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S MacNeil

Publications and source records attributed to S MacNeil.

At least 55 records · Page 3Linked to original sources
PubMed1995

Evidence for chromium acting as an essential trace element in insulin-dependent glucose uptake in cultured mouse myotubes.

Previous work from this and other laboratories has suggested that the trace element chromium plays some role in glucose homeostasis. In this study, we sought to characterise an in vitro cell culture model in which the effects of chromium on insulin-dependent glucose uptake could be studied. Mouse C2C12 myoblasts were differentiated to form myotubes in culture in chromium-replete or chromium-poor media. Chromium levels in standard media were 0.56 +/- 0.01 micrograms/l (mean +/- S.E.M.) compared with 0.09 +/- 0.01 micrograms/l in chromium-poor media. In chromium-poor media, insulin-stimulated uptake of radiolabelled glucose was reduced by almost 50% compared with that found in chromium-replete media. This decreased response could be restored by the addition of physiologically relevant (0.3 micrograms/l) concentrations of inorganic chromium (P < 0.001). The sensitivity of these cells to insulin was reduced dramatically by a reduction in the chromium content of the medium and was again increased (P < 0.001) by chromium addition. The concentrations of chromium required to restore the sensitivity to insulin were of the same order as those found physiologically; much higher concentrations of chromium could also stimulate glucose uptake in the absence of insulin but such concentrations were supra-physiological.

Animals↗
PubMed1995

Effects of pharmacological modulation of intracellular signalling systems on retinal pigment epithelial cell attachment to extracellular matrix proteins.

Complication of retinal detachment by proliferative vitreoretinopathy (PVR) is common. In the contraction of intraocular collagen matrices which occurs in PVR cell proliferation, migration and adhesion seem to be more important than any inherent cellular contractility. The aim of this study was to investigate the pharmacological inhibition of adhesion of retinal pigment epithelial cells to extracellular matrices. The adherence of human RPE lines to a range of ten substrates was assessed to determine their preferred substrates for attachment. The effect of pharmacological inhibitors and stimulators of protein kinase C, cyclic AMP and calcium/calmodulin intracellular signal transduction systems on attachment to substrates was investigated. RPE cells showed a clear substrate preference for fibronectin, and slight preference for collagen type I. Modulation of the protein kinase C and cAMP pathways had relatively minor effects upon RPE attachment. Increasing intracellular calcium concentration reduced RPE attachment to 12% of control, whilst reducing intracellular calcium had a less marked, although significant effect. Down-regulation of calmodulin reduced attachment to 17% of control. The drug tamoxifen, and the experimental calmodulin antagonist J8, produced significant inhibition of attachment even when cells had been allowed to adhere for 24 h prior to exposure to these agents. The adhesion of RPE to extracellular matrices may be markedly affected by drugs which modulate the intracellular calcium and calmodulin signalling systems. Calmodulin antagonists warrant further investigation as possible pharmacological inhibitors of PVR.

Animals↗
PubMed1994

What role does the extracellular matrix serve in skin grafting and wound healing?

Recent research on the structure and composition of the extracellular matrix (ECM) now strongly indicates that the major role of this matrix is in regulating cell/cell communication rather than in passively supporting cells. A wealth of structural data on the ECM suggests that there are specific arrangements of sequences within these proteins which profoundly influence the behaviour of the cells moving in that area, with respect to attachment, migration, differentiation and proliferation. In the skin, the ECM can be argued to promote 'appropriate' communication between the keratinocyte and the fibroblast. Skin ECM can be considered to consist both of the large insoluble proteins produced primarily by the fibroblasts, and soluble proteins which may be produced by fibroblasts or keratinocytes and become attached to the ECM. Both the large insoluble and the smaller soluble proteins may constitute signals which influence the behaviour of the keratinocytes. The clinical awareness of the need for a dermal component in skin grafting highlights the need for further research into the way in which the ECM influences keratinocyte/fibroblast biology. Such research will prove relevant to understanding the problems of graft take, graft contracture and scarring.

Animals↗
PubMed1994

Extracellular matrix derived from hair and skin fibroblasts stimulates human skin melanocyte tyrosinase activity.

