Search PubMed⌕ Search

Biomedical subjects

S W Walker

Publications and source records attributed to S W Walker.

At least 37 records · Page 2Linked to original sources
PubMed1995

Differential control of type-I iodothyronine deiodinase expression by the activation of the cyclic AMP and phosphoinositol signalling pathways in cultured human thyrocytes.

The effects of TSH and the activation of the cyclic AMP (cAMP) and Ca(2+)-phosphatidylinositol (Ca(2+)-PI) cascades on the activity and expression of the selenoenzyme thyroidal type-I iodothyronine deiodinase (ID-I) have been studied using human thyrocytes grown in primary culture. Stimulation of ID-I activity and expression was obtained with TSH and an analogue of cAMP, 8-bromo-cAMP. In the presence or absence of TSH, the addition of the phorbol ester, phorbol 12-myristate 13-acetate (PMA) together with the calcium ionophore A23187, caused a decrease in ID-I activity; a decrease in ID-I expression was also observed as assessed by cell labelling with [75Se]selenite. PMA alone had no effect on ID-I activity in the presence or absence of TSH. A23187 alone produced a small but significant reduction in ID-I activity, but only in TSH-stimulated cells. These data provide evidence that the expression of thyroidal ID-I is negatively regulated by the Ca(2+)-PI cascade, and positively regulated by the cAMP cascade.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

The M3 muscarinic receptor mediates acetylcholine-induced cortisol secretion from bovine adrenocortical zona fasciculata/reticularis cells.

In order to characterize the receptor subtype mediating acetylcholine (ACh)-induced cortisol secretion from purified bovine adrenocortical zona fasciculata/reticularis cells in primary culture, the potencies of a range of selective muscarinic antagonists of ACh-induced steroidogenesis were assessed by Schild analysis. Basal secretion of cortisol was 10.2 +/- 1.4 pmol/well/30 min. ACh stimulated a dose-dependent increase in cortisol secretion and was maximally effective at 10(-5) M, at which concentration cortisol secretion was 143.4 +/- 12.9 pmol/well/30 min. Hexahydro-sila-difenidol and para-fluoro-hexa-hydro-sila-difenidol were potent competitive antagonists of ACh-stimulated cortisol secretion, with pA2 values of 8.68 +/- 0.28 and 7.96 +/- 0.29, respectively. Pirenzepine (pA2 = 6.95 +/- 0.28) and methoctramine (pA2 = 6.06 +/- 0.27) were relatively weak competitive antagonists. The pA2 values determined in this study are characteristic of the M3 muscarinic receptor, and we conclude that this receptor subtype mediates ACh-induced cortisol secretion from bovine zona fasciculata/reticularis cells.

Acetylcholine↗
PubMed1994

Primary cultures of bovine inner zone adrenocortical cells secrete cortisol in response to adenosine triphosphate, adenosine diphosphate, and uridine triphosphate via a nucleotide receptor which may be coupled to two signal generation systems.

Cultured inner zone cells isolated from bovine adrenal cortex secreted cortisol in a dose-dependent fashion in response to ATP and ADP. The threshold response was at 10(-6) M ATP, reaching a maximum by 10(-4) M ATP, at which concentration the n-fold relative to basal was 43.8 +/- 22.3 (mean +/- SD, n = 3). The response to 10(-4) M ATP remained linear for up to 2 h, and the cells appeared morphologically normal after removal of the stimulus. Stimulation of cortisol secretion by ATP was evident after 24 h in primary culture and reached a maximum after 48-72 h, thereafter declining. No response was detected in freshly isolated cells. The possibility that added ATP was degraded over the course of the incubation was investigated by separating ATP, ADP, AMP, and adenosine by high resolution anion exchange chromatography after different times of exposure to the cells. Although there was degradation--largely to ADP--about 50% of the ATP remained at 1 h. The potency order of a range of purines was as follows: ATP = ADP > 2-methyl-S-ATP > alpha, beta-methylene ATP = beta, alpha-methylene ATP = AMP. Cells were also responsive to the pyrimidine nucleotide uridine 5'-triphosphate, which was equipotent with ATP. The purinergic antagonist suramin was relatively ineffective. Cells grown in the presence of [3H]inositol (10 microCi/ml) for 48 h (to prelabel the membrane phosphoinositide pool to isotopic equilibrium) showed a time- and dose-dependent increase in [3H]inositol-labeled total phosphoinositols in response to ATP or ADP; the response was linear for at least 60 min. Cells labelled with the Ca2+ indicator fura-2 showed an increase in intracellular calcium in response to 10(-4) M ATP on days 3 and 4 of culture. Basal intracellular Ca2+ was found to be 57.3 +/- 39.3 nmol/liter (mean +/- SD, n = 12 cell suspensions) rising to 171 +/- 84 nmol/liter (mean +/- SD, n = 12 cell suspensions) in response to ATP (10(-4) M). In response to ATP, bovine inner zone cells also demonstrated a dose-dependent increase in intracellular cAMP measured after 1 min stimulation. It was not possible to account for the cAMP response on the basis of conversion of ATP to adenosine, which then acted at an A2 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1994

