Evaluation of Strategies for the Assessment of Drug-Drug Interactions Involving Cytochrome P450 Enzymes.

BACKGROUND AND OBJECTIVES: Drug-drug interactions (DDIs) can occur when one drug alters the metabolism of another drug. Drug metabolism mediated by cytochrome P450 enzymes (CYPs) is responsible for the majority of metabolism of known drugs and inhibition of CYP enzymes is a well-known cause of DDIs. In the current study, the use of various human liver microsomes (HLM)-based methods to determine occurrence of CYP-mediated metabolism-dependent inhibition (MDI) and possible follow-up studies were evaluated. METHODS: Human CYP inhibition was studied using the following methodologies: direct inhibition and (non-diluted) IC50-shift assays, a ferricyanide-based reversibility assay, a spectrophotometric metabolic intermediate complex (MIC) assay, and recording of reduced carbon monoxide (CO)-difference spectra. HLM incubations in the presence and absence of NADPH and glutathione (GSH) were performed to study the possible formation of CYP-dependent GSH adducts. HLM incubations with the radiolabeled inhibitors mifepristone and paroxetine were performed to study CYP-mediated covalent binding. RESULTS: Dihydralazine and furafylline displayed irreversible MDI of CYP1A2. Paroxetine displayed both quasi-irreversible and irreversible MDI of CYP2D6, formation of CYP-dependent GSH adducts was observed, while CYP-mediated covalent binding occurred which was decreased in the presence of GSH. Mifepristone displayed irreversible MDI of CYP3A4, formation of CYP-dependent GSH adducts was observed, while CYP-mediated covalent binding occurred which was decreased in the presence of GSH. Troleandomycin and verapamil displayed quasi-irreversible MDI of CYP3A4; MIC formation was observed, while no formation of CYP-dependent GSH adducts occurred. CONCLUSIONS: This study gives a representative overview of current methodologies that can be used to study CYP inhibition. The here presented strategy can be applied as a tool during risk evaluation of CYP-mediated DDIs.

The differential effects of recombinant brain natriuretic peptide, nitroglycerine and dihydralazine on systemic oxygen delivery and gastric mucosal microvascular oxygenation in dogs.

Brain natriuretic peptide has vasodilatory properties and may thus increase splanchnic perfusion and oxygenation. We compared the effects of recombinant brain natriuretic peptide on gastric mucosal microvascular haemoglobin oxygenation (reflectance spectrophotometry) and systemic variables with those of equi-hypotensive doses of two other vasodilators (nitroglycerine and dihydralazine). Chronically instrumented, healthy dogs were randomly allocated to receive on different days, one of the three drugs (nitroglycerine and dihydralazine doses titrated to reduce mean arterial pressure by ∼20%). Brain natriuretic peptide significantly increased gastric mucosal microvascular haemoglobin oxygenation selectively, i.e. without concomitant haemodynamic effects. In contrast, the other vasodilators either did not increase gastric mucosal microvascular haemoglobin oxygenation at all (nitroglycerine), or did so only with marked increases in other systemic haemodynamic variables (dihydralazine). Our data suggest a potential role of recombinant brain natriuretic peptide selectively for increasing microvascular mucosal oxygenation. Studies are required to extend these findings to the clinical setting.

Effects of dihydralazine on renal water and aquaporin-2 excretion in humans.

