Elevated Serum Gastrin Is Associated with Melanoma Progression: Putative Role in Increased Migration and Invasion of Melanoma Cells.

Micro-environmental factors, including stromal and immune cells, cytokines, and circulating hormones are well recognized to determine cancer progression. Melanoma cell growth was recently shown to be suppressed by cholecystokinin/gastrin (CCK) receptor antagonists, and our preliminary data suggested that melanoma patients with Helicobacter gastritis (which is associated with elevated serum gastrin) might have an increased risk of cancer progression. Therefore, in the present study, we examined how gastrin may act on melanoma cells. In 89 melanoma patients, we found a statistically significant association between circulating gastrin concentrations and melanoma thickness and metastasis, which are known risk factors of melanoma progression and prognosis. Immunocytochemistry using a validated antibody confirmed weak to moderate CCK2R expression in both primary malignant melanoma cells and the melanoma cell lines SK-MEL-2 and G361. Furthermore, among the 219 tumors in the Skin Cutaneous Melanoma TCGA Pan-Cancer dataset showing gastrin receptor (CCKBR) expression, significantly higher CCKBR mRNA levels were linked to stage III-IV than stage I-II melanomas. In both cell lines, gastrin increased intracellular calcium levels and stimulated cell migration and invasion through mechanisms inhibited by a CCK2 receptor antagonist. Proteomic studies identified increased MMP-2 and reduced TIMP-3 levels in response to gastrin that were likely to contribute to the increased migration of both cell lines. However, the effects of gastrin on tumor cell invasion were relatively weak in the presence of the extracellular matrix. Nevertheless, dermal fibroblasts/myofibroblasts, known also to express CCK2R, increased gastrin-induced cancer cell invasion. Our data suggest that in a subset of melanoma patients, an elevated serum gastrin concentration is a risk factor for melanoma tumor progression, and that gastrin may act on both melanoma and adjacent stromal cells through CCK2 receptors to promote mechanisms of tumor migration and invasion.

Cell cycle dependent expression of the CCK2 receptor by gastrointestinal myofibroblasts: putative role in determining cell migration.

The well-known action of the gastric hormone gastrin in stimulating gastric acid secretion is mediated by activation of cholecystokinin-2 receptors (CCK2R). The latter are expressed by a variety of cell types suggesting that gastrin is implicated in multiple functions. During wound healing in the stomach CCK2R may be expressed by myofibroblasts. We have now characterized CCK2R expression in cultured myofibroblasts. Immunocytochemistry showed that a relatively small proportion (1-6%) of myofibroblasts expressed the receptor regardless of the region of the gut from which they were derived, or whether from cancer or control tissue. Activation of CCK2R by human heptadecapeptide gastrin (hG17) increased intracellular calcium concentrations in a small subset of myofibroblasts indicating the presence of a functional receptor. Unexpectedly, we found over 80% of cells expressing CCK2R were also labeled with 5-ethynyl-2'-deoxyuridine (EdU) which is incorporated into DNA during S-phase of the cell cycle. hG17 did not stimulate EdU incorporation but increased migration of both EdU-labeled and unlabelled myofibroblasts; the migratory response was inhibited by a CCK2R antagonist and by an inhibitor of IGF receptor tyrosine kinase; hG17 also increased IGF-2 transcript abundance. The data suggest myofibroblasts express CCK2R in a restricted period of the cell cycle during S-phase, and that gastrin accelerates migration of these cells; it also stimulates migration of adjacent cells probably through paracrine release of IGF. Together with previous findings, the results raise the prospect that gastrin controls the position of dividing myofibroblasts which may be relevant in wound healing and cancer progression in the gastrointestinal tract.

Fluorescent measurement of [Ca²âº]c: basic practical considerations.

There is a vast array of dyes currently available for measurement of cytosolic calcium. These encompass single and dual excitation and single and dual emission probes. The choice of particular probe depends on the experimental question and the type of equipment to be used. It is therefore extremely difficult to define a universal approach that will suit all potential investigators. Preparations under investigation are loaded with the selected organic indicator dye by incubation with ester derivatives, by micropipet injection or reverse permeabilization. Indicators can also be targeted to a range of intracellular organelles. Calibration of a fluorescent signal into Ca(2+) concentration is in theory relatively simple but the investigator needs to take great care in this process. This chapter describes the theory of these processes and some of the pitfalls users should be aware of. Precise experimental details can be found in the subsequent chapters of this volume.