Primary cultures of bovine inner zone adrenocortical cells secrete cortisol in response to adenosine 5'-triphosphate, adenosine 5'-diphosphate, and uridine 5'-triphosphate via a nucleotide receptor that may be coupled to two signal generation systems.

Cultured inner zone cells isolated from bovine adrenal cortex secreted cortisol in a dose-dependent fashion in response to ATP and ADP. The threshold response was at 10(-6) M ATP, reaching a maximum by 10(-4) M ATP, at which concentration the n-fold increase relative to basal was 43.8 +/- 22.3 (mean +/- SD; n = 3). Cells were also responsive to the pyrimidine nucleotide UTP. EC50 values for ATP, ADP, and UTP were 5.83 +/- 3.98 x 10(-6), 13.7 +/- 5.67 x 10(-6), and 7.33 +/- 4.52 x 10(-7) M, respectively (mean +/- SD; n = 3). The response to 10(-4) M ATP was linear for at least 60 min, and the cells appeared morphologically normal after removal of the stimulus. The purinergic antagonist suramin was relatively ineffective. The potency order of a range of purines was as follows: ATP = UTP > ADP > 2-methyl-S-ATP > alpha, beta-methylene ATP = beta, alpha-methylene ATP = AMP. Stimulation of cortisol secretion by ATP was evident after 24 h in primary culture and reached a maximum after 48-72 h, thereafter declining. No response was detected in static incubations of freshly isolated cells. The possibility that added ATP was degraded over the course of the incubation was investigated by separating ATP, ADP, AMP, and adenosine by high resolution anion exchange chromatography after different times of exposure to the cells. Although there was degradation, largely to ADP, about 50% of the ATP remained at 1 h. Cells grown in the presence of [3H]inositol (10 microCi/ml) for 48 h (to prelabel the membrane phosphoinositide pool to isotopic equilibrium) showed a time- and dose-dependent increase in [3H]inositol-labeled total phosphoinositols to ATP or ADP; the response was linear for at least 20 min. Cells labeled with the Ca2+ indicator fura-2 showed an increase in intracellular calcium to 10(-4) M ATP on days 3 and 4 of culture. The basal intracellular Ca2+ concentration was 57.3 +/- 39.3 nmol/liter (mean +/- SD; n = 12 cell suspensions), rising to 171 +/- 84 nmol/liter (mean +/- SD; n = 12 cell suspensions) after the addition of ATP (10(-4) M). Bovine inner zone cells also demonstrated a dose-dependent increase in intracellular cAMP measured after 1 min of stimulation with ATP. It was not possible to account for the cAMP response on the basis of conversion of ATP to adenosine, which then acted at an A2 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1993

Primary culture of bovine gall bladder epithelial cells.

