Pentagalloylglucose, an antisecretory component of Paeoniae radix, inhibits gastric H+, K(+)-ATPase

We purified a compound with strong inhibitory effect on H+, K(+)-ATPase from Paeoniae radix, which has been used in Japan for the treatment of gastritis and peptic ulcers. The compound was identified as 1,2,3,4,6,-penta-o-galloyl-beta-D-glucose by proton nuclear magnetic resonance, carbon-13 nuclear magnetic resonance, and fast atomic bombardment mass spectrometry. The IC50 of the compound for H+, K(+)-ATPase was 166 nmol/l. Kinetic analyses indicated that the inhibition of the enzyme by pentagalloylglucose was noncompetitive with respect to K+. Pentagalloylglucose had relatively weak inhibitory effects for Mg(+)-ATPase (IC50: > 10 mumol/l) and Na+, K(+)-ATPase (IC50: 2.7 mumol/l). Pentagalloylglucose also inhibited the accumulation of [14C]aminopyrine in parietal cells that had been isolated from guinea pig stomach and stimulated by 10 mumol/l histamine (IC50: 7.8 mumol/l) and 1 mmol/l dbc-AMP (IC50: 10 mumol/l). These results suggest that pentagalloylglucose is a potent inhibitor of H+, K(+)-ATPase and may be responsible for inhibition of acid secretion by Paeoniae radix.     

Mediators of the direct effects of amino acids on the rat kidney.

1. The response of the isolated rat kidney to a mixed amino acid solution was examined in the presence of three renal autacoid inhibitors, indomethacin (a cyclo-oxygenase inhibitor), sulpiride (a dopamine antagonist) and L-nitroarginine methyl ester (an inhibitor of nitric oxide synthesis). 2. Increasing the concentration of the mixed amino acid solution perfusing the kidney from 2 to 8 mmol/l (n = 6) produced a sustained increase in renal perfusate flow (P less than 0.01) and reversed the time-dependent fall in [14C]inulin clearance (P less than 0.01) demonstrated in kidneys perfused with 2 mmol/l mixed amino acids alone. A significant increase in the fractional sodium reabsorption and decrease in the fractional albumin excretion was also observed. 3. Indomethacin (10(-4) mol/l, n = 6) produced partial (50%) inhibition of the effect of mixed amino acids on [14C]inulin clearance, but did not influence their ability to increase renal perfusate flow. 4. Sulpiride (0.7 mumol min-1 kg-1, n = 6) produced partial inhibition of the effect of mixed amino acids on both [14C]inulin clearance and renal perfusate flow by 60% and 50%, respectively. Sulpiride also entirely inhibited the reduction in fractional albumin excretion. 5. L-Nitroarginine methyl ester (10(-4) mol/l, n = 6) completely inhibited the effect of mixed amino acids on [14C]inulin clearance, but did not inhibit the increase in renal perfusate flow, even though the basal vascular resistance was markedly enhanced. L-Nitroarginine methyl ester also inhibited the increase in fractional sodium reabsorption produced by the mixed amino acids. 6. It is concluded that prostaglandins, dopamine and nitric oxide may all have a role to play in the direct effect of mixed amino acids on renal function. This does not, however, preclude further modification by additional stimuli generated in vivo.     

Evidence of an Eicosanoid Contribution to IL-1 Induction of IL-6 in Human Articular Chondrocytes

