Effects of verapamil on gastric acid secretion in vitro and in vivo

The activity of H,K-ATPase, the gastric acid producing enzyme, was concentration dependently inhibited by verapamil in the mM range. Verapamil concentration dependently inhibited acid formation in gastric glands, measured as [14C]aminopyrine accumulation or oxygen consumption. The IC50 values were in the microM range. No inhibition of acid secretion by verapamil was observed in Heidenhain-pouch dogs and stomach-lumen-perfused rats. However, in pylorus-ligated rats an inhibition was observed, this effect is related to its cardiovascular effectiveness. To understand the action of verapamil, its physicochemical properties were considered. Verapamil is a highly lipophilic base with a pKa of 8.7. It accumulates in membranes and in the acidic spaces of the parietal cell. We suggest that the inhibition of vesicular bound H,K-ATPase is dependent on a non-specific accumulation of verapamil in the membrane (detergent effect) and that inhibition of acid production in vitro is due to an additional accumulation of the drug in acidic compartments, leading to an impaired function of the proton pump. Verapamil does not decrease acid secretion in vivo by this mechanism as the required dose would be higher than the dose that causes a strong depression of the cardiovascular system.     

Effects of verapamil, diltiazem, nisoldipine and felodipine on sarcoplasmic reticulum

Verapamil, diltiazem, nisoldipine and felodipine, calcium antagonist drugs with different chemical structures, were studied for their effects on activities of sarcoplasmic reticulum (SR) isolated from dog cardiac and rabbit skeletal muscles. Nisoldipine and felodipine exerted biphasic actions on both cardiac and skeletal SR Ca²⁺-ATPase with maximum activation of 40–60% occurring at 20–40 μM for nisoldipine and 30–40% occurring at 15–30 μM for felodipine. At higher drug concentrations, Ca²⁺-ATPase was inhibited. In the presence of oxalate the maximum activation of the Ca²⁺ uptake rates at 5–20 μM nisoldipine were 30–50% for cardiac SR and 80–100 μM of the drug were 300–500% for skeletal SR. Felodipine inhibited the rate of Ca²⁺ uptake by dog cardiac SR, but activated Ca²⁺ uptake by rabbit skeletal SR with a maximum of 30–50% at 12–25 μM. At higher concentrations of the two drugs the rate of Ca²⁺ uptake was inhibited. In the absence of oxalate, i.e., limited tranport, nisoldipine shortened the duration of time that Ca²⁺ was bound to the cardiac and skeletal SR, while the rate of release of Ca²⁺ from skeletal SR was stimulated. Felodipine at low concentrations similarly caused a premature release of Ca²⁺ from skeletal SR at a rapid rate; at high concentrations both drugs did not alter Ca²⁺ binding but delayed Ca²⁺ release. Unlike nisoldipine and felodipine, verapamil and diltiazem inhibited the rates of Ca²⁺ transport both in cardiac and skeletal SR. The two drugs inhibited Ca²⁺-ATPase in cardiac SR but activated the enzyme in skeletal SR. Thus, these drugs caused complex and different effects on cardiac and skeletal SR, possibly resulting from perturbations of the lipid environment of the SR Ca²⁺-ATPase.     

Expression of matrix metalloproteinase-9 in human platelets: regulation of platelet activation in in vitro and in vivo studies

