Differential inhibition of neutrophil superoxide generation by nonsteroidal antiinflammatory drugs

In order to further characterize the effects of nonsteroidal antiinflammatory drugs on neutrophil superoxide (O₂ ^–) generation, human neutrophils were incubated in the presence of sulfinpyrazone, phenylbutazone, and indomethacin prior to exposure to a variety of oxidative stimuli. Stimuli used included the chemotactic peptideN-formyl-methionyl-leucyl-phenylalanine (FMLP, 5.0 × 10^–7 M), NaF (20 mM), phorbol myristate acetate (PMA, 3.2 × 10^–7 M), and opsonized zymosan (250g/ml). superoxide release induced by FMLP was inhibited by all three drugs with half-maximal inhibition (K_i50) at 2.5, 30, and 120M for sulfinpyrazone, phenylbutazone, and indomethacin, respectively. This inhibition was not due to drug interference with the assay system since comparable inhibition was not observed in a cell-free O₂ ^–-generating system. The neutrophil's response to NaF was blunted by sulfinpyrazone (K _i50=400M) and phenylbutazone (K _i50=65M), but was unaffected by indomethacin. A similar inhibitory pattern was observed when zymosan was used as the oxidative stimulus. Sulfinpyrazone and phenylbutazone inhibited the response to zymosan (K _i50s of 425 and 32M, respectively), whereas indomethacin augmented it. PMA stimulation evoked O₂ ^– production which was inhibited by phenylbutazone (K _i50=350M) but not by sulfinpyrazone or indomethacin in concentrations up to 1 mM. The results support the hypothesis that the enzyme system responsible for neutrophil O₂ ^– generation can be activated by more than one mechanism. The results also emphasize the need to evaluate pharmacologie modulation of neutrophil responses in light of the stimulus used to evoke the response.This work was supported by National Institutes of Health grant T32-AMO7186 and National Institute of Allergy and Infectious Diseases grant AI-17950.     

Regulation of superoxide anion generation in bovine alveolar macrophages by bacterial lipopolysaccharide,serum proteins,and modulators of signal transduction

The respiratory burst of phagocytes in an important leukocyte function which results in generation of oxygen species that are both microbicidal and potentially damaging to host tissues. We investigated regulation of the respiratory burst of alveolar macrophages in response to lipopolysaccharide (LPS) derived from gramnegative bacteria, serum proteins, and several modulators of signal transduction. When employed as a single stimulus, LPS (E. coli 055 B5, 10 ng/ml-1g/ml) was a weak stimulus for generation of superoxide anion (O ₂ ^– ) as compared to the potent effect of the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA; 500 ng/ml). However, when LPS was combined with fetal bovine serum (FBS; 0.4–1.0% vol/vol, equivalent to 128–320g protein/ml), O ₂ ^– generation was enhanced approximately two-fold over LPS alone. A chromatographically-derived bovine serum fraction which contained bovine lipopolysaccharide-binding protein (bLBP; 0.25–1.0g/ ml) was an effective substitute for FBS at a much lower protein concentration than whole FBS, and a similar synergistic effect with LPS on O ₂ ^– generation was observed. Stimulation of macrophages for generation of O ₂ ^– either with LPS alone or with LPS plus serum/serum fraction was suppressed by the protein tyrosine kinase inhibitor herbimycin A (0.2 ng/ml), and the calcium chelator BAPTA (12M), but not by modulators of G-proteins, including pertussis toxin (10 ng/ml) and cholera toxin (5g/ml protein). Essentially complete inhibition of O ₂ ^– synthesis by herbimycin A and BAPTA occurred in the presence of LPS and the bLBP-containing serum fraction (1g/ml protein), but only partial inhibition (46.7% and 64.1%, respectively) was observed in the presence of LPS plus FBS (256g/ml protein). These results indicate that when LPS is used as a sole stimulus it induces modest respiratory burst activity. However, when LPS is combined with appropriate serum components, it stimulates alveolar macrophages to generate larger amounts of O ₂ ^– . Cellular signaling pathways important in stimulation of macrophages by LPS and serum components are protein tyrosine kinase- and Ca⁺⁺-dependent, but do not relay on G-protein-mediated signaling.     