It has been demonstrated previously that interleukin-1 (IL-1) induces articular cartilage explants and chondrocytes in culture to produce elevated levels of inflammatory mediators such as interleukin-6 (IL-6) and prostaglandins. Previous studies have also demonstrated a relationship between IL-6 secretion and the ability of IL-1 to modulate proteoglycan synthesis by chondrocytes. In this study we have utilized an alginate culture system in an effort to investigate a role for eicosanoids in IL-1 induction of IL-6 expression in human articular chondrocytes. IL-1 treatment of chondrocytes cultured in alginate resulted in increased synthesis of IL-6 and prostaglandins, but not leukotrienes. Cyclo-oxygenase inhibitor, indomethacin (5 &mgr;g ml(minus sign1)), was able to inhibit prostaglandin synthesis to below basal levels with no significant effect on the levels of IL-6 released by chondrocytes in response to IL-1. When chondrocytes were treated with 5 &mgr;g ml(minus sign1) indomethacin and 10 &mgr;M of the general lipoxygenase inhibitor, nordihydroguiaretic acid (NDGA), an approximate 50% decrease in IL-1-induced IL-6 expression was observed. Alone, levels of NDGA specific for lipoxygenase inhibition (10 &mgr;M) did not affect IL-1-induced IL-6 expression, but higher levels of NDGA (50 &mgr;M) which inhibited both prostaglandin and leukotriene biosynthesis reduced IL-1-induced IL-6 expression to the same extent as that observed with 5 &mgr;g ml(minus sign1) indomethacin and 10 &mgr;M NDGA. This inhibition of IL-6 expression by NDGA and indomethacin was dose responsive and also reversible with the addition of exogenous prostaglandin E(2) (PGE(2)) or leukotriene B(4) (LTB(4)). Although IL-1-induced IL-6 expression was only affected when both prostaglandin and leukotriene biosynthesis were inhibited, elevated levels of PGE(2) but not leukotriene B(4), C(4), D(4), or E(4) were observed in the culture medium of IL-1-treated chondrocytes. These findings may indicate that cyclo-oxygenase products such as PGE(2) normally contribute to IL-1 induction of IL-6 expression in chondrocytes, and under conditions when cyclo-oxygenase is inhibited, lipoxygenase products alternatively contribute to this response.     

Pentoxifylline inhibits granulocyte and platelet function, including granulocyte priming by platelet activating factor

Pentoxifylline has been claimed to work a beneficial effect in arterial insufficiency by improving erythrocyte deformability and thus improving blood flow. A number of observations, including the drug concentrations required to work the red cell effect, suggested that this was not likely to be a complete explanation. We therefore examined the effect of pentoxifylline on several granulocyte and platelet functions. Pentoxifylline inhibited platelet aggregation in response to 4 mumol/L adenosine diphosphate; although statistically significant inhibition was seen at 1 mumol/L pentoxifylline, over 200 mumol/L was required for 50% inhibition. The adherence of unstimulated platelets to cultured endothelial cells was not strongly inhibited by pentoxifylline; however, the additional increment in adherence seen in the presence of thrombin was strongly inhibited (50% attenuative dose [AD50] = 18 mumol/L). Granulocyte aggregation in response to C5a was modestly inhibited (AD30 approximately equal to 8 mumol/L; AD50 greater than 1 mmol/L), and the adherence of unstimulated polymorphonuclear neutrophils (PMNs) to endothelium was uninhibited. The C5a-mediated augmentation of PMN adherence to endothelium was mildly inhibited (AD50 = 240 mumol/L). Inhibition of PMN chemotaxis to N-Formyl-methionyl-leucyl-phenylalanine (FMLP) or C5a (AD50 = 12 mumol/L) and inhibition of superoxide production in response to FMLP-cytochalasin B (AD50 = 24 mumol/L) were seen at more clinically credible concentrations. Perhaps most important, pentoxifylline blocked the ability of platelet activation factor to prime neutrophils for enhanced response to subsequent stimuli (AD50 approximately equal to 8 mumol/L; AD60 = 10 mumol/L when production was the indicator system); in vivo, this could broaden the drug's effect to include functions that it does not inhibit potently in a primary fashion. Although pentoxifylline is known to be a phosphodiesterase inhibitor, and we found it to elevate intracellular cyclic adenosine monophosphate in stimulated PMNs, we found it to be only marginally more potent than theophylline in this regard; therefore, the failure of theophylline to inhibit PMN priming suggests that this enzyme inhibition is not a complete explanation of the pharmacologic action of pentoxifylline. We suggest that the effects of pentoxifylline on platelet and granulocyte function are likely to contribute to the drug's clinical efficacy.     

