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Biomedical subjects

I M Bird

Publications and source records attributed to I M Bird.

At least 91 records · Page 5Linked to original sources
PubMed1990

Agonist-stimulated turnover of the phosphoinositides and the regulation of adrenocortical steroidogenesis.

Just as the recognition of the role of the phosphoinositides and phosphoinositols as a cellular signalling pathway has seen a dramatic advance in the last 10 years, so parallel investigations in adrenocortical cells have led to an equally dramatic increase in our understanding of the mechanisms involved in the control of adrenal steroidogenesis. In rat and bovine adrenocortical cells, the non-cAMP stimulatory agonists AII, acetylcholine and vasopressin have been shown to promote receptor/G-protein-mediated activation of a polyphosphoinositide-specific phospholipase C. In turn, studies in rat ZG and bovine ZG and ZFR cells have provided strong evidence for a causal relationship between the rapid and sustained formation of inositol 1,4,5-trisphosphate and DG by phospholipase C, and the subsequent increase in steroidogenesis in these cell types. In addition to describing the stimulatory effects of the various agonists on phospholipase C activity, this review has considered whether agonists may act through stimulation of phospholipase A2. No agonist can be said to act exclusively through phospholipase A2, and only AII can be said not to act through phospholipase A2 in adrenocortical cells. It seems unlikely that many studies will focus on this question in future unless an alternative physiological role for phospholipase A2 becomes apparent.

Adrenal Cortex↗
PubMed1989

The preparation of D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,4,5-trisphosphate in milligram quantities from a readily available starting material.

The optimisation of a reaction for the conversion of glycerophosphoinositols to phosphoinositols is described. This reaction has been used in a scheme, described in detail, for the formation of D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,4,5-trisphosphate in mg quantities from a readily available preparation of mixed phosphoinositides. An optimised procedure is also detailed for the recovery of these products to high yield and purity. The identity of the products has been confirmed both by high resolution anion-exchange column chromatography and by 1H nuclear magnetic resonance studies. We report for the first time the 1H nuclear magnetic resonance spectrum for D-myo-inositol 1,4-bisphosphate.

Chromatography, Thin Layer↗
PubMed1989

Angiotensin II-stimulated cortisol secretion is mediated by a hormone-sensitive phospholipase C in bovine adrenal fasciculata/reticularis cells.

Conditions have been established for the incorporation of [3H]inositol ([3H]Ins) into the phosphoinositides of cultured bovine adrenal zona fasciculata/reticularis (ZFR) cells. Stimulation of these prelabelled cells with angiotensin II (10(-11)-10(-7) M AII) resulted in the dose-dependent (max. 16-fold at 10(-7) M AII), time-dependent formation of water-soluble radiolabelled products which show the same chemical and chromatographic properties as [3H]InsP, [3H]InsP2 and [3H]InsP3 standards. The results of the time-course studies of the changes in these products are consistent with the view that AII rapidly (less than 15 s) induces the activation of a polyphosphoinositide-specific phospholipase C. The action of this phospholipase on the polyphosphoinositides is sustained throughout 15 min of stimulation. The dose dependency of this response correlates closely with cortisol output and is reduced (to 52%, P less than 0.00005), but not abolished, in the absence of extracellular Ca2+. To our knowledge these results are the first clear demonstration that AII stimulates a polyphosphoinositide-specific phospholipase C in bovine ZFR cells.

Angiotensin II↗
PubMed1989

Characterization of the steroidogenic responsiveness and ultrastructure of purified zona fasciculata/reticularis cells from bovine adrenal cortex before and after primary culture.