Effects of arylaminobenzoate-type chloride channel blockers on equivalent short-circuit current in rabbit colon

Arylaminobenzoates were examined in rabbit colon mounted in an Ussing chamber. The open-circuit transepithelial voltage (V _te) and resistance (R _te) were measured and the equivalent short-circuit current (I _SC=V _te/ R _te) was calculated. After serosal (s) and mucosal (m) addition of indomethacin (1 mol/l) I _SC was –71±11 (n = 118) A/cm². Amiloride (0.1 mmol/l, m) inhibited this current and reversed the polarity to + 32±4 (n=118) A/cm². In the presence of amiloride and indomethacin, prostaglandin E₂ (1 mol/l, s), known to induce Cl^– secretion, generated an I _SC of -143 ± 8 (n = 92) A/cm². The arylaminobenzoate and Cl^– channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) reduced I _SC reversibly with a half-maximal inhibition (IC₅₀) at approximately 0.35 mmol/l and 0.2 mmol/l for mucosal and serosal application respectively. To test whether the poor effect was caused by mucus covering the luminal surface, dose/response curves of the mucosal effect were repeated after several pretreatments. Acidic pH on the mucosal side reduced IC₅₀ to approximately 0.1 mmol/l. A similar effect was observed after N-acetyl-l-cysteine (m) preincubation. Pretreatment with N-acetyl-l-cysteine (m) and carbachol (s), in order to exhaust mucus secretion, and l-homocysteine (m) were more effective and reduced IC₅₀ to approximately 50 mol/l. To test whether this effect of NPPB was caused by non-specific effects, the two enantiomers of 5-nitro-2-(+/–1-phenylethylamino)-benzoate were tested of which only the (+) form inhibited the Cl^– conductance in the thick ascending limb of the loop of Henle (TAL). In the present study the (+) enantiomer inhibited significantly more strongly than the (–) form. This suggests that the inhibitory effect of NPPB, even though it requires rather high concentrations, is probably due to Cl^– channel inhibition. For other arylaminobenzoates the sequence of potencies was different from that determined for the TAL. The present data indicate that substances that have been designed to block the Cl^– conductance of the TAL segment also inhibit reversibly but with much lower affinity the PGE₂-induced Cl^– secretion in rabbit colon.Supported by DFG Gr 480/10     

Effects of adenosine on guinea pig pulmonary eosinophils

Extracellular adenosine has pharmacological activity on a wide variety of cell types and may play an important role as an inflammatory modulator with both pro- and anti-inflammatory activities. These studies examine the effects of adenosine on guinea pig pulmonary eosinophils. Adenosine alone did not directly induce superoxide (O ₂ ^– ) production. Pretreatment with adenosine primed the O ₂ ^– response of guinea pig pulmonary eosinophils following the addition of 1 or 10M plateletactivating factor (PAF). Priming was seen at adenosine concentrations greater than 1 M and was maximal at 100M. At this maximal dose, adenosine priming increased the O ₂ ^– response to 1M and 10M PAF by 86% and 51%, respectively. Priming by adenosine was not seen when ionomycin or phorbol myristate acid (PMA) were used as agonists. In fura-2 loaded eosinophils, the addition of 100 M adenosine resulted in a small but significant rise in intracellular calcium of 54.4 ±9.2 nM above baseline. In contrast, similar adenosine concentrations had no effect on cytosolic calcium levels in guinea pig neutrophils. These data demonstrate a pro-inflammatory role for adenosine in elicited guinea pig pulmonary eosinophils.     