OBJECTIVE: Dihydralazine is a vasodilator that lowers blood pressure, but often also leads to significant water and sodium retention. To characterize the effect of dihydralazine on renal sodium and water handling, we tested the hypothesis that dihydralazine causes water retention parallel with an increase in urinary excretion of aquaporin-2 (u-AQP2) in healthy humans. MATERIAL AND METHODS: The effect of intravenous infusion of dihydralazine in three doses (3.125 mg, 6.250 mg and 9.375 mg) on urinary AQP2, water and sodium excretion, heart rate (HR), blood pressure (BP) and vasoactive hormones was measured in a randomized, placebo-controlled, double-blind, crossover study in 17 healthy subjects. Glomerular filtration rate (GFR) and renal tubular function were determined with the continuous infusion clearance technique and vasoactive hormones with radioimmunoassays. RESULTS: Dihydralazine compared to placebo had no impact of u-AQP2 (effect of dihydralazine versus placebo +/-SE) (-0.074+/-0.048 ng/min versus -0.015+/-0.034 ng/min; p = 0.42), despite significant reductions in urine output and free water clearance after 9.375 mg of dihydralazine. Dihydralazine significantly lowered BP and increased HR, plasma levels of angiotensin II and (except after 3.125 mg) atrial natriuretic peptide, while plasma levels of vasopressin, GFR and fractional excretions of sodium and lithium were not significantly changed. CONCLUSIONS: These findings suggest that dihydralazine increases water re-absorption in the distal tubules, independently of vasopressin and of sodium re-absorption. Furthermore, our study does not support an effect of the sympathetic nervous system, the renin-angiotensin system and the natriuretic peptide system on u-AQP2 regulation.

Progress curve analysis of CYP1A2 inhibition: a more informative approach to the assessment of mechanism-based inactivation?

Mechanism-based cytochrome P450 inactivation is defined as a time- and NADPH-dependent inactivation that is not reversible upon extensive dialysis. Current methodologies use dilution approaches to estimate the rate of inactivation and offer limited mechanistic insight and are significantly influenced by experimental conditions. We investigated the potential of progress curve analysis because this experimental design allows investigation of both the reversible (K(iapp)) and irreversible (K(i), K(inact)) components of the reaction mechanism. The human liver microsomal CYP1A2 inactivation kinetics of resveratrol, oltipraz, furafylline, and dihydralazine (Fig. 2) were evaluated. The inactivation results for furafylline (K(i), 0.8 microM; K(inact), 0.16 min(-1)) are within 2-fold to published data (K(i), 1.6 microM; K(inact), 0.19 min(-1)). Resveratrol and dihydralazine results are within a 4.3-fold range of published data, which compares well with ranges of estimates of these parameters across publications (e.g., furafylline has estimates ranging of K(i) from 1.6 to 22.3 microM and K(inact) from 0.19 to 0.87 min(-1)). This range of estimates highlights the potential caveats surrounding the existing methodologies that have been previously discussed in depth. In addition to these inactivation parameters, we have been able to demonstrate a variation in balance of reversible versus irreversible inhibition within these inactivators. Oltipraz and resveratrol have K(iapp) values similar to their K(i), indicating a significant early onset reversible inhibition, whereas furafylline and dihydralazine are dominated by irreversible inactivation. This approach allows a more mechanistic investigation of an inactivator and in the future may improve the prediction of clinical drug-drug interactions.

Dihydralazine treatment limits liver injury after hemorrhagic shock in rats.

OBJECTIVE: Impaired hepatic perfusion after hemorrhagic shock frequently results in hepatocellular dysfunction associated with increased mortality. This study characterizes the effect of the vasodilators dihydralazine and urapidil on hepatocellular perfusion and integrity after hemorrhagic shock and resuscitation. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University experimental laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: To register systemic and regional hepatic hemodynamics, rats (n=6 per group) were instrumented and randomly assigned to the following groups: shock+vehicle; shock+dihydralazine (1.5 mg/kg); or shock+urapidil (3 mg/kg). After 1 hr of hemorrhagic shock, animals were resuscitated for 5 hrs and mean arterial pressure was maintained at 70+/-5 mm Hg by administration of dihydralazine or urapidil. To evaluate hepatic heme oxygenase-1 expression and liver injury (determination of levels of alanine and aspartate aminotransferase [ALT, AST] and histology), an additional series of experiments with six animals per group was performed. At the end of each experiment, animals were killed and blood and liver tissue was obtained for subsequent analyses. MEASUREMENTS AND MAIN RESULTS: Dihydralazine increased cardiac output and portal and hepatic microvascular flow (p<.05) and reduced liver injury after shock (lower ALT and AST levels [p<.05]; improvement of histopathological changes). In contrast, urapidil had no effect on portal flow or liver injury. Hepatic heme oxygenase-1 mRNA expression was upregulated in animals subjected to hemorrhagic shock but did not differ among experimental groups. CONCLUSIONS: Dihydralazine increases nutritive portal and hepatic microvascular flow and limits liver injury after hemorrhagic shock. This protective effect appears to be the result of increased cardiac output and increased portal flow. These findings may offer a new strategy for hepatic protection after hemorrhagic shock.