Dynamic changes in cytosolic and mitochondrial ATP levels in pancreatic acinar cells.

BACKGROUND & AIMS: Previous studies of pancreatic acinar cells characterized the effects of Ca(2+)-releasing secretagogues and substances, inducing acute pancreatitis on mitochondrial Ca(2+), transmembrane potential, and NAD(P)H, but dynamic measurements of the crucial intracellular adenosine triphosphate (ATP) levels have not been reported. Here we characterized the effects of these agents on ATP levels in the cytosol and mitochondria. METHODS: ATP levels were monitored using cytosolic- or mitochondrial-targeted luciferases. RESULTS: Inhibition of oxidative phosphorylation produced a substantial decrease in cytosolic ATP comparable to that induced by inhibition of glycolysis. Cholecystokinin-8 (CCK) increased cytosolic ATP in spite of accelerating ATP consumption. Acetylcholine, caerulein, and bombesin had similar effect. A bile acid, taurolithocholic acid 3-sulfate (TLC-S); a fatty acid, palmitoleic acid (POA); and palmitoleic acid ethyl ester (POAEE) reduced cytosolic ATP. The ATP decrease in response to these substances was observed in cells with intact or inhibited oxidative phosphorylation. TLC-S, POA, and POAEE reduced mitochondrial ATP, whereas physiological CCK increased mitochondrial ATP. Supramaximal CCK produced a biphasic response composed of a small initial decline followed by a stronger increase. CONCLUSIONS: Both glycolysis and oxidative phosphorylation make substantial contributions to ATP production in acinar cells. Ca(2+)-releasing secretagogues increased ATP level in the cytosol and mitochondria of intact isolated cells. TLC-S, POA, and POAEE reduced cytosolic and mitochondrial ATP. When cells rely on nonoxidative ATP production, secretagogues as well as TLC-S, POA, and POAEE all diminish cytosolic ATP levels.

Compartmentalized signalling: Ca2+ compartments, microdomains and the many facets of Ca2+ signalling.

Ca(2+) regulates a multitude of cellular processes and does so by partitioning its actions in space and time. In this review, we discuss how Ca(2+) responses are constructed from small quantal (elementary) events that have the potential to propagate to produce large pan-cellular responses. We review how Ca(2+) is compartmentalized both physically and functionally, and describe how each organelle has its own distinct Ca(2+)-handling properties. We explain how coordination of the movement of Ca(2+) between organelles is used to shape and hone Ca(2+) signals. Finally, we provide a number of specific examples of where compartmentation and localization of Ca(2+) are crucial to cell function.

Attenuation of receptor-dependent and -independent vasoconstriction in the human radial artery.

BACKGROUND: Vasodilator strategies used to treat bypass grafts in the operating theatre, such as nitrates, phosphodiesterase inhibitors and calcium channel antagonists have a broad but short-lived effect against a variety of vasoconstrictor stimuli. Treatments that react irreversibly with proteins modulating vasoconstriction have the advantage that their effects can last well into the postoperative period. In addition systemic effects are avoided as the treatment is localised to the treated graft. This study investigated the use of two clinically applied drugs; fluphenazine (SKF7171A, HCl), an irreversible calmodulin antagonist and minoxidil sulphate, an irreversible potassium channel opener. Treatments were tested against receptor and non-receptor-mediated contraction in the human radial artery. METHOD: Isometric tension was measured in response to angiotensin II, KCl and vasopressin in 108 radial artery rings (taken from 31 patients undergoing coronary artery bypass grafting). Control responses were compared with rings pretreated with fluphenazine or minoxidil sulphate. Vasopressin responses were also compared in the presence of glyceryl trinitrate or the reversible Rho kinase inhibitor Y27632. RESULTS: Fluphenazine pretreatment significantly suppressed vasoconstriction to all agonists tested. Maximal responses to angiotensin II, vasopressin and KCl were reduced by 42+/-19%, 35+/-8% and 48+/-15% respectively, without any measurable effect on the EC(50). Minoxidil sulphate showed no discernable effect. Vasopressin-induced contraction was also reduced by high levels of glyceryl trinitrate (220 microM; 50 microg/ml) or 10 microM Y27632. CONCLUSIONS: The irreversible calmodulin antagonist fluphenazine has potential to be developed as an inhibitor of contraction in arterial graft vessels. The involvement of Rho kinase indicates that other vasoconstrictors and surgical stress can sensitize radial artery to vasopressin-induced contraction. Strategies targeting this pathway also have future potential.

