An Improved 3-(4,5-Dimethylthiazol-2-yl)-5-(3-Carboxymethoxyphenyl)-2-(4-Sulfophenyl)-2H-Tetrazolium Proliferation Assay to Overcome the Interference of Hydralazine.

The MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay is one of the most commonly used assays to assess cell proliferation and cytotoxicity, but is subject to interference by testing compounds. Hydralazine, an antihypertensive drug, is commonly investigated in multiple fields such as heart failure, cancer, and blood pressure research. This study reported interference of the MTS assay by hydralazine and a simple modification overcoming this interference. Vascular smooth muscle cells were cultured in the presence or absence of hydralazine (0, 10, 50,100, and 500 µM) for 2 or 24 h. Cell numbers were analyzed using MTS, trypan blue exclusion, or microscopic assays. A modified version of the standard MTS assay was established, in which an additional step was added replacing the test medium, containing hydralazine, with fresh culture medium immediately before the addition of the MTS reagent. Culture with hydralazine at concentrations of 50, 100, and 500 µM for 2 h increased absorbance (p < 0.05) in the standard MTS assay, whereas microscopy suggested no change in cell numbers. Culture with 500 µm hydralazine for 24 h increased absorbance (p < 0.05) in the standard MTS assay, however, trypan blue exclusion and microscopy suggested a decrease in cell numbers. In a cell-free system, hydralazine (≥10 µM) increased absorbance in a concentration-dependent manner. The modified MTS assay produced results consistent with trypan blue exclusion and microscopy. In conclusion, a simple modification of the standard MTS assay overcame the interference of hydralazine and may be useful to avoid interference from other tested compounds.

Inhibition of calcium influx and tonic response to K+ of intestinal smooth muscle by hydralazine.

1. Evidence concerning the mechanism of the inhibition of contraction caused by hydralazine has been sought in ileal longitudinal muscle and taenia coli of guinea-pig. Hydralazine (10(-3)-3 x 10(-3) M) markedly inhibited K+ (60 mM) induced tonic response with smaller effects on the phasic response in the ileal muscle. However, 10(-2) M hydralazine completely abolished both responses. 2. Hydralazine increased the threshold for Ca2+ induced contraction in Ca2+ free, K+ depolarized taenia coli and reduced the maximal response size. A low concentration (3 x 10(-10) M) of nifedipine, an L-type Ca2+ channel blocker, further caused a parallel shift to the right in the presence of hydralazine in the concentration-response curves obtained with Ca2+ 3. Hydralazine caused a significant decrease in Ca2+ uptake measured by the lanthanum method during the K+ induced tonic response in ileal muscle; however, it did not affect the Ca2+ efflux. 4. In ileal muscle fibres treated with Triton-X-100, in which the Ca2+ release sites are destroyed, 10(-3) M hydralazine had no effect on the contractions induced by 10(-5) M Ca2+; however, hydralazine at a higher concentration (10(-2) M) had a slight inhibitory effect on the contraction. 5. The present finding indicates that the inhibitory action on contractions produced by hydralazine may result mainly from the interference of calcium permeability at the cell membrane in ileal muscle. There is the possibility that hydralazine of higher concentrations may have a minor action on the contractile system.

Effects of indomethacin on pulmonary hemodynamics and gas exchange in patients with pulmonary artery hypertension, interference with hydralazine.