Optimal conditions for measurement of Na+,K+-ATPase activity of human leucocytes

The Na+,K+-ATPase activity of human leucocytes was assayed by measuring the release of inorganic phosphate (Pi) from ATP. The maximum enzyme activity was achieved under the following conditions: concentration (mmol/l), Tris/HCl 50, Na 100, K 15, ATP 5, Mg 7, EDTA 1; pH 7.2 and temperature 37 degrees C, were optimal. Ouabain showed maximal inhibition at a concentration of 10-100 mumol/l. Ethanol, the solvent for ouabain, had a dose-related inhibitory effect. Heparin or citrate used as an anticoagulant gave similar results. Leucocyte samples could be stored at -20 degrees C for up to 6 days without loss of activity. Hypotonic lysis had advantages over sonication as the technique for cell disruption. The leucocyte Na+,K+-ATPase enzyme activity in healthy subjects was 186 mumol of Pi h-1g-1 of protein (median) with a range 136-243 mumol of Pi h-1g-1 of protein. The within-batch coefficient of variation was 6.4% and the between-batch precision was 9.6%.     

Simple and simultaneous determination of sulphapyridine and acetylsulphapyridine in human serum by column-switching high-performance liquid chromatography

OBJECTIVE: A high-performance liquid chromatography (HPLC) with an automated on-line column-switching system was used for the simultaneous determination of sulphapyridine and acetylsulphapyridine, two major active metabolites related to the adverse effects of sulphasalazine, in human serum. METHODS: Serum samples were directly injected into the HPLC, with the valve automatically switched on to remove serum proteins and other hydrophilic components remaining in the pre-column after elution of sulphapyridine and acetylsulphapyridine to the analytical column. RESULTS: Serum proteins did not interfere with the analysis of either compound. The recoveries of SLP and Ac-SLP from drug-free human serum were 93.03-99.18% and CV were 2.88-4.34%. The within-run reproducibility of assays was excellent with relative standard deviations (RSD) of 1.01-3.90% (SLP) and 0.77-5.56% (Ac-SLP). The limit of quantification of sulphapyridine and acetylsulphapyridine was 3.13 microg/mL and 0.50 microg/mL, respectively. The serum concentrations in a patient with ulcerative colitis, who took 1.0 g sulphasalazine twice daily, were 31.20 microg/mL for sulphapyridine and 14.64 microg/mL for acetylsulphapyridine at 7 h after ingestion. CONCLUSION: The present simple and reproducible assay was useful for the monitoring of serum sulphapyridine and acetylsulphapyridine.     

Purification and characterization of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human skeletal muscle.

The purification and characterization of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human skeletal muscle are described. The characteristics of leucyl aminopeptidase were as follows: optimum activity was at pH 9.5 in the presence of 5 mmol/l Mg2+ or 0.5 mmol Mn2+. No activation of enzyme activity was obtained following addition of other divalent cations or sulphhydryl reagents. Only the leucyl-AMC and methionyl-AMC derivatives were appreciably hydrolysed. The mol mass was estimated as 280 kDa. Approx. 50% inhibition of activity was obtained following addition of p-hydroxymercuriphenyl sulphonate (10 mumol/l), N-ethyl maleimide (2 mmol/l), o-phenanthroline (5 mmol/l), bacitracin (1 mmol/l), amastatin (1 microgram/ml) and bestatin (0.1 mumol/l); no inhibition of activity was obtained in the presence of phenylmethanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 microgram/ml) or pepstatin (100 microgram/ml). The following oligopeptides were hydrolysed by the enzyme: luliberin 7-10, proctolin and [Leu5]enkephalin; oligopeptides not appreciably hydrolysed included neurotensin, angiotensin-I, substance-P and bradykinin. Pyroglutamyl aminopeptidase had the following characteristics: optimum activity was at pH 8.5 in the presence of 1 mmol/l dithiothreitol (an absolute requirement for maintenance of enzyme activity). Maximum activity was obtained in the absence of divalent cations. Only the pyroglutamyl-AMC derivative was appreciably hydrolysed. The mol mass of this enzyme was estimated as 22 kDa. Approximately 50% inhibition of activity was obtained on addition of phenanthroline (4 mmol/l) and antipain (7 microgram/ml); no inhibition of activity was obtained following addition of phenyl methanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 microgram/ml) or pepstatin (100 microgram/ml). Only oligopeptides with a pyroglutamyl N-terminal residue (thyroliberin, neurotensin, and luliberin) were hydrolysed by the enzyme.     