There is indirect evidence that both skin and hair melanocytes are regulated by the activity of adjacent cells. In hair, the specialized fibroblasts (dermal papilla cells) appear to play a role in the regulation of hair growth. Hair pigmentation may relate to hair growth. In skin, melanocytes are located adjacent to the basement membrane zone. As far as we are aware, direct interactions of fibroblasts with melanocytes have not previously been investigated. Accordingly, the objective of this study was to develop co-culture conditions in which to investigate whether dermal fibroblasts from skin or hair could influence melanocyte differentiation. The influence of fibroblast-conditioned media, co-culture with fibroblasts, and fibroblast-derived extracellular matrix (ECM) on normal human skin melanocyte tyrosinase activity was examined. Fibroblasts from both skin and hair were capable of altering melanocyte morphology and significantly increasing tyrosinase activity when melanocytes were cultured in the absence, but not the presence, of the major proliferative drives. Although stimulation of tyrosinase activity was detectable with conditioned medium and co-culture with fibroblasts, the most striking result was obtained with the fibroblast-produced ECM which, on average, produced a four-fold increase in tyrosinase activity within 6 days. Thus, the study describes co-culture conditions in which the stimulatory effect of the fibroblast on melanocyte differentiation can be examined.

Cell Differentiation↗
PubMed1994

Tamoxifen inhibition of ocular melanoma cell attachment to matrix proteins.

Tamoxifen plays a major role in the management of breast cancer in women and is currently used to a lesser extent in other neoplasias. Many of the pharmacological properties of tamoxifen are consistent with anti-estrogen activity, but it also has significant, although lesser, benefit in patients whose tumours are estrogen-receptor negative. We recently reported that murine B16 melanoma cell attachment to extracellular matrix proteins can be inhibited by calmodulin antagonists. In seeking a calmodulin antagonist that could be used clinically, we investigated tamoxifen, which is known to have calmodulin antagonist activity in vitro, and confirmed that it will inhibit murine melanoma cell attachment in vitro. In the current study, we examined the effect of tamoxifen on the attachment of human ocular melanoma cell lines to a range of extracellular matrix substrates to evaluate the potential relevance of calmodulin antagonists, including tamoxifen, to reducing metastatic spread of these tumours. We report that six ocular melanoma cell lines established from choroidal melanoma tumours showed rapid attachment to a range of substrates and that this attachment can be significantly reduced by an experimental calmodulin antagonist (J8) and by tamoxifen. In summary, we conclude that the ability of calmodulin antagonists, including tamoxifen, to inhibit ocular melanoma cell attachment to matrix proteins in vitro merits further investigation as it may offer another approach to reducing metastatic spread of these tumours.

Animals↗
PubMed1994

An investigation of the ability of TSH and Graves' immunoglobulin G to increase intracellular calcium in human thyroid cells, rat FRTL-5 thyroid cells and eukaryotic cells transfected with the human TSH receptor.

The purpose of this study was to determine if immunoglobulin G preparations (IgGs) from patients with Graves' disease can increase intracellular calcium in thyroid cells, as has been reported for TSH. Both TSH and Graves' IgGs (prepared by protein G affinity chromatography) increased calcium in a range of thyroid cells; however, the response seen, using Fura-2-loaded coverslips of cell monolayers, varied considerably. Chinese hamster ovary (CHO/JPO9) cells transfected with a high number of human TSH receptors showed the greatest response: TSH (10 mU/ml) increased calcium in 46% of experiments and 18 out of 25 (72%) Graves' IgGs increased calcium at 0.1 mg/ml (significantly greater, P < 0.001, than for control IgGs where cells responded to 2 out of 13 preparations). Rat FRTL-5 cells only responded to TSH in 22% of experiments and to 2 out of 8 (25%) of Graves' IgGs. Similarly, human thyroid cells responded to TSH in 22% of experiments and to 2 out of 9 (22%) of Graves' IgGs. (When studying cyclic AMP responses in JPO9 cells, much higher concentrations of Graves' IgGs were required (1-3 mg/ml). However, higher concentrations (0.3 mg/ml) of both Graves' IgGs, and to a lesser extent of control IgGs, were capable of increasing calcium in cells both with and without TSH receptors (control CHO cells and normal human dermal fibroblasts). We conclude that relatively low concentrations of patient IgGs can be distinguished from control IgGs in JPO9 cells on the basis of their ability to increase calcium, but that additionally all IgG preparations possibly contain another factor which can increase calcium in a range of cells independent of the presence of the TSH receptor.

Adolescent↗
PubMed1994

Effect of melanocyte stimulating hormone on human cultured choroidal melanocytes, uveal melanoma cells, and retinal epithelial cells.

