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1.  NO-independent regulatory site of direct sGC stimulators like YC-1 and BAY 41-2272 
BMC Pharmacology  2001;1:13.
The most important receptor for nitic oxide is the soluble guanylate cyclase (sGC), a heme containing heterodimer. Recently, a pyrazolopyridine derivative BAY 41-2272, structurally related to YC-1, was identified stimulating soluble guanylate cyclase in an NO-independent manner, which results in vasodilatation and antiplatelet activity. The study described here addresses the identification of the NO-independent site on soluble guanylate cyclase.
We developed a photoaffinity label (3H-meta-PAL) for the direct and NO-independent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 by introducing an azido-group into the tritium labeled compound. The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity. Irradiation with UV light of 3H-meta-PAL together with the highly purified sGC leads to a covalent binding to the α1-subunit of the enzyme. This binding is blocked by unlabeled meta-PAL, YC-1 and BAY 41-2272. For further identification of the NO-independent regulatory site the 3H-meta-PAL labeled sGC was fragmented by CNBr digest. The 3H-meta-PAL binds to a CNBr fragment, consisting of the amino acids 236–290 of the α1-subunit. Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the 3H-meta-PAL.
Our data demonstrate that the region surrounding the cysteines 238 and 243 in the α1-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators.
PMCID: PMC64637  PMID: 11801189
2.  Pharmacokinetics of artesunate after single oral administration to rats 
BMC Pharmacology  2001;1:12.
Artesunate is a commonly used antimalarial drug derived from artemisinin. It is rapidly converted to dihydroartemisinin. Little is known on this conversion in the GI tract and blood, and how this influences absorption. In order to study the absorption phase of the kinetics of artesunate following oral administration in rats, samples were collected at baseline, and then 0.5, 2, 5, 10, 15, 30, 45, 60 and 120 minutes after a single dose of 150 mg.
Peak concentration of parent artesunate and dihydroartemisinin was achieved within 5 and 37.5 +/- 8.7 min, respectively of start of administration through gavage. The half lives of absorption were 2.73 +/- 0.85 and 12.49 +/- 2.49 min, respectively.
These times were considerably shorter for artesunate than those found in studies which start sampling later. The profiles of parent compound and metabolite result from a complex equation dictated by the pH-dependent rates of hydroxylation of artesunate to dihydroartemisinin, the different rates at which either compounds are absorbed, and the catalytic hydroxylation by esterases. The rate of chemical oxidation of artesunate is pH dependent; this explains its rapid conversion to dihydroartemisinin in the stomach, as compared to its greater stability in other compartments at higher pH and in plasma. We propose that variable proportions of absorption take place in the stomach, and conclude that parent artesunate reaches an early peak within minutes of dosing, and that the early dihydroartemisinin levels result primarily from the absorption of the metabolite as such.
PMCID: PMC65525  PMID: 11835690
3.  Minipig cytochrome P450 3A, 2A and 2C enzymes have similar properties to human analogs 
BMC Pharmacology  2001;1:11.
The search for an optimal experimental model in pharmacology is recently focused on (mini)pigs as they seem not only to be an alternative source of cells and tissues for xenotherapy but also an alternative species for studies on drug metabolism in man due to similarities between (mini) pig and human drug metabolizing systems. The purpose of this work is to characterize minipig liver microsomal cytochromes P450 (CYPs) by comparing their N-terminal sequences with corresponding human orthologs.
The microsomal CYPs exhibit similar activities to their human orthologous enzymes (CYP3A4, nifedipine oxidation; 2A6, coumarin 7-hydroxylation; 2D6, bufuralol 1'-hydroxylation; 2E1, p-nitrophenol hydroxylation; and 2C9, tolbutamide hydroxylation). Specific minipig CYP (2A, 2C and 3A) enzymes were partially purified and proteins identified by immunostaining (using antibodies against the respective human CYPs) were used for N-terminal amino acid sequencing. From comparisons, it can be concluded that the sequence of the first 20 amino acids at the N-terminus of minipig CYP2A is highly similar to human CYP2A6 (70% identity). The N-terminal sequence of CYP2C shared about 50% similarity with human 2C9. The results on the minipig liver microsomal CYP3A yielded identical data with those obtained for amino acid sequences of the pig CYP3A29 showing 60% identity with human CYP3A4.