Human mast cells recovered by bronchoalveolar lavage: their morphology, histamine release and the effects of sodium cromoglycate

Mast cells make up between 0.5 and 3% (mean 1.35%) of total cells recovered by bronchoalveolar lavage (BAL). The majority of these cells have the morphological characteristics of mucosal mast cells in that they fail to stain in the alcian blue-safranin reaction after fixation in formol-saline but stain well after fixation in Carnoy's solution. Cells staining with berberine sulphate were seen in only four of the 26 lavages. BAL cells released histamine in response to anti-human immunoglobulin E (IgE) in a dose-dependent manner that was optimal at a dilution of anti-IgE of 1:100. Maximum release was obtained by 2 min. Histamine release was completely inhibited by a combination of 2-deoxyglucose (5 mmol/l) and antimycin A (1 mumol/l). Disodium cromoglycate (DSCG) significantly inhibited this histamine release at 1 mmol/l (P less than 0.02), 100 mumol/l (P less than 0.002) and 10 mumol/l (P less than 0.003), with maximum inhibition of 50.1% at 10 mumol/l.     

Effect of D-propranolol on growth and motility of flagellate protozoa

Propranolol inhibits sperm motility and has been considered as a spermicide contraceptive. In view of the inhibitory effects of D-propranolol on sperm flagellar activity, we have investigated its effect on motility and growth of two human flagellate, protozoan parasites. D-propranolol had a dose-dependant inhibitory effect on motility of Giardia lamblia and Trichomonas vaginalis with ED50s of 0.38 and 0.66 mmol/l respectively, D-propranolol also inhibited growth of both parasites with ED50s of 0.18 and 0.23 mmol/l for Giardia and Trichomonas respectively. D-propranolol, unlike DL-propranolol is devoid of the unwanted effects of beta-blockade, and thus may be a useful antiprotozoal drug particularly after vaginal placement when it is concentrated in vaginal mucus.     

Effect of aspirin infusions on platelet function in humans

1. The inhibitory effects of aspirin on platelet function in vitro have been shown to be both time (over 3 h) and concentration (1-10 mumol/l) dependent. 2. To determine if these effects occurred in vivo, four volunteers received intravenous infusions on four occasions, to give constant plasma aspirin concentrations of 0, 1, 2 and 4 mumol/l over 3 h. Infusions were performed at intervals of at least 2 weeks. 3. Before and during the infusions, blood was taken for assay of aspirin concentrations, and measurements of platelet aggregation in response to collagen, adenosine 5'-pyrophosphate and arachidonate. Thromboxane generation after stimulated platelet aggregation and whole-blood coagulation was also measured. 4. At each aspirin concentration, both platelet aggregation and thromboxane generation in response to collagen and arachidonate were inhibited progressively over the 3 h infusion period. Greatest inhibition was seen during the 4 mumol/l infusion, which produced maximal or near-maximal inhibition by the third hour. 5. Thromboxane generated during whole-blood coagulation was similarly inhibited in both a time- and concentration-dependent manner throughout all aspirin infusions. 6. The progressive nature of the inhibition of platelet function with these low aspirin concentrations may be due to either slow aspirin transport across the platelet membrane or delayed interaction with cyclo-oxygenase.     

