Interleukin-6 and tumor necrosis factor alpha levels after bisphosphonates treatment in vitro and in patients with malignancy.

Bisphosphonates are potent inhibitors of bone resorption and are widely used in the treatment of bone diseases. One of the side effects of administered aminobisphosphonates is transient fever and some biological changes that are suggestive of an acute phase response. Pamidronate [(3-amino-1-hydroxypropylidene).1, 1-bisphosphonate] and ibandronate [1-hydroxy-3-(methylpentylamino) propylidenebisphosphonate] incubated in heparinized whole blood at doses of 10(-4) and 10(-5) mol/L, induced the production of tumor necrosis factor alpha (TNFalpha). Moreover, pamidronate was found to slightly stimulate interleukin-6 IL-6 production. In contrast, clodronate (dichloromethylenebisphosphonate) did not increase IL-6 or TNFalpha. To investigate these phenomena in vivo, acute phase reaction was assessed in patients with malignant disease treated with 60 mg of pamidronate (n = 29), 1500 mg of clodronate (n = 8), or 0.5-2 mg of ibandronate (n = 6), all given intravenously. A significant decrease in lymphocyte and leukocyte count was observed in the pamidronate group. In the same group, seven patients (24%) showed a transient increase of body temperature above 37 degrees C with an increase > or = 0.5 degrees C at 24 h. These changes were not found in the patients treated with clodronate or ibandronate. Plasma IL-6 and TNFalpha levels increased significantly after pamidronate treatment, whereas no change was seen after clodronate infusion. The peak of IL-6 level (53.7 +/- 14.1 [SEM] pg/mL) was observed at 24 h, and that of TNFalpha level (26.9 +/- 3.4 pg/mL) at 48 h after the beginning of pamidronate administration (values before treatment, respectively: 28.6 +/- 7.1 pg/mL, p < 0.006; and 13.1 +/- 1.5 pg/mL, p = 0.0001). The peak of C-reactive protein (CRP) level was found at 48 h (41.0 +/- 7.8 vs. 25.5 +/- 5.6 mg/L before treatment, p < 0.01) and CRP levels were strongly correlated with IL-6 levels (p = 0.65,p < 0.001). Only one patient treated with ibandronate showed an increase in IL-6 and CRP levels. Patients treated with pamidronate, whose body temperatures were increased at 24 h, had a greater increases of circulating IL-6, TNFalpha, and CRP at 24 h and 48 h than patients without temperature increase. These results suggest that pamidronate treatment, but not clodronate and possibly not ibandronate at the doses used, induced an increase in the plasma levels of IL-6 and TNFalpha, which may be responsible for the acute phase reaction observed clinically.

Astrocytes secrete a factor inducing the expression of HT7-protein and neurothelin in endothelial cells of chorioallantoic vessels.

Among the most specific markers of the blood-brain barrier phenotype of endothelial cells are the well-characterized immunoglobulin-like surface glycoprotein HT7 and the probably related or identical glycoprotein neurothelin. Both can be induced in chorioallantoic vessels by transplants of embryonic mouse brain. Other blood-brain barrier markers have been shown to be inducible by type-1 astrocytes in endothelial cells of non-neural origin. In the present work we tested the hypothesis that this cellular interaction between astrocytes and endothelial cells is mediated by a soluble factor(s). Chorioallantoic vessels of embryonic day 9 chick embryos were exposed for 4 or 10 days to a constant and localized delivery of astrocyte-conditioned medium by using a piece of gelfoam posed onto the chorioallantoic membrane, as a localized reservoir, which was connected to a miniosmotic pump system delivering astrocyte-conditioned medium at a steady rate. We found that in a significant number of chorioallantoic vessels located near the gelfoam, endothelial cells exposed to astrocyte-conditioned medium for a period of 4 or 10 days, but not to glioma-, fibroblast- or endothelial cell-derived conditioned medium, expressed the HT7 antigen and neurothelin. These results provide evidence that type-1-astrocytes are capable of inducing blood-brain barrier related properties in endothelial cells of non-neural origin through a soluble factor(s).

