Effects of equol on gene expression in female cynomolgus monkey iliac arteries.

BACKGROUND AND AIMS: To examine effects of equol, the soy phytoestrogen metabolite, on gene expression in the monkey iliac artery. METHODS AND RESULTS: A high fat/high cholesterol diet was fed to eight ovariectomized cynomolgus monkeys for 6.5 years. After biopsy of the left iliac artery, the animals were randomized to two treatment groups for 8 months; the treatment groups were equol (23.7 mg/100 g diet, n = 4) and vehicle (n = 4). The right iliac artery was removed at necropsy. Gene expression in the iliac arteries in response to equol was determined by DNA microarray. Comparison of atherosclerotic lesions and plasma lipids at pre-versus post-equol treatment time points and in vehicle versus equol treatment groups did not identify any significant differences. Despite the lack of effect of equol on these parameters, 59 genes were down-regulated and 279 were up-regulated in response to equol. Comparison of these data to previous work identified 10 genes regulated in opposite directions by equol compared to presence of atherosclerosis plaque (Menopause 2011; 18:1087-1095) and 55 genes differentially expressed in the same direction in response to both equol and estradiol (Eyster et al., Menopause 2014;21:143-152.). CONCLUSIONS: Similar responses of genes to both equol and estradiol may reflect the extent to which equol serves as a natural selective estrogen receptor modulator in the arteries. Opposite responses of 10 genes to equol versus the presence of atherosclerosis implicates those genes in the potential protective effects of equol in arteries.

Sexual dimorphism in angiotensin II-induced hypertension and vascular alterations.

Sex differences in the degree of high blood pressure have been described in several forms of experimental animal models of hypertension. However, the influence of sex on angiotensin II-induced hypertension has not been studied. In the present study, we investigated and compared the effects of chronic angiotensin II treatment on blood pressure and vascular function in male and female rats. Chronic treatment with angiotensin II (0.7 mg/kg daily for 10 d) significantly raised arterial blood pressure in male but not female Sprague-Dawley rats; it upregulated the NAD(P)H oxidase gp67 phox subunit in the aorta of male but not female rats; and it exaggerated the vasoconstrictor responses to norepinephrine and serotonin in the mesenteric vascular bed (MVB) of male but not female rats. Vasodilator responses to acetylcholine (ACh) but not papaverine (PPV) or isoprenaline (ISO) were reduced in the MVB of angiotensin II-treated male but not female rats. ACh, but not PPV or ISO dilatory responses were potentiated in the MVB of angiotensin II-treated female rats. The present findings demonstrate that exogenous angiotensin II upregulates aortic NAD(P)H oxidase gp67 phox subunit, and induces hypertension and mesenteric vascular dysfunction only in male rats.

Estrogen receptor-alpha antisense decreases brain estrogen receptor levels and affects ventilation in male and female rats.

We hypothesized that administration of an antisense oligodeoxynucleotide (ODN) to estrogen receptor (ER)-alpha mRNA decreases the ER protein in the neonatal rat brain, alters the sex-specific ventilatory responses to aspartic acid in rats, and counteracts the effects of testosterone proportionate (TP) in females. One-day-old rat pups were injected intraventricularly with vehicle, antisense ER ODN, or scrambled ODN control. Additional groups of females received TP or vehicle and one of the three treatments. Brain ER protein levels were decreased by 65% at 6 h and 35% at 24 h after antisense ODN. Aspartic acid decreased ventilation in all groups of weanling males and females except ER ODN-treated females and TP-vehicle-treated females. Aspartic acid decreased ventilation in all groups of adult females except those given TP and in males. Weanling ER ODN-treated rats were shorter and weighed less than controls. Only adult ER ODN-treated males exhibited these traits. Thus neonatal ER affects aspartic acid modulation of breathing and body growth in a sex-specific and developmental manner.

Dietary soy exerts an antihypertensive effect in spontaneously hypertensive female rats.