Diadenosine polyphosphates are selective vasoconstrictors in human coronary artery bypass grafts.

Diadenosine polyphosphates (Ap(n)A) are released by degranulating platelets and high, local concentrations may form at sites of platelet activation. Radial artery grafts, now often used alongside the internal mammary artery in coronary artery bypass surgery, are particularly reactive to several vasoconstrictors but the response to Ap(n)A has not been investigated. This study compared the vasoconstrictor activity of Ap(n)A in human radial artery with other vessels commonly used as bypass grafts. Radial artery demonstrated robust concentration-dependent vasoconstriction to Ap(n)A (n=4-6) at concentrations in the micromolar range. In contrast, average responses in internal mammary artery were negligible. Cross-desensitization revealed that Ap(n)A-mediated vasoconstriction occurred via an alphabetamethyleneATP-sensitive receptor. Responses to both Ap(5)A and alphabetamethyleneATP were inhibited by suramin but were insensitive to the P2X(1) receptor antagonist 8,8'-[Carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid (NF279). Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) enhanced responses to Ap(5)A. Similar responses were obtained in saphenous vein. In conclusion, diadenosine polyphosphates contract radial artery and saphenous vein by an as yet uncharacterized P2X receptor but have only limited activity in internal mammary artery. The selective activity of diadenosine polyphosphates in radial artery would implicate them as potential mediators of post-operative contraction in this graft.

Potentiation of ATP- and bradykinin-induced [Ca2+]c responses by PTHrP peptides in the HaCaT cell line.

In the epidermis, local and systemic factors including extracellular nucleotides and parathyroid hormone-related protein (PTHrP) regulate keratinocyte proliferation and differentiation. Extracellular nucleotides increase proliferation via activation of P2 receptors and induction of calcium transients, while endoproteases cleave PTHrP, resulting in fragments with different cellular functions. We investigated the effects of adenosine 5'-triphosphate (ATP) alone and in combination with synthetic PTHrP peptides on calcium transients in HaCaT cells. ATP induced calcium transients, while PTHrP peptides did not. C-terminal and mid-molecule PTHrP peptides (1-100 pM) potentiated ATP-induced calcium transients independently of calcium influx. 3-Isobutyl-1-methylxanthine potentiated ATP-induced calcium transients, suggesting that a cyclic monophosphate is responsible. Cyclic AMP is not involved, but cyclic GMP is a likely candidate since the protein kinase G inhibitor, KT5823, inhibited potentiation. Co-stimulation with ATP and either PTHrP (43-52) or PTHrP (70-77) increased proliferation, suggesting that this is important in the regulation of cell turnover and wound healing and may be a mechanism for hyperproliferation in skin disorders such as psoriasis. Finally, PTHrP fragments potentiated bradykinin-induced calcium transients, suggesting a role in inflammation in the skin. Since PTHrP is found in many normal and malignant cells, potentiation is likely to have a wider role in modulating signal transduction events.

Atrial natriuretic peptide attenuates elevations in Ca2+ and protects hepatocytes by stimulating net plasma membrane Ca2+ efflux.