Analysis by electron microscopy indicated that after 3 days of primary culture, purified bovine adrenal zonal fasciculata/reticularis (ZF/ZR) cells showed improved integrity of their ultrastructure, with an increased density of lipid droplets and smooth endoplasmic reticulum. The basal cortisol output was significantly (P less than 0.05) greater on day 3 of culture than for the freshly isolated cells in six out of seven experiments. Similarly, in six experiments with ACTH (1 nmol/l) and five experiments with angiotensin II (10 nmol/l), the stimulated cortisol secretion was significantly (P less than 0.01 for all 11 experiments) higher on day 3 of culture than in freshly isolated cells. No significant increase in cortisol secretion above basal was observed with noradrenaline at any concentration in the freshly isolated cells, whereas a dose-dependent increase in cortisol secretion was observed on day 3 of culture in all of four experiments. These findings were supported by cyclic (c) AMP output measured in one such experiment. Thus the basal cAMP output and that stimulated by ACTH (1 nmol/l) were significantly higher after culture (P less than 0.001, n = five wells for basal comparison; P less than 0.05, n = three wells for ACTH at 1 nmol/l). In agreement with the cortisol results, cAMP production was unaffected by any concentration of noradrenaline in the freshly isolated cells, whereas a dose-dependent rise was found after culture. Angiotensin II at all concentrations had no effect on cAMP production in freshly isolated or cultured cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1989

Role of phosphoinositol metabolism and phospholipases C and A2/A1 in signal transduction in isolated rat adrenal cells.

Isolated rat adrenal glomerulosa cells were prelabelled with [3H]inositol and stimulated with 25 nM-angiotensin II in the presence of Li+. The resulting inositol monophosphates were separated using h.p.l.c. and 2 major peaks of radioactivity were detected. These showed the same characteristics as inositol 4-phosphate and inositol 1-phosphate and stimulation with angiotensin II increased activity 4-5 fold and 7-8-fold respectively. A minor peak with the characteristics of inositol 1:2 cyclic phosphate increased 1.5-fold after stimulation. No material corresponding to inositol 2-phosphate or inositol 5-phosphate was detected. The results establish the identify of the main inositol phosphate products in angiotensin II-stimulated rat glomerulosa cells. Analysis by h.p.l.c. has been similarly used to assess the inositol phosphates produced after ACTH1-39-stimulation of isolated rat adrenal fasiculata-reticularis cells. A low dose of ACTH1-39 (10(-12) M) stimulated production of small but significant amounts of both glycerophosphoinositol and inositol monophosphate. Using superfused isolated fasiculata-reticularis cells it was also found that ACTH1-39 (10(-9) M and 10(-12) M) rapidly increased efflux of 45Ca+ from 45Ca2+-prelabelled cells. It is concluded that although the results are consistent with a role for phospholipase C in the action of angiotensin II on adrenal glomerulosa cells, in the action of ACTH on adrenal fasciculata-reticularis cells a role for phospholipase A2/A1 is implicated.

Adrenal Glands↗
PubMed1987

H.p.l.c. analysis of inositol monophosphate isomers formed on angiotensin II stimulation of rat adrenal glomerulosa cells.

[3H]Inositol-prelabelled isolated rat adrenal glomerulosa cells were stimulated with 25 nM-AII ([Asp1, Ile5]-angiotensin II) in the presence of 10 mM-Li+, and the resulting inositol monophosphate isomers were separated successfully by using a recently developed h.p.l.c. methodology. Two major peaks of radioactivity were detected which showed the same retention characteristics on h.p.l.c. as inositol 4-phosphate and inositol 1-phosphate and which increased 5-fold and 8-fold respectively on stimulation with AII. In addition, a relatively small peak with the retention characteristics of inositol 1:2-cyclic phosphate was seen to undergo a 1.5-fold increase on stimulation. This was not considered sufficient to suggest that cyclic phosphoinositols were a major product of AII-stimulated phosphoinositide turnover. No peaks of radioactive material were detected in the regions expected for inositol 2-phosphate (an acid hydrolysis product of inositol 1:2-cyclic phosphate) or inositol 5-phosphate. These results establish the identity of the major inositol phosphate products in AII-stimulated glomerulosa cells and confirm and extend the previous observations of Balla, Baukal, Guillemette, Morgan & Catt [(1986) Proc. Natl. Acad. Sci. 83, 9323-9327].