Gall bladder epithelial cells serve numerous biological functions and abnormalities in their function are important in the pathogenesis of several gall bladder diseases. Direct studies on cell function are rare due to lack of reliable methods to culture this epithelium. This study reports a reliable and reproducible method of harvesting and culturing gall bladder epithelial cells. Normal bovine gall bladder epithelium, obtained within 20 minutes of slaughter, was rinsed with modified Hanks's balanced salt solution, the mucosa separated and incubated in trypsin--EDTA solution at 37 degrees C. The cells were isolated and resuspended in Dulbeco's modification of Eagles' medium containing 10% fetal calf serum and, after filtration and centrifugation, were plated under aseptic conditions. The growth rate was established by flow cytometry and the morphological characteristics of the growing cells by electron microscopy. Gall bladder epithelial cells grew successfully and visible clusters of cells were present by day two, confluency being reached at 8 to 10 days in collagen coated plates and 12 to 14 days in uncoated plates. Electron microscopy showed typical gall bladder epithelia with microvilli, tight junctions, and mucus droplets. This method proved reliable and reproducible for the culture of gall bladder epithelial cells and should allow direct studies of the biological properties of these cells in human tissue.

Animals↗
PubMed1993

Angiotensin II stimulates growth and steroidogenesis in zona fasciculata/reticularis cells from bovine adrenal cortex via the AT1 receptor subtype.

Primary cultures of bovine adrenocortical zona fasciculata/reticularis (zfr) cells responded to angiotensin II (AII) with a dose-dependent increase in [3H]thymidine incorporation into DNA. The effect was maximal at 100 nmol/liter AII, and was dose dependently inhibited by (sar1, ala8)-AII (saralasin) and DuP753, but not by PD123177. Both AII-stimulated cortisol secretion and phosphoinositidase C activity were also inhibited by saralasin and DuP753, but not by PD123177. Pharmacological analysis of the antagonism of AII-stimulated cortisol secretion by saralasin and DuP753 produced pA2 values of 8.79 and 7.02, respectively. Whereas the pA2 for saralasin agreed closely with previous measurements in other systems, DuP753 was at least one order of magnitude less potent in inhibiting the action of AII in bovine zfr cells compared to previous measurements in rabbit vascular smooth muscle. We conclude that the steroidogenic and mitogenic effects of AII in bovine zfr cells are mediated by the AT1 receptor.

Angiotensin II↗
PubMed1993

The role of thyroidal type-I iodothyronine deiodinase in tri-iodothyronine production by human and sheep thyrocytes in primary culture.

We have studied the origin of tri-iodothyronine (T3) secreted by human and sheep thyrocytes in primary culture and also the expression of type-I thyroidal iodothyronine deiodinase (ID-I) in the thyroid and liver of man and various other animals. Inhibitors of ID-I reduced T3 secretion from human but not sheep thyrocytes. In contrast, inhibitors of de-novo thyroid hormone synthesis reduced both thyroxine (T4) and T3 production in sheep thyrocytes, but had no effect on the T3 secreted by human thyrocytes. Human thyrocytes did not produce T4 under the culture conditions used, although some endogenous T4 was present in the cells following their isolation. Although thyrotrophin (TSH) stimulated T3 production in both human and sheep thyrocytes, iodine in the form of potassium iodide was only essential for T3 and T4 production by the sheep cells. Although 125I from Na125I was incorporated into T3 and T4 in TSH-stimulated sheep thyrocytes, no 125I incorporation into T3 or T4 was detected in TSH-stimulated human thyrocytes. Using activity measurements and affinity labelling, ID-I was present in the livers of all species studied, but ID-I could not be detected in thyroid tissue from cattle, pigs, sheep, goats, rabbits, deer or llamas. In contrast, thyroid tissue from man, mice, guinea-pigs and rats had significant ID-I activity and expressed an affinity-labelled protein with a molecular mass of approximately 28.1 kDa on SDS-PAGE. These data show that under the culture conditions used, sheep thyrocytes produced T3 by de-novo synthesis, whilst human thyrocytes produced T3 by deiodination of endogenous T4. We conclude that thyroidal ID-I shows marked species difference in its expression and that, in those species which express the enzyme (man, mice, guinea-pigs and rats, in this study), it appears that it may make an important contribution to thyroidal T3 production.

