpreted as the result of HSA coat formation on the liposome surface. This direct interaction is significant for small liposomes. The interpretation is fully supported by differential scanning calorimetry experiments.     

The effects of drug complexation on the stability and conformation of human serum albumin

We report different analytical methods used to study the effects of 3\'-azido-3\'-deoxythymidine, aspirin, taxol, cisplatin, atrazine, 2,4-dichlorophenoxyacetic, biogenic polyamines, chlorophyll, chlorophyllin, poly(ethylene glycol), vanadyl cation, vanadate anion, cobalt-hexamine cation, and As2O3, on the stability and secondary structure of human serum albumin (HSA) in aqueous solution, using capillary electrophoresis, Fourier transform infrared, ultraviolet visible, and circular dichroism (CD) spectroscopic methods. The concentrations of HSA used were 4% to 2% or 0.6 to 0.3 mM, while different ligand concentrations were 1 microM to 1 mM. Structural data showed drugs are mostly located along the polypeptide chains with both specific and nonspecific interactions. The stability of drug-protein complexes were in the order K(VO(2+)) 1.2 x 10(8) M(-1) > K(AZT) 1.9 x 10(6) M(-)1 > K(PEG) 4.1 x 10(5) M(-1) > K(atrazine) 3.5 x 10(4) M(-1) > K(chlorophyll) 2.9 x 10(4) M(-1) > K2,4-D 2.5 x 10(4) M-1 > K(spermine) 1.7 x 10(4) M(-1) > K(taxol) 1.43 x 10(4) M(-1) > K(Co(3+)) > 1.1 x 10(4) M(-1) > K(aspirin) 1.04 x 10(4)i(-1) > K(chlorophyllin) 7.0 x 10(3) M(-1) > K(VO(3)(-)) 6.0 x 103 M(-1) > K(spermidine) 5.4 x 10(3) M(-1) > K(putrescine) 3.9 x 10(3) M(-1) > K(As(2)O(3)) 2.2 x 10(3) M(-1)> K(cisplatin) 1.2 x 10(2) M(-1). The protein conformation was altered (infrared and CD results) with major reduction of alpha-helix from 60 to 55% (free HSA) to 49 to 40% and increase of beta-structure from 22 to 15% (free HSA) to 33 to 23% in the drug-protein complexes. The alterations of protein secondary structure are attributed to a partial unfolding of HSA on drug complexation.     

Distribution and function of muscarinic receptor subtypes in the ovine submandibular gland.

The effects of muscarinic receptor antagonists on responses to electrical stimulation of the chorda-lingual nerve were determined in pentobarbitone-anesthetized sheep and correlated to the morphology of tissue specimens. Stimulation at 2 Hz continuously, or in bursts of 1 s at 20 Hz every 10 s, for 10 min induced similar submandibular fluid responses (19 +/- 3 vs. 21 +/- 3 microl x min(-1) x g gland(-1)), whereas vasodilatation was greater during stimulation in bursts (-52 +/- 4 vs. -43 +/- 5%; P < 0.01). Continuous stimulation at 8 Hz induced substantially greater responses (66 +/- 9 microl x min(-1) x g gland(-1) and -77 +/- 3%). While atropine (0.5 mg/kg iv) abolished the secretory response at 2 and 20 Hz (1:10 s), a small response persisted at 8 Hz (<5%). The "M1-selective" antagonist pirenzepine (40 microg/kg iv) reduced the fluid response at all frequencies tested (P < 0.05-0.01), most conspicuously at 2 Hz (reduced by 69%). Methoctramine ("M2/M4-selective"; 100 microg/kg iv; n = 5) had no effect on fluid or the vascular responses but increased the protein output at 2 (+90%, P < 0.05) and 8 Hz (+45%, P < 0.05). The immunoblotting showed distinct bands for muscarinic M1, M3, M4, and M5 receptors, and immunohistochemistry showed muscarinic M1 and M3 receptors to occur in the parenchyma. Thus muscarinic M1 receptors contribute to the secretory response to parasympathetic stimulation but have little effect on the vasodilatation in the ovine submandibular gland. Increased transmitter release caused by blockade of neuronal inhibitory receptors of the M4 subtype would explain the increase in protein output.     