Effect of the renin response during renin inhibition: oral Ro 42-5892 in normal humans.

The effect of the new renin inhibitor Ro 42-5892 was evaluated in healthy volunteers after both intravenous and oral administration. In a preliminary study, 14 subjects received a 10-min infusion of Ro 42-5892 at doses ranging from 0.001 to 1 mg/kg. Plasma renin activity (PRA) and angiotensin (Ang) II levels were maximally suppressed in a dose-dependent manner at the end of the infusion. Plasma active renin concentration increased up to threefold. In a second study, 24 volunteers received placebo or 100, 600, or 1,200 mg of Ro 42-5892 p.o. in a single-blind, randomized fashion. Within 30 min after drug intake, PRA and plasma Ang I and Ang II levels fell to their nadir. Both Ang I and Ang II were measured specifically after extraction on phenylsilylsilica and separation by isocratic HPLC. The degree as well as the duration of inhibition were dose related. The decrease in plasma Ang lasted maximally for 2 h. Active renin increased dose dependently and remained elevated for more than 8 h after the 1,200 mg dose. A theoretical generation rate of Ang I was calculated for individual plasma samples assuming Michaelis-Menten kinetics for competitive inhibition and steady-state conditions. This calculated Ang I generation rate, based on plasma active renin concentrations and drug levels, closely correlated with actually measured Ang I and Ang II levels (r = 0.90, n = 88) over the whole 8 h time period. Thus, a sustained renin inhibition by Ro 42-5892, as indicated by increased plasma active renin levels, induces a much shorter fall in plasma Ang I and II apparently because of a rise in renin secretion.

Determinants of angiotensin II generation during converting enzyme inhibition.

The reaction of the renin-angiotensin system to acute angiotensin converting enzyme inhibition was investigated in a single-blind, crossover study in nine normal volunteers receiving two out of three regimens in random order: the new converting enzyme inhibitor benazepril (20 mg once or 5 mg four times at 6-hour intervals) or enalapril (20 mg). Plasma converting enzyme activity, drug levels, angiotensin I and angiotensin II, active renin, and aldosterone were measured before and 1-4 hours and 14-30 hours after drug intake. Baseline in vitro plasma converting enzyme activity was 97 +/- 15 nmol/ml/min (mean +/- SD) when Hip-Gly-Gly was used as substrate, but with carbobenzoxy-Phe-His-Leu (Z-Phe-His-Leu) or angiotensin I as substrate it was only 20 +/- 4 and 1.7 +/- 0.3 nmol/ml/min, respectively. Discriminating power at peak converting enzyme inhibition was enhanced with the two latter substrates. In vivo converting enzyme activity was estimated by the plasma angiotensin II/angiotensin I ratio, which correlated well with in vitro converting enzyme activity using Z-Phe-His-Leu as substrate (r = 0.76, n = 252). Angiotensin II levels returned to baseline less than 24 hours after drug administration, whereas in vitro and in vivo converting enzyme activity remained considerably inhibited and active renin together with angiotensin I levels were still elevated. A close linear relation was found between plasma angiotensin II and the angiotensin I/drug level ratio (r = 0.91 for benazeprilat and r = 0.88 for enalaprilat, p less than 0.001). Thus, plasma angiotensin II truly reflects the resetting of the renin-angiotensin system at any degree of converting enzyme inhibition. The ratio of plasma angiotensin II to angiotensin I represents converting enzyme inhibition more accurately than in vitro assays, which vary considerably depending on substrates and assay conditions used.

Caffeine-induced diuresis and atrial natriuretic peptides.