Changes in metabolism and blood flow following catecholamine stimulation in the synovial membrane measured with microdialysis.

Adrenergic reactions could mediate metabolic and circulatory changes in the synovial membrane following knee surgery. The interstitial fluid of the synovial membrane and subcutaneous adipose tissue (reference) was monitored in vivo with microdialysis following knee arthroscopy with adrenaline added to the dialysis solvent, adrenaline together with a local anestetic added intra-articularly and without. Local metabolism and blood flow were measured. There was a similar increase, about two fold, in dialysate lactate in all three experimental conditions in the synovial membrane but no change in adipose tissue. Glucose and blood flow decreased by approximately 50% and 10% in both tissues following addition of adrenaline to the dialysate but no changes in the glucose concentrations or blood flow were observed in the other two experimental situations. As regards glycerol the addition of adrenaline caused an approximate 20% increase of the concentration in adipose tissue but an approximate 20% decrease in the synovial membrane. The intra-articular injection caused an approximate 50% increase of the glycerol level in the synovial membrane but no change in adipose glycerol. Thus, the hypermetabolic state in the synovial membrane following standard arthroscopy and the tissue damage (increased glycerol level) in the synovial membrane following postoperative pain relief by intra-articularly injected local anesthetics together with adrenaline doesn't enhance the hypermetabolic state seen postoperatively without adrenaline. However, catecholamines have pronounced in vivo effects on metabolism and blood flow in the synovial membrane.

Acute treatment of hypertension increases infarct sizes in spontaneously hypertensive rats.

We studied the effect of dihydralazine treatment of hypertension in spontaneously hypertensive stroke-prone rats in a model of permanent focal cerebral ischemia (stroke). After occlusion of the middle cerebral artery systemic arterial pressure (SAP) was lowered with a computer controlled infusion device from 163 to 135 or 117 mm Hg for 24h. In the control group SAP was not manipulated. Reduction of SAP to normotension (117 mm Hg) significantly worsened outcome and increased infarct volume measured 7 days after induction of ischemia, whereas a mild reduction of SAP (to 137 mm Hg) had no statistically significant effect on outcome or infarct volume. We conclude that pharmacological treatment of hypertension may negatively affect neurological outcome and infarct volume in a rat stroke model.

Mechanism-based inactivation of cytochrome P450s 1A2 and 3A4 by dihydralazine in human liver microsomes.

