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Biomedical subjects

I M Bird

Publications and source records attributed to I M Bird.

At least 37 records · Page 2Linked to original sources
PubMed1999

Activation of the mitogen-activated protein kinase cascade is necessary but not sufficient for basic fibroblast growth factor- and epidermal growth factor-stimulated expression of endothelial nitric oxide synthase in ovine fetoplacental artery endothelial cells.

Basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) may play important roles in the placental vasculature, not only by controlling cell growth and differentiation, but also by mediating production of local vasodilators such as nitric oxide. As the mitogen-activated protein kinase (MAPK) signal cascade has been widely associated with cell growth in response to growth factors, herein we investigate whether bFGF, EGF, and VEGF also stimulate expression of endothelial nitric oxide synthase (eNOS) via activation of the MAPK cascade in ovine fetoplacental artery endothelial cells. The presence of the receptors for all three growth factors was confirmed by both immunocytochemistry and a functional cell proliferation assay. All three growth factors at 10 ng/ml rapidly (<10 min) activated MAPK. This activation was inhibited by PD 98059, a specific MAPK kinase inhibitor. bFGF and EGF, but not VEGF, dose- and time-dependently increased eNOS protein levels. Maximal stimulatory effects of bFGF and EGF on eNOS protein expression were observed at 10 ng/ml for 24 h of treatment and were associated with elevated eNOS messenger RNA. PD 98059 also significantly inhibited bFGF- and EGF-induced increases in eNOS protein expression. Because treatment with all three growth factors resulted in activation of the MAPK cascade, while bFGF and EGF, but not VEGF, increased eNOS expression, we conclude that activation of the MAPK cascade is necessary, but not sufficient, for bFGF- and EGF-induced increases in eNOS protein expression in ovine fetoplacental artery endothelial cells. Thus, additional signaling pathways are implicated in the different controls of eNOS expression and mitogenesis by growth factors.

Animals↗
PubMed1998

Specific pregnancy-induced angiotensin II type-1 receptor expression in ovine uterine artery does not involve formation of alternate splice variants or alternate promoter usage.

Recently we reported that pregnancy is associated with a dramatic increase in angiotensin II type-1 receptor (AT1-R; both protein and mRNA) in ovine uterine artery endothelial cells (UAEC), which far exceeds that seen in omental (systemic) arteries. Recent reports also suggest that alternate splicing of AT1-R mRNA may play a role in regulation of AT1-R expression in humans. Herein, we have investigated the possibility of alternate transcript splicing/promoter usage in UAEC from pregnant ewes by 5'-RACE (rapid amplification of cDNA 5'-ends). To provide our control "reference" sequences, we first performed 5'-RACE analysis of AT1-R mRNA transcripts in liver, kidney, and adrenal cortex. Analysis of 17 resultant clones showed exceptional homology, indicating that a single identically spliced mRNA product is observed in all three ovine tissues. Homology of the 5'-untranslated region to that of the human was low (34.2%), but four in-context start/stop codons and the beginning of human exons 1 and 5 were highly conserved. Subsequently we isolated 30 individual clones using UAEC RNA from three pregnant ewes and found no evidence of any sequence formed through unique splicing or promoter usage. We conclude that the pregnancy-induced increase in AT1-R expression unique to UAEC during pregnancy is not mediated by splicing of a unique transcript or unique promoter usage.

Angiotensin II↗
PubMed1998

Endothelial vasodilator production by uterine and systemic arteries. III. Ovarian and estrogen effects on NO synthase.

During the follicular phase of the ovarian cycle, when the local estrogen-to-progesterone ratio is elevated, uterine blood flow is elevated. This vasodilatory response is reproduced by exogenous 17beta-estradiol (E2beta) administration via a nitric oxide (NO)-mediated mechanism. We hypothesized that endogenous ovarian estrogen and exogenous E2beta treatment elevate expression of endothelial cell-derived NO synthase (eNOS) in uterine, but not in systemic, arteries. Uterine, mammary, and systemic (renal and/or omental) arteries were collected from 1) ewes synchronized to the follicular (day -1 to day 0) or luteal (day 10) phases of the ovarian cycle (n = 4 per phase), 2) ovariectomized ewes 120 min after systemic vehicle or E2beta (5 micrograms/kg iv) treatment, and 3) ovariectomized ewes on days 0, 3, 6, 8, and 10 of E2beta (5 micrograms/kg iv, followed by 6 micrograms/kg per day) treatment. Expression of eNOS was localized primarily to the endothelium rather than vascular smooth muscle (VSM) in all arteries examined by immunohistochemistry and Western analysis; inducible NOS was not detected in either endothelium or VSM. Expression of eNOS protein was greater (P < 0.05) in uterine, but not in systemic, artery endothelium-isolated protein collected from follicular versus luteal phase ewes. Acute systemic E2beta treatment of ovariectomized ewes increased (P < 0.05) eNOS protein levels in uterine artery endothelium. Prolonged E2beta administration progressively increased uterine, but not systemic, artery endothelial eNOS protein expression. Therefore, the increased local estrogen-to-progesterone ratio during the follicular phase locally elevates eNOS expression, which possibly elevates uterine blood flow. These responses can be partly reproduced with E2beta administration.