PURPOSE: To establish methodology for the culture of human choroidal melanocytes to compare their responsiveness to melanocyte stimulating hormone (MSH) with that of their transformed melanoma counterparts and with that of the retinal epithelial cell. METHODS: Choroidal melanocytes from the choroid of eyes enucleated for the presence of malignant melanoma were cultured in MCDB 153 medium supplemented with insulin, transferrin, hydrocortisone, glutamine, nystatin, vitamin E, phorbol myristate acetate, bovine hypothalamic extract, cholera toxin, and chelexed fetal calf serum. RESULTS: High yields of pure spindle-shaped bipolar melanocytes were obtained with a doubling time of 3 to 4 days in nine consecutive eyes. Cells continued to divide after 4 months in culture. In contrast, uveal malignant melanoma cells grew rapidly in a relatively simple medium of Ham's F12:DMEM (1:1) supplemented with fetal calf serum, insulin, transferrin and glutamine. This medium was unable to support choroidal melanocytes. Choroidal melanocytes were DOPA-positive with appreciable tyrosinase activity that significantly increased with treatment with MSH. MSH also significantly altered the size, local density, and distribution of primary and mature melanosomes of ocular melanocytes. In contrast, uveal melanoma cells had a low level of tyrosinase activity and failed to respond to MSH with either an increase in enzyme activity or melanosome size. Retinal epithelial cells failed to show significant tyrosinase activity under the conditions studied or any increase in melanosome size in response to MSH. CONCLUSION: Ocular melanocytes show evidence of regulation by MSH and a range of mitogenic stimuli unlike the transformed melanoma cells, implying a loss of regulatory control in the latter.

Animals↗
PubMed1993

The inter-relationship between insulin and chromium in hyperinsulinaemic euglycaemic clamps in healthy volunteers.

Evidence in the literature suggests that the trace element chromium may have a role in glucose homeostasis through the regulation of insulin action. We have previously reported a significant reduction in plasma chromium levels in healthy individuals, following a 75 g oral glucose load, and after meals and glucose-dependent uptake of chromium in insulin-dependent tissues in vitro. However, in vivo it is unclear whether the changes in plasma chromium relate to changes in plasma glucose or insulin. The present study describes a series of euglycaemic hyperinsulinaemic clamps designed to attempt to define the initiator of changes in plasma chromium levels in ten healthy individuals. The data showed a significant (P < 0.01) reduction in fasting plasma chromium levels following glucose infusion and an initial bolus of insulin. Significant (P < 0.02) increases in post-clamp urinary chromium excretion were insufficient to explain the decrease in plasma levels. During the recovery phase of an extended two-phase clamp protocol we found plasma insulin levels decreased by 70% within 10 min, associated with an increase in plasma chromium levels of 30% and no significant change in plasma glucose level. These data indicate that alterations in plasma glucose are unlikely to be directly related to changes in plasma chromium, whilst supporting the hypothesis that plasma insulin may influence plasma levels of this trace element. In contrast, plasma zinc was unaffected throughout these clamp studies.

Adult↗
PubMed1993

MSH receptors and function in amelanotic B16 melanoma cells.

The possible mechanisms for the reduced melanin content and poor melanogenic response to MSH was investigated in B16-F10DD differentiation deficient melanoma cells. In particular, the MSH receptor status and associated signal transduction pathway linking to tyrosinase activity in these cells was studied for evidence of any defects. F10DD cells contained high-affinity binding sites for alpha-MSH, with KD values similar to those previously reported for other variants of the B16 melanoma. SDS-PAGE analysis after radioactive ligand cross-linking showed no evidence of gross structural alterations of the receptor. The F10DD cells expressed approximately twice as many receptors as the F10 parent cell line, suggesting a possible feedback response attempting to compensate for the amelanotic condition. The functional integrity of the MSH receptors in F10DD cells was confirmed by the presence of increased levels of cAMP in response to MSH stimulation. These results, coupled with the observation that F10 and F10DD cells express similar levels of tyrosinase mRNA and protein, point to a structural defect in tyrosinase or in the post-translational control mechanisms by which the activity of this enzyme is regulated.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Calcium-calmodulin-dependent activation of adenylate cyclase in prostaglandin-induced electrically-monitored intestinal secretion in the rat.