To assess the ability of indomethacin (Indo) to influence pulmonary vascular tone in patients with chronic lung disease, we studied the hemodynamic and gas exchange alterations induced by a 50-mg indomethacin infusion in 10 patients suffering from varying degrees of pulmonary artery hypertension and hypoxemia. The most pronounced effects were observed 3 h after Indo administration. Mean systemic arterial pressure (Psa) increased from 76 +/- 4 to 86 +/- 4 mm Hg (p less than 0.01), whereas mean pulmonary arterial pressure (Ppa) was unchanged. The cardiac index (CI) decreased from 3.1 +/- 0.2 to 2.8 +/- 0.2 L/min/m2 (p less than 0.02) because of the reduced heart rate, which decreased from 86 +/- 5 to 80 +/- 4 beats/min (p less than 0.05). Systemic and pulmonary vascular resistance indexes increased, respectively, from 22 +/- 2 to 27.5 +/- 2 U/m2 (p less than 0.001) and from 11.9 +/- 2 to 13.4 +/- 2 U/m2 (p less than 0.05). We measured an increase in PaO2, from 49.5 +/- 4 to 57.5 +/- 4 mm Hg (p less than 0.001) simultaneously with a reduced venous admixture, from 39.5 +/- 4 to 30.5 +/- 3% (p less than 0.001). The calculated PO2 uptake was unchanged, but mixed venous O2 tension increased from 30.5 to 33.5 mm Hg (p less than 0.01). Because Indo may interfere with the hypotensive effect of hydralazine and because hydralazine has been proposed in the treatment of patients with pulmonary hypertension, 7 of these patients also received 0.35 mg/kg hydralazine and Indo plus hydralazine (Indo + H) injected simultaneously.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemodynamic interaction of nonselective vs. beta-1-selective beta-blockade with hydralazine in normal humans.

To assess the effects of nonselective vs. beta 1-selective beta-blockade on the hyperdynamic circulation induced by hydralazine, eight healthy volunteers received placebo, propranolol, 20 and 40 mg, and atenolol, 25 and 50 mg, on 5 separate days, followed by hydralazine (range 75 to 150 mg). Hydralazine decreased afterload (end-systolic wall stress) and increased venous return and left ventricular performance (by M-mode echocardiography). Both beta-blockers blunted the increases in heart rate, cardiac output, and venous return similarly, although heart rate and cardiac output were not completely normalized. Atenolol did not affect the hydralazine-induced decrease in afterload, whereas propranolol significantly opposed this change (P less than 0.03). The hyperdynamic circulation seen with hydralazine is mostly beta mediated, primarily beta 1. When given with hydralazine the two beta-blocker types differ primarily in their effects on afterload.

Hydralazine inhibits vascular reactivity by a mechanism independent of vascular prostaglandin biosynthesis: role of thromboxane synthetase in blocking hydralazine actions.

To explore the mechanism of action of hydralazine on vascular reactivity of small vessels we have examined its actions in the isolated perfused mesenteric vascular bed. In buffer perfused preparations hydralazine inhibited responses to nondepolarizing stimuli at concentrations comparable to those achieved in vivo. Inhibition of cyclo-oxygenase activity enhanced hydralazine's action as did inhibition of thromboxane synthetase. Hydralazine stimulated mesenteric vascular bed prostaglandin biosynthesis (6-keto PGF1a and PGE2 determined by radio-immunoassay) and stimulated aorta PG12 synthesis (monitored by platelet bioassay). Extracellular calcium opposed hydralazine's action by a mechanism sensitive to cyclo-oxygenase inhibition. Concentrations of hydralazine substantially greater than those effective in the perfused vascular bed were required to demonstrate inhibition of platelet aggregation and ram seminal vesicles cyclooxygenase activity. These data indicate: Hydralazine acts directly on the smooth muscle to attentuate responses to nondepolarizing stimuli. Hydralazine does not inhibit vascular reactivity by a PG12 dependent mechanism although it stimulates prostaglandin biosynthesis. Reduction of vascular bed prostaglandin and thromboxane A2 biosynthesis enhances hydralazine actions. Hydralazine appears to act at a thromboxane A2 sensitive site however it is not a nonselective prostaglandin antagonist. Hydralazine is effective at concentrations which do not inhibit either platelet aggregation or ram seminal vesicle cyclooxygenase. These data suggest that hydralazine is a potent direct acting vasodilator which stimulates prostaglandin biosynthesis and whose potency may in turn be attenuated by the production of proconstrictory prostaglandins.

[Effect of an antihypertensive drug, budralazine, on the cerebral circulation in spontaneously hypertensive rats].

