J. Wemer

Delayed Antibiotic-Induced Lysis of Escherichia coli in vitro is Correlated with Enhancement of LPS Release

A kinetic turbidimetric Limulus amebocyte lysate (LAL) assay was used to study the effects of gentamicin, amoxycillin and ciprofloxacin (16 x MIC) upon release of lipopolysaccharide at different stages of a growing Escherichia coli 055:B5:H culture in vitro. In this model a linear correlation was present between the logarithms of colony counts and free LAL activities. Untreated E. coli grew from log values of 4.9 +/- 0.15 (low inoculum) and 6.8 +/- 0.08 cfu/ml (high inoculum) at t = 0 to 8.9 +/- 0.05 and 9.1 +/- 0.13 cfu/ml at t = 6 h, respectively. The log values of basal free LAL activities at low and high inoculum sizes were 1.9 +/- 0.07 and 3.3 +/- 0.14 endotoxin units/ml, increasing 2100- and 69-fold, respectively during a 6-h growth. Amoxycillin-induced lysis was not significantly associated with an increase in free LAL activity. Efficacy of bacterial killing by gentamicin was high, but free LAL activity increased only 3.2- and 7.7-fold at the low and high inoculum experiments, respectively. Ciprofloxacin induced cell filamentation during the experiments. At low and high inoculum conditions exposure to ciprofloxacin induced a 43- and 68-fold increase in free LAL activities, respectively. Our data indicate that (a) LPS is released as long as E. coli remain structurally intact; (b) LPS release is enhanced when bacterial biomass increases; and (c) are taken as evidence against the concept of lysis-correlated LPS release.

The mechanism of fluoride-induced hypocalcaemia

1 Fluoride intoxication leads to sudden cardiac death which has been assumed to result from the accompanying severe hypocalcaemia. The aim of this study has been to investigate the suggestion that fluorapatite formation rather than CaF2 precipitation is responsible for this low calcium. 2 Measurements of free Ca2+ and F- ion concentrations in HEPES buffered solutions containing F-, Ca2+, and phosphate ions at different concentrations in the absence and presence of hydroxyapatite showed that the presence of hydroxyapatite enhanced the decrease of Ca2+ and F- concentration. 3 The ratio of Ca2+:F- clearance was 5:1 which is consistent with formation of fluorapatite. These results support the hypothesis that hydroxyapatite acts as a nucleation catalyst for fluorapatite formation and this process is responsible for the hypocalcaemia induced by fluoride intoxication. 4 The proposed mechanism explains also the metabolic acidosis which is frequently seen in cases of fluoride intoxication.

Comparison of the antiatherogenic effects of isradipine and ramipril in cholesterol-fed rabbits. I: Effect on progression of atherosclerosis and endothelial dysfunction

This study was designed to compare the effects of a calcium antagonist (isradipine) and a converting enzyme inhibitor (ramipril) on progression and regression of atherosclerosis in hypercholesterolemic rabbits. Sixty rabbits in three groups were fed a 0.3% cholesterol diet for 4 weeks. After this induction period, group II received the 0.3% cholesterol diet, group III received cholesterol diet with isradipine (0.33 mg/kg/day), and group IV received cholesterol with ramipril (0.33 mg/kg/day) for 12 more weeks. A group of 20 rabbits received a standard diet throughout the study (group I). After 16 weeks, 10 rabbits were randomly chosen from each group and used in the progression study. The other rabbits were placed on a standard diet and remained on their respective drug regimen for 12 more weeks. In the progression phase of the study, ramipril significantly attenuated the percentage of aortic lesions in group IV (35 +/- 6%) as compared with group II (56 +/- 6%, p < 0.05), whereas isradipine had no effect. Acetylcholine (ACh)-induced maximum endothelium-dependent relaxations (EDR) of aortic rings were significantly reduced by the atherogenic diet to 37 +/- 4 versus 77 +/- 2% in group I (p < 0.05). Treatment with ramipril significantly improved maximum EDR to 53 +/- 3% (p < 0.05 vs. group II). Isradipine had no significant effect on impaired EDR. Aortic rings with endothelium from group II developed supersensitivity to sodium nitroprusside (SNP) and had significantly reduced basal cyclic GMP levels as compared with those of group I. Both drugs prevented development of supersensitivity to SNP and blunted the cholesterol-induced reduction in basal cyclic GMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the antiatherogenic effects of isradipine and ramipril in cholesterol-fed rabbits:II. Effect on regression of atherosclerosis and restoration of endothelial function