Docosahexaenoic acid--induced vasorelaxation in hypertensive rats: mechanisms of action

The authors investigated the vasorelaxant properties of the omega-3 fatty acid, docosahexaenoic (DHA, 22:6n-3), and the possible involvement of endothelium-derived nitric oxide, prostanoids, opening of K+ channels, and/or modulation of calcium-mediated events. Isolated aorta from male spontaneously hypertensive rats (SHR) (age 16-17 weeks) were used to measure isometric tension. DHA-induced (1-100 mumol/l) relaxation was examined following contraction to norepinephrine (NE) (10(-6) mol/l) or high-K+ (80 mmol/l) solution in the presence and absence of various inhibitors and calcium-containing solution. DHA acid induced a significant vasorelaxant effect in both NE and high-K(+)-induced contracted SHR aortic rings, although DHA relaxations were greater in high-K(+)-induced contracted rings. In the absence of extracellular calcium, DHA (5-30 mumol/l) inhibited the initial phasic and sustained components of NE-induced contraction under different conditions. Inhibition of nitric oxide synthesis by N omega-nitro-L-arginine methyl ester hydrochloride (100 mumol/l) had no effect on DHA relaxations; however, indomethacin or nifedipine caused significant inhibition at > or = 30 mumol/l DHA. The K+ channel blocker, glibenclamide, but not tetraethyl-ammonium, also had an inhibitory effect on DHA-induced relaxation. These results indicate that DHA's vasorelaxant actions in SHR aorta are independent of endothelium-derived nitric oxide; however, at DHA concentrations > or = 30 mumol/l, vasodilatory prostanoids that activate ATP-sensitive K+ channels (KATP) may be involved. At lower concentrations, DHA-induced relaxation appears to be attributed to modulation of intracellular Ca2+ release and L-type Ca2+ channels in vascular smooth muscle cells. The vasorelaxant properties of DHA may contribute, in part, to the blood pressure-lowering effect of dietary fish oil in this hypertensive model.     

Sulphasalazine-induced leucopenia in a patient with renal dysfunction

BACKGROUND: Sulphasalazine is used for the long-term maintenance therapy of ulcerative colitis to prevent the relapse of symptoms. However, its clinical use is often restricted by its serious adverse effects. OBJECTIVE: Leucopenia occurred in a patient with severe renal dysfunction after administration of sulphasalazine. The present study was designed to examine whether the adverse event was associated with a disability in the metabolism of sulphasalazine. SUBJECT: A 29-year-old male patient with ulcerative colitis, who underwent haemodialysis thrice a week because of severe renal dysfunction. The chief complaint was diarrhoea. METHODS: Serum concentrations of three major metabolites of sulphasalazine, (5-aminosalicylic acid, sulphapyridine and N-acetyl-sulphapyridine), were measured. The polymorphism of N-acetyltransferase 2, an enzyme that metabolizes sulphapyridine, was also determined by polymerase chain reaction. RESULTS: The trough levels of 5-aminosalicylic acid, sulphapyridine and N-acetyl-sulphapyridine were 0.77-1.45 microg/mL, 31.20-39.25 microg/mL and 14.19-15.03 microg/mL, respectively. The gene diagnosis of N-acetyltransferase 2 suggested that the type was classified as NAT2*6A/*7B, indicating that the patient was a slow acetylator. CONCLUSION: The patient was a slow acetylator, which might lead to a rise in the serum sulphapyridine concentration. Moreover, the decrease in protein binding of sulphasalazine as a result of severe renal dysfunction might have potentiated the effect because of the extremely high protein binding of this compound. Thus, it is most likely that these two factors contributed to the sulphasalazine-induced leucopenia.     