The calcium-calmodulin antagonist 5-iodo-C8-W7 inhibited the PGE2-induced stimulation of cAMP production by isolated enterocytes from rat small intestine. It also reduced the secretory response of intestinal sheets to PGE2, measured as a rise in short-circuit current. It did not however, inhibit the electrical responses to forskolin and dibutyryl cAMP, nor to acetylcholine, a secretagogue whose effect is not mediated by cAMP. It is concluded that the receptor-mediated activation of adenylate cyclase and the subsequent secretory response are dependent upon calcium-calmodulin.

Acetylcholine↗
PubMed1992

A simple bioassay for epidermal growth factor using pig thyrocytes.

A bioassay for epidermal growth factor (EGF) is described using an eluted stain assay (ESTA) of dehydrogenase activity in pig thyrocytes. Optimal responsiveness to EGF was obtained in confluent cultures of primary pig thyrocytes cultured with EGF or biological samples containing EGF for either 24 or 48 h. Dehydrogenase activity was determined by measuring the production and then the release of a coloured formazan product produced by reduction of a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide substrate added to the cells. The assay responded equally to mouse, human and recombinant EGF and was suitable for measuring EGF activity in some but not all biological fluids. Specificity of detection of EGF activity was confirmed using antibody to EGF. The ESTA assay compared favourably with the radioreceptor assay for EGF in terms of sensitivity to EGF with half-maximal activation at 0.24 +/- 0.06 nmol/l (mean +/- S.E.M., n = 22 experiments) for the ESTA assay and 0.60 +/- 0.13 nmol/l (n = 7 experiments) for the radioreceptor assay.

Animals↗
PubMed1991

1 alpha,25(OH)2 vitamin D3 increases intracellular calcium in human keratinocytes.

Vitamin D3 metabolites have been found to improve psoriasis but their mechanism of action is not clear. Keratinocyte proliferation and differentiation are known to be dependent on calcium concentrations in vitro. The aim of this study was to examine whether 1 alpha,25(OH)2 vitamin D3 had any direct effect on intracellular free calcium concentrations in cultured keratinocytes. A response to 1 alpha,25(OH)2 vitamin D3 was seen in 88% of monolayers of normal human keratinocytes attached to glass coverslips. An increase in intracellular free calcium was seen in 80% of the reactive cultures, with over half the responses occurring within 30 s of exposure to 1 alpha,25(OH)2 vitamin D3 and the remainder occurring within minutes. Responses could be seen at physiological concentrations of 1 alpha,25(OH)2 vitamin D3 and were not blocked by the protein synthesis inhibitor cycloheximide. The response to 1 alpha,25(OH)2 vitamin D3 took the form of rapid transient increases in intracellular free calcium in 29 out of 59 coverslips. The basal intracellular free calcium was calculated to be 245 +/- 47 nM rising to a maximum of 834 +/- 267 nM (mean +/- SEM; n = 20) following exposure to 1 alpha,25(OH)2 vitamin D3. We conclude that 1 alpha,25(OH)2 vitamin D3 acts directly on keratinocytes to increase intracellular free calcium and that this may be relevant to its mechanism of action in psoriasis.

Calcitriol↗
PubMed1990

Elevated platelet-free calcium in uraemia.

Bleeding complications in uraemia are not uncommon. The pathogenesis of haemorrhage in uraemia is still a matter of controversy and the pattern of bleeding suggests a defect of primary haemostasis. Platelet aggregation and biochemistry, including calcium levels, have been studied; however, the results are controversial. We have examined platelet aggregation, platelet-free calcium and calmodulin in platelet-rich plasma because of the significant role of calcium and calmodulin in regulating platelet and other cells' functions. Platelet aggregation in uraemic subjects was similar to that of controls. Platelet basal free cytosolic calcium and platelet calcium in response to 10 microM Ca++ ionophore A23187 in eight subjects with uraemia were 117 +/- 33 nM and 2025 +/- 398 nM (mean +/- SEM) respectively. By contrast in seven matched healthy controls basal calcium and ionophore-stimulated calcium values were 47 +/- 14 nM and 1354 +/- 414 nM, significantly less than in the patients with uraemia (P less than 0.05). The sensitivity of uraemic platelets to A23187 was similar to that of controls. Calmodulin activity in platelet-rich plasma of 12 subjects with uraemia showed no significant difference from that of controls [1.86 +/- 0.29 micrograms/ml (mean +/- SEM) and 2.0 +/- 0.37 micrograms/ml (mean +/- SEM) respectively]. We conclude that despite elevation of platelet calcium in uraemia, which may be due to a plasma factor such as parathyroid hormone, platelet aggregation is normal and bleeding in uraemia is more likely to be due to other factors, including the effect of reduced haematocrit on platelet endothelial interaction. Disturbances in platelet calcium cannot explain the bleeding manifestations in uraemia but warrant further investigation in order to identify the pathogenic mechanisms responsible.