Animals↗
PubMed1998

Pregnancy increases ovine uterine artery endothelial cyclooxygenase-1 expression.

During normal pregnancy, and especially in the third trimester, both uterine blood flow and prostacyclin production by ovine uterine arteries are dramatically increased. We sought to determine if this is due, in part, to an increase in cyclooxygenase (COX) expression in the uterine artery endothelium. In this study we compared COX expression in uterine artery endothelium from nonpregnant and third-trimester pregnant (110-142 days' gestation) ewes. COX-2 expression was not detectable by Western blotting in uterine artery endothelium or vascular smooth muscle (VSM). In contrast, COX-1 expression was clearly observed in uterine artery. Immunohistochemical localization of COX-1 was endothelium > VSM, with both cell types showing an increase in COX-1 during the third trimester of pregnancy. COX-1 protein and messenger RNA (mRNA) levels were also detectable in collagenase dispersed endothelial cells, with expression of COX-1 in uterine artery endothelial cells dramatically increased during the third trimester of pregnancy at both the level of protein (346.4 +/- 28% of nonpregnant controls, P < 0.0005) and mRNA (51.04 +/- 7.98-fold of nonpregnant controls, P < 0.001). We conclude that the pregnancy-induced increases in prostacyclin production by uterine arteries is largely due to a dramatic increase in expression of COX-1 mRNA and associated protein predominantly occurring in the uterine artery endothelium and, to a lesser extent, in the VSM.

Animals↗
PubMed1998

Pregnancy increases soluble and particulate guanylate cyclases and decreases the clearance receptor of natriuretic peptides in ovine uterine, but not systemic, arteries.

Pregnancy increases uterine blood flow by 30- to 50-fold and uterine production of cGMP by 38-fold. Moreover, cGMP causes potent vasodilatation. We hypothesized that pregnancy up-regulates soluble and particulate guanylate cyclases (sGC and pGC) in ovine uterine arteries. Activities of sGC and pGC were compared by measuring cGMP production (37 C; 10 min) by uterine arteries from nonpregnant (n = 5) and pregnant (n = 4, 120 +/- 2 days' gestation; term = 145 +/- 3 days; mean +/- SE) ewes after sodium nitroprusside (100 microM), atrial natriuretic peptide (1 microM), or C-type natriuretic peptide (CNP; 1 microM) treatment. The protein and/or messenger RNA expressions of sGC beta1-subunit, pGC-A, pGC-B, the clearance receptor of natriuretic peptide (CR), and CNP were investigated in uterine and systemic (renal and/or omental) arteries from nonpregnant (n = 29) and pregnant (n = 21; 125 +/- 2 days' gestation) ewes. The potencies of uterine arterial GC activities were sGC >> pGC-A > pGC-B. Activities as well as protein expression of sGC, pGC-A, and pGC-B in pregnant uterine arteries were increased 48-128% above those in nonpregnant controls concomitant with a 34% down-regulation of CR protein expression; systemic arterial protein expressions were unaltered. These changes in uterine arterial GC-B and CR were confirmed using RT-PCR. Immunohistochemical staining of CNP in uterine, but not systemic, arterial endothelium from pregnant ewes was much stronger than that from nonpregnant ewes. Thus, two distinct GC pathways are present in ovine uterine artery, and both may be specifically upregulated during pregnancy and so contribute to the tremendous local increase in cGMP production during pregnancy.

Animals↗
PubMed1998

Protein kinase A, protein kinase C, and Ca(2+)-regulated expression of 21-hydroxylase cytochrome P450 in H295R human adrenocortical cells.

