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S MacNeil

Publications and source records attributed to S MacNeil.

At least 19 recordsLinked to original sources
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↗
PubMed1988

Calmodulin antagonists of improved potency and specificity for use in the study of calmodulin biochemistry.

Syntheses are described for a range of N-(omega-aminoalkyl)-5-iodo- and -5-cyanonaphthalene-1-sulphonamides. The selective activity of these compounds as inhibitors for calmodulin-dependent phosphodiesterase (EC 3.1.4.17) is compared with their activity for the calmodulin-independent but calcium-dependent enzymes protein kinase C and transglutaminase (EC 2.3.2.13). The results show a drastic improvement in the selectivity of effect for the 5-iodo-compounds compared with the widely-used drug, W7, N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide.

Calmodulin↗
PubMed1988

Calcium/calmodulin regulation of the proliferation of human epidermal keratinocytes, dermal fibroblasts and mouse B16 melanoma cells in culture.

We have investigated the relationship between extracellular calcium, intracellular calmodulin and proliferation in normal keratinocytes. Keratinocyte proliferation and its sensitivity to calmodulin antagonists was compared with that of normal human dermal fibroblasts and neoplastic mouse B16 melanoma cells. Keratinocytes were similar to fibroblasts in showing reduced proliferation in low (0.15 mM) calcium medium and unlike B16 cells which continued to proliferate until calcium was reduced to submicromolar levels. Intracellular calmodulin was significantly higher in rapidly dividing keratinocytes in normal (1.15 mM) calcium medium than in slower dividing cells in low (0.15 mM) calcium. Fibroblasts and B16 cells maintained similar calmodulin levels in both low and normal calcium media. Calmodulin antagonists inhibited proliferation of all three cell types equally. Thus, keratinocyte calmodulin seems related to the proliferative state of the cell (unlike fibroblast calmodulin) and calmodulin antagonists may be of use in controlling the hyperproliferation of the psoriatic epidermis.

Animals↗
PubMed1988

Extracellular calmodulin and its association with epidermal growth factor in normal human body fluids.

In this study we describe the occurrence of a calmodulin-like protein in normal human biological fluids. Extraction of the calmodulin-like protein from breast milk, saliva, serum and urine provided an extract with enhanced calmodulin immunoreactivity which, in the case of milk and saliva, showed a protein band comigrating with authentic calmodulin (Mr 17,000) on sodium dodecylsulphate-polyacrylamide gel electrophoresis. However, in milk, saliva and serum a major protein band of Mr 14,000-15,000 was always observed, which we speculate may be related to calmodulin, possibly as a partially degraded form. Estimates of biologically active calmodulin in most normal extracellular fluids were of the order which we have found will stimulate cell division when added to the extracellular medium of cells in culture. Levels ranged from 0.03 nmol/l in urine to 18.6 nmol/l in breast milk, and exhibited a quantitative relationship (r = 0.79, P less than 0.01) to epidermal growth factor (EGF) levels in fluids. Where EGF concentrations varied from normal (increased in saliva 24 h after oral surgery and reduced in the urine of patients with renal failure) calmodulin concentrations were similarly affected. The presence of calmodulin in serum may in part be attributable to its release from platelets which are particularly rich in calmodulin. Release of calmodulin from the platelet was associated with that of EGF and other platelet products.

Blood Platelets↗
PubMed1987

Evidence for the involvement of calmodulin in natural cytotoxicity using a range of calmodulin antagonists of varying potency and improved specificity.

The calmodulin antagonist W7 and 4 of its analogues were examined for their ability to inhibit human NK cell mediated cytotoxicity. With the exception of one of these compounds, which is extremely hydrophobic, there was a good correlation between the ability of drugs to inhibit human NK antitumour cytotoxicity and calmodulin-dependent phosphodiesterase activity in vitro. The most potent of the compounds, 5-iodo-1-C8, an analogue of W7, has an IC50 of 3 microM upon biological and biochemical assay. This particular compound is both more potent and specific than the parent compound W7, is non-toxic to cells over the range used and is also capable of inhibiting the biological activity of NK cells upon pre-treatment of the effector cells, inferring the mechanism of NK cytotoxicity to be calmodulin dependent.