Tubular transport processes in proximal tubules of hypothyroid rats. Lack of relationship between thyroidal dependent rise of isotonic fluid reabsorption and Na+−K+-ATPase activity

Hypothyroid rats reconstituted with 10 g/kg b.w. per day of tri-iodothironine (T₃) for 4 days resulted in normal free T₃ and TSH levels. FT₃ levels were: 0.53±0.3 pg/ml in hypothyroid rats; 2.78±1.21 pg/ml in hormone reconstituted rats and 2.90±0.90 pg/ml in euthyroid rats. TSH levels were 3,508±513 g/ml in hypothyroid rats; 1,008±204 g/ml in reconstituted rats and 270±184 ng/ml in euthyroid rats.When hypothyroid rats were reconstituted with 50 g T₃/kg b.w. per day, TSH levels were nearly normal after 4 days (1,157±621 ng/ml). However FT₃ levels after 1–4 days were always higher than in euthyroid rats.Hypothyroid rats show a decrease in isotonic fluid reabsorption (J _v) in the proximal tubule (1.50±0.08 versus 4.96±0.23 10^–2 nl·mm^–1·s^–1 in euthyroid animals). 1 day after T₃ (10 g/kg b.w./day) injectionJ _v was increased significantly to 2.05±0.20 10^–2 nl·mm^–1·s^–1 and continued to increase during 4 days of T₃ reconstitution.When 50 g T₃/kg b.w./day was used,J _v increased to 2.75±0.07 after 1 day and to 3.10±0.42 10^–2 nl·mm^–1·s^–1 after 4 days.J _v was never reaching a value close to that of euthyroid rats because the tubular radius in hypothyroid rats (14.7±1.8 m) is less than that of euthyroid rats (19.2±0.5 m). The radius in hypothyroid rats treated with T₃ was unchanged over a 4 day course with either high or low doses of T₃.Na⁺–K⁺-ATPase activity was found to be 2.91±0.16 M P_i/h×mg protein in homogenates of kidney cortex from hypothyroid rats. Treatment of hypothyroid rats with 10 g or 50 g of T₃ resulted in an initial decrease in ATPase activity, followed by an increase to base level in hypothyroid rats with 10 g and a significantly higher level with 50 g. This decrease in ATPase activity was contrasted to the increase inJ _v.These data indicate that there is a dissociation between the effects of physiological doses of thyroid hormones on proximal tubular reabsorption and the effects of T₃ on Na⁺–K⁺-ATPase activity of kidney cortex. This leads to question the relationship between sodium transport and ATPase activity under physiological doses of thyroid hormones. An early effect of physiological doses of thyroid hormones on brush border Na⁺ permeability is suggested.     

Effects of muscarinic blockade on the thermic effect of oral or intravenous carbohydrate

Summary Muscarinic blockade by atropine has been shown to decrease the thermic effect of a mixed meal, but not of intravenous glucose. To further delineate the mechanisms involved in the atropine-induced inhibition of thermogenesis after a meal, plasma substrate and hormone concentrations, energy expenditure (EE) and substrate oxidation rates were measured before and during a continuous glucose infusion (44.4 mol·kg^–1·min^–1) with or without atropine. After 2 h of glucose infusion, a 20-g oral fructose load was administered while the glucose infusion was continued. Plasma insulin concentrations attained a plateau at 596 (SEM 100) pmol·l^–1 after 120 min of glucose infusion and were not affected by muscarinic blockade; plasma glucose concentrations peaked at 13.3 (SEM 0.5) mmol·l^–1 at 90 min and decreased progressively thereafter; no difference was observed with or without atropine. Plasma free fatty acid and glucagon concentrations, with or without atropine, were both decreased to 201 (SEM 18) mol·l^–1 and 74 (SEM 4) ng·l^–1, respectively, after 2 h of glucose infusion, and were not further suppressed after oral fructose. Carbohydrate oxidation rates (CHO_ox) increased to 20.8 (SEM 1.4) mol·kg^–1·min^–1 and lipid oxidation rates (L_ox) decreased to 1.5 (SEM 0.3) mol·kg^–1·min^–1 between 90 and 120 min after the beginning of glucose infusion and were not affected by atropine. Glucose-induced thermogenesis was similar with [6.5% (SEM 1.4%) of basal EE] or without [6.0% (SEM 1.0%), NS) muscarinic blockade during the 30 min preceding fructose ingestion. During the second half-hour after fructose ingestion, atropine infusion inhibited markedly the stimulation of CHO_ox [+2.8 (SEM 1.0) mol·kg^–1·min^–1 vs +6.9 (SEM 1.0) mol·kg^–1·min^–1, saline, P<0.02] and the suppression of L_ox [–0.8 (SEM 0.2) mol·kg^–1·min^–1 vs –1.4 (SEM 0.2) mol·kg^–1·min^–1, saline, P<0.05]. Carbohydrate-induced thermogenesis during the second half-hour after fructose ingestion, increased to 13.0% (SEM 2.0%) without atropine and was suppressed to 7.7% (SEM 1.9%) (P< 0.05, vs saline) with atropine. It was concluded that muscarinic blockade suppressed the increase of thermogenesis observed after oral fructose, but not during intravenous glucose infusion and that this suppression occurred independently of alterations of plasma insulin concentrations.     