The physiological importance of adrenal 21-hydroxylase cytochrome P450 (CYP21) expression is clearly demonstrated by 21-hydroxylase deficiency, which results in adrenal hyperplasia and over-production of C19 steroids, leading to virilization. The mechanisms regulating normal expression of this key enzyme in human adrenocortical cells are ill defined. Herein we examine the role of the calcium, protein kinase C, and protein kinase A signaling pathways in the expression of CYP21 messenger ribonucleic acid (mRNA) using the H295R human adrenocortical cell model. Forskolin (10 mumol/L) treatment caused a progressive increase in CYP21 mRNA levels (maximum, 4-fold; P < 0.05) over 36 h of treatment, whereas angiotensin II (AII; 10 nmol/L) produced a smaller, biphasic rise (maximum, 1.8-fold at 12 h; P < 0.05). K+ (14 mmol/L) also induced a time-dependent (maximal, 1.5-fold at 12 h; P < 0.05) and dose-dependent (P < 0.05 12 mmol/L or above at 20 h) rise in CYP21 mRNA levels. The action of forskolin was reproduced by dibutyryl cAMP, confirming the involvement of cAMP in this response. The action of AII was greater than that of K+ or the calcium channel agonist BAYK8644, suggesting that AII action was not solely through the Ca2+ signaling pathway. The action of AII was reproduced and indeed exceeded by the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA; 10 nmol/L; 5.5-fold increase; P < 0.05). The actions of forskolin alone were not significantly increased by combined treatment with AII, suggesting neither synergy nor attenuation of the effects of protein kinase A activation. This was further demonstrated at the level of mRNA and 21-hydroxylase activity by the observation that the effect of forskolin and TPA in combination did not exceed that of TPA alone. Inhibition of protein synthesis with cycloheximide blocked induction of CYP21 as well as type II 3 beta-hydroxysteroid dehydrogenase (3 beta HSDII) mRNA expression in response to AII, forskolin, and dibutyryl cAMP, but had no effect on 17 alpha-hydroxylase cytochrome P450 (CYP17) or cholesterol side-chain cleavage cytochrome P450 (CYP11A) mRNA. Together, these findings were remarkably similar to those of our previous studies regarding mechanisms regulating 3 beta HSDII expression and underline the existence of a subset of steroidogenic enzymes regulated positively (CYP21 and 3 beta HSDII) as opposed to negatively (CYP17 and CYP11A) by the protein kinase C signaling pathway. The additional finding of a small induction of CYP21 expression in response to increased Ca2+, as previously reported for CYP17, but not 3 beta HSDII, expression, also demonstrates that the mechanisms of control of CYP21 and 3 beta HSDII are not identical. This latter finding may also relate to how CYP21 as well as CYP17 expression continues in the zona reticularis after adrenarche, whereas 3 beta HSD expression declines.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1997

The regulation of 3 beta-hydroxysteroid dehydrogenase expression.

3 beta-Hydroxysteroid dehydrogenasel delta 5-->4-isomerase (3 beta-HSD) catalyzes the formation of delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids, an obligate step in the biosynthesis not only of androgens and estrogens but also of mineralocorticoids and glucocorticoids. The enzyme is expressed in the adrenal cortex and in steroidogenic cells of the gonads, consistent with this role. However, 3 beta-HSD is also expressed in many other tissues, such as the liver and kidney, where its function is not entirely clear. It is established that a family of closely related genes encode for 3 beta-HSD. The various 3 beta-HSD isoforms are expressed in a tissue-specific manner involving separate mechanisms of regulation. The human type I 3 beta-HSD is expressed at high levels in syncytial trophoblast and in sebaceous glands, and the type II isoform is almost exclusively expressed in the adrenal cortex and gonads. An important feature in liver and kidney (at least of hamster, mouse, rabbit, and rat) is the sexual dimorphic nature of 3 beta-HSD expression. We briefly review studies on the regulation of the human 3 beta-HSD I and II genes in human trophoblast and adrenal cortex and extend this to discuss the rat 3 beta-HSD I gene expressed in adrenals and gonads. The complexity of 3 beta-HSD expression through multiple signaling pathways acting on a multigene family of enzymes may contribute importantly to the diverse patterns and locations of steroid hormone biosynthesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1997

Expression of basic fibroblast growth factor, endothelial mitogenic activity, and angiotensin II type-1 receptors in the ovine placenta during the third trimester of pregnancy.

To evaluate expression of basic fibroblast growth factor (FGF), endothelial mitogenic activity, and angiotensin II type-1 receptors (AT1r), as well as the role of angiotensin II (ANG II) in regulating basic FGF production/secretion, placentae were obtained from ewes on Days 110, 120, 130, and 142 of pregnancy and were separated into fetal cotyledonary (COT) and intercotyledonary (ICOT), as well as maternal caruncular (CAR) and intercaruncular (ICAR) components. Using immunohistochemistry, basic FGF and AT1r were found for the most part to be colocalized in all placental components, primarily in epithelium, stroma, endothelium, and vascular smooth muscle. Changes in basic FGF levels in placental explant-conditioned media were observed in fetal, but not maternal, components. In COT, basic FGF levels increased 2.4-fold (r2 = 0.48, p < 0.04) from Day 110 to 130 and then declined at term. In ICOT, basic FGF levels increased 6.4-fold (r2 = 0.33, p < 0.05) from Day 110 to 142. The rank order of averaged basic FGF levels was COT > CAR > ICOT = ICAR (p < 0.05). Endothelial mitogenic activity of conditioned media was observed in COT from Day 130 pregnant ewes (232.5 +/- 38.7% of control; p < 0.05) but not in other components, and it was neutralized by a basic FGF antibody. ANG II did not alter basic FGF levels in any placental component. Thus, throughout the third trimester, 1) basic FGF and AT1r are present in placentae, 2) both basic FGF levels and endothelial mitogenic activity in COT increase, 3) basic FGF levels are associated with endothelial mitogenic activity of COT, and 4) ANG II has no effect on production/secretion of basic FGF by the placenta.

Angiotensin II↗