1. The aim of this study was to identify the presence of matrix metalloproteinase-9 (MMP-9) in human platelets and systematically examine its inhibitory mechanisms of platelet activation. 2. In this study, we report on an efficient method for the quantitative analysis of pro-MMP-9 in human platelets using capillary zone electrophoresis (CZE). To elucidate subcellular localization of MMP-9 in human platelets, we investigated intraplatelet MMP-9 by immunogold labeling and visualized it using electron microscopy. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium. 3. MMP-9-gold labeling was observed on the plasma membrane, alpha-granules, open canalicular system, and within the cytoplasma both in resting and activated platelets. Furthermore, activated MMP-9 concentration-dependently (15-90 ng ml(-1)) inhibited platelet aggregation stimulated by agonists. Activated MMP-9 (21 and 90 ng ml(-1)) inhibited phosphoinositide breakdown, intracellular Ca(2+) mobilization, and thromboxane A(2) formation in human platelets stimulated by collagen (1 microg ml(-1)). In addition, activated MMP-9 (21 and 90 ng ml(-1)) significantly increased the formation of nitric oxide/cyclic GMP. 4. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12, 13-dibutyrate (PDBu) (60 nm). This phosphorylation was markedly inhibited by activated MMP-9 (21 and 90 ng ml(-1)). Activated MMP-9 (1 microg g(-1)) significantly prolonged the latency period of inducing platelet plug formation in mesenteric venules. 5. These results indicate that the antiplatelet activity of activated MMP-9 may be involved in the following pathways. (1) Activated MMP-9 may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown, protein kinase C activation, and thromboxane A(2) formation, thereby leading to inhibition of intracellular Ca(2+) mobilization. (2) Activated MMP-9 also activated the formation of nitric oxide/cyclic GMP, resulting in inhibition of platelet aggregation. These results strongly indicate that MMP-9 is a potent inhibitor of aggregation. It may play an important role as a negative feedback regulator during platelet activation.     

Dose-dependent inhibition of phosphoinositide metabolism in human platelets by aspirin in vitro and in vivo.

The effects of aspirin on the metabolism of phosphoinositides in human platelets were studied in vitro and in vivo. Eight volunteers received, at two-weekly intervals, a single dose of 10, 30, 100 or 600 mg aspirin. Before the first dose platelets were taken and incubated in vitro with a range of concentrations (10 nM-100 microM) of aspirin. Formation of inositol phosphates (IP) was measured in [3H]-inositol labelled platelets after incubation with collagen and thrombin for 30 min, a time at which a maximal increase in [3H]-IP was observed. The in vitro IC50 for inhibition of the response to collagen by aspirin was approximately 1 microM; the in vivo ID50 was 40-50 mg. Aspirin did not fully inhibit the collagen stimulated IP formation either in vitro or in vivo, and the response to thrombin was unaffected. The ID50 and IC50 of aspirin is thus in accord with the doses of aspirin associated with inhibition of platelet aggregation and thromboxane production in other studies. The possible relevance of these data to the clinical uses of aspirin is discussed.     

间尼索地平和尼索地平对兔离体心肌和血管平滑肌的作用(英文)

应用离体兔心肌和血管比较间尼索地平(m—Nis)和尼索地平(Nis)的负性变时、负性变力及对动、静脉收缩的抑制作用.m—Nis对离体右心房自发收缩频率和左心房及右心室乳头肌由电刺激所致收缩的抑制作用均较Nis为弱.两药对胸主动脉环收缩的抑制作用相似,而m—Nis对门-肠系膜静脉的抑制作用弱于Nis.表明m—Nis对血管选择性高,对动脉收缩的抑制作用强于静脉.     

Lipolytic activity of cis-beta-carboxyacrylamidine: studies in vivo and in vitro.

cis-β-Carboxyacrylamidine, a product isolated from the fermentation broth of the actinomycete, Streptomyces fur longus var.fur longus nov. sp., stimulated lipolysis in rat adipose tissue and isolated fat cells. Lipid-mobilizing activity in vivo was indicated by the increase in plasma FFA concentrations that occurred after the compound was administered orally. Daily doses reduced body weight gain but after approximately 5 days the rats became resistant to this effect of the compound. Resistant animals still responded to the lipid-mobilizing activity of the agent with an increase in plasma FFA levels. cis-β-Carboxyacrylamidine did not increase cyclic AMP concentrations in incubated adipose tissue. Also it neither stimulated adenylate cyclase or cyclic AMP-dependent protein kinase nor inhibited cyclic AMP phosphodiesterase or phosphoprotein phosphatase. The lipolytic mechanism was found to differ from the mechanism of N-ethylmaleimide. Also cis-β-carboxyacrylamidine inhibited soluble hormone-sensitive lipase, suggesting that soluble hormone sensitive lipase may not be the lipase mainly responsible for the response of adipose tissue and isolated fat cells to lipolytic hormones.     