Differential effects of sulindac and indomethacin on blood pressure in treated essential hypertensive subjects

Attenuation of the effectiveness of antihypertensive therapy by non-steroidal anti-inflammatory (NSAI) drugs has been attributed to inhibition of systemic or renal vasodilator prostaglandin synthesis, or a combination of both. Indomethacin is a NSAI drug with both renal and extrarenal cyclo-oxygenase inhibition properties. Sulindac is a relatively selective cyclo-oxygenase inhibitor said not to affect urinary prostaglandin excretion. This study examines the relative effect on blood pressure of 4 weeks' treatment, with indomethacin 25 mg three times daily and sulindac 200 mg twice daily, in a randomized placebo controlled trial in 26 hypertensive subjects. In nine patients treated with indomethacin, supine blood pressure rose 11 mmHg systolic and 4 mmHg diastolic by the end of the first week, whereas nine subjects treated with sulindac showed a fall in blood pressure similar to the trend seen in placebo-treated subjects. Indomethacin treatment inhibited renal cyclo-oxygenase with a 78% reduction in urinary prostaglandin E2 excretion and 89% suppression of plasma renin activity. Neither measurement was affected by sulindac. Extrarenal cyclo-oxygenase activity was inhibited by both indomethacin and sulindac with serum thromboxane B2 decreasing by 96% and 69% respectively. The results suggest that the pressor effect of NSAI drugs is predominantly related to renal cyclo-oxygenase inhibition. the lack of effect of sulindac on blood pressure may make it a safer therapeutic option if NSAI drug therapy is necessary in the hypertensive patient.     

A new screening system for nonsteroidal anti-inflammatory drugs based upon inhibition of chemiluminescence produced from human cells (granulocytes).

A new screening system for nonsteroidal anti-inflammatory drugs that involves use of human phagocytic cells has been developed in which chemiluminescence measurement is used. Luminol-dependent chemiluminescence is measured after the addition of opsonized (coated with antibodies and complement) zymosan particles to human granulocytic leukocytes in the presence or absence of drugs. Of all the compounds tested, indomethacin was the most potent in blocking chemiluminescence, with measurable inhibitory activity at 5 mumol/L. The order of inhibitory potency at 0.1 mmol/L and in the presence of Ca2+ and Mg2+ was indomethacin greater than sodium salicylate greater than fenoprofen Ca greater than tolmetin greater than naproxen greater than ibuprofen. It is likely that the active compound itself must be added to the system because aspirin did not inhibit chemiluminescence, whereas its metabolite, sodium salicylate, was markedly inhibitory. Dexamethasone and methylprednisolone also did not inhibit chemiluminescence. The drugs that inhibit chemiluminescence are also known inhibitors of prostaglandin synthase (cyclooxygenase portion).     

Interaction of amino acids with glycl-L-leucine hydrolysis and transport in monkey small intestine.

1. There are two saturable transport processes in the monkey small intestine for glycyl-L-leucine, one with Vmax. 1 mumol min-1 g-1 wet weight of tissue and an affinity constant (kt) of 5 mmol/l, and the other with Vmax 3.9 mumol min-1 g-1 wet weight of tissue and kt 33 mmol/l. 2. Glycyl-L-leucine uptake is inhibited by a wide variety of amino acids, although to a variable extent. The inhibition was shown to be competitive with leucine used as the representative amino acid. Phenylalanine, methionine, alanine and leucine are the most potent in their inhibitory action. 3. The effect of various amino acids on the hydrolysis of glycyl-L-leucine by particulate and cytosol fractions of monkey small intestine was studied. All the amino acids, except glycine, proline, alanine and glutamic acid, inhibit both the particulate and cytosol glycyl-L-leucine hydrolase activities. In general, the cytosol enzyme is more susceptible to amino acid inhibition than the particulate enzyme. 4. There is no correlation between the effects of amino acids on glycyl-L-leucine uptake and hydrolysis of glycyl-L-leucine by either particulate or cytosol fraction.     

