Ion channel phenotype of melanoma cell lines.

Melanoma cells are transformed melanocytes of neural crest origin. K+ channel blockers have been reported to inhibit melanoma cell proliferation. We used whole-cell recording to characterize ion channels in four different human melanoma cell lines (C8161, C832C, C8146, and SK28). Protocols were used to identify voltage-gated (KV), Ca2+-activated (KCa), and inwardly rectifying (KIR) K+ channels; swelling-sensitive Cl- channels (Clswell); voltage-gated Ca2+ channels (CaV) and Ca2+ channels activated by depletion of intracellular Ca2+ stores (CRAC); and voltage-gated Na+ channels (NaV). The presence of Ca2+ channels activated by intracellular store depletion was further tested using thapsigargin to elicit a rise in [Ca2+]i. The expression of K+ channels varied widely between different cell lines and was also influenced by culture conditions. KIR channels were found in all cell lines, but with varying abundance. Whole-cell conductance levels for KIR differed between C8161 (100 pS/pF) and SK28 (360 pS/pF). KCa channels in C8161 cells were blocked by 10 nm apamin, but were unaffected by charybdotoxin (CTX). KCa channels in C8146 and SK28 cells were sensitive to CTX (Kd = 4 nm), but were unaffected by apamin. KV channels, found only in C8146 cells, activated at approximately -20 mV and showed use dependence. All melanoma lines tested expressed CRAC channels and a novel Clswell channel. Clswell current developed at 30 pS/sec when the cells were bathed in 80% Ringer solution, and was strongly outwardly rectifying (4:1 in symmetrical Cl-). We conclude that different melanoma cell lines express a diversity of ion channel types.

Evidence for opioid modulation and generation of prostaglandins in sulphur dioxide (SO)2-induced bronchoconstriction.

BACKGROUND: Inhalation of sulphur dioxide (SO2) provokes bronchoconstriction in asthmatic subjects. Cholinergic mechanisms contribute, but other mechanisms remain undefined. The effect of morphine, an opioid agonist, on the cholinergic component of SO2-induced bronchoconstriction was investigated, and the effect of indomethacin, a cyclooxygenase inhibitor, on SO2-induced bronchoconstriction and tachyphylaxis was studied. METHODS: In the first study 16 asthmatic subjects inhaled either ipratropium bromide or placebo 60 minutes before an SO2 challenge on days 1 and 2. On day 3 an SO2 challenge was performed immediately after intravenous morphine. In the second study 15 asthmatic subjects took either placebo or indomethacin for three days before each study day when two SO2 challenges were performed 30 minutes apart. The response was measured as the cumulative dose causing a 35% fall in specific airways conductance (sGaw; PDsGaw35). RESULTS: Ipratropium bromide significantly inhibited SO2 responsiveness, reducing PDsGaw35 by 0.89 (95% CI 0.46 to 1.31) doubling doses. This effect persisted after correction for bronchodilatation induced by ipratropium bromide. The effect of ipratropium bromide and morphine on SO2 responsiveness also correlated (r2 = 0.71). In the second study SO2 tachyphylaxis developed with PDsGaw35 on repeated testing, being reduced by 0.62 (95% CI 0.17 to 1.07) doubling doses. Indomethacin attenuated baseline SO2 responsiveness, increasing PDsGaw35 by 0.5 (95% CI 0.06 to 0.93) doubling doses. CONCLUSIONS: These results suggest that opioids modulate the cholinergic component of SO2 responsiveness and that cyclooxygenase products contribute to the immediate response to SO2.

Monocyte/macrophage regulation of coagulant events.

Monocytes/macrophages actively regulate both the assembly and function, as well as the substrate specificity, of various coagulation enzymes at their membrane surface. Regulation is effected through a variety of mechanisms, not limited to, but including the expression of receptors (or 'binding sites') for the various protein constituents of the complexes, the expression of different receptors which may alter the function of the protease, and the expression of membrane proteases which may affect protein cofactor function. Monocyte stimulation with various agonists modulates many of these responses as does their adherence to and differentiation on various substrates.

Human coagulation factor V is activated to the functional cofactor by elastase and cathepsin G expressed at the monocyte surface.