Adolescent↗
PubMed1990

Cyclic GMP release and vasodilatation induced by EDRF and atrial natriuretic factor in the isolated perfused kidney of the rat.

1. Guanosine 3':5'-cyclic monophosphate (cyclic GMP) release and vascular tone was measured in the isolated kidney of the rat perfused at constant flow with Krebs-Henseleit solution. The effects of 3 vasodilators, acetylcholine (ACh), atrial natriuretic factor (ANF) and sodium nitroprusside (SNP) on the renal release of cyclic GMP and vascular tone were examined. The ability of the endothelial-derived relaxing factor (EDRF) inhibitors, haemoglobin and gossypol, to modify vasodilatation and vasodilator-induced changes in cyclic GMP releases from the kidney was also investigated. 2. Renal cyclic GMP release was elevated 8 fold by ANF (0.01 microM), 5 fold by SNP (1 microM) and 3 fold by ACh (0.3 microM). 3. For ACh, both the increase in renal cyclic GMP release and the vasodilatation were reduced by the EDRF inhibitors, haemoglobin (1 microM) and gossypol (15 microM). For SNP, neither the increase in renal cyclic GMP release nor vasodilatation were inhibited by gossypol (15 microM). 4. For ANF, neither the increase in cyclic GMP release from the kidney nor its vasodilator activity were affected by haemoglobin (1 microM). 5. EDRF inhibitors reduced the basal release of cyclic GMP from 0.32 +/- 0.06 pmol min-1 to 0.18 +/- 0.03 pmol min-1, gossypol being more effective than haemoglobin. 6. The results are consistent with the ability of ACh to induce EDRF-mediated vasodilatation in the isolated perfused kidney of the rat. Basal EDRF release appears to contribute approximately 50% to the basal release of cyclic GMP from this preparation. The renal vasodilator action of ANF however, is independent of EDRF, although the renal vascular endothelium cannot be discounted as a site at which ANF stimulates cyclic GMP production.

Acetylcholine↗
PubMed1989

I alpha-25-dihydroxyvitamin D3 increases intracellular free calcium in murine B16 melanoma.

Vitamin D3 and its active metabolite I alpha-25-dihydroxyvitamin D3 (I alpha-25-(OH)2D3) have been reported to play a role in melanogenesis. Physiological concentrations of I alpha-25-(OH)2D3 were found to acutely elevate intracellular free calcium (using Fura 2) in B16 primary (Io) cells. Membrane phosphoinositide turnover was unaffected by I alpha-25-(OH)2D3. The rise in intracellular free calcium was entirely dependent on extracellular calcium and was not mimicked by vitamin D3. However, in neither B16-Io nor B16-F1 melanoma cells did vitamin D3 or I alpha-25-(OH)2D3 increase melanin production.

Animals↗
PubMed1988

An extracellular role for calmodulin-like activity in cell proliferation.

1. Addition of extracellular pure pig brain calmodulin was found to modulate DNA synthesis and cell proliferation in K562 human leukaemic lymphocytes. At lower cell densities calmodulin significantly stimulated [3H]thymidine uptake; at higher densities it decreased it. 2. A protein biochemically indistinguishable from calmodulin was detected in the cell-conditioned media of rapidly dividing K562 cells. The concentration of calmodulin-like activity found in the conditioned media of these and a range of other normal and neoplastic cells (250-1636 ng/ml) was of the same order as would stimulate DNA synthesis in subconfluent cells. 3. Amounts of extracellular calmodulin-like activity and immunoreactivity varied during cell growth from low to high density, a peak of extracellular calmodulin preceding DNA synthesis in synchronized K562 cells. Extracellular calmodulin concentrations did not correlate with the presence of lactate dehydrogenase in the medium. 4. Inhibition of extracellular calmodulin activity by calmodulin antagonist immobilized on agarose beads, or by antibody to calmodulin, significantly decreased DNA synthesis. 5. These data strongly suggest that calmodulin or a very closely related protein can influence mitosis through an extracellular mechanism.

Animals↗