Stimulus interactions in release of superoxide anion (O2 −) from human neutrophils

A wide variety of agents stimulate superoxide anion (O₂ ^–) release from human neutrophils. To determine whether the same or different cellular pathways are utilized, neutrophils were stimulated to release O₂ ^– with combinations of f-Met-LeuPhe (FMLP) (10^–7 M), C5a (25 nM), Con A (100 g/ml), arachidonic acid (100 M), and PMA (1 g/ml). These concentrations produced maximal O₂ ^– production when used alone. A synergistic response was observed when Con A was used in combination with FMLP or C5a. This response was twice the expected release in cytochalasin B-treated cells and three to five times the expected release in untreated cells. Additional studies showed that synergism was dependent upon the simultaneous presence of both agents. Additive O₂ ^– responses were observed when either FMLP, Con A, or C5a was tested in combination with arachidonic acid and when FMLP and C5a were tested together. When PMA was tested with C5a, FMLP, or Con A, a nonadditive O₂ ^– response resulted, whereas mixtures of PMA and arachidonic acid resulted in a less than additive response. These contrasting results using different soluble stimuli in combination suggest that multiple pathways exist for the stimulation of neutrophil O₂ ^– release, with some stimuli being totally independent of each other (possibly activating separate pools of oxidase), while other stimuli show cooperative effects on oxidase activation.     

In vitro effect of N-methoxy-3-(3,5-ditert-butyl-4-hydroxybenzylidene)-2-pyrrolidone (E-5110), a novel nonsteroidal anti-inflammatory agent,on generation of some inflammatory mediators

Cultured rat synovial cells generate PGE₂ upon stimulation with a factor derived from rate polymorphonuclear cells (Prostaglandins27, 697, 1984). E-5110 inhibited PGE₂ generation by the synovial cells. The IC₅₀ values (M) for inhibition of PGE₂ generation were 0.026 for E-5110, 0.008 for indomethacin, 0.112 for piroxicam, 0.003 for R-830, 0.667 for BW-755C and 2.05 for benoxaprofen. Calcium ionophore A-23187-stimulated LTB₄ generation by human neutrophils was inhibited by E-5110 with an IC₅₀ value of 0.20 M, which was similar to NDGA. The inhibition of LTB₄ by E-5110 was more potent than that of R-830, BW-755C or benoxaprofen. E-5110 also inhibited superoxide generation by human neutrophils stimulated with opsonized zymosan, f-Met-Leu-Phe and phorbol myristate acetate. These results indicate that E-5110 is a potent dual inhibitor that suppresses superoxide generation.     

Inhibitory effects ofγ-interferon on bradykinin-induced bone resorption and prostaglandin formation in cultured mouse calvarial bones