Dihydralazine is known to induce immunoallergic hepatitis. Since anti-liver microsome (anti-LM) autoantibodies found in the serum of the patients react with P450 1A2, it is suggested that dihydralazine is biotransformed into a reactive metabolite, which covalently binds to cytochrome P450 1A2 and triggers an immunological response as a neoantigen. We investigated inactivation of P450 enzymes, including P450 1A2, during the metabolism of dihydralazine to evaluate the selectivity of P450 1A2 as a catalyst and a target of dihydralazine. Human liver microsomes or microsomes from lymphoblastoid cells expressing P450 enzymes were preincubated with dihydralazine in the presence of NADPH, followed by an assay of several monooxygenase activities. Preincubation of human liver microsomes with dihydralazine in the presence of NADPH resulted in decreases in phenacetin O-deethylase activity (an indicator of P450 1A2 activity) and testosterone 6beta-hydroxylase activity (P450 3A4), but not in diclofenac 4'-hydroxylase activity (P450 2C9), an indication of inactivation of P450s 1A2 and 3A4 during the dihydralazine metabolism. The inactivation of both of the P450s followed pseudo-first-order kinetics and was saturable with increasing dihydralazine concentrations. Similar time-dependent decreases in the activities were obtained in the case for use in microsomes expressing P450 1A2 and P450 3A4 instead of the human liver microsomes. The data presented here demonstrated that dihydralazine was metabolically activated not only by P450 1A2 but also by P450 3A4, and the chemically reactive metabolite bound to and inactivated the enzyme themselves, suggesting that dihydralazine is a mechanism-based inactivator of P450s 1A2 and 3A4. The data support the postulated covalent binding of a reactive metabolite of dihydralazine to P450 1A2 as a step in the formation of anti-LM antibodies in dihydralazine hepatitis, but it is not the unique factor for determining the specificity of the autoantibodies.

Dihydralazine-induced inactivation of cytochrome P450 enzymes in rat liver microsomes.

Dihydralazine is known to induce immunoallergic hepatitis, and the anti-liver microsome (anti-LM) autoantibodies found in the serum of the patients have been reported to react with cytochrome P450 1A2 (CYP1A2). It is thus suggested that a reactive metabolite of dihydralazine covalently binds to the P450 protein and triggers an immunological response as a neoantigen. We investigated the selectivity of inactivation of P450 enzymes during the metabolism of dihydralazine to evaluate the target protein of its reactive metabolite. Liver microsomes from male Wistar rats were preincubated with dihydralazine in the presence of NADPH, followed by assays of several monooxygenase activities. Preincubation of microsomes of beta-naphthoflavone-treated rats with dihydralazine resulted in time-dependent loss of phenacetin O-deethylase activity (an indicator of CYP1A2 activity), showing inactivation of CYP1A2 during the dihydralazine metabolism. The preincubation with dihydralazine was less effective on ethoxyresorufin O-deethylase activity in microsomes of beta-naphthoflavone-treated rats (CYP1A1) and pentoxyresorufin O-depentylase activity in microsomes of phenobarbital-treated rats (CYP2B). On the other hand, preincubation of microsomes of untreated rats with dihydralazine caused time-dependent loss of testosterone 2alpha-, 16alpha- (CYP2C11), and 6beta- (CYP3A) hydroxylase activities. These results demonstrated that dihydralazine was metabolically activated by CYP1A2, and the chemically reactive metabolite bound to the enzyme itself and inactivated it, as was suggested by the appearance of anti-LM antibodies in dihydralazine-hepatitis, whereas CYP2C and -3A enzymes were also suggested to be the enzymes that activate dihydralazine and lead to the target of the reactive intermediates.

Drug actions in preeclampsia: aspirin, but not magnesium chloride or dihydralazine, differentially inhibits cultured human trophoblast release of thromboxane and prostacyclin without affecting angiotensin II, endothelin-1, or leukotriene B4 secretion.