Elevations in intracellular Ca(2+) concentration and calpain activity are common early events in cellular injury, including that of hepatocytes. Atrial natriuretic peptide is a circulating hormone that has been shown to be hepatoprotective. The aim of this study was to examine the effects of atrial natriuretic peptide on potentially harmful elevations in cytosolic free Ca(2+) and calpain activity induced by extracellular ATP in rat hepatocytes. We show that atrial natriuretic peptide, through protein kinase G, attenuated both the amplitude and duration of ATP-induced cytosolic Ca(2+) rises in single hepatocytes. Atrial natriuretic peptide also prevented stimulation of calpain activity by ATP, taurolithocholate, or Ca(2+) mobilization by thapsigargin and ionomycin. We therefore investigated the cellular Ca(2+) handling mechanisms through which ANP attenuates this sustained elevation in cytosolic Ca(2+). We show that atrial natriuretic peptide does not modulate the release from or re-uptake of Ca(2+) into intracellular stores but, through protein kinase G, both stimulates plasma membrane Ca(2+) efflux from and inhibits ATP-stimulated Ca(2+) influx into hepatocytes. These findings suggest that stimulation of net plasma membrane Ca(2+) efflux (to which both Ca(2+) efflux stimulation and Ca(2+) influx inhibition contribute) is the key process through which atrial natriuretic peptide attenuates elevations in cytosolic Ca(2+) and calpain activity. Moreover we propose that plasma membrane Ca(2+) efflux is a valuable, previously undiscovered, mechanism through which atrial natriuretic peptide protects rat hepatocytes, and perhaps other cell types, against Ca(2+)-dependent injury.

Cell shape-dependent Control of Ca2+ influx and cell cycle progression in Swiss 3T3 fibroblasts.

The ability of adherent cells such as fibroblasts to enter the cell cycle and progress to S phase is strictly dependent on the extent to which individual cells can attach to and spread on a substratum. Here we have used microengineered adhesive islands of 22 and 45 mum diameter surrounded by a nonadhesive substratum of polyhydroxyl methacrylate to accurately control the extent to which individual Swiss 3T3 fibroblasts may spread. The effect of cell shape on mitogen-evoked Ca2+ signaling events that accompany entry into the cell cycle was investigated. In unrestricted cells, the mitogens bombesin and fetal calf serum evoked a typical biphasic change in the cytoplasmic free Ca2+ concentration. However, when the spreading of individual cells was restricted, such that progression to S phase was substantially reduced, both bombesin and fetal calf serum caused a rapid transient rise in the cytoplasmic free Ca2+ concentration but failed to elicit the normal sustained influx of Ca2+ that follows Ca2+ release. As expected, restricting cell spreading led to the loss of actin stress fibers and the formation of a ring of cortical actin. Restricting cell shape did not appear to influence mitogen-receptor interactions, nor did it influence the presence of focal adhesions. Because Ca2+ signaling is an essential component of mitogen responses, these findings implicate Ca2+ influx as a necessary component of cell shape-dependent control of the cell cycle.

Temperature changes stimulate contraction in the human radial artery and affect response to vasoconstrictors.

BACKGROUND: Radial artery conduits are increasingly used in coronary artery bypass grafting as an additional arterial graft to the internal thoracic artery. Their reactive nature remains a concern, often necessitating the routine use of topically applied vasodilators, such as glyceryl trinitrate, papaverine, phenoxybenzamine, or calcium channel antagonists, in theatre. During preparation prior to surgery and grafting, radial artery conduits are exposed to cooling and rewarming. We investigated how these temperature changes would affect radial artery contractility and how commonly used topical treatments might be used to prevent this. METHODS: Human radial artery was obtained excess to surgery and arterial sections used in organ bath tension experiments or for the culture of smooth muscle cells from medial explants. RESULTS: The radial artery responded to rapid cooling by the addition of 22 degrees C buffer with contraction. Gradual cooling, over a 20 to 30 minute period, reduced basal tension and the response to potassium chloride (KCl) and noradrenaline. Subsequent rewarming from 22 degrees C to 37 degrees C reestablished contraction at precooled levels and led to an elevation of the basal tension. Increases in tension measured in the radial artery were paralleled by increases in intracellular calcium in smooth muscle cells. Contraction induced by rapid temperature changes could be blocked by glyceryl trinitrate but not by phenoxybenzamine. Papaverine and calcium channel blockers had only limited activity. CONCLUSIONS: Temperature changes commonly encountered in theatre during the preparation of radial artery grafts are likely to cause contraction. If rapid temperature change cannot be avoided during graft preparation, then topically applied glyceryl trinitrate will block these responses.

