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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.

Normal human peripheral blood mononuclear cells (PBMC) and leukaemic cell lines (three of human and one of gibbon origin) were found to contain similar levels of calmodulin (CaM) when expressed relative to the total cell protein. Two of the cell lines examined further were found to contain much higher amounts of CaM per cell (up to 5-fold) than PBMC but this was readily explained by their much greater cell size. Variations in CaM levels were noted during culture of both PBMC and leukaemic cells which were apparently independent of the percentage of cells undergoing active division in these cultures. These results do not support, the contention that transformed cells contain a higher proportion of CaM than normal cells.

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-l-C8, an analogue of W7, has an IC50 of 3 μM 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.

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.

A protein which shared several characteristics with authentic calmodulin was extracted from human thyroid homogenates. The protein bound to fluphenazine–Sepharose and could be specifically eluted using EGTA. The eluted protein had a u.v. spectrum characteristic of calmodulin and migrated like authentic calmodulin with a calcium-dependent shift on sodium dodecyl sulphate polyacrylamide-gel electrophoresis. Calmodulin in thyroid cell extracts was shown to be biologically active, measured by its ability to activate a calmodulin-deficient cyclic GMP phosphodiesterase; this activation could be inhibited by trifluoperazine. A possible role for calmodulin in the action of TSH on the thyroid was demonstrated by studying the effects of phenothiazines and the naphthalene sulphonamide, W7, a more specific calmodulin inhibitor, on TSH-stimulated cyclic AMP levels in cultured thyroid cells. The phenothiazines and W7 were found to inhibit the accumulation of cyclic AMP in response to TSH in a concentration-dependent manner although low concentrations of W7 enhanced TSH-stimulated cyclic AMP accumulation.

1. The addition of 50 000g cytosol preparations of isolated human platelets, cultured rat osteogenic sarcoma or cultured bone cells to particulate preparations of adenylate cyclase, from the same or unrelated tissues, caused marked enhancement of the hormone-stimulated enzyme activities. 2. The degree of enhancement obtained by addition of the cytosol preparations was similar to that observed on addition of GTP. 3. The enhancing activity of the three cytosol types was found to be sensitive to digestion by trypsin and alkaline phosphatase, partially heat-labile and partially inactivated by exposure to charcoal. 4. Gel filtration studies indicated an apparent molecular weight of 20 000–30 000. Further, the 20000-30000-mol.wt. fractions obtained by gel filtration could enhance the adenylate cyclase activity of particulate preparations derived from unrelated cell types. 5. The results suggest a common or similar adenylate-cyclase-enhancing factor or factors, protein in nature, present in the three cytosol types.

Introduction: The importance of calcium in the regulation of cell function has become increasingly recognized in the past 20 years. It is now known that changes in intracellular calcium concentration, like changes in adenosine 3′:5′-monophosphate (cyclic AMP), are of crucial importance in stimulus-response coupling. Thus, the second messenger theory originally proposed by Sutherland et al. [1], in which a hormone or nerve impulse is first messenger and cyclic AMP the second intracellular messenger, has been expanded to include calcium ions as well as cyclic nucleotides. Moreover, there is now evidence in many cellular systems that calcium ions and cyclic nucleotides act as dual interrelated messengers [2], and, therefore, some cellular processes are regulated by calcium as well as by cyclic nucleotides, as shown in Table 1. Evidence has accumulated that it is changes in free intracellular calcium concentration that are, in some way, responsible for activation of the enzymes involved in the processes shown in this Table.

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.

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.