Thus, our results further support the view that minipigs may serve as model animals in pharmacological/toxicological studies with substrates of human CYP enzymes, namely, of the CYP3A and CYP2A forms.
PMCID: PMC60991  PMID: 11737866
4.  Peroxisome proliferator-activated receptor agonists prevent 25-OH-cholesterol induced c-jun activation and cell death 
BMC Pharmacology  2001;1:10.
Cholesterol oxides, the oxygenated derivatives of cholesterol, have been shown to cause programmed cell death in a variety of cell types. Using N9 microglia, this study was designed to investigate the molecular events induced by cholesterol oxides prior to the execution of programmed cell death.
Microglia were very sensitive to 25-OH-cholesterol, such that a 2-day treatment of the cells with 5 μM 25-OH-cholesterol reduced cell viability to 5–10% of controls. There was a dose- and time-dependent increase in c-jun and phospho-c-jun levels in microglia prior to this 25-OH-cholesterol induced cell death. In contrast, 7-β-OH-cholesterol, which was relatively non-toxic to microglia, did not increase phospho-c-jun levels. Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptors that have important roles in atherogenesis. Results from this study indicate that PPAR agonists such as 15d-PGJ2, indomethacin and WY14643 can attenuate cholesterol oxide induced c-jun activation and cell death in microglia.
Peroxisome proliferator-activated receptor agonists may be useful in future development of pharmacological agents against cholesterol oxide induced cytotoxicity.
PMCID: PMC60650  PMID: 11737865
5.  Receptorphin: A conserved peptide derived from the sequence of the opioid receptor, with opioid displacement activity and potent antiproliferative actions in tumor cells 
BMC Pharmacology  2001;1:9.
In addition to endogenous opioids, a number of peptide sequences, derived from endogenous (hemorphins, alphaS1-casomorphin), and exogenous proteins (casomorphins, exorphins) have been reported, possessing opioid activity. In the present work, we report the identification of a new peptide, receptorphin (Tyr-Ile-Phe-Asn-Leu), derived from the sequence of the second transmembrane loop of the opioid receptor. This sequence is unique for the opioid receptor, and conserved in all species and receptor-types.
Results and Discussion
Receptorphin competes for opioid binding, presenting a kappa-receptor interaction, while it binds equally to delta- and mu- opioid and somatostatin-binding sites, and inhibits the cell proliferation of a number of human cancer cell lines, in a dose-dependent and reversible manner, at the picomolar or the nanomolar range. Receptorphin shows a preferential action on prostate cancer cells.
Our work identifies, for the first time a peptide, in a receptor sequence, possessing ligand-agonistic activities. A hypothesis, based on receptorphin liberation after cell death, is presented, which could tentatively explain the time-lag observed during opioid antiproliferative action.
PMCID: PMC60649  PMID: 11737867
6.  Inability of Serotonin to Activate the c-Jun N-terminal Kinase and p38 Kinase Pathways in Rat Aortic Vascular Smooth Muscle Cells 
BMC Pharmacology  2001;1:8.
Serotonin (5-HT, 5-hydroxytryptamine) activates the Extracellular Signal-Regulated Kinase (ERK)/ Mitogen-Activated Protein Kinase (MAPK) pathways, in vascular smooth muscle cells. Parallel MAPK pathways, the c-Jun N-terminal Kinase (JNK) and p38 pathway, are activated by stimulators of the ERK/MAPK pathway. We hypothesized that 5-HT would activate the JNK and p38 pathways in rat vascular smooth muscle cells.
Results were determined using standard Western analysis and phosphospecific JNK and p38 antibodies. No significant activation by 5-HT (10-9 – 10-5 M; 30 min) of the JNK or p38 pathways, as measured by protein phosphorylation, was observed in any of these experiments. These experiments were repeated in the presence of the serine/threonine phosphatase inhibitor okadaic acid (1 uM) and the tyrosine phosphatase inhibitor sodium orthovanadate (1 uM) to maximize any observable signal. Even under these optimized conditions, no activation of the JNK or p38 pathways by 5-HT was observed. Time course experiments (5-HT 10-5 M; 5 min, 15 min, 30 min and 60 min) showed no significant activation of JNK after incubation with 5-HT at any time point. However, we detected strong activation of JNK p54 and p46 (5- and 7 fold increases in bands p54 and p46, respectively over control levels) by anisomycin (500 ng/ml, 30 min). Similarly, a JNK activity assay failed to reveal activation of JNK by 5-HT, in contrast to the strong stimulation by anisomycin.