Animals↗
PubMed1992

Studies of hormone-sensitive and -insensitive pools of phosphoinositides in cultured bovine zona fasciculata/reticularis cells. Evidence that acetylcholine and angiotensin II stimulate the breakdown of a common pool of phosphoinositides.

The effects of acetylcholine (ACh) and manganese pre-incubation on angiotensin II (AII)-stimulated incorporation of [3H]inositol into phosphoinositide, phosphoinositol and free inositol fractions of adrenocortical cells isolated from the bovine zona fasciculata/reticularis (zfr) were investigated. In cells pre-labelled for 6 hr with [3H]inositol, ACh and AII stimulated the incorporation of cytosolic [3H]inositol into a common hormone-sensitive pool of phosphoinositides, which was distinct from the non-hormone-sensitive pool labelled in the presence of manganese. Regression analysis of the cortisol versus [3H]inositol headgroup responses for both AII (10(-11)-10(-7) M) and ACh (10(-9)-10(-3) M) showed that the gradients of these responses were not significantly different. These data provide strong evidence that in cultured bovine zfr cells, ACh and AII stimulate the breakdown and resynthesis of a common pool of phosphoinositides.

Acetylcholine↗
PubMed1992

Identification and metabolism of phosphoinositol species formed on angiotensin II stimulation of zona fasciculata-reticularis cells from the bovine adrenal cortex.

The identity of phosphoinositol isomers accumulating on stimulation of primary cultures of bovine adrenocortical zona fasciculata/reticularis cells with angiotensin II (AII), in the presence of Li+, has been established by chromatographic separation on a MonoQ HR5/5 column. The metabolism of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in a broken cell preparation has also been studied in the absence or presence of added ATP. Our results show that Ins(1,4,5)P3 is formed within 5 s of stimulation of whole cells, but is rapidly converted to Ins(1,3,4)P3 through an Ins(1,3,4,5)P4 intermediate. All the phosphoinositol products accumulating on prolonged (15 min) stimulation of whole cells (Ins1P, Ins4P, Ins(1,3)P2, Ins(1,4)P2, Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4) can be accounted for by the metabolism of Ins(1,4,5)P3 in broken cells, either through direct dephosphorylation in the absence of added ATP (Ins(1,4)P2, Ins4P) or through dephosphorylation of Ins(1,3,4,5)P4 formed in the presence of added ATP (Ins(1,3,4)P3, Ins(1,3)P2 and Ins1P). Our results provide further evidence to suggest that AII stimulates the rapid and sustained breakdown of phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P2) to form Ins(1,4,5)P3.

Angiotensin II↗
PubMed1992

The relationship of blood lactate concentrations, oxygen delivery and oxygen consumption in septic shock and the adult respiratory distress syndrome.

Tissue hypoxia is thought to be pivotal to the development of multiple organ failure, but cannot be measured directly in clinical practice. We assessed the relationship between initial arterial blood lactate concentrations and the presence of the phenomenon of delivery-dependent oxygen consumption, both of which may indicate tissue hypoxia. Twenty-three critically ill patients with septic shock and adult respiratory distress syndrome were studied prospectively and allocated to one of two groups according to blood lactate concentrations. In group 1, blood lactate concentration was less than the level widely accepted as significant (2 mmol.l-1); in group 2, the concentration exceeded 2 mmol.l-1. In both groups, resuscitation with colloid, blood and vasoactive drugs resulted in significant increases in oxygen delivery; in group 1 (n = 13), mean (SEM) oxygen delivery increased from 484 (36) to 730 (44) ml.min-1.m-2 (p < 0.005) and in group 2 (n = 10) from 550 (54) to 780 (54) ml.min-1.m-2 (p < 0.05). In neither group was there a significant change in oxygen consumption. However, there were individuals in both groups who exhibited pathological delivery dependence. This suggests that the absence of hyperlactataemia does not preclude delivery dependence of oxygen consumption with the attendant potential for tissue hypoxia.

Adult↗
PubMed1992

Interleukin-1 beta stimulates growth of adrenocortical cells in primary culture.