Urinary inhibitor of the ammoniagenic response to acute acidosis is a prostaglandin

Both acute respiratory acidosis and acute metabolic acidosis stimulate NH3 production by the isolated perfused rat kidney. This stimulatory effect is abolished if the urine is drained back into the recirculating perfusate rather than collected. To determine whether the urinary inhibitor is a cyclooxygenase product, studies were carried out using prostaglandin synthetase inhibitors. Kidneys perfused with 0.5 mmol/L glutamine and urine reinfusion were subjected to acute respiratory acidosis (30% CO2, pH 6.8). With either indomethacin (20 mumol/L) or meclofenamate (20 mumol/L) in the perfusate, NH3 production increased significantly in response to acute respiratory acidosis despite urine reinfusion. The increment in NH3 production was comparable to that in studies with urine collection, indicating that a cyclooxygenase product can account completely for the urinary inhibitor. To further characterize the urinary prostaglandin inhibitor, studies were performed with both the isolated perfused kidney and renal cortical tubules. Prostaglandin E2 (PGE2) did not exhibit an inhibitory effect on NH3 production with either experimental model. Prostaglandin F2 alpha at low doses inhibited NH3 production in response to acute acidosis by the isolated kidney, but an effect was not apparent with higher concentrations. PGF2 alpha inhibited the stimulatory effect of a low pH (7.1) on NH3 production by isolated tubules, and had no effect on ammoniagenesis at pH 7.4. Thus a prostaglandin, which is not PGE2 and may be PGF2 alpha, appears to be the previously unidentified urinary inhibitor of the ammoniagenic response to acute acidosis found with the isolated perfused kidney.     

Inhibition of 1α-hydroxy-vitamin D3 stimulated bone resorption in tissue culture by the calcium antagonist verapamil

The effect of the calcium antagonist verapamil on 1 alpha-hydroxy-vitamin D3 (1 alpha (OH)D3) stimulated bone resorption in tissue culture has been investigated. It was found that verapamil in concentrations above 20 mumol/l reduced 1 alpha (OH)D3-stimulated mineral mobilization, as measured by release of in vivo incorporated 45Ca from mouse calvarial bones. The inhibition of verapamil could be seen already 3 h after exposure to the drug. The increased degradation by 1 alpha (OH)D3 of the organic matrix in the calvaria, as assessed by the release of in vivo 3H-proline labelled collagen, was also reduced by verapamil. The inhibitory effect of the drug on 45Ca release was reversible after withdrawal. 1 alpha (OH)D3 increased the release of stable calcium and beta-glucuronidase, and these effects could be blocked by verapamil. Increasing medium calcium concentration from 1.8 to 5 mmol/l slightly reduced the inhibitory capacity of 50 mumol/l verapamil on 1 alpha (OH)D3-stimulated 45Ca release. These data indicate that stimulation of osteoclasts by hydroxylated metabolites of vitamin D to resorb bone and secrete lysosomal enzyme is dependent on an increased availability of free intracellular calcium.     

Inhibition of platelet aggregation and thromboxane production by low concentrations of aspirin in vitro

1. The aspirin concentrations previously reported to inhibit platelet aggregation in vitro (40-500 mumol/l) are much greater than those required in vivo in man (5 mumol/l). 2. Human platelet-rich plasma was incubated with buffer or various aspirin concentrations at 37 degrees C for up to 4.5 h. Platelet aggregation and thromboxane generation were measured in response to collagen (0.4-6.3 micrograms/ml) and adenosine 5'-pyrophosphate (0.5-4 mumol/l). 3. The concentration of aspirin needed to inhibit platelet aggregation in response to a critical concentration of aggregating agent (lowest concentration to cause greater than 50% aggregation) was lower than that required for higher concentrations of aggregating agent. 4. With more prolonged incubation times with aspirin, lower concentrations of aspirin inhibited platelet aggregation. 5. Inhibition of platelet aggregation and thromboxane formation by 10 mumol/l aspirin was maximal by 90 min. There was progressive inhibition by 3 mumol/l aspirin during incubation for 270 min. By the end of this time there was also significant inhibition by 1 mumol/l aspirin. 6. The apparent discrepancy between inhibitory aspirin concentrations in vivo and those observed in vitro in previous studies appears to have been resolved by extending the incubation time of platelets with low aspirin concentrations, thus mimicking the conditions in vivo.     