Effects of rociverine on the human ureter: in vivo and in vitro experimental study

An in vitro and in vivo study was conducted to verify the effects of rociverine--a new spasmolytic agent--on peristalsis in the human ureter. In vitro (human ureter strips), rociverine exerted a spasmolytic activity both on the baseline motility and on the spasm induced by direct contractants (eledoisin, KCl) or by histamine. It may therefore be concluded that rociverine is a predominantly myotropic spasmolytic. In the in vivo study, conducted in patients with cutaneous ureterostomy, the drug showed a marked inhibitory effect on the amplitude and frequency of the ureteral rhythmic spikes, without affecting the baseline tone.     

Inhibition of platelet aggregation by verapamil: quantification by in vivo and in vitro techniques

Platelet aggregation appears to play a prominent role in myocardial ischemia. Verapamil, a slow-channel blocking agent with important antiarrhythmic and vasodilating actions, has been shown to inhibit in vitro platelet aggregation. We used an electronic particle size analyzer to evaluate the effects of verapamil on platelet aggregation in vitro and in vivo in 88 rats. The intravenous injection of verapamil (0.4 mg/kg) did not change the platelet count compared to control animals receiving an equal volume of normal saline (verapamil, 1.1 +/- 0.04 x 10(6)/mm3, vs. control, 1.2 +/- 0.09 x 10(6)/mm3, (p greater than 0.05). The mean size of platelet aggregates induced by adenosine diphosphate (0.2 microM), was reduced by verapamil (verapamil, 15.3 +/- 1.2 x 10(3) micron3 vs. control 24.4 +/- 2.7 x 10(3) micron; p less than 0.01). Platelet aggregates induced in vivo, following a standardized technique of extravasation of right iliac artery blood into the peritoneal cavity, were also smaller following verapamil infusion (verapamil, 12.6 +/- 1.1 x 10(3)micron3, vs control, 17.3 +/- 0.9 x 10(3) micron3 p less than 0.001). We conclude that verapamil exerts and inhibitory effect on platelet aggregation both in vitro and in vivo. This property may add an important new dimension to its potential therapeutic usefulness in ischemic heart disease.     

尼索地平微球在家兔体内释药的研究

目的:研究尼索地平微球在家兔体内的释药行为。方法:建立检测体内尼索地平血药浓度的高效液相色谱法,研究家兔肌注自制微球后的血药浓度经时变化情况。结果:微球在第1天有明显的突释效应,此后18 d内每天的血药浓度维持在4 μg·L~(-1)左右,波动范围较小。结论:微球在体内有良好的控释作用。     

In vitro and in vivo studies on the metabolism of tirofiban.

Tirofiban hydrochloride [L-tyrosine-N-(butylsulfonyl)-O-[4-(4-piperidinebutyl)] monohydrochloride, is a potent and specific fibrinogen receptor antagonist. Radiolabeled tirofiban was synthesized with either (3)H-label incorporated into the phenyl ring of the tyrosinyl residue or (14)C-label in the butane sulfonyl moiety. Neither human liver microsomes nor liver slices metabolized [(14)C]tirofiban. However, male rat liver microsomes converted a limited amount of the substrate to a more polar metabolite (I) and a relatively less polar metabolite (II). The formation of I was sex dependent and resulted from an O-dealkylation reaction catalyzed by CYP3A2. Metabolite II was identified as a 2-piperidone analog of tirofiban. There was no evidence for Phase II biotransformation of tirofiban by microsomes fortified with uridine-5'-diphospho-alpha-D-glucuronic acid. After a 1 mg/kg i.v. dose of [(14)C]tirofiban, recoveries of radioactivity in rat urine and bile were 23 and 73%, respectively. Metabolite I and unchanged tirofiban represented 70 and 30% of the urinary radioactivity, respectively. Tirofiban represented >90% of the biliary radioactivity. At least three minor biliary metabolites represented the remainder of the radioactivity. One of them was identified as I. Another was identified as II. When dogs received 1 mg/kg i.v. of [(3)H]tirofiban, most of the radioactivity was recovered in the feces as unchanged tirofiban. The plasma half-life of tirofiban was short in both rats and dogs, and tirofiban was not concentrated in tissues other than those of the vasculature and excretory organs.     