Calmodulin↗
PubMed1987

Activation of calmodulin by the essential trace element chromium.

Chromium at very low concentrations is an essential trace element--at higher concentrations it is associated with contact dermatitis and other toxicity problems. Its ionic radius is just outside that of other metal cations which have been found to activate calmodulin in vitro. We found that chromium was able to activate calmodulin at two different concentration ranges--over the micromolar range (which would probably never be achieved in man) a small degree of activation was found--but a much greater activation (76% of the maximum possible) was also found at nanomolar concentrations of chromium. In welders, who work with stainless steel and who were not reporting any physical symptoms of chromium toxicity, red cell chromium levels were 28.2 +/- 3.3 nM (n = 22) compared to 7.5 +/- 0.7 nM (n = 11) for normal controls. Thus, the concentration of chromium experienced within the cell can be of the order which will activate calmodulin in vitro. The possibility exists, therefore, that inappropriate activation of calmodulin could be relevant to chromium biology possibly contributing to the symptoms of chromium toxicity.

Animals↗
PubMed1987

A comparative study of the anti-proliferative effects of calmodulin antagonists in cultured cells--W7 derivatives of improved cytostatic potential.

We have compared the effects of the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide (W7) and relatives of the parent compound, modified by substituting the 5-chloro- for an iodo-residue and increasing stepwise the length of the carbon chain from 6 to 12, for their ability to inhibit cell proliferation in tissue culture. These species showed improved specificity and potency, as determined against a calcium calmodulin-dependent beef heart phosphodiesterase in vitro, exhibited time courses of inhibition similar to the parent compound W7, but were more potent at inhibiting DNA synthesis in K562 human leukaemic cells cultured in serum-free medium. The elevation observed in the percentage of cells in G1 of the cell cycle following drug exposure indicated that these drugs, like W7, arrested growth by inhibiting entry of cells into and through S phase. Higher doses of all drugs were irreversibly cytotoxic as determined by the inability of the cells to recover DNA synthetic capacity and to form colonies in soft agar or to recover their normal cell cycle distribution. We also discuss the possible implications of extracellular calmodulin and antagonism thereof on cell proliferation.

Animals↗
PubMed1986

Psoriasis.

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Calmodulin↗
PubMed1986

An investigation of the ability of antipsoriatic drugs to inhibit calmodulin activity: a possible mode of action of dithranol (anthralin).

Epidermal calmodulin (CaM) has been reported to be elevated in psoriasis and to decrease following clearance of psoriasis with treatment. We set out to investigate whether any of the principle drugs used in the treatment of psoriasis had inherent CaM antagonist activity. Utilizing a CaM-activated phosphodiesterase we have demonstrated that even at very high concentrations, the systemic drugs etretinate, methotrexate, and 8-methoxypsoralen, and the topical agents hydrocortisone and crude coal tar showed minimal CaM inhibitory activity. Dithranol (anthralin), however, whether freshly prepared or oxidized, produced substantial inhibition of CaM activity and was demonstrated to be a potent competitive antagonist of CaM, suggesting another possible therapeutic mode of action of dithranol in psoriasis.

Animals↗
PubMed1986

Calmodulin levels in psoriasis: the effect of treatment.

Epidermal calmodulin levels were measured in lesional and non-lesional skin in 20 psoriatics before and after treatment by a variety of established topical and systemic regimes. Pretreatment, 10 out of 20 patients showed elevated calmodulin levels in lesional epidermis. Clearance of psoriatic plaques was accompanied by a significant overall reduction (p less than 0.05) in epidermal calmodulin, irrespective of the treatment regime used. For the non-lesional epidermis, only 5 out of 17 patients initially showed elevated calmodulin levels and there was no significant reduction in those levels following treatment.

Adult↗