OBJECTIVE: We hypothesized that aspirin Mg++, and dihydralazine affect the release of vasoactive agents from cultured human placental trophoblast. STUDY DESIGN: Cytotrophoblasts isolated from placentas of preterm or term deliveries of 14 healthy control women and 15 preeclamptic women were cultured in Dulbecco's modified Eagle's medium for 5 days in the presence or absence of either 0.1 mmol/L aspirin, 3 mmol/L magnesium chloride, or 136 ng/ ml dihydralazine. Vasoactive substances were quantitated by radioimmunoassay with mean +/- SEM percentage of untreated cells (= 100%) compared by the Mann-Whitney U test and analysis of variance. RESULTS: Aspirin inhibited (p < 0.01) both thromboxane and prostacyclin on days 1 and 2 in culture but not on days 3 to 5 unless the Dulbecco's modified Eagle's medium was supplemented with arachidonic acid. Aspirin inhibition was greater (p < 0.01) for thromboxane in cells cultured 24 hours after preeclamptic pregnancy (preterm 29.9% +/- 6.8%, term 20.1% +/- 5.9%) compared with normal controls (preterm 66.3% +/- 10.6%, term 68.9% +/- 11.6%). Aspirin reduced (p < 0.01) the ratio of thromboxane to prostacyclin in media of cells from preeclampsia (untreated 27.8 +/- 7.2, aspirin 13.3 +/- 4.4), but aspirin had no effect on this ratio in cultures from control normal pregnancies (untreated 6.8 +/- 2.9, aspirin 4.8 +/- 1.1). Neither magnesium chloride nor dihydralazine affected trophoblast prostanoid production, and no drug altered the media levels of angiotensin II, endothelin-1, or leukotriene B4. CONCLUSION: Aspirin selectively inhibits trophoblast prostanoid production. This inhibition depends on the availability of arachidonic acid and the presence or absence of preeclampsia. Magnesium and dihydralazine effects in pregnancy are not related to altered release of trophoblast vasoactive compounds.

Urinary excretion of 2,3-dinor-6-keto-PGF1 alpha and 11-dehydro-TXB2 by the gravid spontaneously hypertensive rat.

Little is known about the pathophysiological processes leading to superimposed preeclampsia. We present an animal model where the uteroplacental blood flow in spontaneously hypertensive rats (SHR) was reduced by a silver clip. Thus, a superimposed preeclampsia-like syndrome could be studied under defined conditions. Urinary excretion of 2,3-dinor-6-keto PGF1 alpha and 11-dehydro-TxB2 were measured by enzyme immunoassays at day 16 and 20 of pregnancy. In gravid, sham-operated animals excretion of 2,3-dinor-6-keto-PGF1 alpha was largely elevated compared to non gravid control animals (day 16: 1259 vs. 258 ng/kg 24h; day 20: 471 vs. 269 ng/kg.24h). However, in the gravid rats with reduced uteroplacental blood flow urinary excretion of 2,3-dinor-6-keto-PGF1 alpha decreased to non gravid levels (day 16: 335 ng/kg.24h; day 20: 238 ng/kg.24h). By antihypertensive therapy with dihydralazin this effect was largely abolished. Only minor alterations were found in the excretion of 11-dehydro-TxB2. Our findings suggest, that a reduction of uteroplacental blood flow in the spontaneously hypertensive rat decreases the systemic prostacyclin synthesis.

A comparison between the haemodynamic effects of oral nifedipine and intravenous dihydralazine in patients with severe pre-eclampsia.

OBJECTIVE: To compare the effects of a single oral dose of nifedipine with those of intravenous dihydralazine on central haemodynamics in pregnant women with severe pre-eclampsia. DESIGN: A prospective comparative study. SETTING: The High Risk Obstetric Unit, University Hospital Rotterdam Dijkzigt, Rotterdam. SUBJECTS: Twenty patients with severe pre-eclampsia between 27 and 35 weeks gestation with normal cardiac filling pressures and without fetal distress. INTERVENTIONS: A pulmonary artery thermodilution catheter and a radial artery line were placed. Ten patients chewed a 10-mg capsule of nifedipine and 10 patients received dihydralazine by intravenous infusion at a rate of 1-3 mg/h. Arterial pressures, heart rate, cardiac output and pulmonary capillary wedge pressure were determined before and after drug administration. Fetal condition was continuously monitored by cardiotocography. RESULTS: The reduction in arterial blood pressure obtained with both drugs was similar, and was associated with a similar rise in heart rate and cardiac output and a similar reduction in systemic vascular resistance. Pulmonary capillary wedge pressures decreased significantly less with nifedipine than with dihydralazine. Signs of fetal distress occurred in none of the nifedipine-treated patients, but in five of the patients treated with dihydralazine. CONCLUSION: From the haemodynamic viewpoint nifedipine seems to be a useful agent in the treatment of hypertensive emergencies in pregnancy.