The effects of mouse recombinant-interferon (-IFN) and indomethacin on bone resorption stimulated by bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg⁹-bradykinin and prostaglandin E₂ (PGE₂) have been studied using cultures of neonatal calvarial bones and analyzing the release of⁴⁵Ca from prelabelled bones as a paramenter of bone resorption. In addition, the effects of-IFN and indomethacin on formation of PGE₂ in bone cultures stimulated by bradykinin was analyzed. Indomethacin (1 mol/l) totally abolished bradykinin (1 mol/l) induced⁴⁵Ca release. The inhibitory effect of indomethacin could be fully reversed by addition of PGE₂ (1 mol/l).-IFN (1000 U/ml) almost totally inhibited⁴⁵Ca release stimulated by bradykinin (1 mol/l), but the inhibitory effect could only be partially overcome by PGE₂.-IFN and indomethacin also inhibited the stimulatory effects of Lys-bradykinin, Met-Lys-bradykinin and des-Arg⁹-bradykinin (1 mol/l) on⁴⁵Ca release. The stimulatory effects of PGE₂ (1 mol/l) on radioactive calcium mobilization was partially inhibited by-IFN (1000 U/ml), whereas indomethacin (1 mol/l) was without effect. The inhibitory effect of-IFN on⁴⁵Ca release stimulated by bradykinin and PGE₂ was dose-dependent with threshold for action at 3–30 U/ml. Comparative dose-response curves showed that-IFN was most potent as inhibitor of bradykinin induced⁴⁵Ca release. Bradykinin (1 mol/l) significantly stimulated PGE₂ formation by a mechanism that was completely inhibited by indomethacin (1 mol/l).-IFN (1000 U/ml) partially inhibited the stimulatory effect of bradykinin on PGE₂ formation. These data show that i)-IFN is a potent inhibitor of bone resorption induced by bradykinin and bradykinin analogues and ii) that the mechanism of action can be mainly explained by an inhibition of kinin induced prostaglandin biosynthesis. The results, however, also show that-IFN can inhibit bone resorption by mechanisms unrelated to prostaglandin formation.     

Intracellular Cl− concentration in striated intralobular ducts from rabbit mandibular salivary glands

Intralobular striated ducts have been isolated from rabbit mandibular salivary glands and maintained in primary culture for up to 2 days. Such ducts were loaded with the Cl^–-sensitive fluorescent dyeN-(ethoxycarbonylmethyl)-(6-methoxyquinolinium bromide) (MQAE) and intracellular Cl^– concentration ([Cl^–]_i monitored using a fluorescence microscope. Intracellular Cl^– could be rapidly and reversibly emptied from striated duct cells by replacing Cl^– in the superfusing solution with NO ₃ ^– . [Cl^–]_i could be lowered by removal of external Na⁺, exposure to 10 M amiloride or to 10 M 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS). Both amiloride and DIDS were able to inhibit the recovery of [Cl^–]_i after an initial exposure to Na⁺- or Cl^–-free solution. The amiloride derivatives, benzamil (2 M) and N-isobutyl-N-methylamiloride (MIBA), (10 M) also lowered [Cl^–]_i by similar amounts as 10 M amiloride. Varying external K⁺ concentration ([K⁺]_o) also affected [Cl^–]_i. Increasing [K⁺]_o increased [Cl^–]_i, but decreasing [K⁺]_o did not decrease [Cl^–]_i. Instead, [Cl^–]_i was also increased when [K⁺]_o was lowered below the control value. Bumetanide (0.1 mM) lowered [Cl^–]_i by only a small amount, while ouabain (1 mM) had no significant effect on [Cl^–]_i. These data are consistent with current models of electrolyte transport in salivary ducts which include Cl^– channels, Na⁺ channels, and Na⁺/H⁺ exchangers in the apical membrane. The effects of low [K⁺]_o can be interpreted in terms of a K⁺-dependent exit mechanism for Cl^–.     

Effects of gold compounds on function of phagocytic cells

The effect of sodium aurothiomalate and auranofin on the generation of superoxide anions (O ₂ ^– ) by polymorphonuclear leukocytes (PMNLs) and adherent mononuclear phagocytic cells (AMNCs) has been investigated. Sodium aurothiomalate at final concentrations of 1, 10, and 100 g Au/ml and auranofin ranging from 0.1 to 2.0 g Au/ml were used in the reactions involving all celt types. Results have been compared between cells drawn from normal controls and patients with active rheumatoid disease. The effect of gold compounds on both cell types was assessed following activation by phorbyl myristate acetate (1×10^–8 M) and N-formyl-methionylleucyl-phenylalanine (1×10^–4 M) using a cytochromec reduction method. Sodium aurothiomalate at the maximum concentration modestly inhibited O ₂ ^– generation by PMNLs but not AMNCs. Auranofin inhibits O ₂ ^– generation by both cell types. Inhibition of cells from patients with rheumatoid arthritis was greater than that seen with cells from normal controls.     