We report the effects of isradipine and ramipril on regression of diet-induced atherosclerosis in rabbits. Regression of diet-induced atherosclerosis was not significantly affected by ramipril, but isradipine significantly retarded regression. Thirty rabbits in three groups were fed a 0.3% cholesterol diet for 4 weeks. After this induction period, group IIr received the 0.3% cholesterol diet, group IIIr received the 0.3% cholesterol diet with isradipine (0.33 mg/kg/day), and group IVr received the 0.3% cholesterol diet with ramipril (0.33 mg/kg/day) for 12 more weeks. The rabbits then received a standard diet and remained on their respective drug regimen for 12 more weeks. Group Ir (10 rabbits) received a standard diet for 28 weeks. Acetylcholine (ACh)-induced maximal endothelium-dependent relaxations (EDR) of aortic rings were significantly less in group IIr (22.8 +/- 3.2%) than in group Ir (66.4 +/- 4.0%; p < 0.05). Ramipril and isradipine did not improve EDR as compared with group IIr. Regression of atherosclerosis was accompanied by an improved endothelium-dependent releasing factor (EDRF) release from the endothelium, but ramipril and isradipine did not promote this process. In addition, regression was associated with increasing sensitivity of vascular smooth muscle to EDRF that was significantly retarded by isradipine but not ramipril. Basal cyclic GMP levels were significantly reduced in aortic rings from group IIr as compared with group Ir. Ramipril, but not isradipine, restored basal cyclic GMP levels to control values. Both isradipine and ramipril protect against endothelial degeneration in hypercholesterolemic rabbits. However, isradipine but not ramipril inhibits regression of diet-induced atherosclerosis in rabbits.

Funcdonal characterization of the muscarinic receptor in rat lungs

The effects of various muscarinic antagonists on antigen- and acetylcholine-induced bronchoconstriction were studied. In isolated and ventilated lungs of naive rats, the pA2 values with respect to acetylcholine-induced bronchoconstriction were 9.01 (atropine), 8.39 (ipratropium bromide), 7.39 (pirenzepine), 5.94 (AF-DX 116, a M2-selective muscarinic antagonist), 6.91 (UH-AH 37, a novel muscarinic antagonist) and 9.37 (4-DAMP: 4-diphenylacetoxy-N-methylpiperidine methobromide). Except for ipratropium bromide, the slopes of the Schild plots were not significantly different from unity. None of the drugs were potent or effective in inhibiting bronchoconstriction or histamine release evoked by antigen challenge in actively sensitized rats. However, in vivo, in anesthetized spontaneously breathing rats, vagotomy and atropine (1 mg/kg) did reduce antigen-induced bronchoconstriction. It is concluded that functional muscarinic receptors in isolated rat lungs are probably of the M3 receptor subtype. With respect to antigen-induced bronchoconstriction and mediator release in a denervated model such as the isolated lung, they are of little, if any, importance. In vivo, vagotomy and atropine reduced antigen-induced bronchoconstriction, probably by blockade of a vagal reflex which is thought to play a role in antigen-evoked bronchoconstriction.

Experimental study of the detrimental effect of dopamine/glucagon combination in d,l- propranolol intoxication

1 Respiratory and cardiovascular failure are the princi ple toxic effects of β-blocker overdose. Respiratory arrest is the primary cause of death in β-blocker intoxicated rats. 2 The effect of glucagon, dopamine and the combination of glucagon/dopamine on respiratory and cardiovas cular function and survival time in β-blocker overdose was investigated in a model of acute d,l-propranolol (resp. 30 and 15 mg kg-1 h-1 in rat and rabbit) intoxication in spontaneously breathing rats and artifically ventilated rats and rabbits. 3 Glucagon (initial dose of 100 μg kg-1 (bolus), followed by 1 μg kg -1 min-1), dopamine (25 μg kg-1 min-1 ) or the combination of glucagon/dopamine did not im prove survival time (ST) in d,l-propranolol intoxicated spontaneously breathing rats and artificially venti lated rats and rabbits, although some haemodynamic variables i.e. heart rate (HR), mean arterial blood pressure (MAP), left ventricular pressure (LVPmax) and the differentiated left ventricular pressure (LVdp/ dtmax) temporarily improved. 4 Survival time was considerably reduced in d,l- propranolol intoxicated spontaneously breathing and artifically ventilated rats treated with a combination of glucagon /dopamine, which induced a decrease in PaO2 and pH and an increase in PaCO2 partly due to ventilation/perfusion mismatch. 5 The combination of glucagon/dopamine should be used carefully in the treatment of β-blocker overdose in man.