Alpha-melanocyte stimulating hormone (alpha-MSH) has pigmentary, anti-inflammatory, antipyretic, and general immunomodulatory roles. It can oppose several cytokines including tumor necrosis factor-alpha in a number of tissues, including skin. We have previously shown that alpha-MSH can inhibit tumor necrosis factor-alpha stimulated intercellular adhesion molecule 1 upregulation and nuclear factor kappaB (NFkappaB) transcription factor activation in melanocyte and melanoma cells. It is thought, however, that this MSH biology may also extend to other cells of the skin and in this study we extend our work to keratinocytes. We have investigated in detail the ability of three alpha-MSH peptides to inhibit tumor necrosis factor alpha stimulated NFkappaB activation in nonpigmentary HaCaT keratinocytes (alpha-MSH, L-Lys-L-Pro-L-Val, and L-Lys-L-Pro-D-Val) and two adrenocorticotropic hormone (ACTH) peptides (1-17 and 1-39), reported to be present in skin tissue. NFkappaB/p65 activation was analyzed by electrophoretic mobility shift assay and immunofluorescent microscopy. alpha-MSH, L-Lys-L-Pro-L-Val, and L-Lys-L-Pro-D-Val all significantly inhibited tumor necrosis factor alpha stimulated NFkappaB activation, whereas ACTH 1-17 and 1-39 did not, in the HaCaT keratinocytes. MSH peptides and ACTH 1-39 were effective, however, at inhibiting NFkappaB activation in normal human keratinocytes. Immunolabeling of inhibitor kappaBalpha of NFkappaB (IkappaBalpha) revealed an abnormal localization to the nucleus of HaCaT cells, which was unaffected by MSH/ACTH peptides. In contrast, normal human keratinocytes showed a normal IkappaBalpha distribution that responded to MSH/ACTH with nuclear translocation. Our data support previous work on the role of MSH/ACTH peptides as immunomodulatory/anti-inflammatory regulators, and extend this work to keratinocytes identifying a novel IkappaBalpha mechanism and extends findings to ACTH peptides, identifying an abnormal IkappaBalpha mechanism in the immortal HaCaT versus normal keratinocyte.

1 The effect of intravenous infusion of lithium, 2.56 mumol/min on the antidiuretic responses to antidiuretic hormone (ADH), adenosine triphosphate (ATP), 3'-5' adenosine cylic monophosphate (cyclic AMP) and theophyline was studied in water-loaded, alcohol-anaesthetized rats. 2 Lithium reversibly inhibits the antidiuretic response to all concentrations of ADH, depressing the maximum response but not changing the amount required for half maximal response. 3 The rate of increase of serum lithium relates more clearly to the inhibitory effect than does the serum concentration. 4 Sodium concentrations in the renal papilla seem to fall when serum lithium levels are rising. 5 Lithium inhibits the antidiuretic response to ATP and cyclic AMP but does not inhibit the response to theophyline.

The aim of this study was to determine whether levels of biologically active calmodulin are elevated in both lesional and uninvolved epidermis in psoriasis. Epidermal shave biopsies were obtained from normal controls and from both psoriatic plaques and nonlesional psoriatic skin. Following determination of the protein content, the calmodulin activity of the homogenized samples was then measured using a calmodulin-sensitive phosphodiesterase enzyme bioassay. In normal skin, calmodulin activity was 1.29 +/- 0.35 micrograms calmodulin mg-1 epidermal protein (mean +/- SEM, n = 12 volunteers) compared to 7.88 +/- 1.59 micrograms calmodulin mg-1 epidermal protein for plaque (n = 16 patients) and 10.19 +/- 2.35 micrograms calmodulin mg-1 epidermal protein for the uninvolved skin of 12 of these patients. The levels of biologically active calmodulin were therefore elevated in both plaque and uninvolved epidermis of patients with psoriasis compared to epidermis from normal volunteers. These results suggest that an abnormality in the regulation of calmodulin activity may be involved in the pathogenesis of psoriasis.

The 34-amino acid NH2-terminal fragment of human parathyroid hormone synthesized according to the sequence described by Niall et al. (1) is approximately 140 times more potent than the fragment synthesized according to Brewer et al (2) in activating human renal cortex adenylate cyclase. The potencies of the two peptides, relative to the effect of MRC standard bovine parathyroid hormone preparation 67342 in this system, were 5600 ± 600 (S.E.M.) units/mg and 40 ± 5 units/mg respectively. The potencies of the more active peptide and the corresponding bovine parathyroid hormone sequence were similar in this system and also in assays based upon the production of cyclic AMP by chick kidney both in vivo and in vitro.