Using tritiated-thymidine incorporation as a measure of cell growth, interleukin-1 beta stimulated the growth of bovine zona fasciculata/reticularis adrenocortical cells after 72 h in primary culture. Within the range of 10-1000 pg/ml, interleukin-1 beta produced over 40% of angiotensin II-stimulated [3H]thymidine incorporation (P less than 0.005 compared with basal for 10 pg/ml and 1000 pg/ml; P less than 0.05 for 100 pg/ml; two-tailed unpaired Student's t-test). Interleukin-1 beta did not directly stimulate cortisol secretion. By stimulating adrenocortical growth, the increase in interleukin-1 during fever provides a potential mechanism for chronically raising glucocorticoid output. This study is the first demonstration of a long-term effect involving interleukin-1 beta on the adrenal cortex.

Animals↗
PubMed1992

Evidence for two distinct hormone-sensitive [3H]phosphoinositide pools in bovine adrenocortical zona fasciculata/reticularis cells stimulated with angiotensin II.

Bovine adrenocortical cells from the zona fasciculata/reticularis were isolated and their phosphoinositides labelled to a steady state with [3H]inositol in primary culture. Experiments performed on these cells in the presence of Li+ have shown that, over a period of 60 min, angiotensin II (AII; 10(-7) M) stimulated a linear increase in [3H]inositol phosphates that was sustained through the utilization of two hormone-sensitive subpools of prelabelled lipid (30% and 45% respectively), and a rapid resynthesis of [3H]phosphoinositide into one of these pools using cytosolic [3H]inositol. The 30% pool was used immediately on stimulation, and was sustained at a steady-state size of 10-15% during the first 30 min of stimulation through rapid resynthesis using cytosolic [3H]inositol. Only after 30 min, when the cytosolic [3H]inositol was depleted and resynthesis could no longer occur, did the additional 45% pool start to supply further substrate to the phospholipase C, thereby further sustaining the generation of [3H]inositol phosphates. Once this pool was depleted however (by approximately 60 min), [3H]inositol phosphate generation finally ceased. These findings establish the differential use of two metabolically distinct hormone-sensitive pools of phosphoinositide following AII stimulation in bovine adrenocortical cells, events which are dependent upon the availability of cytosolic inositol for phosphoinositide resynthesis.

Angiotensin II↗
PubMed1992

Expression of an abundant alpha-class glutathione S-transferase in bovine and human adrenal cortex tissues.

Bovine adrenal cortex tissue expresses high levels of glutathione S-transferase (GST) from each of the alpha, mu and pi gene families. We describe the purification and characterization of an abundant alpha-class GST from this tissue that has not been identified previously because of its failure to bind to S-hexylglutathione-Sepharose 6B (S-hexG-Ag). This enzyme has been affinity purified on glutathione-Sepharose 6B (GSH-Ag) and was obtained in a highly purified form by employing S-hexG-Ag to remove the bulk of GST before chromatography on GSH-Ag. The purified GST eluted from GSH-Ag was found to exhibit marked peroxidase and delta 5-ketosteroid isomerase activities (19.2 and 1.67 U/mg respectively). The bovine enzyme also showed high GST activity towards 4-hydroxynonenal (5.09 U/mg). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the bovine GST contains two distinct polypeptides, one with an Mr of 25,900 and the other with an Mr of 26,500. An abundant alpha-class GST was also purified from human adrenal cortex that possessed properties which were similar to the bovine alpha-class GST described above; however, unlike the bovine enzyme, the corresponding human alpha-class GST bound to S-hexG-Ag. As with the bovine enzyme, the purified human GST displayed marked peroxidase and isomerase activities (27 and 4.02 U/mg respectively). Further analysis on SDS-PAGE (Mr 25,800) and reverse-phase high-performance liquid chromatography established that this abundant alpha-class GST in human adrenal cortex is equivalent to the human liver GST B1B1 enzyme. As both human and bovine adrenal cortex contain high levels of alpha-class GST with similar catalytic properties, we discuss the possible functions of these enzymes in this tissue.

Adrenal Cortex↗
PubMed1992

Further characterization of the steroidogenic responsiveness of purified zona fasciculata/reticularis cells from bovine adrenal cortex before and after primary culture: changing responsiveness to phosphoinositidase C agonists.