Collectively, these data support the conclusion that 5-HT does not activate the JNK or p38 pathways in rat vascular smooth muscle cells.
PMCID: PMC58586  PMID: 11667949
7.  Effect of a short-term in vitro exposure to the marine toxin domoic acid on viability, tumor necrosis factor-alpha, matrix metalloproteinase-9 and superoxide anion release by rat neonatal microglia 
BMC Pharmacology  2001;1:7.
The excitatory amino acid domoic acid, a glutamate and kainic acid analog, is the causative agent of amnesic shellfish poisoning in humans. No studies to our knowledge have investigated the potential contribution to short-term neurotoxicity of the brain microglia, a cell type that constitutes circa 10% of the total glial population in the brain. We tested the hypothesis that a short-term in vitro exposure to domoic acid, might lead to the activation of rat neonatal microglia and the concomitant release of the putative neurotoxic mediators tumor necrosis factor-α (TNF-α), matrix metalloproteinases-2 and-9 (MMP-2 and -9) and superoxide anion (O2-).
In vitro, domoic acid [10 μM-1 mM] was significantly neurotoxic to primary cerebellar granule neurons. Although neonatal rat microglia expressed ionotropic glutamate GluR4 receptors, exposure during 6 hours to domoic acid [10 μM-1 mM] had no significant effect on viability. By four hours, LPS (10 ng/mL) stimulated an increase in TNF-α mRNA and a 2,233 % increase in TNF-α protein In contrast, domoic acid (1 mM) induced a slight rise in TNF-α expression and a 53 % increase (p < 0.01) of immunoreactive TNF-α protein. Furthermore, though less potent than LPS, a 4-hour treatment with domoic acid (1 mM) yielded a 757% (p < 0.01) increase in MMP-9 release, but had no effect on MMP-2. Finally, while PMA (phorbol 12-myristate 13-acetate) stimulated O2- generation was elevated in 6 hour LPS-primed microglia, a similar pretreatment with domoic acid (1 mM) did not prime O2- release.
To our knowledge this is the first experimental evidence that domoic acid, at in vitro concentrations that are toxic to neuronal cells, can trigger a release of statistically significant amounts of TNF-α and MMP-9 by brain microglia. These observations are of considerable pathophysiological significance because domoic acid activates rat microglia several days after in vivo administration.
PMCID: PMC59507  PMID: 11686853
8.  Antinociceptive and antiedematogenic properties and acute toxicity of Tabebuia avellanedae Lor. ex Griseb. inner bark aqueous extract 
BMC Pharmacology  2001;1:6.
Tabebuia avellanedae is a tree from the Bignoniaceae family. Commonly know as "pau d'arco" in Brazil, its inner bark is used as analgesic, anti-inflammatory, antineoplasic and diuretic at the Brazilian northeast. A validation of the plant usage has not been previously performed.
Antinociceptive and antiedematogenic effects of Tabebuia avellanedae Lor. ex Griseb. inner bark were measured by nociceptive experimental models in mice. A rat paw edema test induced by carrageenan (1%) was also performed in rats to access the plant's antiedematogenic effect. The inner bark aqueous extract, administered via oral in three different concentration, namely 100, 200 and 400 mg/Kg, reduced the nociception produced by acetic acid (0.6% in water, i.p.) by 49.9%, 63.7% and 43.8%, respectively. The aqueous extract (200 and 400 mg/Kg, p.o.) reduced formalin (1%) effects only at the second phase of the experiment by 49.3% and 53.7%, respectively. Naloxone (5 mg/Kg, i.p.) was not able to revert the extract effect, however caffeine (10 mg/Kg, i.p.) reverted its effect by 19.8% at the second phase of the formalin test. The aqueous extract (200 mg/Kg, p.o.) inhibited edema by 12.9% when we used the rat paw edema model. The acute toxicity was low in mice.
The T. avellanedae inner bark aqueous extract presented antinociceptive and antiedematogenic activities at the used models, with a possible antinociceptive effect associated to the adenosine system.
PMCID: PMC56902  PMID: 11574048
9.  Omapatrilat normalizes renal function curve in spontaneously hypertensive rats 
BMC Pharmacology  2001;1:5.