The effect of lithium on the urine concentrating response to antidiuretic hormone (ADH) and the excretion of ADH has been studied in rats and man. The maximum urine osmolarity following 18 h dehydration and Pitressin (5 u) was decreased in three out of four patients during lithium treatment compared to their response to the same test in the absence of lithium. In a fifth patient, tested only during lithium treatment, the urine remained hypotonic to plasma throughout this test. Lithium increased the excretion of ADH in non-polyuric patients from 9-22 mu/24 h in the absence of lithium to 36-202 mu/24 - during lithium treatment. In four patients with lithium-induced polyuria, a diuretic acting on the distal tubules, clorexolone, reduced the polyuria. Lithium increased urine volume and the excretion of ADH in four rats receiving lithium in their diet. The response to exogenous ADH was decreased during lithium administration.

1 The relationship between the diuretic effectiveness and the effect on the renal adenylate cyclase of three diuretics, acetazolamide, frusemide and ethacrynic acid, was examined. The hypothesis that acetazolamide and parathyroid hormone (PTH), inhibit renal carbonic anhydrase by a cyclic adenosine 3′,5′-monophosphate (cyclic AMP)-dependent mechanism was also tested. 2 In vitro, acetazolamide, frusemide and ethacrynic acid at high concentrations (10-3M) all produced some inhibition of basal and stimulated rat kidney plasma membrane adenylate cyclase. The effect of acetazolamide was much less than that of frusemide and ethacrynic acid. These plasma membrane effects were reproduced in studies of cyclic AMP formation in isolated kidney tubules of rats. 3 Intravenous injections of acetazolamide did not change the total cyclic AMP content of the kidneys of rats killed by microwave irradiation. 4 Acetazolamide produced a diuresis in the rat and a slight inhibition of the antidiuretic effect of Pitressin. Frusemide produced a diuresis and greatly reduced the antidiuretic response to Pitressin. Ethacrynic acid was ineffective as a diuretic in the rat and actually enhanced the antidiuretic response to Pitressin. 5 In investigating the possible influence of diuretics and PTH on the activity and state of phosphorylation of carbonic anhydrase it was found that: there was no correlation between the ability of diuretics to inhibit carbonic anhydrase activity and to inhibit carbonic anhydrase phosphorylation; neither PTH nor cyclic AMP (in the presence of adenosine triphosphate, Mg2+, K+ and incubation at 37°C) inhibited rat cortex homogenate carbonic anhydrase activity. 6 It seems unlikely that any of the tested diuretics exerts its pharmacological effect by means of changes in kidney cyclic AMP metabolism.

1. Homogenates of whole tissues were shown to contain both intracellular and extracellular factors that affected particulate adenylate cyclase activity in vitro. Factors present in the extracellular fluids produced an inhibition of basal, hormone- and fluoride-stimulated enzyme activity but factors present in the cell cytosol increased hormone-stimulated activity with relatively little effect on basal or fluoride-stimulated enzyme activity. 2. The existence of this cytosol factor or factors was investigated using freshly isolated human platelets, freshly isolated rat hepatocytes, and cultured cells derived from rat osteogenic sarcoma, rat calvaria, mouse melanoma, pig aortic endothelium, human articular cartilage chondrocytes and human bronchial carcinoma (BEN) cells. 3. The stimulation of the hormone response by the cytosol factor ranged from 60 to 890% depending on the tissue of origin of the adenylate cyclase. 4. In each case the behaviour of the factor was similar to the action of GTP on that particular adenylate cyclase preparation. 5. No evidence of tissue or species specificity was found, as cytosols stimulated adenylate cyclase from their own and unrelated tissues to the same degree. 6. In the human platelet, the inclusion of the cytosol in the assay of adenylate cyclase increased the rate of enzyme activity in response to stimulation by prostaglandin E1 without affecting the amount of prostaglandin E1 required for half-maximal stimulation or the characteristics of enzyme activation by prostaglandin E.