The ability of intact peripheral blood monocytes to modulate factor V procoagulant activity was studied using electrophoretic and autoradiographic techniques coupled to functional assessment of cofactor activity. Incubation of plasma concentrations of factor V with monocytes (5 x 10(6)/ml) resulted in the time-dependent cleavage of the 330-kDa protein. Activation occurred via several high molecular mass intermediates (> or = 200 kDa) to yield peptides of 150, 140, 120, 94, 91, 82, and 80 kDa, which paralleled the expression of cofactor activity. The cleavage pattern observed differed from that obtained with either thrombin or factor Xa as an activator. The incubation time required to achieve full cofactor activity was dependent on the monocyte donor and ranged from 10 min to 1 h and was consistently slightly lower than that obtained with thrombin-activated factor Va. Cofactor activity was not diminished by additional incubation. The cofactor activity generated bound to the monocyte such that a competent prothrombinase complex was formed at the monocyte membrane surface. Furthermore, within 5 min of factor V addition to monocytes, near maximal cofactor activity (approximately 70%) was bound and expressed on the monocyte membrane. The proteolytic activity toward factor V was associated primarily with the monocyte membrane, as little proteolytic activity was released into the cell-free supernatant. Proteolytic activity was inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride. However, the inhibitor profile obtained with alpha 1-antiproteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin suggested membrane-bound forms of elastase and cathepsin G were mediating, in large part, the proteolysis observed. These data were confirmed using purified preparations of both proteases and a specific anti-human leukocyte elastase antibody. Thus, expression of these proteases at the monocyte surface may contribute to thrombin generation at extravascular tissue sites by catalyzing the activation of the essential cofactor, factor Va, which binds to the monocyte surface and supports the factor Xa-catalyzed activation of prothrombin.

Procoagulant activities expressed by peripheral blood mononuclear cells.

These combined data support the concept that the procoagulant response elicited by mononuclear cells, particularly monocytes, is accomplished through regulated binding site-mediated (or perhaps "receptor"-mediated) assembly of proteolytic activities at their membrane surface. Because the work of several laboratories indicate that the monocytes provide the appropriate membrane surface for the assembly and function of all the coagulation complexes required for thrombin production in vivo, monocytes may provide a unique opportunity to investigate how coagulant reactions are regulated on cell surfaces through both receptor-mediated events as well as by channeling a product of one reaction to serve as a mediator of a second reaction.

A controlled study of the effects of aerobic exercise on antihypertensive drug requirements of essential hypertensive patients in the general practice setting.

1. To investigate the effect of an exercise training programme on antihypertensive drug requirements, 19 sedentary subjects (14 men and five women) with mild essential hypertension (systolic blood pressure greater than 140 mmHg but less than 180 mmHg), aged 29-55 years, were randomly assigned to 16 weeks of moderate intensity exercise or light intensity exercise (control), and titrated on antihypertensive drug therapy (captopril and hydrochlorothiazide) until resting systolic blood pressure (sitting) of less than 140 mmHg was achieved. 2. The moderate exercise group (n = 11) followed a graded walk-jog programme to greater than 60% age-predicted maximum heart rate (HRmax), attending 45 min sessions, three times each week. The light exercise group (n = 8) followed a programme of flexibility (stretching exercises) and walking to less than 60% age-predicted HRmax, attending 45 min sessions, three times each week. 3. There was no difference between treatment groups for the level of reduction in both resting systolic and diastolic blood pressure over the 16 weeks (15.64 +/- 10.6/12.81 +/- 9.97 mmHg in the moderate intensity group and 15.31 +/- 10.9/7.7 +/- 7.1 mmHg in the light intensity group). The change in 24 h ambulatory blood pressure profile was also similar in both treatment groups. The final antihypertensive drug requirements to achieve these changes in blood pressure was not significantly different between the two treatment groups. This was despite a significant improvement in submaximal exercise performance in the moderate intensity exercise group (P less than 0.001). 4.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of L-glutamic acid on airway function and reactivity in the rabbit.

The ingestion of monosodium glutamate in sensitive individuals has been reported to cause severe asthma. We therefore studied the effects of L-glu on airway function and histamine (H) responsiveness in the rabbit. Histamine dose response curves (HDR's) were performed by measuring total lung resistance (RL) after inhalation of saline and increasing concentrations of H (1-30 mg/ml). The concentration of H producing a 20% increase in RL (PC20H) was obtained by interpolation. To assess the effects of L-glu, 8 rabbits were infused with L-glu (0.2 g/kg/hr) or saline in random order (14 days apart) for 4 hours followed by an HDRC. To look at possible late effects, a repeat HDRC was also performed in 6 rabbits 12 hours after completion of the L-glu infusion. In order to see whether rabbits rendered hyperresponsive responded to L-glu, the above protocol was performed in 7 rabbits following the inhalation of 3 micrograms of the activated complement fragment C5a des Arg. The L-glu infusions increased the plasma levels approx. ten-fold (mean +/- SEM 0.119 +/- 0.012 base-line, 1.272 +/- 0.061 mmol/l post infusion). L-glu did not increase the PC20H or baseline RL in either the normal rabbits at 4 or 12 hours or in the C5a des Arg treated rabbits at 4 hours. It is concluded that L-glu does not cause bronchoconstriction or an increase in airway responsiveness to H in the rabbit.

Monosodium L-glutamate-induced asthma.