Enhanced inhibition of estrogen receptor nuclear binding in the uterus of aged mice

We have studied why uteri from aging mice show a decrease nuclear concentration of estrogen receptors (UER). While 50-60% of available cytosolic UER from ovariectomized (OVX) mice aged 4-8 months, upon physiochemical activation, are able to bind either to DNA-cellulose or nuclear suspensions from young animals, only 20-30% of comparable concentrations of cytosolic UER from mice aged 15-18 months did so under identical experimental conditions. Nuclear dilutions with uterine cytosolic fractions from estrogen treated OVX mice prior to determination of [3H] UER binding sites in nuclear suspensions decreased the number of nuclear ER sites in both age groups. However, we observed that cytosols from aged animals showed a greater ability to prevent [3H]E2 binding to nuclear sites when compared to young ones (inhibition index: 0.286 +/- 0.013 (SE) vs. 0.137 +/- 0.025, P less than 0.05). These changes occur independently of protein concentration and result from dilution of a specific endogenous inhibitor of [3H]E2 binding to nuclear sites. The significance of these observed differences is discussed.     

Thionamides and arsenite inhibit specific T3 binding to the hepatic nuclear receptor

Methimazole (MMI) and propylthiouracil (PTU) are widely used for the treatment of Graves' disease. However, no studies have been reported on the action of these drugs on binding of L-triiodothyronine (T3) to the nuclear receptor. T3 receptors of rat liver nuclei, prepared by differential centrifugation, were extracted with 0.4 M KCl and 5 mM dithiothreitol (DTT). In the assessment of T3 binding to the DTT-reduced receptor, the hepatic nuclear extract was chromatographed on Superose 6 to remove DTT and isolate proteins of relative mass approximately 50,000 (chromatographed nuclear receptors (CNRs)), prior to the addition of [125I]T3 of high specific activity (3300 microCi/micrograms; 1 Ci = 37 GBq). MMI or PTU at 2 mM reduced specific T3 binding to CNR by 84% and 85%, respectively. The inhibitory effects of these reagents and 2 mM sodium arsenite (which complexes dithiols) were additive. Scatchard analyses indicated that neither MMI nor PTU (at 2 mM) significantly altered the affinity constant (Ka) (from 2.41 x 10(9) to 1.74 x 10(9) M-1 for PTU and 1.79 x 10(9) M-1 for MMI), while they both decreased (p less than 0.02) maximal binding capacity (from 0.36 +/- 0.02 to 0.19 +/- 0.02 pmol/mg protein for MMI and 0.17 +/- 0.02 pmol/mg protein for PTU). Dose-response curves showed that 50% inhibition was attained at 0.6 mM PTU or 1.0 mM MMI with approximately 25% inhibition by both at 0.1 mM. Artefactual binding effects by MMI and PTU on [125I]T3 were excluded by chromatography experiments. Similar results were obtained using nuclear receptors prepared from livers of hyperthyroid rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of estriol, estriol-3-sulfate and estriol-17-sulfate on progesterone and estrogen receptors of MCF-7 human breast cancer cells