The possible role of calmodulin in the control of prolactin secretion was examined. The effects of a wide variety of agents on inhibition of prolactin secretion from isolated rat anterior pituitary cells and inhibition of calmodulin activity in an in-vitro system (calmodulin-activated cyclic GMP phosphodiesterase) were compared. A number of phenothiazines showed a close correlation of potencies in the two systems, as did the more specific calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W7), suggesting that calmodulin may be involved in the control of prolactin secretion. Some other drugs also inhibited both prolactin secretion and calmodulin activity in addition to their other well-characterized biochemical effects. However, many of these drugs (including two phenothiazines) were more potent inhibitors of prolactin secretion than of calmodulin activity, suggesting that other intracellular systems in addition to calmodulin may be involved in the control of the secretory process.

Recent evidence suggests that epidermal growth factor (EGF) may play an important role in the regulation of thyroid growth and function. We have examined the interaction of murine EGF (mEGF) with human and porcine thyroid membranes and compared this with the binding of bovine TSH (bTSH) using 125I-labelled hormones as tracers. The characteristics of the binding of mEGF were found to be similar for human and porcine thyroid membranes. Epidermal growth factor bound with high affinity (affinity constant = 1·4 × 109 l/mol); the density of binding sites was low compared with the TSH receptor. At 37 °C, the binding of 125I-labelled EGF was maximal at 1 h and was saturable in the presence of unlabelled EGF; half-maximal inhibition was at 1 ng EGF/tube (0·5 nmol/l) using 0·5 mg membrane protein/tube. Unlabelled bTSH had no effect on the binding of labelled EGF. Similarly, unlabelled EGF did not affect the binding of labelled TSH; hence it was concluded that mEGF and bTSH bound to independent sites. Epidermal growth factor had no effect on adenylate cyclase activity in membranes prepared from human non-toxic goitre; increasing concentrations of EGF did not affect basal, TSH-stimulated or fluoride-stimulated enzyme activity. J. Endocr. (1984) 102, 57–61

The production of prostacyclin (PGI2) by cultured porcine aortic endothelial cells, in response to serum and the calcium ionophore A23187, was inhibited by TMB-8, an antagonist of intracellular calcium mobilization. The calcium-channel blocker methoxyverapamil (D600) inhibited serum-induced PGI2 production in but had little effect on A23187-induced PGI2 production. Calmodulin activity was detected in endothelial-cell Jysates and was inhibited by the calmodulin antagonist W7, which also inhibited PGI2 production in response to both agonists. Calcium and calmodulin appear to play an important role in mediating PGI2 production by the vascular endothelium.

University Department of Obstetrics & Gynaecology, Jessop Hospital, Shefield S3 7RE, U.K.; *MRC Unit of Reproductive Biology, 37 Chalmers Street, University of Edinburgh, Edinburgh EH3 9EW, U.K.; ?Department of Anatomy, Monash University, Clayton, Victoria 3168, Australia; and IDepartment of Medicine, Clinical Sciences Centre, Northern General Hospital, Shefield S5 7AU. U.K. Keywords

Invasion of melanoma cells from the primary tumor involves interaction with adjacent tissues and extracellular matrix. The extent of this interaction is not fully understood. In this study Raman spectroscopy was applied to cryo-sections of established 3D models of melanoma in human skin. Principal component analysis was used to investigate differences between the tumor and normal tissue and between the peri-tumor area and the normal skin. Two human melanoma cells lines A375SM and C8161 were investigated and compared in 3D melanoma models. Changes were found in protein conformations and tryptophan configurations across the entire melanoma samples, in tyrosine orientation and in more fluid lipid packing only in tumor dense areas, and in increased glycogen content in the peri-tumor areas of melanoma. Raman spectroscopy revealed changes around the perimeter of a melanoma tumor as well as detecting differences between the tumor and the normal tissue.

A 9-year-old girl with severe unilateral ocular surface disease due to limbal stem cell deficiency following chemical injury underwent autologous cultivated limbal epithelial transplantation (CLET) twice in her right eye that failed to improve the condition of the ocular surface. On both occasions, limbal biopsies were obtained from the unaffected left eye. Since further attempts at CLET were not considered to be safe, deep anterior lamellar keratoplasty (DALK) was performed instead. DALK was complicated by postoperative Descemet9s membrane detachment, which was successfully treated. Two years post-DALK the patient had a clear graft, stable ocular surface and visual acuity of 20/80. The mechanism by which CLET allows a patient to repopulate the ocular surface is as yet unknown. This case highlights that although clinically little had changed following two failed CLET surgeries, some subclinical improvement must have taken place for a DALK to remain successful after 2 years.