This study tested the hypothesis that dietary soy would attenuate the development of hypertension in female spontaneously hypertensive rats (SHR). Female SHR and control Wistar-Kyoto rats were obtained at 4 wk of age, randomly assigned to either an ovariectomized (OVX) group or a sham-operated group, and placed on a soy diet or control casein diet. After a minimum of 8 wk on their respective diets, mean arterial pressure (MAP) and heart rate (HR) were recorded before and after inhibition of nitric oxide synthase, air-jet stress, or ganglionic blockade. The major finding of this study is that MAP was reduced in the OVX SHR consuming soy diet compared with the casein-fed controls (150 +/- 4 vs. 164 +/- 3 mmHg). Plasma genistein concentrations were increased in the soy-fed OVX SHR (1.23 +/- 0.31 microM) compared with the casein-fed OVX SHR (nondetectable). However, there was no difference in plasma genistein concentrations between sham-operated and OVX SHR (1.37 +/- 0.42 vs. 1.23 +/- 0.31 microM). Inhibition of nitric oxide synthase increased MAP and decreased HR in all groups; diet did not affect this response. Air-jet stress increased MAP and HR in all groups. However, these responses were exaggerated in the soy-fed SHR. Finally, ganglionic blockade abolished the antihypertensive effect of soy diet in the OVX SHR. These findings indicate that dietary soy exerts an antihypertensive effect in OVX SHR. This effect does not involve the nitric oxide system but may be related to an as yet undefined interaction with the autonomic nervous system.

Molecular medicine: a primer for clinicians. Part XII: DNA microarrays and their application to clinical medicine.

Previous papers in our Molecular Medicine series have described how the many tools of the molecular biologist are being used to develop practical bedside applications of modern molecular biology. We have discussed molecular diagnostics, gene therapy, and applications in clinical genetics. In this paper we discuss DNA microarray technology which provides a genome-wide profile of gene expression. We then describe some current and potential clinical applications in the disease diagnosis, prognosis and treatment. Particularly exciting is the potential of DNA array technology to provide individualized treatment for a wide variety of clinical conditions.

Effect of genistein on cardiovascular responses to angiotensin II in conscious unrestrained rats.

Soybeans contain genistein, a phytoestrogen that may have beneficial effects in coronary artery disease, osteoporosis, and breast cancer. In vitro studies demonstrated that genistein reduced vascular smooth muscle contractions to angiotensin II. We tested the hypothesis that genistein attenuates the overall cardiovascular responses to angiotensin II via nongenomic mechanisms in conscious rats. Mean arterial blood pressure (MAP) was recorded from conscious unrestrained Sprague-Dawley rats (n = 26) approximately 48 hours after surgery. Cumulative dose response curves to angiotensin II (10-200 ng/kg/min) were constructed before and after i.v. treatment with genistein given as a single bolus dose of 160 microg/kg or 1500 microg/kg, or as a loading dose of 160 microg/kg followed by an infusion at a rate of 20 microg/kg. Angiotensin II infusions were associated with graded increases in arterial pressure ranging between 0+/-1 and 35+/-4 mm Hg. These pressor responses were accompanied by significant dose-dependent decreases in heart rate. None of the genistein treatment regimens significantly affected the pressor responses to angiotensin II. Accordingly, we conclude that short-term i.v. treatment with genistein does not depress pressor responsiveness to angiotensin II.

Introduction to signal transduction: a primer for untangling the web of intracellular messengers.

The field of signal transduction developed from our need to understand the specific mechanisms involved in the transmission of extracellular signals to intracellular actions in target cells. As our comprehension of cellular function has grown, it has become clear that the need to understand signal transduction events has invaded all fields of biological science. Many scientists learned about adenylate cyclase and cyclic AMP when they were students but went on to focus on some other aspect of science; now they find the need to apply studies of signal transduction to their own work. However, the field of signal transduction has progressed so rapidly that from starting with a rudimentary knowledge of adenylate cyclase and cyclic AMP to understanding the field and applying the knowledge to one's own work seems insurmountable. The goal of this commentary is to provide a starting point for those who recognize the need to understand the mechanisms of signal transduction but do not know where or how to begin.

Effect of developmental age and hyperoxia exposure on kinase and phosphatase activities in newborn rat lungs.