After a single-blind, randomized, cross-over protocol using decaffeinated coffee in a control experiment, the effect of an oral 250-mg caffeine dose on plasma immunoreactive atrial natriuretic peptide (ANF) was assessed in eight healthy students who had been on a methylxanthine-free diet for 1 week. One to 2 h after caffeine ingestion, both systolic blood pressure (SBP) and diastolic BP (DBP) increased by 12 mm Hg while heart rate (HR) also tended to increase. An increase in diuresis and in urinary sodium, potassium, and osmol excretion was observed within 1 h. Decaffeinated coffee induced no change in any of these parameters. Plasma epinephrine (EPI) increased gradually from 16.6 +/- 3.2 pg/ml (mean +/- SEM) to 45.1 +/- 7.9 pg/ml within 2 h after caffeine ingestion, but did not change after decaffeinated coffee (p less than 0.001). Plasma norepinephrine (NE), renin activity (PRA), aldosterone, and vasopressin remained unchanged. Plasma ANF was measured by radioimmunoassay (RIA) using an extremely sensitive antiserum (Kd = 10(-12) M) after rapid and virtually complete (90-103%) extraction from plasma. In 0.2 ml plasma, the theoretical detection limit is 1.1 fmol/ml. Normal plasma ANF concentrations in supine subjects were 17.9 +/- 8.1 fmol/ml (mean +/- SD) and 11.0 +/- 3.3 fmol/ml in subjects in the upright position. Plasma ANF levels were not affected by coffee drinking. In conclusion, by using a new and sensitive assay for plasma ANF, we did not find that caffeine-induced diuresis is mediated by ANF.

Reactive hyperreninemia is a major determinant of plasma angiotensin II during ACE inhibition.

The new ACE inhibitor trandolapril was administered to normal volunteers at daily doses of 0.5, 2, and 8 mg for 10 days. Twenty-one volunteers, aged 21-30 years, were included in the study. To randomly selected groups of seven subjects, each dose was administered in a single-blind fashion. None of the doses induced a consistent fall in blood pressure. Angiotensin-converting enzyme activity (ACE) was measured in vitro using three different synthetic substrates (i.e., Hip-Gly-Gly, Z-Phe-His-Leu, or angiotensin I). Although the degree of ACE inhibition assessed with the three methods varied widely, all methods clearly indicated dose-dependent ACE inhibition. These in vitro results were confirmed by measuring ACE inhibition in vivo using the ratio of plasma angiotensin II (ANG II) to blood angiotensin I (ANG I). The dose-dependent ACE inhibition was paralleled by a dose-dependent rise in active renin and blood angiotensin I levels, most evident on day 10. In contrast, plasma ANG II levels on day 10 were not different whether the volunteers received 0.5 or 8 mg trandolapril. Thus, whereas increasing doses of this new ACE inhibitor progressively enhanced the blockade of ACE activity, this was not reflected by additional reductions of plasma ANG II levels. The progressive enhancement of ACE inhibition seemed to be offset by the accentuation of the compensatory rise in renin and ANG I, which was still partially converted to ANG II.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of renal hypertension in the rat by neuropeptide Y.

Neuropeptide Y is known to enhance blood pressure responsiveness to various constrictors, including angiotensin II, and to suppress renin secretion. This study was undertaken to assess the effect of neuropeptide Y on the development of two-kidney, one clip renal hypertension. Normotensive rats either had a silver clip placed on the left renal artery or were sham-operated upon. An osmotic minipump, which was connected via a catheter to a jugular vein, was implanted subcutaneously in all rats. These pumps delivered either neuropeptide Y (0.001 microgram/min) or saline intravenously. Eight days later, an intra-arterial catheter was inserted and the rats were studied while not anesthetized on the following day. Neuropeptide Y did not affect body weight. In clipped rats, neuropeptide Y prevented the development of hypertension and suppressed renin secretion. Neuropeptide Y significantly decreased blood pressure also in sham-operated rats, although it had no effect on plasma renin activity. These data indicate that prolonged neuropeptide Y infusion may lower blood pressure by different mechanisms, one of which is probably a suppression of renin release.

Need for plasma angiotensin measurements to investigate converting-enzyme inhibition in humans.