Budralazine was evaluated for its effect on the cerebral blood flow (CBF) in comparison with some antihypertensive drugs in spontaneously hypertensive rats (SHR). The oral doses for each drug were selected to reduce arterial blood pressure to near normotensive levels. Equihypotensive rats were also produced by a controlled-hemorrhage and used as a control group. The blood flows in the parietal cortex and caudate nucleus were measured using the hydrogen clearance method at the time points when the pressure levels averaged about 120 mmHg after administration of each of the drugs or controlled-hemorrhage. Budralazine (40 mg/kg) significantly increased the regional CBF by approximately 60% with a significant decrease in the cerebral vascular resistance. A similar effect was also observed with hydralazine (9 mg/kg). The CBF-increasing effect of nifedipine (7 mg/kg) was less potent than that of budralazine. Neither prazosin (6 mg/kg) nor alpha-methyldopa (1,000 mg/kg) increased the regional CBF. Such cerebrovascular responses were also observed with controlled-hemorrhage. Furthermore, budralazine given intravenously (3-10 mg/kg) caused a significant and dose-dependent increase (50-250%) in the regional CBF without affecting the arterial blood pressure. This effect of budralazine was partially attenuated by the pretreatment with either reserpine or 6-hydroxydopamine. It is possible that catecholaminergic control of cerebrovascular tone is at least in part involved in the mechanism whereby budralazine increases the CBF.

Pharmacological analyses of hydralazine-induced cardiac action in intact dogs and isolated, blood-perfused canine atria.

The effects of hydralazine (HYD) on heart rate and blood pressure in the intact dog and on atrial rate and contractile force in the isolated atrium were investigated. HYD (0.1-1 mg) injected into the sinus node artery produced double peaked positive inotropic and negative chronotropic effects in a dose-related manner. The initial positive inotropic and negative chronotropic responses were not affected by propranolol and atropine, respectively. The second positive inotropic response was inhibited by propranolol or reserpine, but it was not suppressed by imipramine or tetrodotoxin. When HYD (0.1-1 mg/kg) was administered intravenously to the donor dog, an initial increase followed by a decrease in blood pressure and an increase in heart rate were observed. In the isolated atrium, an increase in contractile force was induced. The increases of blood pressure and heart rate in the donor dog and the positive inotropic effect in the isolated atrium after HYD treatment were suppressed by reserpine. These results suggest that HYD has direct positive inotropic and negative chronotropic effects and indirect cardiac stimulating effects caused by a release of catecholamines from sympathetic nerve terminals, and that HYD-induced catecholamine release is not mediated by a tyramine-like action or via nerve excitation.

Vasodilative effect of hydralazine in awake dogs: the roles of prostaglandins and the sympathetic nervous system.

The relative roles of prostaglandins and the sympathetic nervous system in mediating the hypotensive effects of hydralazine were studied in awake dogs with and without pretreatment with indomethacin, propranolol, and phentolamine. In normal dogs, mean aortic pressure decreased 23 +/- 4 mm Hg after administration of hydralazine (cumulative dose of 0.8 mg/kg). This hypotensive effect of hydralazine was potentiated by phentolamine but was abolished by propranolol. Indomethacin caused a paradoxic pressor response (11 +/- 3 mm Hg) to hydralazine, which also was abolished by addition of phentolamine. Hydralazine produced vasodilation in the coronary, skeletal muscle (quadriceps), splanchnic, and renal circulations in normal dogs. The increase in coronary blood flow was associated with increased cardiac oxygen consumption and narrowed arteriovenous oxygen difference across the heart. Propranolol reduced the increases in cardiac oxygen consumption and coronary blood flow, but only indomethacin abolished the narrowed arteriovenous oxygen difference, suggesting that the increase in coronary blood flow was related to both the increased cardiac oxygen demand and prostaglandin-mediated active coronary vasodilation. The decrease in skeletal muscle vascular resistance after hydralazine was abolished by propranolol. Skeletal muscle vascular resistance actually increased after administration of hydralazine in dogs pretreated with both propranolol and indomethacin. These effects were blocked by the addition of phentolamine. Unlike the normal response, renal and splanchnic vascular resistances increased after administration of hydralazine in dogs pretreated with indomethacin. The splanchnic vasoconstriction was abolished by phentolamine, but the renal vascular change was affected by neither phentolamine nor propranolol. The results indicate that hydralazine does not produce uniform vasodilation in all organs and that the cardiovascular actions of hydralazine involve both prostaglandins and the sympathetic nervous system.