Activation of Ca(2+)-dependent K(+) current by nordihydroguaiaretic acid in porcine coronary arterial smooth muscle cells.

The effects of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor and an antioxidant, on membrane currents were examined in single smooth muscle cells isolated from porcine coronary artery. Spontaneous transient outward currents (STOCs) recorded at -30 mV were markedly enhanced by NDGA (>/=10 microM). Pretreatment with caffeine and ryanodine abolished STOCs and reduced NDGA-induced increase in outward current at -30 mV by approximately 60%. NDGA showed dual action on an outward current elicited by step depolarization from -60 to 0 mV: inhibition and enhancement at concentrations of 3 and >/=10 microM, respectively. In the presence of Cd(2+), the inhibition of outward current by NDGA disappeared and the enhancement remained. NDGA inhibited both the voltage-dependent Ca(2+) channel current (IC(50) = 2.5 microM) and the delayed rectifier K(+) current (IC(50) = 9.8 microM). The NDGA-induced enhancement of STOCs and outward currents on depolarization was abolished by 100 nM iberiotoxin but was not affected by glibenclamide or apamin. Under current clamp mode, 30 microM NDGA significantly hyperpolarized myocytes. The application of lipoxygenase inhibitors (caffeic acid and esculetin), a cyclooxygenase inhibitor (indomethacin), antioxidants (ascorbic acid and erythorbic acid), and structural-related compounds of NDGA (catechol and dopamine) did not enhance K(+) currents. These results indicate that the opening of the large conductance Ca(2+)-dependent K(+) channel by NDGA, which is independent of its lipoxygenase inhibition or antioxidant effect, results in membrane hyperpolarization.     

Kinetics of the human lymphocyte Na(+)-H+ exchanger

1. A human lymphocyte preparation, obtained by Percoll gradient centrifugation and free of contaminating monocytes and granulocytes, was used to study the kinetics of the Na(+)-H+ exchanger through activation by nigericin-induced acidic loading. The fluorescent probe biscarboxyethylcarboxyfluorescein (acetoxymethyl ester) was used to determine cell pH and buffering capacity and to measure Na(+)-H+ exchange activity as external Na(+)-dependent H+ efflux. 2. At a cell pH of 6.2, H+ efflux was stimulated by external Na+ with a Km of 30 mmol/l (SEM 6, n = 3) and a calculated Vmax. of 0.73 mmol s-1 l-1 of cell water (SEM 0.06, n = 6). External Na(+)-dependent H+ efflux was more than 98% inhibited and half-maximally inhibited by 200 mumol/l and 17 mumol/l amiloride, respectively. The external pH also inhibited Na(+)-H+ exchange, with a Ki of 93 nmol/l. 3. Na(+)-H+ exchange was sigmoidally activated by an internal pH lower than 7.0 with a Hill coefficient of 2.14 (SEM 0.15, n = 6) and a pK of 6.57 (SEM 0.03, n = 6). Cell buffering capacity was also measured as a function of cell pH and found to gradually increase from 14 to 26 mmol l-1 of cell water pH-1 when cell pH fell below 6.6. The maximal transport rate (cell pH 6.0-6.2) was 0.50 mmol (s l of cell water)-1 (SEM 0.08, n = 12) and ranged between 0.25 and 1.10.(ABSTRACT TRUNCATED AT 250 WORDS)