Fluorescent measurement of [Ca2+]c: basic practical considerations.

During the last decade or so, the range of fluorescent indicators for Ca2+ has increased dramatically, so that there are now a host of probes available. Each may offer particular advantages depending on the design of the experiment and the fluorometric equipment available. Careful choice of the indicator is therefore central to achieve a successful outcome. The probe that is chosen will of course depend on the aims of the experiment, how the indicator will be introduced into the cell(s), and the excitation source and detection equipment that are available. I hope that this chapter will not only help investigators choose the most appropriate indicator but, in addition, give an insight into what can be achieved using fluorescent Ca2+ indicators.

Human keratinocytes release ATP and utilize three mechanisms for nucleotide interconversion at the cell surface.

Nucleotide activation of P2 receptors is important in autocrine and paracrine regulation in many tissues. In the epidermis, nucleotides are involved in proliferation, differentiation, and apoptosis. In this study, we have used a combination of luciferin-luciferase luminometry, pharmacological inhibitors, and confocal microscopy to demonstrate that HaCaT keratinocytes release ATP into the culture medium, and that there are three mechanisms for nucleotide interconversion, resulting in ATP generation at the cell surface. Addition of ADP, GTP, or UTP to culture medium elevated the ATP concentration. ADP to ATP conversion was inhibited by diadenosine pentaphosphate, oligomycin, and UDP, suggesting the involvement of cell surface adenylate kinase, F(1)F(0) ATP synthase, and nucleoside diphosphokinase (NDPK), respectively, which was supported by immunohistochemistry. Simultaneous addition of ADP and GTP elevated ATP above that for each nucleotide alone indicating that GTP acts as a phosphate donor. However, the activity of NDPK, F(1)F(0) ATP synthase or the forward reaction of adenylate kinase could not fully account for the culture medium ATP content. We postulate that this discrepancy is due to the reverse reaction of adenylate kinase utilizing AMP. In normal human skin, F(1)F(0) ATP synthase and NDPK were differentially localized, with mitochondrial expression in the basal layer, and cell surface expression in the differentiated layers. We and others have previously demonstrated that keratinocytes express multiple P2 receptors. In this study we now identify the potential sources of extracellular ATP required to activate these receptors and provide better understanding of the role of nucleotides in normal epidermal homeostasis and wound healing.

Fluorescent measurement of [Ca²+]c: basic practical considerations.

It is extremely difficult to write a prescriptive account of how to measure cytosolic-free Ca(2+) ([Ca(2+)](c)) that will suit all potential investigators. The problem arises because of the wide diversity of fluorescent Ca(2+) indicators that are now available, the variety of cells to be investigated, and the range of detection equipment that can be used. Consequently, this chapter is designed to provide the user with an overview of the technology in order that he or she can move toward developing a protocol that will suit the experimental objectives, the cells, and the equipment available to the investigator.

A nonradioactive assay for nitric oxide synthase activity in tissue extracts.

We describe a fluorescence assay for nitric oxide synthase activity based on a new indicator, 4,5-diaminofluorescein (DAF-2). The method offers the advantage of being safer and more convenient than the citrulline radioassay in common use. The rapid and irreversible binding of DAF-2 to oxidized nitric oxide (NO) enables NO production to be measured in real time. The protocol is applied to the measurement of nitric oxide synthase in crude extracts of skeletal muscle.

Phenoxybenzamine treatment is insufficient to prevent spasm in the radial artery: the effect of other vasodilators.