Deleterious effects of inhibition of the renin-angiotensin system in neonatal rats.

The angiotensin I converting enzyme inhibitor (ACEI) perindopril (2 mg/kg body weight), the peripheral vasodilator dihydralazine (DHL) (25 mg/kg body weight) or distilled water was given daily from birth to day 14 to neonatal rats. Blood pressure, plasma creatinine, plasma renin activity (PRA), substrate (PRS) and concentration (PRC) and renin content of kidney tissue sections were evaluated on days 14 and 28. By day 14, a high mortality in the ACEI group was observed. ACEI, but not DHL, led to a significant fall (P < 0.01) in blood pressure, 57 +/- 11 versus 89 +/- 25 in the DHL group and 103 +/- 24 mmHg in controls, and to a dramatic increase in plasma creatinine. PRA and PRS were undetectable in ACEI-treated rats; in contrast, PRC and renal staining with anti-renin antibody were significantly increased in ACEI rats. On day 28, the blood pressure was normal in all groups and plasma creatinine returned to the normal range in ACEI rats. PRA, PRS and PRC were not significantly different in the ACEI group and controls. These results suggest that the renin-angiotensin system (RAS) plays a major postnatal role in the neonatal rat. Inhibition of the RAS during the first 2 weeks of life leads to high mortality, severe hypotension, reversible renal failure and a defect in circulating angiotensinogen.

Differential gene expression of renin and angiotensinogen in the TGR(mREN-2)27 transgenic rat.

Transgenic rats carrying the murine Ren-2 gene represent a monogenetic model of hypertension characterized by low plasma renin and high extrarenal expression of the transgene. The hypothesis has been raised that stimulated local reninangiotensin systems may be responsible for the development of hypertension in this model. This study analyzes the effects of the converting enzyme inhibitor lisinopril, which specifically interferes with the renin-angiotensin system, and the direct vasodilator dihydralazine on the renal and extrarenal expression of renin and angiotensinogen. A comparison of gene expression between heterozygous and homozygous transgenic and normal Sprague-Dawley rats was also performed. We demonstrate high sensitivity of blood pressure toward converting enzyme inhibition in transgenic TGR(mREN-2)27 rats. In the kidney, expression of the transgene and the endogenous renin gene increased, suggesting that both are modulated by lisinopril in a similar manner. On the other hand, blood pressure reduction by dihydralazine did not abolish renal renin suppression in transgenic rats, indicating that mechanisms different from direct effects of blood pressure account for renin suppression. Homozygosity for the transgene led to increased Ren-2 expression and higher blood pressure and had opposite effects on angiotensinogen expression compared with heterozygous rats. Cardiac hypertrophy was reduced by lisinopril but not dihydralazine and was positively correlated with cardiac angiotensinogen expression. Increased angiotensin II in the adrenal gland of TGR(mREN-2)27 rats, which overexpresses the transgene, provides evidence that this leads to enhanced generation of tissue angiotensin II. We conclude that expression of the mouse transgene, the endogenous rat renin gene, and the angiotensinogen gene is subject to differential tissue-specific regulation. Reversal of cardiovascular damage with the converting enzyme inhibitor but not dihydralazine suggests that angiotensin II generated locally may be involved in the pathogenesis of hypertension and structural changes in TGR(mREN-2)27 rats.

[Chronosensitivity to adelphane-esidrex and sinepres and effectiveness of treatment of patients with hypertension]. / Khronochuvstvitel'nost' k adel'fanu-ézidreksu i sinepresu i éffektivnost' lecheniia bol'nykh gipertonicheskoi bolezn'iu.