Ingested chemicals, including aspirin and sulfites, are becoming increasingly recognized as provokers of acute severe asthma. In order to investigate the asthma-provoking potential of the widely used flavor enhancer, monosodium L-glutamate (MSG), we challenged 32 subjects with asthma, a number of whom gave histories of severe asthma after Chinese restaurant meals or similarly spiced meals. The subjects received an additive-free diet for 5 days before challenge and were challenged in hospital, after an overnight fast, with 500 mg capsules of MSG. They were challenged in a single-blind, placebo-controlled fashion with increasing doses of MSG from 0.5 gm to 5.0 gm. Thirteen subjects reacted. Seven subjects (group 1) developed asthma and symptoms of the Chinese restaurant syndrome 1 to 2 hours after ingestion of MSG. Six subjects (group 2) did not develop symptoms of Chinese restaurant syndrome, and their asthma developed 6 to 12 hours after ingestion of MSG. These challenge studies confirm that MSG can provoke asthma. The reaction to MSG is dose dependent and may be delayed up to 12 hours, making recognition difficult for both patient and physician.

The relationship of inhaled sulfur dioxide reactivity to ingested metabisulfite sensitivity in patients with asthma.

When ingested in acid solution, the preservative sodium metabisulfite (MB) provokes asthma within minutes of ingestion in a proportion of asthmatic subjects. Freshly prepared acid solutions of MB liberate significant quantities of gaseous SO2. In order to test the hypothesis that asthma provoked by ingestion of acidified solutions of MB was due to supersensitivity to SO2 inhaled during swallowing, 3 groups of 10 subjects were studied. Groups 1 and 2 were asthmatics. Group 1 subjects were reactors and Group 2 were nonreactors to ingested MB. Group 3 subjects were nonasthmatic control subjects. Subjects were challenged, on separate days, with 50 mg of MB in citric acid and SO2 gas, via a steady-state system, at increasing concentrations (0.5, 1.5, 3, 5 ppm). The mean percent fall in peak expiratory flow rates after ingestion of MB for Group 1 was 35 +/- 14, Group 2 was 6 +/- 6, and Group 3 was 5 +/- 3. The mean SO2 provocation concentration (Pc20.SO2) for Group 1 was 1.19 +/- 0.78 ppm, for Group 2 it was 2.25 +/- 1.42 ppm, and for Group 3 it was greater than 5 ppm. The means for Pc20.SO2 of Groups 1 and 2 were not statistically different (p = 0.075), although the possibility of a Type 2 error is recognized. Five MB-sensitive subjects were subsequently challenged with the MB solution by mouthwash and nasogastric tube. Asthma was provoked in all 5 subjects by mouthwash but not by gastric challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Adverse reactions to foods.

The discovery of IgE in the mid-1960s resulted in a widespread view that allergy was the basis of most adverse reactions to food, but it is becoming increasingly clear that other, as yet poorly understood, mechanisms are responsible in the overwhelming majority of cases. This, together with the proliferation of popular literature on "food allergy" has resulted in considerable confusion in the minds of both the public and the medical profession on the subject. In the majority of patients presenting with food intolerance, recognized or otherwise, symptoms are precipitated by various small, non-immunogenic organic molecules present in the food as natural or added ingredients. These reactions are pharmacological rather than immunological in nature, although in some situations they may share a final common pathway with true allergic reactions, resulting in similar symptoms.

In vitro releasability of histamine and serotonin: studies of atopic patients.

Sixteen atopic patients with anaphylaxis to food, eczema, asthma and/or rhinitis were investigated for in vitro reactivity of their leukocytes and platelets to various stimuli. Leukocytes from two patients with anaphylaxis to foods had very high spontaneous release of histamine. Compared to non-atopic volunteers without respiratory disease, leukocytes from the other atopic patients showed increased histamine release after stimulation with methacholine in concentrations between 10(-2) and 10(-6) M. Histamine release induced by anti-IgE or anti-kappa chain serum was slightly decreased in atopics compared to controls, and was significantly decreased at low concentrations of anti-IgE (p less than 0.05). There was no significant difference of means for histamine release induced by the calcium ionophore A-23815. The uptake of 3H serotonin from platelet-rich plasma of atopic patients appeared to occur more slowly than in non-atopics. Serotonin release from washed platelets after stimulation with aggregated IgG was significantly lower in the atopic group (p less than 0.01). There was no significant difference in serotonin release induced by thrombin, epinephrine, ionophore or methacholine. Alterations in releasability of mediator containing cells to immunologic and non-immunologic stimuli may play a role in the expression of atopic disease.

HLA and bird breeder's hypersensitivity pneumonitis.

HLA antigen status was assessed in non-related individuals and related family members with bird breeder's hypersensitivity pneumonitis (HP). 35% of 20 non-related patients carried HLABW40 compared to 10% of 861 normal controls (p less than 0.001, Pc less than 0.05, respectively). A consistent association of an HLA haplotype with disease was demonstrated in three of four families. Selected HLA antisera contained antibodies which blocked the blastogenic response to bird antigen. This study suggests the existence of an HLA associated Ir gene in bird breeder's HP.