The levels of progesterone receptors (PR [cytosol (Cy) and nuclear (N)] and estrogen receptors (ER) [cytosol and nuclear; occupied and unoccupied specific binding sites] were evaluated in the MCF-7 cancer cell line incubated with estriol (E3), estriol-3-sulfate (E3-3-S) or estriol-17-sulfate (E3-17-S) for 7 days in culture. Cells were grown in MEM medium containing 2 mM glutamine, 10% v/v dialysed calf serum and penicillin-streptomycin (100 U/ml) in the absence (control) or in the presence of 5 X 10(-8) M E3, E3-3-S or E3-17-S. The total PR (Cy + N) concentration which was 0.47 +/- 0.10 (SE) pmol/mg DNA in the non-treated cells, increased to 1.95 +/- 0.48 in the E3 and to 1.55 +/- 0.26 in the E3-3-S treated cells. No effect (PR: 0.47 +/- 0.15 pmol/mg DNA) was observed with the E3-17-S treatment. Total ER (Cy + N, occupied + unoccupied binding sites) in pmol/mg DNA +/- SE, were as follows: control 0.79 +/- 0.17; + E3: 0.33 +/- 0.09; +E3-3-S: 0.90 +/- 0.18 and +E3-17-S: 1.82 +/- 0.58. The measurement by radioimmunoassay of unconjugated estriol in the culture medium indicated that after incubation with E3-3-S, a fraction (0.5-1%) of the sulfate was hydrolyzed but no hydrolysis was observed in the incubations with E3-17-S. It is concluded that in the MCF-7 human mammary cancer cell line E3 and E3-3-S stimulate PR very significantly, but it is suggested that E3-3-S acts through the hydrolyzed E3. On the other hand, E3-17-S is inactive because it is not hydrolyzed. Consequently, E3-3-S can play an important role in the biological responses of this mammary cancer cell line.     

Binding study of desethyloxybutynin using high-performance frontal analysis method

Plasma protein binding of N-desethyloxybytynin (DEOXY), a major active metabolite of oxybutynin (OXY), was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of DEOXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. DEOXY is bound in human plasma strongly and enantioselectively. The unbound drug fraction in human plasma samples containing 5 microM (R)- or (S)-DEOXY was 1.19 +/- 0.001 and 2.33 +/- 0.044%, respectively. AGP plays the dominant role in this strong and enantioselective plasma protein binding of DEOXY. The total binding affinity (nK) of (R)-DEOXY and (S)-DEOXY to AGP was 2.97 x 10(7) and 1.31 x 10(7) M(-1), respectively, while the nK values of (R)-DEOXY and (S)-DEOXY to HSA were 7.77 x 10(3) and 8.44 x 10(3) M(-1), respectively. While the nK value of (S)-DEOXY is weaker than that of (S)-OXY (1.53 x 10(7) M(-1)), the nK value of (R)-DEOXY is 4.33 times stronger than that of (R)-OXY (6.86 x I0(6) M(-1)). This suggests that the elimination of an ethyl group weakens the binding affinity of the (S)-isomer because of the decrease in hydrophobicity, while the binding affinity of the (R)-isomer is enhanced by the decrease in steric hindrance. The total binding affinity of DEOXY to HSA is much lower than that of DEOXY-AGP binding as well as OXY-HSA binding (2.64 x 10(4) and 2.19 x 10(4) M(-1) for (R)-OXY and (S)-OXY, respectively). The study on competitive binding between OXY and DEOXY indicated that DEOXY enantiomers and OXY enantiomers are all bound competitively at the same binding site of AGP molecule.     

Spasmolytic action of the methanol extract and isojuripidine from Solanum asterophorum Mart. (Solanaceae) leaves in guinea-pig ileum