Since only a minute proportion of total angiotensin-converting enzyme (ACE) is present in plasma, the reliability of conventional in vitro measurements of ACE activity has been questioned. Data presented here demonstrate that the definition of ACE inhibition depends on the methodology used, with different results obtained with different substrates. We have developed a method that provides accurate and precise determinations of "true" angiotensin levels and in vivo ACE activity was estimated by measuring the plasma angiotensin II/angiotensin I ratio. Since the initial interruption of angiotensin II production by an ACE inhibitor stimulates renal renin release, the response can be quantitated by measuring changes in plasma levels of angiotensin I. The actual state of the renin-angiotensin system during ACE inhibition is represented by the plasma angiotensin II level. When ACE inhibition is no longer complete, increased angiotensin I levels bring the system back toward initial angiotensin II concentrations.

Effects of smoking and physical exercise on platelet free cytosolic calcium in healthy normotensive volunteers.

Platelet free cytosolic calcium (PFCC) was measured in 21 healthy volunteers before and after cigarette smoking or physical exercise. The aim was to investigate whether acute blood pressure changes and increases in circulating levels of catecholamines and vasopressin modify PFCC. PFCC was determined using the Quin-2 method. Following cigarette smoking, significant increases in blood pressure, heart rate, plasma epinephrine (35 +/- 18 pg/ml before versus 51 +/- 31 pg/ml after smoking, P less than 0.05, mean +/- s.d.) and vasopressin levels (0.8 +/- 0.3 pg/ml before and 4.2 +/- 4.1 pg/ml after smoking, P less than 0.001) were observed. However, despite these acute hormonal and hemodynamic changes, PFCC remained stable at 156 +/- 55 nmol/l prior to the study and 157 +/- 29 nmol/l and 156 +/- 38 nmol/l at 20 and 80 min post-smoking, respectively. Acute physical exercise led to an increase in heart rate and systolic blood pressure but to a decrease in diastolic pressure. Moreover, a marked increase in plasma norepinephrine levels was observed after exercise (213 +/- 71 pg/ml before versus 747 +/- 501 pg/ml after exercise, P +/- 0.001). Again, PFCC was stable at 185 +/- 56 nmol/l at baseline versus 188 +/- 51 nmol/l at 20 min and 155 +/- 26 nmol/l at 80 min after exercise. These results therefore demonstrate that PFCC is not influenced acutely either by blood pressure increases, or by elevations in circulating catecholamine and vasopressin concentrations.

Angiotensin converting enzyme inhibition: discrepancy between antihypertensive effect and suppression of enzyme activity.

With chronic angiotensin converting enzyme (ACE) inhibition, blood pressure remains controlled throughout the day despite intermittent recovery of normal function of the renin-angiotensin system. This has been taken as evidence to suggest that the disappearance of angiotensin II (Ang II) from the circulation is not the main mechanism involved in the blood pressure-lowering action of ACE inhibitors. However, the degree of ACE inhibition is often not reliably estimated by the commonly used measurements of plasma ACE activity in vitro or plasma immunoreactive Ang II levels. The most appropriate method to assess ACE activity seems to be the measurement of the ratio between circulating angiotensin-(1-8)octapeptide and angiotensin I (Ang I) concentrations. The octapeptide and angiotensin I (Ang I) concentrations. The octapeptide Ang II can be measured precisely using high pressure liquid chromatography followed by a radioimmunoassay. Even using such improved methods, with long-term ACE inhibition, there exists a clear dissociation between the time course of ACE inhibition and that of the antihypertensive effect. Although it is attractive to speculate on this basis that other pathways such as blockade of tissue renin or enhanced vasodilator activity are responsible for the antihypertensive effect of ACE inhibitors, it is important to remember that the dissociation between the pharmacokinetic profile and the time course of the antihypertensive effect is not specific for ACE inhibitors and is well known with other agents. Since intermittent reduction of circulating Ang II is still an omnipresent feature of ACE inhibition, it seems at present that ACE inhibitors reduce blood pressure predominantly by this mechanism.