Direct measurement of K movement by39K NMR in perfused rat mandibular salivary gland stimulated with acetylcholine

The intracellular K content (K_in) of the isolated perfused rat mandibular gland was measured by³⁹K NMR spectroscopy at 25°C, using an inversion recovery technique based on the fact that the spin-lattice (T₁) relaxation of K_in is much faster than that of the extracellular K. K_in decreased by 30–34% of the resting level and reached a plateau level during secretion evoked by a sustained infusion of 1 mol/l acetylcholine. Addition of 1 mmol/l ouabain decreased K_in by an additional 41% of the resting level. The K net flux to the blood and saliva was calculated from the K concentrations and flow rates of the effluent and the saliva. At an initial stage of secretion the gland lost K to the vascular side at a rate of 12.6±1.8 mol/g-min (mean ± SEM,n=7). During sustained secretion, the gland took K up from the vascular side at a rate of 3.3±0.7 mol/g-min (n=7), and the same amount of K was secreted into the saliva (4.7±1.1 at 5–10 min, 2.8±0.8 mol/g-min at 20–30 min), resulting in no net K movement from the gland. Addition of 1 mmol/l ouabain stopped salivary secretion and caused a transient K release to the vascular side at a maximum rate of 12.8±1.1 mol/g-min. Withdrawal of acetylcholine and ouabain induced K uptake from the vascular side (6.5±0.7 mol/g-min) and the amount of K released was completely restored when K_in recovered completely. The ratio (0.38) of the acetylcholine-induced K loss (30 mol/g) to the ouabain-induced total K loss (80 mol/g) was very similar to the ratio (0.41–0.45) measured by³⁹K NMR. The present observations lead to the conclusion that the changes in³⁹L-NMR-visible intracellular K directly relate to K movement across both the basolateral and the luminal membranes of salivary epithelia.     

Free radical profiles in an encapsulated pancreatic cell matrix model

The survival of encapsulated pancreatic cells or islets is often limited because of nutrient deficiency, fibrotic overgrowth, and immune attack. Activated immune cells, such as macrophages, release nitric oxide (NO) and superoxide O ₂ ^- These species or their reactive intermediates, such as peroxynitrite, can be cytotoxic, mutagenic, and/or carcinogenic. The transport of these free radicals to encapsulated pancreatic cells cannot be impeded by the present immunoisolation technology. A model has been developed simulating free radical profiles within an encapsulation matrix due to macrophage immune cells attached to the surface of an encapsulation matrix. The model incorporates the transport and reactions of NO,O ₂ ^- O² and total peroxynitrite (PER). The model predictions of NO, O ₂ ^- and PER concentrations to which pancreatic cells are potentially exposed are in the range of 8–42 M, 0.5–8 nM, and 0.1–0.8 M, respectively, for a 100–500 m radius encapsulation matrix. The results demonstrate that the potential exists for free radical damage of encapsulated pancreatic cells and also demonstrates that additional exposure studies may be necessary for assessing free radical effects on pancreatic cell function. Also, care must be taken in assuming that encapsulated cell systems are completely protected from immunological action. © 2002 Biomedical Engineering Society. PAC2002: 8716Ac, 8239Rt, 8716Uv     

Protein kinase C as mediator of arachidonic acid-induced decrease of neuronal M current

The M current, I _M, of NG108-15 neuroblastoma×glioma hybrid cells, a non-inactivating K⁺ current, is decreased by arachidonic acid (5–25 M), often after an initial transitory increase. To test the possibility that the decrease is caused by activation of protein kinase C (PKC) we used the PKC 19–31 peptide, which is an effective inhibitor of PKC. With 1 M peptide in the pipette solution the normally observed strong reduction of I _M by 1 M phorbol 12,13-dibutyrate (PDB) was almost totally prevented, indicating that PKC is completely inhibited; also the voltage dependence of the M conductance, g _M(V), was shifted to more negative membrane potentials. In the presence of 1 M peptide the effect of 25 M arachidonic acid on I _M was significantly reduced, suggesting that the effect, or at least a large part of it, is mediated by PKC.     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].     