Adrenal Glands↗
PubMed1986

Two species of phospholipase C isolated from lymphocytes produce specific ratios of inositol phosphate products.

Chromatography of the soluble porcine lymphocyte phospholipase C on cellulose phosphate resolves the activity into two species. An HPLC method is described for separating the enzyme products, Ins 1,2 greater than P and Ins 1P. Use of these methods reveals that the two iso-enzymes liberate, with a high degree of reproducibility, characteristic ratios of the two products. This suggests that the amount of each product produced is an inherent property of the enzyme mechanism.

Animals↗
PubMed1985

Identification of multiple variants of fast muscle troponin T in the chicken using monoclonal antibodies.

Two monoclonal antibodies, T1/7 and T1/61, have been prepared which are specific for chicken fast muscle troponin T. Both are of the IgG gamma 1 subclass. Both antibodies cross-react strongly with human fast and chicken cardiac troponin T, but while T1/7 reacts weakly with rabbit fast troponin T, T1/61 does not. The antibodies can be used for fibre typing of both chicken and human muscle. The antibodies have been used to identify fast troponin T on two-dimensional maps of proteins from a variety of chicken muscles by electrophoretic transfer to nitrocellulose followed by immunoperoxidase staining. Using this technique five variant forms of fast troponin T have been identified. Two variants, fBT1 and fBT2, are expressed in breast muscle, while the other three, fLT1, fLT2 and fLT3 are expressed in leg muscle. Of the leg muscle variants, fLT1 and fLT2 correspond to the two forms described previously [Wilkinson, J.M., Moir, A.J.G. and Waterfield, M.D. (1984) Eur. J. Biochem. 143, 47-56]. The third variant, fLT3, has not been described before and is expressed in muscles which have a high content of slow fibres. In addition to these clearly defined variant forms immunostaining reveals multiple minor variants of troponin T present in leg muscle which may reflect complex RNA processing of the troponin T gene transcript.

Animals↗
PubMedDate not supplied

Pregnancy induces expression of cPLA2 in ovine uterine artery but not systemic artery endothelium.

OBJECTIVE: To determine whether pregnancy increases cytosolic phospholipase A2 (cPLA2) expression in uterine artery (UA) endothelial cells and vascular smooth-muscle (VSM) cells, and whether ovarian steroids mediate this effect. METHODS: Uterine arteries and omental arteries (systemic control) were isolated from pregnant (120-130 days' gestation) ewes and from nonpregnant ewes synchronized to the corresponding phases of the ovarian cycle (follicular or luteal) or ovariectomized. In addition, ovariectomized ewes were treated with vehicle, estradiol-17 beta (E2 beta), progesterone (P4), or combined E2 beta/P4 for 10 days, and UAs were collected. Arteries from all studies were separated into mechanically isolated endothelial and VSM fractions. Proteins were then solubilized and separated on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western immunoblotting using an affinity-purified mouse monoclonal antibody for cPLA2. RESULTS: Both UA endothelium and VSM fractions were seen to express cPLA2, detected as a single band with a molecular mass similar to that observed in myometrium (about 100 kD). Distribution of cPLA2, when expressed per microgram of protein, was observed as 40% in UA endothelium compared with 60% in VSM fractions. Uterine artery endothelial cPLA2 expression was specifically increased 1.9-fold in pregnancy (P < .05), whereas there was no significant change from VSM. Furthermore, in ovariectomized sheep versus intact luteal or follicular phase animals, there was no significant change in cPLA2 expression in endothelium or VSM. Administration of E2 beta, P4, or their combination in ovariectomized sheep also failed to reproduce the pregnancy-induced increase in cPLA2 expression in UA endothelium. Omental artery endothelial and VSM cPLA2 expression was observed at similar magnitudes as UA expression, but levels were consistently unchanged by pregnancy, the ovarian cycle, or ovariectomy. CONCLUSIONS: cPLA2 was expressed throughout the endothelium and VSM of both uterine and omental arteries, but only in UA endothelium was pregnancy associated with elevated cPLA2 expression. The lack of change in cPLA2 expression with the ovarian cycle or in ovariectomized animals, even after prolonged treatment with E2 beta, P4, or E2 beta/P4 combined suggests this pregnancy-induced increase in cPLA2 may not be estrogen and/or progesterone dependent.