To better understand the biochemical events accompanying lung alveolarization and development, we studied the specific activity of the cAMP-dependent protein kinase (PKA) and the type 2A protein phosphastase (PP2A), and the activity and protein content of the calcium- and lipid-dependent protein kinase (PKC) in cytosolic preparations of lungs. Lungs were obtained from rat pups on day 2 of life and on days 7, 14, and 27 from pups exposed to hyperoxia (> 95% O2, days 4-14; 65% O2 days 15-27) or normoxia from day 4 onwards. There were no significant changes in PKA specific activity with developmental age or hyperoxic exposure. PKC specific activity increased significantly (P < .05) in normoxic animals from day 2 (64 +/- 13.5 pmol phosphate released/min/mg protein) to day 14 (105 +/- 9). The increase was sustained to day 27. There was no effect on PKC activity due to hyperoxia alone (ANOVA). This increase in PKC activity was accompanied by an increase in the mass of the delta, epsilon and zeta isoforms of PKC in normoxic pups. The gamma isoform of PKC was undetectable in all samples whereas the alpha and beta isoforms were detectable but showed no changes with developmental age. PP2A specific activity increased significantly (P < .05) from 13.3 +/- 0.5 nmol phosphate released/min/mg protein on day 2 to 17.7 +/- 0.9 on day 7 in normoxic pups, then returned to day 2 level at advanced developmental age. Hyperoxia exposure prevented the increase in enzyme activity observed on day 7 in normoxic animals. These data suggest that protein phosphorylation may be one mechanism by which alveolarization is regulated in developing lungs.

Regulation of prostaglandin H2 synthase-2 expression in primary human amnion cells by tyrosine kinase dependent mechanisms.

Prostaglandin H2 synthase (PGHS)-1 and PGHS-2 expression was examined in primary cultures of human amnion cells, an in vitro model of amnion tissue. Epidermal growth factor (EGF), the protein kinase C (PKC) activating phorbol ester TPA, and the protein phosphatase inhibitor, okadaic acid (OA), stimulated PGHS activity and the level of PGHS-2 mRNA, but did not affect the level of PGHS-1 mRNA. In situ hybridization suggested that the same population of cells responded to EGF, TPA and OA. Okadaic acid promoted PGHS activity independently of PKC. EGF stimulated the activity of extracellular signal-regulated protein kinase (Erk) and N-terminal c-Jun kinase (Jnk). OA increased Jnk activity but had no effect on Erk activity, while TPA had no influence on either Erk or Jnk activity. PD098059, a selective inhibitor of the Erk-activating kinase MEK, blocked the stimulation of PGHS expression by EGF, but did not decrease stimulation in response to OA. Herbimycin A, a tyrosine kinase inhibitor, suppressed the stimulation of PGHS activity and PGHS-2 mRNA abundance by all three stimulants, and blocked signalling via the Erk and Jnk mitogen-activated protein kinase pathways. Thus, growth factor stimulation, PKC activation and protein phosphatase inhibition induced the expression of PGHS-2 in primary amnion cells by distinct regulatory mechanisms involving tyrosine kinase(s). Tyrosine kinase inhibitors may constitute a new category of PGHS-2 inhibitors that act by blocking the expression of the enzyme.

Protein phosphatase activity in the rat ovary throughout pregnancy and pseudopregnancy.

Specific protein phosphatase activity against protein kinase C-phosphorylated substrate was measured in the rat ovary during pseudopregnancy and pregnancy. Tissues were processed in the presence of sodium fluoride and inorganic phosphate to inhibit the phosphatase and thereby prevent autodephosphorylation of the type 2A protein phosphatase (PP2A) during homogenization. Manganese was added at the time of enzyme assay to reactivate the phosphatase. The specific activity of the protein phosphatase did not vary significantly across pseudopregnancy (p > 0.05). In contrast, the specific activity of protein phosphatase decreased significantly between Day 7 and Day 10 of pregnancy (28.8 +/- 5 pmol/min x microg protein and 20.7 +/- 2 pmol/min x microg protein, respectively; p < 0.05) and remained at the decreased value for the remainder of pregnancy. To determine whether hormones of pregnancy could regulate PP2A activity in the ovaries, pseudopregnant rats were treated with prolactin (3 IU twice a day), bromocriptine (100 microg twice a day), or estradiol benzoate (50 microg). Bromocriptine and estradiol treatments caused a decrease in PP2A-specific activity, but prolactin had no effect. Bromocriptine treatment caused a decrease in the protein content of the PP2A catalytic subunit, but prolactin and estradiol treatments had no effect. The data suggest that the specific activity and protein content of PP2A in the rat ovary are hormonally regulated.