兔血小板释放的内源性5—HT对凝血酶诱导的血小板聚集和ATP释放的？…

AIM: To study the effects of arachidonic acid (AA)-induced endogenous serotonin (5-HT) release on platelet aggregation and ATP release by thrombin (Thr). METHODS: Platelet aggregation and release reaction were quantified by light transmission in platelet-rich-plasma (PRP) and the amount of ATP in medium. The effects of endogenous 5-HT were evaluated by the filtration of content in cuvette A (content A) containing endogenous 5-HT into cuvette B in which Thr-induced aggregation was observed in the absence/presence of ?(+/-)-5 (Z)-7-[3-endophenylsulfonylamino [2.2.1] bicyclohept-2-exo-yl]heptanoic acid, sodium salt? (S-145) or/and methysergide (Met). RESULTS: (1) AA 100 and 200 mumol.L-1 induced aggregation and ATP release in cuvette A. When the aggregation reached a peak, the content A directly caused platelet aggregation in cuvette B, and it was inhibited by S-145 100 nmol.L-1, Met 30 mumol.L-1, and inhibited more potently by S-145 + Met. (2) In the presence of S-145 100 nmol.L-1 in cuvette B, aggregations by Thr 0.1 and 0.3 IU.L-1 were enhanced (P < 0.01) by the filtrate, while Thr 0.5 IU.L-1-caused ATP release was suppressed (P < 0.01) without the effect on aggregation. Preincubation with S-145 and Met, the effects of the filtrate on aggregation and ATP release were abolished. (3) By prolongation of the time intervals between filtration and addition of Thr, the aggregation was enhanced and ATP release was reduced. CONCLUSION: Endogenous 5-HT was released from activated platelet and plays, in turn, a role in the regulation of platelet aggregation by the superimposition of cytosolic-free calcium ([Ca2+]i) and the feedback loop to regulate release reaction and calcium.     

Pathophysiology of cyanoginosin-LR: in vivo and in vitro studies

Cyanoginosin-LR, one of the group of virulent cyclic heptapeptide toxins (cyanoginosins) isolated from some strains of the cyanobacterium, Microcystis aeruginosa, kills mice within 1-2 hr after iv or ip injection. Although the liver is a target organ of the toxin, the rapidity of lethality is incompatible with metabolic death from failure of hepatocellular function. However, disintegration of sinusoidal endothelium causes massive intrahepatic hemorrhage. The loss of the structural integrity of hepatic sinusoids provides a previously undescribed mechanism for embolization of disintegrating cells from the liver to the lung. No injury to either cultured bovine pulmonary artery endothelial cells or mouse peritoneal macrophages was observed following prolonged incubation with high concentrations of the toxin, and there was no increase in vascular permeability to 125I-labeled albumin detected before intrahepatic hemorrhage. However, plasma fibronectin increased transiently after toxin injection. Acute, severe thrombocytopenia, a characteristic of cyanoginosin-LR toxicity, remains unexplained since platelets did not concentrate in the lungs, liver, or spleen. There are similarities between the effects of cyanoginosin-LR and of the lipopolysaccharide endotoxins, such as elevations of plasma levels of thromboxane B2 and 6-keto-prostaglandin F1 alpha.     

Effects of dihydropyridines and pyridines on multidrug resistance mediated by breast cancer resistance protein: in vitro and in vivo studies.