Increased response of brachial artery diameter to norepinephrine in hypertensive patients.

The hyperresponsiveness of small arteries to norepinephrine is well documented in essential hypertensive patients. Our objective was to investigate in situ the reactivity to norepinephrine of the diameter of large arteries, which are involved in the arterial disease of hypertension as well as small arteries. Brachial artery diameter, blood flow velocity, local volumic blood flow, and local vascular resistances were determined noninvasively with a pulsed Doppler system in 19 patients with essential hypertension and 9 normotensive subjects, before and after the administration of placebo (glucose) or increasing doses of norepinephrine (10, 20, and 40 ng.kg-1.min-1 iv) given in a single-blind fashion. In hypertensive patients, norepinephrine (40 ng.kg-1.min-1) induced 1) a significant decrease in brachial artery diameter, local blood velocity, volumic flow, and conductance and 2) a small increase in mean arterial pressure. These hemodynamic changes did not occur in the placebo group and were significantly greater in hypertensive patients than in normotensive subjects, although plasma norepinephrine increased to the same extent in both groups. We conclude that in hypertensive patients the increase in vascular reactivity to norepinephrine involves not only the resistive vessels but also the large arteries thus decreasing their conducting and buffering function.

[Hyperreactivity of the humeral artery to noradrenaline in essential hypertension patients]. / Hyperréactivité de l'artère humérale à la noradrénaline chez l'hypertendu essentiel.

The hyperresponsiveness of small arteries to norepinephrine is well documented in essential hypertensive patients. Our objective was to investigate in situ the reactivity to norepinephrine of the diameter of large arteries which are involved in the arterial disease of hypertension as well as small arteries. Brachial artery diameter, blood flow velocity, local volumic blood flow and local vascular resistances were determined non invasively using a pulsed Doppler system in 19 patients with essential hypertension and 9 normotensive subjects, before and after placebo (glucose) or increasing doses of norepinephrine (10, 20 and 40 ng/kg/min; i.v.) given in a single blind fashion. In hypertensive patients, norepinephrine (40 ng/kg/min) induced (i) a significant decrease in brachial artery diameter, local blood velocity, volumic flow and conductance and (ii) a small increase in mean arterial pressure. These hemodynamic changes did not occurred in the placebo group and were significantly greater in hypertensive patients than in normotensive subjects although plasma norepinephrine increased to the same extent in both groups. We conclude that in hypertensive patients, the increase in vascular reactivity to norepinephrine involves not only the resistive vessels, but also the large arteries thus decreasing their conducting and buffering function.

Rapid measurement of total and active renin: plasma concentrations during acute and sustained converting enzyme inhibition with CGS 14824A.

Total and active renin were measured in plasma of 6 normal volunteers before and after acute and sustained angiotensin converting enzyme (ACE) inhibition with CGS 14824A (2 mg and 10 mg p.o. q.d.) or placebo treatment. The same sandwich technique was used for the measurement of both total and active renin using a polyacrylamide-iron-oxide linked monoclonal antibody to trap renin and 125I-labelled second monoclonal antirenin antibodies without or with specificity for active renin. Normal values for supine subjects ranged for active renin between less than 3 pg/ml and 28 pg/ml and for total renin between 73 and 263 pg/ml. Plasma ACE activity was clearly suppressed during 24 hours following both 2 mg and 10 mg CGS 14824A. Active plasma renin reached 6- and 12-fold normal values on days 1 and 7 of treatment with the 10 mg dose. Total renin rose to 150% and 228% respectively at the same time. Inactive renin continued rising during the first day of 10 mg CGS treatment to a final 141% at 24 hours post-drug and didn't change on day 7. Plasma renin activity correlated well with active renin levels (r = 0.92). We conclude that both total and active plasma renin concentrations can now be directly measured with great accuracy within 6 hours.