Protein kinase and phosphatase activity in the lungs of normoxic versus hyperoxic rats.

Studies were designed to examine aspects of phosphorylation and dephosphorylation in rat lung cells in response to hyperoxic exposure. Protein kinase and phosphatase activities were measured in preparations of lungs from normoxic rats, hyperoxia-exposed rats (95% O2 for 60h), and rats recovering in room air for 1 and 3 days. Protein kinase C (PKC) activity immediately postexposure was significantly lower than in normoxic controls (normoxia 127.1 +/- 13 vs. hyperoxia 101.5 +/- 6 pmol/min mg-1) and continued to decline during the recovery period (85.3 +/- 4 and 78.2 +/- 6 pmol/min mg-1 at 1 and 3 days recovery, respectively). The PKC activity did not translocate from cytoplasm to the membranes. In contrast, PKA activity did not change in response to hyperoxia exposure or recovery. Protein phosphatase activity was decreased significantly by hyperoxia exposure (normoxia 30.7 +/- 3 vs. hyperoxia 21.9 +/- 1 pmol/min microgram-1) but returned to normoxic control levels by 1 and 3 days (24.1 +/- and 31.5 +/- 1 pmol/min microgram -1, respectively). Protein phosphatase activity was inhibited by okadaic acid (Ki = 1 nM) and calyculin A (Ki = 0.61 pM), indicating a type 2A protein phosphatase. Enzyme activities in cultured type II alveolar cells paralleled those observed in whole lung preparations. Decreased enzyme activities in the lung may be located to the development of acute lung injury during hyperoxic exposure.

Production of an endogenous inhibitor of protein kinase C by embryonic myoblasts undergoing differentiation.

The current studies were undertaken to determine whether embryonic myoblasts or myogenic satellite cells undergoing differentiation and fusion contained endogenous modulators of protein kinase C (PKC). Clonal-derived turkey embryonic myoblast and satellite cell cultures were harvested at confluency and at approximately 40% fusion (embryonic myoblasts) or 75% fusion (satellite cells). PKC activity in cystosolic preparations of the cells and myotubes was undetectable. Cytosolic extracts (0.065 mg protein) of confluent and fused satellite cell cultures and confluent embryonic myoblasts had no effect on control PKC activity (control: 14.9 pmol/min, control + cytosols: 15.2, 13.9 and 13.5 pmol/min, respectively). Cytosolic preparations (0.065 mg protein) of embryonic myoblast-derived myotubes inhibited control PKC activity (4.0 pmol/min). In a time-course study, PKC-inhibitory activity was present in embryonic myoblasts at the earliest time point examined (30% fusion). Additionally, protein phosphatase activity correlated with PKC inhibitory activity. Thus, PKC-inhibitory activity appears as embryonic myoblasts begin to undergo fusion to form myotubes, but is not present in differentiating satellite cells.

The heat-stable protein kinase C (PKC) stimulatory factor in the rat ovary may allow PKC to be active independent of cell membrane lipids.

A protein kinase C (PKC) stimulatory factor in heat-treated ovarian cytosol appears to be protein in nature as it was susceptible to proteinase K digestion and strong acid, and its lipid-substituting properties were retained after extraction of lipids. Phosphorylation of substrate by PKC stimulated by the factor from heat-treated ovarian cytosol in the absence of lipids was subject to dephosphorylation by the ovarian phosphatase. The factor stimulated purified PKC obtained from a commercial source as well as PKC partially purified from rat brain. Heat-treated ovarian cytosol stimulated PKC phosphorylation of myelin basic protein, but not protamine sulphate, in a calcium-dependent manner.

Protein phosphatase activity against protein kinase C-phosphorylated substrates in human placenta.