Breast cancer resistance protein (BCRP, ABCG2) is a recently identified member of the ATP-binding cassette family of cell surface transport proteins. This study was conducted to investigate the effect of a series of newly synthesized 1,4-dihydropyridines and pyridines, designed as potent P-glycoprotein inhibitors, on BCRP-mediated drug efflux both in vitro and in vivo. The effects of 25 synthesized dihydropyridines and corresponding pyridines along with 4 commercially available dihydropyridines (niguldipine, nicardipine, nifedipine, and nitrendipine) on the intracellular accumulation of the BCRP substrate mitoxantrone were evaluated in BCRP-expressing human breast cancer MCF-7/MX100 and human non-small cell lung cancer H460/MX20 cells. At a 2.5 microM concentration, 24 of 25 newly synthesized dihydropyridines and pyridines produced a significant increase of mitoxantrone accumulation in both cell lines. The most potent compound was able to enhance mitoxantrone accumulation approximately 4.5-fold, greater than that obtained with 10 microM fumitremorgin C, which is a specific BCRP inhibitor. The results from the two cell lines showed good correlation (r(2) = 0.71, p < 0.01). Niguldipine, nicardipine, and nitrendipine also demonstrated potent BCRP inhibition, whereas nifedipine had no effect. The effects of the dihydropyridine and pyridine compounds on mitoxantrone cytotoxicity paralleled their effects on mitoxantrone accumulation. Coadministration of a selected dihydropyridine compound, I(m) [DHP-014; 3-(3-(4,4-diphenylpiperidin-1-yl)propyl) 5-methyl 4-(3,4-dimethoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate)] with topotecan, a good BCRP substrate and a moderate to poor P-glycoprotein substrate, resulted in significant increases in the systemic exposure and peak concentration of topotecan in Sprague-Dawley rats when oral topotecan (2 mg/kg) was combined with 20 mg/kg DHP-014. The observed increase of topotecan exposure provides proof-of-concept for in vivo inhibition of BCRP by these agents.     

Effects of polyunsaturated fatty acids on human and rat cells. In vitro versus in vivo experiments

Human infant skin fibroblasts and liver cells were subcultured with 250 microM PUFA (polyunsaturated fatty acid), and primary cultures of glial brain cells from new-born rats with 100 microM; oleic acid was added to controls. Minimum essential medium (MEM) supplemented with bovine serum was used as a reference. During the short-term experiment (18-24 h), control liver cells showed a regular increase in protein level, while protein increment was more rapid in linoleic and especially in arachidonic acid-treated cells, but only for the first 3 hours. During the long-term experiment (7 d), control skin fibroblasts showed a faster growth rate (increase in number of cells) than reference or fibroblasts cultured with the added PUFAs. Lipid droplets were seen in the PUFA-treated liver cells and skin fibroblasts, and ultrastructural modifications were observed in fibroblasts, but without growth rate alteration. During the long-term experiment (2 w), control glial brain cells showed faster protein increment (measuring growth rate) than PUFA-treated cells, particularly than arachidonic acid-treated cells. HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) activity, determined after 6 h (liver cells) or 1 and 2 w (brain cells) of culture, was low in controls and reference, whilst higher in PUFA-treated-cells, and was especially high in arachidonic acid-treated brain cells. The present study indicates than the high HMGR activity may correspond to cultures of cells rapidly stopped in their protein increment, and to cultures of cells showing a slow rate of proliferation. This contrasts with results obtained from in vivo experiments; it also emphasizes the high mevalonate (MVA) level as a possible sign of nutritional medium imbalance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transdermal drug delivery systems of albuterol: in vitro and in vivo studies.

In vitro and in vivo experiments were conducted with double- and single-layer albuterol transdermal pads designed for once-a-day application. In the in vitro experiments, dissolution of albuterol from pads and permeation of albuterol through hairless mouse skin were monitored. In the in vivo experiments, pads were applied to the chest area of four female rhesus monkeys (Macaca mulata), and an albuterol aqueous solution was injected into the saphenous vein of the same animals in a crossover design. The amount lost from pads applied to monkeys was monitored by analysis of pad residue. Blood samples were withdrawn at regular intervals and analyzed by a high-performance liquid chromatography-fluorescence method. Skin irritation due to the pad was measured by a modified Draize score test. The amounts released from the two formulations were similar. The amount released was, however, dependent on the technique used and decreased in the following manner: pad dissolution greater than in vivo amount lost from pads applied to monkeys greater than in vitro permeation through hairless mouse skin. The pharmacokinetic parameters determined after intravenous and transdermal administration were as follows: terminal half-life, 2.26 +/- 0.45 h; apparent volume of distribution, 1935 +/- 37.2 mL.kg-1; and total body clearance, 612.0 +/- 118 mL.h-1.kg-1. The average concentrations in serum after application of single- and double-layer pads were 44.60 +/- 16.40 and 62.50 +/- 8.00 ng/mL, respectively. Further, the amount lost from pads applied to monkeys correlated with the respective amount absorbed in monkeys, as calculated from the average concentration in serum and clearance.(ABSTRACT TRUNCATED AT 250 WORDS)