Animals↗
PubMedDate not supplied

Expression of endothelial and inducible nitric oxide synthases and nitric oxide production in ovine placental and uterine tissues during late pregnancy.

We evaluated the expression of endothelial (eNOS) and inducible (iNOS) nitric oxide (NO) synthases, NO production, and the role of angiotensin II (ANG II) in regulating NO production during late ovine pregnancy (day 110-142). Samples of the following tissues were obtained: fetal [cotyledonary (COT)] and maternal [caruncular (CAR)] portions of the placentoma, intercotyledonary fetal chorioallantoic membrane (ICOT) and intercaruncular maternal endometrium (ICAR). Using immunohistochemistry, eNOS positive staining was detected in all four tissues, primarily in the endothelium, chorioallantoic membrane, and luminal and glandular epithelium. For iNOS, the positive staining was observed primarily in stromal cells in ICOT and ICAR. Expression of eNOS and iNOS proteins was confirmed in COT using Western immunoblot. eNOS protein levels increased (P< 0.05) approximately 3.5-fold from day 110 to 130 and then declined at term, whereas no change in iNOS protein levels was observed throughout the days studied. The tissue explants of COT, CAR, ICOT and ICAR were cultured in media in the absence or presence of ANG II (10(-9)or 10(-7) m) for 24 h. Total NO (nitrate and nitrite) levels in the explant-conditioned media were determined by chemiluminesence. In fetal COT, total NO levels increased (P< 0.05) 3.5-fold from day 110 to 130 and then declined (P< 0.05) at term. In ICOT, total NO levels exhibited a gradually increasing trend (r(2)=0.96, P< 0.01) from day 110 to days 130 and 142. In maternal CAR, total NO levels were higher (P< 0.05) on day 130 than those on days 120 and 142, whereas no change in total NO levels was observed in ICAR. ANG II at 10(-7) m treatment decreased (P< 0.05) total NO levels in COT on day 130. Thus, during late ovine pregnancy: (1) eNOS is expressed in COT, CAR, ICOT and ICAR while iNOS is primarily seen in stromal cells of ICOT and ICAR; (2) NO production by COT exhibits a biphasic pattern and parallels the changes in eNOS, but not iNOS protein levels, suggesting that eNOS is a predominant NOS isoform for the NO production; and (3) ANG II may contribute partially to decreases in NO production by COT at term.

Angiotensin II↗
PubMedDate not supplied

Acetylcholine induces oscillations in intracellular calcium in isolated bovine adrenal zona fasciculata/reticularis cells.

The effects of acetylcholine (ACh) on intracellular free calcium were studied in primary cultures of purified bovine adrenal zona fasciculata/reticularis (ZFR) cells. In fura-2 loaded single cells, concentrations of ACh which stimulated cortisol secretion and phosphoinositol production were found to promote an increase in free cytosolic calcium ([Ca2+]i). This response was heterogeneous, showing either (i) an initial increase in [Ca2+]i followed by a fall to a level above that in unstimulated cells, or (ii) an initial increase followed by oscillations about the original resting level or a higher resting level. The frequencies of [Ca2+]i oscillations to ACh showed a dose-dependent trend. The sustained [Ca2+]i oscillations were abolished by the muscarinic antagonist atropine, or by removal of extracellular Ca2+. These data demonstrate for the first time in adrenocortical cells that: (i) ACh can induce [Ca2+]i oscillations in single ZFR cells; (ii) these oscillations occur in a dose-dependent manner; (iii) the sustained oscillatory phase is dependent on influx of extracellular Ca2+. Thus, like cells of the zona glomerulosa, bovine ZFR cells are also capable of sustained dose-dependent oscillatory responses to agonists which activate phosphoinositidase C.