The presence of endogenous modulators of protein kinase C (PKC) in human placenta has not been reported. The specific activity of PKC in human placental cytosol was 20.52 +/- 1.8 pmol/min x mg protein. Partial purification of placental cytosol on diethylaminoethyl cellulose (DEAE) resulted in recovery of 145 per cent of original enzyme activity. Placental cytosol mixed with a control preparation of PKC significantly inhibited the control enzyme activity (control 42.42 +/- 2.8 pmol/min; control+placental cytosol 27.44 +/- 2.8 pmol/min, P < 0.05). The PKC-inhibitory activity was abolished by the addition of phosphatase inhibitors calyculin A (0.09 nM), microcystin LR (0.8 nM), and okadaic acid (0.4 nM). Protein substrates phosphorylated by PKC were rapidly dephosphorylated upon the addition of placental cytosol; this dephosphorylation was prevented by the presence of calyculin A and was removed by fractionation of placental cytosol on DEAE. Protein but not peptide substrate supported both the PKC-inhibitory activity and the dephosphorylation of PKC-phosphorylated substrates. The placental serine-threonine protein phosphatase was active against phosphorylase a, but not against substrate phosphorylated by cAMP-dependent protein kinase. These data indicate that the human placenta contains an endogenous inhibitor of PKC which interacts with substrate rather than with the PKC and that the inhibitor is a protein phosphatase.

Endogenous modulation of protein kinase C in the rhesus monkey corpus luteum.

The activity of protein kinase C (PKC) and the presence of endogenous PKC-modulatory factors in monkey CL have not been previously reported. The specific activity of Ca(2+)- and lipid-dependent PKC activity in cytosols prepared from CL obtained from rhesus monkeys at midluteal phase was 515.1 +/- 79 pmol/min x mg; Ca(2+)-independent PKC activity was 233.2 +/- 31 pmol/min x mg. No PKC-inhibitory activity was seen when luteal cytosol was mixed with a control preparation of PKC (control PKC 10.2 +/- 2.0 pmol/min; luteal cytosol plus control PKC 26.0 +/- 2.1 pmol/min). However, the monkey CL contained PKC-phosphatase activity (the endogenous inhibitor of PKC in the rat ovary is a protein phosphatase). Specific activity of luteal PKC phosphatase activity was 609.0 +/- 94 pmol/min x mg. Kinetic studies suggested that the monkey CL contained a competitive inhibitor of the phosphatase. Heat-treated cytosol mixed with control PKC (9.0 +/- 0.7 pmol/min) resulted in enzyme activity that was significantly greater than that of the control (13.7 +/- 1.2 pmol/min). Luteal cytosol that had been heat-treated and extracted with petroleum ether substituted for lipids in the activation of PKC. In summary, the activity of PKC in the monkey CL was readily measurable; it was not masked by an endogenous PKC-inhibitory factor. The monkey CL did contain a heat-stable PKC-stimulatory activity.

The endogenous inhibitor of protein kinase-C in the rat ovary is a protein phosphatase.

Calcium- and lipid-dependent protein kinase (PKC) activity in the ovary of the pseudopregnant rat is masked by an endogenous inhibitor of PKC. These studies were undertaken to examine the mechanism of action of the endogenous inhibitor of PKC in the rat ovary. The addition of the phosphatase inhibitors calyculin-A (0.09 nM), microcystin-LR (6.4 nM), and okadaic acid (10 nM) resulted in the loss of PKC inhibitory activity and an increase in basal PKC activity in rat ovarian cytosol. In phosphatase assays, significant dephosphorylation of histone-III-S or myelin basic protein that had been phosphorylated by PKC occurred within 4 min after the addition of ovarian cytosol from the pseudopregnant rat. This dephosphorylation was prevented from the pseudopregnant rat. This dephosphorylation was prevented by the addition of calyculin-A (0.73 nM) and was removed by fractionation of ovarian cytosol on diethylaminoethyl cellulose. No inhibition of PKC activity was observed when the PKC-specific peptides AcMBP-(4-14) and [Ser25]PKC-(19-31) were used as the substrate for phosphorylation. In addition, rat ovarian cytosol did not exhibit phosphatase activity when the peptide AcMBP-(4-14) was used as the substrate. Addition of ovarian cytosol resulted in dephosphorylation of phosphorylase-alpha phosphorylated by phosphorylase kinase, but not dephosphorylation of histone-II-A or histone-VIII-S phosphorylated by PKA. The data suggest that the endogenous inhibitor of PKC in the rat ovary is a protein phosphatase.