When bovine adrenocortical cells from the zona fasciculata/reticularis (zfr) are maintained in primary culture, cortisol secretion in response to acute stimulation with ACTH and adrenaline (which activate adenylate cyclase) is seen to increase steadily over the first 48 h, while secretion in response to angiotensin II and acetylcholine (which activate phosphoinositidase C) shows an initial decline in the first 24 h and a recovery to maximum after 48 h. We have investigated whether these discrepant changes in cortisol secretory response to the different agonists are due to changes in formation of the associated second messengers (cAMP or inositol phosphates), or altered coupling of these second messenger signals to steroid secretion. Increases in steroid secretion in response to ACTH and adrenaline were paralleled by increased cAMP. Steroid secretion in response to exogenous 8-bromoadenosine 3':5'-cyclic monophosphate also increased steadily during this 48-h period. Thus increased responsiveness was due to both increased second messenger formation and increased coupling to the steroid secretory response. The decreased steroid secretory response to angiotensin and acetylcholine after 24 h, and subsequent recovery after 48 h in culture, were accompanied by an increased formation of phosphoinositols after 24 h and a further increase by 48 h. However, the steroid secretory response to a combination of calcium ionophore and the protein kinase C activator, phorbol 12-myristate 13-acetate, was reduced after 24 h and recovered by 48 h of culture. Fura-2-loaded cells also showed an increase in intracellular [Ca2+] after 24 h in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The apoptosis endonuclease and its regulation.

Activation of an endogenous endonuclease has been observed in conjunction with the structural changes of apoptosis in a wide variety of cell types and circumstances. The endonuclease is present constitutively in some cells (e.g. rodent cortical thymocytes) in which apoptosis is readily triggered by many unrelated stimuli, but is inducible in others. Purification of this enzyme is an objective of some importance in apoptosis research, as it might act as a marker of susceptibility to apoptosis and lead to better understanding of the regulation of the process as a whole. Early data suggest that the thymocyte endonuclease is an anionic protein of molecular weight greater than 110 kDa, with a pH optimum of 7.5 and a double-strand cleavage preference. Its activity, and the induction of apoptosis as a whole, is regulated by several familiar cellular proto-oncogenes and oncosuppressor genes, including c-myc, Ha-ras, bcl-2 and p53.

Animals↗
PubMed1991

Adrenergic and cholinergic regulation of cortisol secretion from the zona fasciculata/reticularis of bovine adrenal cortex.

Inner zone cells, isolated from bovine adrenal cortex, secrete cortisol in response to both adrenergic and cholinergic agonists. The response to adrenaline (and other catecholamines) appears during culture, is evident by 24 h and reaches a maximum by 48-72 h, but is absent in freshly isolated cells. Pre-incubation of cultured cells with adrenaline leads to homologous desensitisation; the possibility that this may explain the absent response in freshly isolated cells is discussed. Cells show a dose-dependent cyclic AMP response but no increased membrane phosphoinositide turnover. In agreement, cortisol secretion is blocked by beta-receptor, but not alpha-receptor, antagonists. Schild analysis established that the response occurs through binding to a beta 1-receptor subtype, consistent with adrenergic innervation as opposed to an effect of circulating catecholamines. In contrast, cortisol secretion to AcCh was present in both freshly isolated cells and those in culture, reaching a maximum by 48-72 h in culture. The response was specifically blocked by muscarinic, but not nicotinic, antagonists. No effect on cyclic AMP formation was observed, but dose-dependent stimulation of phosphoinositide turnover occurred. HPLC analysis of the time-course of appearance of 3H-inositol labelled head groups (from cells pre-labelled with 3H-inositol) confirmed that AcCh activates a phosphoinositidase C. Intracellular Ca2+ oscillations were also measured from fura-2 loaded single cells in response to AcCh. Together with other pharmacological studies, these observations establish that AcCh acts through a M3 muscarinic receptor subtype in these cells. The possible significance of these findings in vivo is discussed.

Acetylcholine↗