24-h trends in central and peripheral circulation in response to pharmacological tests were registered in 40 patients with stage II essential hypertension (EH). Rhythmic patterns of hemodynamic parameters (heart rate, systolic pressure, stroke volume, etc.) sensitivity to adelphane-esidrex were found. Hypotensive effect of this drug was maximal in morning hours because of the fall in arterial pressure due to reduced peripheral resistance. Sinepres chronotherapy given to 18 EH patients turned out superior to conventional treatment (1 pill 3 times a day) given to 20 EH patients. When taken into consideration, chronosensitivity to the above drugs may bring about a reduction in daily and course doses and earlier occurrence of a hypotensive effect.

The effects of dihydralazine, labetalol and magnesium sulphate on the isolated, perfused human placental cotyledon.

OBJECTIVE: To assess the effects of dihydralazine, labetalol and magnesium sulphate on the vascular tone in the isolated, perfused human placental cotyledon. METHODS: In vitro perfusion of the fetal compartment of isolated, human placental cotyledons. RESULTS: None of the drugs affected basal vascular tone. The thromboxane A2-mimic U46619 and endothelin-1 induced a concentration-dependent increment in perfusion pressure, while 5-hydroxytryptamine induced a variable increase, and norepinephrine induced a small, transient increase in perfusion pressure. After preconstriction with U46619, magnesium sulphate (1.5 x 10(-3) to 6 x 10(-3) mol/l) induced a decrease in perfusion pressure, while dihydralazine (10(-6) to 10(-4) mol/l) or labetalol (10(-7) to 10(-4) mol/l) enhanced the perfusion pressure. These effects of dihydralazine and labetalol were unaffected by treatment with indomethacin 10(-6) mol/l, but could be reversed by addition of magnesium sulphate 6 x 10(-3) mol/l. Labetalol 10(-6) to 10(-4) mol/l also caused an increase in the perfusion pressure induced by endothelin-1, but showed no effects after preconstriction with 5-hydroxytryptamine. Pretreatment with labetalol 10(-4) mol/l inhibited the transient increase in perfusion pressure induced by norepinephrine 3 x 10(-5) mol/l. CONCLUSIONS: The present data demonstrated that the commonly used vasodilating agents labetalol and dihydralazine do not produce vasodilatation in the human perfused cotyledon after vasoconstriction induced by agents of suggested importance for maintenance of fetal placental vascular tone, and that high concentrations of these drugs may even enhance vasoconstriction induced by thromboxane and endothelin-1 in this area. Magnesium sulphate may show the potential to reverse such unwanted effects of dihydralazine and labetalol.

Neuromodulatory effects of the renin-angiotensin system on the cat electroretinogram.

PURPOSE: Angiotensin-converting-enzyme (ACE) catalyses the formation of angiotensin II (ANGII), which presumably acts as a central neurotransmitter/modulator. ANGII-related effects have also been observed in the retina. Present in vivo experiments were designed to investigate further ANGII-related effects on retinal neuromodulation. METHODS: In 12 anesthetized cats, electroretinographic measurements were carried out in the dark-adapted state using corneal contact lens electrodes and a Ganzfeld stimulator. Quinapril was used to inhibit ACE. RESULTS: Reducing ANGII-concentration increased sensitivity (0.5 log units) and gain (50%) of the rod b-wave amplitude. The b-wave implicit time was stimulus dependent, shortening at high intensities. The scotopic threshold response and the oscillatory potentials were also influenced by ACE inhibition. However, a-wave and 30 Hz flicker remained unaffected. Effects of Quinapril on ERG-amplitudes were reversed by subsequent ANGII administration, except for the implicit time of the b-wave and scotopic threshold response. CONCLUSIONS: Although these results are accompanied by alterations in systemic blood pressure, several findings support the evidence that the renin-angiotensin system might have a neurophysiologic effect on retinal neurons outside the vascular system. These results are in accordance with immunohistochemical data found by others that point to angiotensinergic cell involvement and thereby further support the concept of angiotensinergic processes in the inner retina from a functional point of view.