Protein kinase C activity in the rat ovary: increasing enzyme activity and presence of an endogenous inhibitor during pregnancy.

1. Protein kinase C (PKC) activity was measured in corpora lutea and ovarian interstitial tissues obtained from rats on days 7, 13/14 and 20 of normal pregnancy. 2. PKC activity increased steadily from day 7 to day 20 of pregnancy in luteal tissue but not in ovarian interstitial tissues. 3. Cytosolic fractions of both tissue types inhibited PKC activity in a control preparation of the enzyme, suggesting that both tissues contain an endogenous inhibitor of PKC throughout pregnancy.

Protein kinase-C stimulatory activity in human amnion cytosol.

Cytosolic preparations from human amnions at term were tested for the presence of endogenous modulators of protein kinase-C (PKC) activity. Tissues were obtained from 14 patients undergoing cesarean section (CS) and 14 patients after spontaneous delivery (SL). PKC activity was significantly greater in cytosols from CS than SL amnion (1.65 +/- 0.04 vs 0.73 +/- 0.2 pmol/min, respectively; mean +/- SE; n = 14 CS; n = 14 SL; P < 0.05). When amnion cytosols were mixed with a control preparation of PKC (rat brain cytosol partially purified on diethylaminoethyl), PKC activity was significantly increased compared to the control value (control, 12.81 +/- 2.1; control + CS, 22.19 +/- 1.5; control + SL, 21.98 +/- 0.7 pmol/min). The stimulation of PKC was dose dependent. The PKC stimulatory factor in amnion cytosol was stable to heat treatment at 80-90 C for 2 min (control + heat-treated CS, 23.20 +/- 1.2; control + heat-treated SL, 24.49 +/- 1.0 pmol/min) and substituted for phosphatidylserine and diacylglycerol in the PKC assay (control, no lipids, 0.05 +/- 0.04 pmol/min; control + amnion cytosol, no lipids, 9.60 +/- 1.06 pmol/min). The PKC stimulatory factor was calcium dependent, was not extractable in organic solvents, and was greater then 100,000 mol wt. Thus, the human amnion contains a PKC stimulatory factor which may modify or mediate the cellular response to extracellular stimulators of the PKC pathway.

Protein kinase C stimulatory activity in the pseudopregnant rat ovary.

Ovarian cytosol from pseudopregnant rats was heated to 80-90 degrees C for 2 min and precipitated proteins removed by centrifugation. The supernatant of the heated ovarian cytosol contained no protein kinase C activity but when added to a control preparation containing protein kinase C, enzyme activity was increased to 200% of control. The stimulatory activity was stable to heating for 10 min, was retained on a centrifugal filtration device with a 100,000 M(r) cut-off, did not affect cAMP-dependent protein kinase, was not extractable in petroleum ether or chloroform/methanol (2:1), and enhanced the phosphorylation of protein kinase C-specific peptide substrates. The stimulatory factor was calcium-dependent and could substitute for phosphatidylserine and diacylglycerol in the protein kinase C assay. This stimulatory factor may provide a mechanism whereby the response of protein kinase C to hormonal activation could be regulated by the cell.

An endogenous inhibitor of protein kinase C in the pseudopregnant rat ovary.

Removal of contaminating proteins from rat ovarian cytosol by DEAE chromatography results in a 358% recovery of protein kinase C activity. The data suggest that rat ovaries contain an endogenous inhibitor of protein kinase C whose activity dominates that of protein kinase C in the cytosol. The inhibitor is specific for protein kinase C and does not activate the enzyme through proteolysis. This endogenous inhibitor may be important in the hormonal control of protein kinase C in the rat ovary, and may become an important tool in the study of the role of protein kinase C in other cell functions.