Acetylcholine↗
PubMedDate not supplied

Hormonal regulation of angiotensin II type 1 receptor expression and AT1-R mRNA levels in human adrenocortical cells.

Human adrenocortical H295R cells express AII receptors which are predominantly of the AT1 but not AT2 subclass. These receptors are functionally coupled to phosphoinositidase C in a manner similar to that seen in fetal human, sheep and bovine adrenocortical cells. Treatment of H295R cells with forskolin or dbcAMP to activate the protein kinase A pathway caused a rapid (maximal by 3 h) and sustained decrease in AT1-R mRNA levels which in turn preceded a time-dependent (maximal by 12 h) and dose-dependent loss of [125I]AII binding and phosphoinositidase C activation on subsequent AII challenge. Thus, both decreased AT1-R mRNA levels and functional receptor expression appear to parallel each other in response to activation of protein kinase A. Activation of the Ca2+/protein kinase C pathways by treatment with AII also caused a rapid (maximal by 3 h) and dose-dependent loss in AT1-R mRNA, but mRNA levels subsequently rose again, approaching control levels by 36 h. Treatment with AII for 48 h had little effect on either [125I]AII binding or the subsequent phosphoinositidase C response. The effect of AII, but not forskolin, was blocked by the presence of cycloheximide. The action of AII on AT1-R mRNA was probably mediated through both protein kinase C and Ca(2+)-sensitive protein kinases as the effect at 4 h was not completely reproduced by phorbol ester alone, but was fully reproduced by a combination of phorbol ester and Ca2+ ionophore. However, increased Ca2+ influx alone, due to treatment with BAYK8644 or elevated extracellular K+, also resulted in a decrease in AT1-R mRNA levels. Thus in the H295R cell, control of AT1-R expression appears to be complex, being achieved at least in part through control of the level of AT1-R mRNA by multiple independent signaling pathways including protein kinase A, protein kinase C and Ca2+.

Adenylyl Cyclases↗
PubMedDate not supplied

Battle of the kinases: integration of adrenal responses to cAMP, DG and Ca2+ at the level of steroidogenic cytochromes P450 and 3betaHSD expression in H295R cells.

While ACTH receptors (activating the protein kinase A pathway) are expressed throughout the human/bovine/ovine zona glomerulosa (zg) and zona fasciculata (zf), there are clear zonal differences in AII Type-1 receptor levels (activating protein kinase C/Ca2+), as well as resting membrane potential. Thus zg is most responsive to AII and K+ (Ca2+ signalling), while zf is less responsive to AII with no K+ response. Zonal function in turn requires differential expression of CYP17/3betaHSD and CYP11B2/CYP11B1. We have used the H295R cell to study how differential activation of kinase A, kinase C and Ca2+/calmodulin (CaM) kinases may alter the relative expression of the steroidogenic P450s and 3betaHSDII. While CYP11A, CYP17, 3betaHSDII, CYP21, and CYP11B1 are all induced by increases in cAMP, studies with TPA alone or in combination with forskolin reveal subsets of steroidogenic enzymes regulated either positively (CYP21, 3betaHSDII) or negatively (CYP17, CYP11A) by protein kinase C. Thus adrenal 3betaHSDII and CYP21 expression is high in zg and zf, but CYP17 is not expressed in the zg where AII activation of kinase C is highest. In turn both K+ and AII-induced elevation of Ca2+ strongly induces CYP11B2 but not CYP11B1, consistent with preferential expression of CYP11B2 in the zg. We conclude that differential signaling through kinase C and CaM kinases in addition to kinase A underlies zonal differences in both the early and late pathways involved in steroid hormone production within the adrenocortical zones.