Antihypertensive treatment with beta blockers and gabaergic transmission in rat brain.

The effects of chronic oral administration of propanolol and metoprolol on blood pressure and GABAergic function were investigated in spontaneously hypertensive rats (SHR) and compared with the effect of dihydralazine. Under the experiment conditions employed all drugs reduced significantly (p < 0.01) arterial pressure. The beta blockers elevated GABA turnover in the hypothalamus and the pons-medulla. Dihydralazine, however had not such an effect. Our result suggest that the antihypertensive action of beta blockers may be related in part to the enhanced cerebral GABAergic transmission.

Enhancement of GABAergic system activity by metoprolol in spontaneously hypertensive rats.

The relevance of the GABAergic system for the antihypertensive action of metoprolol in spontaneously hypertensive rats was studied by comparing the effect of metoprolol with the effect of dihydralazine. Chronic oral treatment with metoprolol produced the maximum effect after 49 days (-delta 34 mm Hg). This effect persisted on the same level for up to 55 days. The measurements of gamma-aminobutyric acid (GABA) synthesis and specific [3H]GABA binding were performed in the hypothalamus, the pons-medulla, the hippocampus and the striatum. Significant stimulation of GABA synthesis and turnover appeared in the hypothalamus and the pons medulla. In contrast, chronic administration of dihydralazine had no influence on GABA synthesis rate. It was also shown that metoprolol elevated significantly (P < 0.01) specific [3H]GABA binding in the hypothalamus and the pons-medulla. In the striatum this effect of metoprolol was less pronounced. Binding constant analysis revealed changes in both the receptor density and affinity. Our results suggest that the hypotensive response to chronic treatment with metoprolol might be attributed to an enhancement of GABAergic system activity.

Effects of clonidine, dihydralazine and splanchnic nerve stimulation on the release of neuropeptide Y, MET-enkephalin and catecholamines from dog adrenal medulla.

Various neuropeptides are costored together with catecholamines in the adrenal medulla. The concurrent release (evaluated by adrenal vein plasma levels) of these neuropeptides [neuropeptide Y (NPY), met-enkephaline (ME)] and catecholamines [adrenaline (A) and noradrenaline (NA)] from the adrenal gland was examined in chloralose-anesthetized dogs after intravenous administration of clonidine (10 micrograms/kg) and dihydralazine (1 mg/kg). These results were compared to those obtained after the stimulation of the right splanchnic nerve at 1, 5 and 10 Hz frequencies. The increment in the release of catecholamines and neuropeptides was evaluated for dihydralazine and splanchnic nerve stimulation. Dihydralazine (at its maximal effect) induced a significant preferential increase in catecholamines (expressed as mean (SEM): NA: 17.3 (5.4) fold, A: 13.1 (2.6) fold) and ME (16.0 (7.1) fold) versus basal values. However, the significant increase in NPY-LI was only 2.0 (0.4) times the baseline. Splanchnic nerve stimulation induced a frequency-dependent increase in catecholamines and neuropeptides. When the stimulation frequency was increased from 1 Hz to 5 Hz, NA and A levels increased 17.9 (4.3) and 14.0 (2.2) fold, respectively and ME levels 14.1 (3.0) fold. By contrast, NPY-LI was increased only 2.3 (0.3) fold under the same conditions. Increasing the stimulation frequency from 5 Hz to 10 Hz resulted in similar elevations of NA, ME, and NPY-LI adrenal plasma levels (about 4 times) whereas A only increased twice. Clonidine decreased catecholamine and ME adrenal plasma levels (the maximal percent decrease when compared with control values was about 75%) whereas NPY adrenal plasma levels remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)