Hydralazine: mode of action at the neuroarterial junction.

1. Hydralazine relaxes the rat tail artery by a direct action on vascular smooth muscle cells, which appears to be modulated by the action of sympathetic nerve terminals. 2. There is a gradient of response to hydralazine in arteries from normotensive Wistar rats, the proximal segments being poorly responsive. This gradient disappears after denervation with 6-hydroxydopamine in vitro. 3. Exogenously added purines inhibit noncompetitively the vasodilator response to hydralazine in denervated segments from normotensive Wistar rats. Their order of potency is 2-Cl-adenosine > adenosine > ATP > inosine. 4. The effect of hydralazine in innervated, poorly responsive segments is greatly potentiated by theophylline (50 mumol/l) and propranolol (5 mumol/l). These results, together with the effect of denervation, suggest that there are endogenous purines leaking from the nerve terminals under our experimental conditions. 5. Hydralazine produces a marked inhibition of stimulus-induced contraction and 3H release after [3H]noradrenaline loading. The mechanism of this prejunctional action appears to be different from the mechanism of the postjunctional effect.

Inhibition of hydralazine-induced renin release by indomethacin in the rat.

Renal prostaglandins (PG) appear to mediate the release of renin due to activation of the intrarenal baroreceptor and stimulation of the renal sympathetic nerves. Since the vasodilator hydralazine is thought to stimulate renin release by both of these mechanisms, we examined the effect of indomethacin, a PG synthetase inhibitor, on hydralazine-induced renin release. Hydralazine increased the serum renin levels from 3.3 +/- 0.5 to 13.7 +/- 3.1 and 41.9 +/- 2.4 ng/ml/hr at the 1 and 10 mg/kg doses, respectively. Indomethacin inhibited this hydralazine-induced renin release by 100% at the 1 mg/kg dose and 36% at the 10 mg/kg dose even though the hypotensive effect of the drug was unaltered. Indomethacin (5 mg/kg) also suppressed urinary PGE2 excretion by 60% (p < 0.001). The beta-blocker, propranolol, was as effective as indomethacin in attenuating hydralazine-induced renin release. Additionally, propranolol blocked the tachycardia associated with hydralazine and slightly enhanced the hypotensive action of the drug. When indomethacin and propranolol were combined, no further inhibition of hydralazine-induced renin release was observed. Thus, renal PG's appear to be important as mediators of hydralazine-stimulated renin release but no hydralazine-induced vasodilatation.

Cardiovascular responses to mianserin hydrochloride: a comparison with tricyclic antidepressant drugs.

1. The cardiovascular responses of mianserin hydrochloride and tricyclic antidepressant drugs were investigated using non-invasive methods of cardiac investigation. A study of the interaction of mianserin and antihypertensive drug therapy is reported. 2. In six normal volunteers, mianserin hydrochloride 20 mg was shown to prolong the corrected Q-T interval at 150 min (P less than 0.001). It did not affect heart rate, systolic time intervals or the peak normalized derivative of the apexcardiogram. Amitriptyline 50 mg increased the corrected pre-ejection period interval (PEPI) and the PEP/left ventricular ejection time (LVET) ratio of the systolic time intervals at 150 min (P less than 0.001). Q-T interval was shortened at 90 minutes. 3. In a double-blind patient study, clomipramine increased heart rate, P-R interval, QRS and corrected Q-T interval in one patient at 2 weeks. Mianserin prolonged corrected Q-T interval at 1 week but this returned to the pretreatment time by 2 weeks in two patients. 4. In an open study, mianserin 20 mg three times daily did not antagonize the hypotensive action of propranolol or propranolol and hydrallazine in three patients. 5. In a double-blind study in three patients with desmethylimipramine 25 mg three times daily, mianserin 20 mg three times daily did not antagonize the hypotensive action of either guanethidine or bethanidine.