Solanum asterophorum Mart. (Solanaceae) is a shrub popularly known as "jurubeba-defogo" in the northeast of Brazil. In the present work, the methanol extract (SA-MeOH, 3750 microg/mL) and isojuripidine (10(-7) - 3 x 10(-4) M), a steroidal alkaloid obtained from S. asterophorum Mart. leaves, inhibited phasic contractions induced by both 1 microM histamine [IC50 = (225.8 +/- 47.4), g/mL and (3.5 +/- 0.8) x 10(-5) M] or 1 microm acetylcholine [IC50 = (112.5 +/- 20.6) microg/mL and (2.3 +/- 0.4) x 10(-5) M] in guinea-pig ileum, respectively. The extract and isojuripidine also relaxed the ileum (SA-MeOH, 1-750 microg/mL, and isojuripidine, 10(-9) - 3 x 10(-4) M) pre-contracted with 1 M histamine [EC50 = (101.1 +/- 17.4) microg/mL and (1.2 +/- 0.3) x 10(-6) M] or 1 microM acetylcholine [EC50 = (136.8 +/- 21.1) microg/mL and (1.9 +/- 0.4) x 10(-6) M] or 40 mm KCl [EC50 = (149.4 +/- 19.5) microg/mL and (1.8 +/- 0.7) x 10(-6) M], respectively, in an equipotent and concentration-dependent manner. This effect is probably due to inhibition of calcium influx through voltage-operated calcium (Ca(v)) channels. To confirm this hypothesis, we evaluated their effect on cumulative CaCl2 curves in depolarizing medium nominally without Ca2+. SA-MeOH (27, 243, 500, and 750 microg/mL) and isojuripidine (3 x 10(-8), 10(-6), 3 x 10(-5), and 3 x 10(-4) M) inhibited the contractions induced by CaCl2, in a concentration-dependent manner. The concentration-response curves to CaCl2, in the presence of SA-MeOH and isojuripidine, were shifted downward in relation to a control curve in a non-parallel manner resulting in reduction of the maximum effect [E(max) = (71.2 +/- 9.2); (57.4 +/- 9.2); (43.8 +/- 3.4); (41.5 +/- 2.4) and (90.6 +/- 4.8); (74.7 +/- 8.7); (66.4 +/- 3.9); (31.3 +/- 4.1)%, respectively]. SA-MeOH and isojuripidine present spasmolytic action in guinea-pig ileum due to a partially blockade of calcium influx through Ca(v) channels.     

Effects of autonomic blockers on linear and nonlinear indexes of blood pressure and heart rate in SHR

Endothelium-derived hyperpolarizing factor (EDHF) is released in response to agonists such as ACh and bradykinin and regulates vascular smooth muscle tone. Several studies have indicated that ouabain blocks agonist-induced, endothelium-dependent hyperpolarization of smooth muscle. We have demonstrated that epoxyeicosatrienoic acids (EETs), cytochrome P-450 metabolites of arachidonic acid, function as EDHFs. To further test the hypothesis that EETs represent EDHFs, we have examined the effects of ouabain on the electrical and mechanical effects of 14,15- and 11,12-EET in bovine coronary arteries. These arteries are relaxed in a concentration-dependent manner to 14,15- and 11,12-EET (EC(50) = 6 x 10(-7) M), bradykinin (EC(50) = 1 x 10(-9) M), sodium nitroprusside (SNP; EC(50) = 2 x 10(-7) M), and bimakalim (BMK; EC(50) = 1 x 10(-7) M). 11,12-EET-induced relaxations were identical in vessels with and without an endothelium. Potassium chloride (1-15 x 10(-3) M) inhibited [(3)H]ouabain binding to smooth muscle cells but failed to relax the arteries. Ouabain (10(-5) to 10(-4) M) increased basal tone and inhibited the relaxations to bradykinin, 11,12-EET, and 14,15-EET, but not to SNP or BMK. Barium (3 x 10(-5) M) did not alter EET-induced relaxations and ouabain plus barium was similar to ouabain alone. Resting membrane potential (E(m)) of isolated smooth muscle cells was -50.2 +/- 0.5 mV. Ouabain (3 x 10(-5) and 1 x 10(-4) M) decreased E(m) (-48.4 +/- 0.2 mV), whereas 11,12-EET (10(-7) M) increased E(m) (-59.2 +/- 2.2 mV). Ouabain inhibited the 11,12-EET-induced increase in E(m). In cell-attached patch clamp studies, 11,12-EET significantly increased the open-state probability (NP(o)) of a calcium-activated potassium channel compared with control cells (0.26 +/- 0.06 vs. 0.02 +/- 0.01). Ouabain did not change NP(o) but blocked the 14,15-EET-induced increase in NP(o). These results indicate that: 1) EETs relax coronary arteries in an endothelium-independent manner, 2) unlike EETs, potassium chloride does not relax the coronary artery, and 3) ouabain inhibits bradykinin- and EET-induced relaxations as has been reported for EDHF. These findings provide further evidence that EETs are EDHFs.