Identification of phospholipase D (PLD) activity in mouse peritoneal macrophages

It is now believed that PLD may contribute to the sustained generation of diacylglycerol (DAG) within activated cells. DAG can be formed from phosphatidylcholine by the sequential actions of PLD and phosphatidic acid phosphohydrolase. Phorbal myristate acetate (PMA, 1 M), A23187 (10 M) or platelet-activating factor (PAF, 100 nM) caused significant enhancement of intracellular¹⁴C-phosphatidic acid levels 2–5 min after the addition of stimulus, in cultures of peritoneal macrophages pre-labelled with¹⁴C-palmitate. Bacterial lipopolysaccharide (LPS) (5 g/ml) or zymosan (375 g/ml) also stimulated the production of¹⁴C-phosphatidic acid, but over a longer time course (15–60 min). In the presence of 1% ethanol each stimulus caused significant production of⁴C-phosphatidylethanol at the expense of¹⁴C-phosphatidic acid, thus confirming a contribution of PLD in these reactions. This is the first report of PLD activity in this cell type.     

Nonsteroidal antiinflammatory agents inhibit stimulated neutrophil adhesion to endothelium: Adenosine dependent and independent mechanisms

All nonsteroidal antiinflammatory drugs (NSAIDs) inhibit neutrophil aggregation (homotypic cell-cell adhesion) and do so without affecting expression of CD11b/CD18. Since the first step in acute inflammation is a critical interaction between neutrophils and the vascular endothelium (heterotypic cell-cell adhesion), we determined whether NSAIDs diminish the adherence of neutrophils to the endothelium. At antiinflammatory concentrations (0.5–5 mM) sodium salicylate, an NSAID that does not inhibit prostaglandin synthesis, inhibited stimulated but not unstimulated neutrophil adherence to endothelial cells (IC₅₀ < 1 mM,P < 0.00001). Salicylates have previously been shown to inhibit oxidative phosphorylation and, predictably, sodium salicylate inhibited oxidative phosphorylation, as evidenced by depletion of ATP stores (875±75 pmol/10⁶ PMN, [2.92±0.25 mM]) in stimulated (FMLP, 0.1M) but not resting neutrophils treated with antiinflammatory doses of sodium salicylate (EC₅₀=1 mM,P < 0.00001). Indomethacin and piroxicam (10 and 30M) only minimally decreased ATP concentrations in stimulated and resting neutrophils. ATP is metabolized to adenosine, and we have previously demonstrated that both endogenously released (180–200 nM) and exogenous adenosine (IC₅₀=250 nM) inhibit stimulated neutrophil adherence to endothelial cells. To determine whether the increased metabolism of ATP and the resultant increase in adenosine release were responsible for inhibition of neutrophil adhesion to endothelium, we determined whether addition of adenosine deaminase (ADA, 0.125 IU/ml), an enzyme that converts extracellular adenosine to its inactive metabolite, inosine, affected inhibition of neutrophil adhesion to endothelium by stimulated neutrophils. ADA significantly reversed inhibition of neutrophil adherence to endothelium by sodium salicylate (0.5–5 mM,P < 0.00001). This suggests that sodium salicylate inhibits neutrophil adherence by increasing adenosine release. Whereas indomethacin and piroxicam (10–50M) also inhibited stimulated neutrophil adherence to endothelial cells, ADA did not affect their inhibition of adherence. These studies demonstrate a heretofore unexpected antiinflammatory mechanism for salicylates: salicylates increase ATP hydrolysis and thereby enhance release of adenosine. Moreover, these data are consistent with the hypothesis that NSAIDs differ from one another with respect to their mechanisms of action.     