3-Hydroxysteroid Dehydrogenases↗
PubMedDate not supplied

Isolation of an ovine genomic sequence containing the full-length angiotensin II type-1 receptor.

We have isolated from a genomic library using PCR amplification an 1171 base sequence containing a putative ovine AT1-R protein coding sequence of 1080 bases. As expected the protein coding sequence is of greater than 99% homology to the partial protein coding sequence reported by Robillard et al, with only one base difference. Relative to other species, highest homology at the level of the cDNA protein coding sequence is to bovine (97.6%) and lowest homology to rat Type 1a (83.3%). The predicted protein amino acid sequence in turn encodes a protein with the properties of a seven alpha-helix transmembrane receptor (by TMPred) sharing closest homology (98.6%) to the bovine receptor and lowest to the rat Type 1a (90.2%). As expected from such a high degree of interspecies homology, amino acids identified by site-directed mutagenesis of the human or rat AT1A-R as involved in binding and action of AII are very highly conserved in the ovine sequence. In addition, both bovine and ovine AT1-R are known to exhibit lower affinity for DuP753 than human AT1-R, and in bovine AT1-R this has been suggested to coincide with the amino acid substitutions Ala->Thr (163) and Leu->Met (262) relative to the human sequence. Our ovine AT1-R cDNA sequence shares these same bovine substitutions.

Amino Acid Sequence↗
PubMedDate not supplied

Dopaminergic stimulation of cortisol secretion from bovine zfr cells occurs through nonspecific stimulation of adrenergic beta-receptors.

Previous reports have suggested a possible dopaminergic inhibition of the actions of AII on aldosterone secretion via adenylate-cyclase inhibitory 'D2' receptors. Others suggest a possible stimulation of aldosterone secretion via a stimulatory 'D1' receptor/cAMP pathway. We have examined the actions of dopamine on basal and AII-stimulated cortisol secretion by cultured bovine zfr cells. Dopamine alone caused a dose-dependent increase in cortisol secretion at doses >10(-5) M, and also enhanced steroidogenic output in response to submaximal (10(-10) M) but not maximal (10(-8) M) stimulatory doses of AII. The stimulatory action of dopamine alone on cortisol secretion was not, however, reproduced by the 'D1' agonist fenoldopam, and was fully blocked by propranolol. Dopamine had neither a stimulatory effect on basal phosphoinositol production nor an inhibitory effect on AII-stimulated phosphoinositol production. Our findings are therefore inconsistent with the activation of a 'D1' or 'D2' class receptor, and suggest the stimulation of cortisol secretion occurred nonspecifically through a beta-adrenergic receptor.

Angiotensin II↗
PubMedDate not supplied

Antenatal steroid treatment and adverse fetal effects: what is the evidence?

This article reviews current animal and human data regarding possible adverse fetal effects from antenatal steroid treatment. Although it is now well accepted that such treatment is of benefit to fetal lung development, the potential for adverse fetal outcomes as a result of single or multiple glucocorticoid dosing has not been widely recognized. There are now growing concerns, based on animal and some human data, that repeated antenatal doses could lead to a decrease in birth weight, a decrease in fetal brain and other organ size, and abnormal neuronal development. Previous investigations have been hampered by nonstandardization in the type of glucocorticoid, route of delivery, timing of administration, and number of treatment courses. It is recommended that these concerns be addressed through large randomized, controlled clinical trials. In the meantime, it would be prudent to minimize antenatal steroid treatments to a single course with repeated dosing only if there is a persistent threat of preterm delivery. The practice of giving weekly injections of steroids starting at fetal viability and continuing into the third trimester is not supported.

Adrenal Cortex Hormones↗