Reduced survival after isoprenaline/ dopamine in d,l-propranolol intoxicated rats:

1 Respiratory and cardiovascular failure are principle toxic effects of β-blocker overdose. Respiratory arrest is the primary cause of death in β-blocker intoxicated rats. 2 The effect of β-adrenoceptor agonists on respiratory and cardiovascular failure in β-blocker overdose was investigated in a model of acute d,l-propranolol (30 mg kg -1 h-1) intoxication in spontaneously breathing rats. 3 Neither the aselective, hydrophilic β-agonist isoprena line (10, 25, 50 μg kg-1 min-1), nor the β 1-selective, lipophilic β-agonist flerobuterol (1,3,10 μ g kg-1 min-1) and the β2-selective, lipophilic β-agonist clenbuterol (10, 25, 50 μg kg-1 min-1) had any beneficial effect on cardiovascular and respiratory variables or survival time in d,l-propranolol intoxicated spontaneously breathing rats. 4 Isoprenaline (10 μg kg-1 min-1) had no favourable effect on haemodynamic and respiratory variables in artificially ventilated d,l -propranolol intoxicated rats either. 5 Addition of dopamine to isoprenaline resulted in a significant reduction of survival time, primarily caused by a decrease in mean arterial blood pressure, in artificially ventilated d,l-propranolol intoxicated rats. Addition of glucagon to isoprenaline did not affect survival time. 6 Artificial ventilation is the most important supportive measure in d,l-propranolol intoxication in the rat.

Effects of hypercholesterolemia on the contractions to angiotensin II in the isolated aorta and iliac artery of the rabbit: role of arachidonic acid metabolites.

The aim of this study was to investigate the effect of hypercholesterolemia on the angiotensin II-induced contractions in the isolated aorta and iliac artery of the rabbit, with respect to the role of arachidonate metabolites. Furthermore, the effect of the angiotensin-converting enzyme inhibitor ramipril was studied on the responses to angiotensin II in the cholesterol-fed rabbit. After 12 weeks of cholesterol diet (0.3%), endothelium-dependent relaxations to acetylcholine were significantly fewer compared with control (30.2 +/- 5.9% vs. 73.0 +/- 1.7%) in the aorta but not in the iliac artery of the rabbit. The angiotensin II- and methoxamine-induced contractions were also significantly lower compared with control in the aorta (101.4 +/- 6.7% vs. 60.9 +/- 4.2% and 160.2 +/- 5.7% vs. 135.8 +/- 8.0%, respectively) but not in the iliac artery. The lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) selectively attenuated the angiotensin II-induced contractions in rabbit aortic rings from the control group only in the presence of the endothelium, whereas it had no effect on the responses to angiotensin II in the cholesterol group (with or without endothelium). In the iliac artery, NDGA inhibited the responses to angiotensin II in both the control and cholesterol groups. Treatment with ramipril (0.33 mg/kg/day) significantly improved the maximal angiotensin II-induced contraction in the aorta of rabbits fed a cholesterol diet for 16 weeks to 61.0 +/- 7.3% (vs. 32.7 +/- 9.0% in the cholesterol group). We conclude that hypercholesterolemia leads to a reduction of angiotensin II-induced contractions in the aorta and not in the iliac artery of the rabbit. This reduction might be related to loss of endothelium-dependent lipoxygenase products and is partially reversed by ramipril.

Septic shock: no correlation between plasma levels of nitric oxide metabolites and hypotension or lethality

In the Wistar rat (Riv:TOX strain), Escherichia coli-derived lipopolysaccharide, up to 100 mg/kg, did not affect blood pressure. However, 6 h after administration of live E. coli or Staphylococcus aureus (a microorganism without lipopolysaccharide), both dosed at 12 x 10(9) colony forming units/kg, mean arterial blood pressure significantly decreased to 64% and 48% compared to control, respectively. In contrast to lipopolysaccharide, bacteria produced a dose-dependent lethality within 24 h. Live S. aureus increased plasma levels of nitric oxide metabolites (NOx) only four-fold, while both lipopolysaccharide and live E. coli approximately 20-fold. In conclusion, we demonstrated a lack of correlation between plasma NOx levels and hypotension or lethality.

Cardiovascular responses to the stereoisomers of dobutamine in isolated rat hearts 48 hours after acute myocardial infarction.

Effects of acute myocardial infarction (48 h) oil cardiovascular responses to and dobutamme were studied in isolated perfused rat hearts. The effects of the racemate and the isomers on ventrienlat pressure were measured simultancolusly in infarctcd left ventricle and noninfarcted right ventricle. Administration of and dobutamine resulted in dose-dependent increase, in inotropic parameters and coronary flow (CF) in both control and in infareted hearts. As compared with the (-) isomer, the racemate and the (-)-isomer in both control and infarcted hearts were 1.5 and 15 times weaker with respect to inotropic parameter,. for and -dobutamine, there was no significant difference in pl)2 values in any of the motropic measurements between the infarcted group and the corresponding control group. The maximal obtainable response, were significantly lower in the infarcted group, as compared with their corresponding control group. In control hearts. effects of isoproterenol and methoxamine as compared with (-)-dobutamine were 2 log units more and 2 log units less in potency, respectively. The inotropic effects of (-)-dobutamine but not those of methoxamine were completely antagonized by propranolol (0.3 μM). Although the results provide evidence for the existence of myocardial α1-adrenoceptors. all forms of dohutamine everted positive isotropic effects through activation of β-adrenoceptors both in control and in infarcted rat hearts.