Zinc uptake by proximal cells isolated from rabbit kidney: Effects of cysteine and histidine

The aim of this study was to characterize the mechanisms of zinc transport in proximal cells isolated from rabbit kidney cortex. Uptakes of ⁶⁵Zn were assessed under initial rate conditions, after 0.5 min of incubation. The kinetic parameters obtained at 20°C were a K _m of 15.0±1.5 M, a J _max of 208.0±8.4 pmol min^–1 (mg protein)^–1, and an unsaturable constant of 0.259±0.104 (n=8). Cadmium competitively inhibited the zinc uptake, with a K _i value of 13.0±2.8 M, while zinc competitively inhibited ¹⁰⁹Cd uptake by isolated cells. Cysteine and histidine stimulated zinc transport at an amino acidzinc molar ratio ranging from 11 to 81. This stimulation was not observed in the absence of a sodium gradient. At a molar ratio greater than 161 (i.e., 400 M cysteine or histidine and 25 M Zn), there was evidence of inhibition. These data suggest that zinc enters renal proximal cells (a) as a free ion via a saturable carrier-mediated process or an unsaturable pathway and (b) complexed with cysteine or histidine, by means of a sodium/amino acid cotransport mechanism.     

Characterization of the ATP-inhibited K+ current in canine coronary smooth muscle cells

Intracellular adenosine triphosphate (ATP)-inhibited K⁺ currents (I _{K, ATP} ) in canine coronary artery smooth muscle cells were characterized in the wholecell configuration using the suction pipette method. Cells dialysed internally with solutions containing 5 mM ATP (ATP_i) showed little detectable whole-cell current at potentials more negative than –30 mV. However, cells dialysed with ATP_i-free solutions developed a time- and voltage-independent current which reached a maximum of 132±25 pA at –40 mV about 10 min following patch rupture. After run-up, the current showed little run-down. Concentration-dependent inhibition by ATP_i yielded an inhibition constant (K _i of 350 M and a Hill coefficient of 2.3. In ATP_i-free solutions, the large current at –40 mV was reduced by glibenclamide with aK _i of 20 nM and a Hill coefficient of 0.95. Conversely, in 1 mM ATP_i solutions, the small current at –40 mV was increased by P-1075 from 8±2 pA to 143±33 pA, with a dissociation constant (K _d) of 0.16 M and a Hill coefficient of 1.7. The effect of P-1075 was antagonized by glibenclamide. Maximal current density elicited by either ATP_i depletion or external application of the channel opener P-1075 was similar with slope conductances of 81±10 pS/pF and 76±13 pS/pF respectively in the potential range of –90 to –40 mV. External Ca²⁺ had no effect on this current. Finally, in 1 mM ATP_i, 20 and 50 M adenosine increased the current slope conductance by 36±15% and 73±10% respectively between –90 to –40 mV. TheI _{K, ATP} although very small in these cells, was extremely effective in causing membrane potential hyperpolarization.     

Carbohydrate and fat metabolism in contracting canine skeletal muscle

Summary Isolated canine gracilis muscles were perfused in situ with a free flow (systemic blood flow; FF) or a constant flow (blood from a reservoir; CF). The nervous supply was stimulated electrically for 60 min. A-V-concentration differences for glucose, pyruvate, lactate, glycerol, FFA and O₂ were obtained as well as the concentrations of ATP, CP, glycogen and lactate in the muscle.Resting O₂ uptake ranged from 4 to 11 moles×100 g^–1×min^–1 (FF; CF). A 30- and 5-fold increase in O₂ uptake occurred during stimulation in the FF and CF-experiments, respectively. The release of lactate was, however, the same (20–40 moles×100 g×min^–1) although the muscle lactate concentration was much higher in the CF experiments. In the CF experiment stimulation did not significantly increase glucose uptake which ranged from 0.3 to 3.3 moles×100 g^–1 ×min^–1 at rest. Conversely, stimulation resulted in a 6-fold increase in glucose uptake in the FF experiments. No definite tendency for a FFA uptake or a glycerol release was found in either experiment (FF, CF). Glycogen depletion during the stimulation period amounted to 20–30 moles×g^–1. Thus the glucose uptake could account for only 12% of the carbohydrate utilized during the stimulation period.