The purpose of this study was to assess chromium handling in non-insulin dependent diabetic patients (NIDDM) compared to healthy volunteers. Chromium handling was evaluated using fasting blood and second morning void urine samples from 93 NIDDM patients and 33 healthy volunteers. Significant differences in chromium homeostasis were seen between patients and controls. NIDDM patients had mean levels of plasma chromium around 33% lower and urine values almost 100% higher than those found in health. Healthy volunteers showed a significant negative correlation between fasting levels of plasma chromium and insulin. This was not evident in NIDDM patients. In the early years of onset of NIDDM, plasma chromium values were inversely correlated with plasma glucose. This was lost in patients with diabetes of more than 2 years duration. We suggest large losses of chromium over many years may exacerbate an already compromised chromium status in NIDDM patients and might contribute to the developing insulin resistance seen in patients with type 2 diabetes.

Invasion through stromal extracellular matrix (ECM) is part of the complex, multistep process of tumour cell invasion and metastasis. Our group has previously demonstrated that calcium and calmodulin are important in another step in the metastatic cascade - that of attachment of cells to ECM. Interestingly, the non-steroidal anti-oestrogen tamoxifen (which also has calmodulin antagonist activity), used in the treatment of breast cancer and now in metastatic cutaneous melanoma, can inhibit the attachment of normal and neoplastic cells to ECM. In this study, we investigated whether such drugs, known to inhibit cell attachment, could also subsequently reduce their invasion through a layer of human fibronectin. We examined the ability of the specific calmodulin antagonist J8, tamoxifen and its two major metabolites, N-desmethyltamoxifen (N-des) and 4-hydroxytamoxifen (4-OH), as well as the pure anti-oestrogen ICI 182,780 and 17beta-oestradiol to inhibit invasion of the human cutaneous melanoma cell line, A375-SM, uveal melanoma cells and uveal melanocytes. A375-SM cells and uveal melanoma cells showed a high level of invasion (15.2% and 33.7% respectively) compared with melanocytes (around 5%) under the experimental conditions used. Submicromolar concentrations of N-des, tamoxifen, J8 and 17beta-oestradiol significantly reduced the invasiveness of the A375-SM cell line. The uveal melanoma cells also showed similar inhibition, although at higher concentrations of these agents. 4-OH and ICI 182, 780 had little or no effect on invasion of A375-SM cells (these were not tested on uveal melanoma cells). All cells used in this study were found to be negative for type I nuclear oestrogen receptors, reinforcing the possibility that tamoxifen and 17beta-oestradiol can act via mechanisms unrelated to binding to classical oestrogen receptors to inhibit tumour cell invasion.

Our aim was to develop a biodegradable fibrous dressing to act as a tissue guide for in situ wound repair while releasing Ibuprofen to reduce inflammation in wounds and reduce pain for patients on dressing changes. Dissolving the acid form of Ibuprofen (from 1% to 10% by weight) in the same solvent as 75% polylactide, 25% polyglycolide (PLGA) polymers gave uniformly loaded electrospun fibers which gave rapid release of drug within the first 8 h and then slower release over several days. Scaffolds with 10% Ibuprofen degraded within 6 days. The Ibuprofen released from these scaffolds significantly reduced the response of fibroblasts to major pro-inflammatory stimulators. Fibroblast attachment and proliferation on scaffolds was unaffected by the addition of 1-5% Ibuprofen. Scaffolds loaded with 10% Ibuprofen initially showed reduced cell attachment but this was restored by soaking scaffolds in media for 24 h. In summary, addition of Ibuprofen to electrospun biodegradable scaffolds can give acute protection of adjacent cells to inflammation while the scaffolds provide an open 3D fibrous network to which cells can attach and migrate. By 6 days, such scaffolds will have completely dissolved into the wound bed obviating any need for dressing removal.