OBJECTIVES: After its reintroduction as an arterial graft in coronary artery surgery, the radial artery is now established as an alternative arterial conduit, with good early and midterm patency. However, because of the concern about its vasospasticity, numerous vasodilator strategies have been used. Recently the use of the irreversible alpha-adrenergic antagonist phenoxybenzamine has been proposed. Although this treatment is effective in eliminating the vasoconstriction mediated by noradrenaline, the contribution of other circulating vasoconstrictors to vasospasm could be as important. This study investigates the response of radial arteries treated with phenoxybenzamine to vasoconstrictor stimuli and possible preventative strategies. METHODS: In vitro, sections of radial artery, pretreated with phenoxybenzamine after harvesting, were stimulated with maximal concentrations of the vasoconstrictors noradrenaline, vasopressin, angiotensin II, KCl, and endothelin-1. In matched segments of artery, vasoconstrictor responses were recorded in the presence of diltiazem, glyceryl trinitrate, and papaverine and compared with phenoxybenzamine-treated samples. RESULTS: Phenoxybenzamine-treated radial artery failed to respond to noradrenaline but did respond to vasopressin, angiotensin II, endothelin-1, and KCl. Diltiazem was largely ineffective against contractile stimuli apart from KCl. Glyceryl trinitrate and papaverine significantly reduced responses to all of the vasoconstrictors tested. CONCLUSION: In phenoxybenzamine-treated sections of radial artery, circulating vasoconstrictor agonists may still contribute to the induction of spasm. Additional vasodilator strategies may be required to completely prevent vasospasm.

Atrial natriuretic peptide attenuates Ca2+ oscillations and modulates plasma membrane Ca2+ fluxes in rat hepatocytes.

BACKGROUND & AIMS: Oscillations in cytosolic free Ca2+ concentration are a fundamental mechanism of intracellular signaling in hepatocytes. The aim of this study was to examine the effects of atrial natriuretic peptide (ANP) on cytosolic Ca2+ oscillations in rat hepatocytes. METHODS: Cyclic guanosine monophosphate (cGMP) was measured by enzyme immunoassay. Cytosolic Ca2+ oscillations were recorded from single aequorin-injected hepatocytes. Ca2+ efflux from hepatocyte populations was measured by using extracellular fura-2. Ca2+ influx was estimated by Mn2+ quench of fluorescence of fura-2 dextran injected into single hepatocytes. RESULTS: ANP attenuated cytosolic Ca2+ oscillations through a decrease in their frequency. In addition, ANP dramatically stimulated plasma membrane Ca2+ efflux and modestly inhibited basal Ca2+ influx. All of the observed effects of ANP were mimicked by the cGMP analogue 8-bromo-cGMP (8-Br-cGMP), and were prevented by inhibition of protein kinase G. In contrast, activation of cytosolic guanylyl cyclase by sodium nitroprusside had no effect on Ca2+ efflux, Ca2+ influx, or Ca2+ oscillations. CONCLUSIONS: ANP decreases the frequency of Ca2+ oscillations and modulates plasma membrane Ca2+ fluxes in rat hepatocytes. Attenuation of oscillatory Ca2+ signaling in hepatocytes may represent a key role for ANP in vivo.

Endothelin receptors in cultured and native human radial artery smooth muscle.

In human vascular smooth muscle cells endothelin-1, acting at both endothelin A and endothelin B receptors, has been demonstrated to be both a potent vasoconstrictor and mitogen. Our aim was to study the functional expression of endothelin receptors in human radial artery smooth muscle using both native tissue and cultured cells (RASMCs). Radial artery smooth muscle cells were cultured from arterial explants and loaded with the calcium fluorescent dye fura-2. Cells responded to endothelin-1 and a variety of other vasoconstrictors with rises in cytoplasmic calcium ([Ca2+]c). Arterial rings responded to endothelin-1 with an increase in tension. The response of both cells and arterial rings to endothelin-1 was characterized using the selective endothelin A receptor antagonist BQ123 and the endothelin B receptor antagonist BQ788. The RASMCs were found to express [Ca2+]c responses consistent with the expression of only the endothelin A receptor. Endothelin-1-mediated vasoconstriction in radial artery rings was unaffected by BQ788 but was completely blocked by BQ123. Using the selective radioligands [125I]-PD151242 and [125I]-BQ3020 and a combination of in vitro receptor autoradiography and isolated cell preparations, endothelin A receptors were confirmed to be present on RASMCs and on arterial sections, whereas endothelin B binding was barely detectable on native smooth muscle and on RASMCs.