The present study was designed to analyze the chronic renal response to omapatrilat, a new vasopeptidase inhibitor, in spontaneously hypertensive rats (SHR). To that end, the renal and blood pressure response to a 4-day salt loading protocol was analyzed and the respective chronic renal curves constructed.
In non treated animals, and under normal sodium intake (around 2 mEq/day), mean arterial pressure (MAP), was significantly higher in the SHR as compared with the controls (WKY). After increasing salt intake (8 times normal), MAP did not change significantly in any group and the animals reached a normal sodium balance in four days. In a second group of animals, omapatrilat was given orally for 15 days at the dose of 40 mg/kg/day in the drinking water. In these omapatrilat-treated animals, and under normal sodium intake, MAP was significantly lower in both groups, although the antihypertensive effect was much greater in the SHR, so that the MAP of the SHR group was completely normalized and similar to the WKY-treated group. The subsequent elevation of sodium intake did not significantly elevate MAP in any group and the animals could manage the sodium excess as well as the non treated groups.
These results indicate that chronic treatment with omapatrilat normalizes blood pressure in SHR without affecting adversely the renal ability to eliminate a sodium load. Chronic treatment with omapatrilat resets the chronic pressure natriuresis relationship of the SHR to a normal level, thus without altering the normal salt-independence of this arterial hypertension model.
PMCID: PMC57752  PMID: 11592920
10.  Effects of two atypical neuroleptics, olanzapine and risperidone, on the function of the urinary bladder and the external urethral sphincter in anesthetized rats 
BMC Pharmacology  2001;1:4.
A previous report showed that the atypical neuroleptic clozapine resulted in marked changes in urodynamic parameters and greatly inhibited the activity of the external urethral sphincter in anesthetized rats. Such findings may help explain the high incidence of urinary disturbances reported during clozapine therapy. In an effort to extend our observations to other atypical neuroleptic agents, the present study investigated the effects of two newer atypical antipsychotics, olanzapine and risperidone, on the bladder and external urethral sphincter during cystometry in anesthetized rats.
At a dose of 0.1 mg/kg (i.v.), olanzapine decreased the micturition volume and increased the residual volume. In addition, olanzapine decreased the expulsion time and the amplitude of the high frequency oscillations observed during the expulsion phase. Larger doses (1 mg/kg) had a greater effect. Olanzapine also reduced the activity recorded from the external urethral sphincter, and the bursting observed during the expulsion phase was abolished by 1.0 mg/kg. Risperidone had similar effects although the maximal effects were smaller than those observed with olanzapine. The amplitude of bladder contractions elicited by electrical stimulation of the pelvic nerve was reduced by olanzapine but not risperidone suggesting a possible anti-muscarinic peripheral effect of olanzapine.
Olanzapine and risperidone significantly altered several voiding parameters and decreased the activity of the external urethral sphincter in the anesthetized rat. We propose that these effects are due to the central action of these drugs and not to peripheral effects. These findings may explain some of the clinical reports of urinary incontinence with risperidone and may predict similar occurrences with olanzapine therapy.
PMCID: PMC57001  PMID: 11580866
11.  Phorbol ester impairs electrical excitation of rat pancreatic beta-cells through PKC-independent activation of KATP channels 
BMC Pharmacology  2001;1:3.
Phorbol 12-myristate 13-acetate (PMA) is often used as an activating phorbol ester of protein kinase C (PKC) to investigate the roles of the kinase in cellular functions. Accumulating lines of evidence indicate that in addition to activating PKC, PMA also produces some regulatory effects in a PKC-independent manner. In this study, we investigated the non-PKC effects of PMA on electrical excitability of rat pancreatic β-cells by using patch-clamp techniques.
In current-clamp recording, PMA (80 nM) reversibly inhibited 15 mM glucose-induced action potential spikes superimposed on a slow membrane depolarization and this inhibition can not be prevented by pre-treatment of the cell with a specific PKC inhibitor, bisindolylmaleimide (BIM, 1 μM). In the presence of a subthreshold concentration (5.5 mM) of glucose, PMA hyperpolarized β-cells in a concentration-dependent manner (0.8–240 nM), even in the presence of BIM. Based on cell-attached single channel recordings, PMA increased ATP-sensitive K+ channel (KATP) activity. Based on inside-out patch-clamp recordings, PMA had little effect on KATP activity if no ATP was in the bath, while PMA restored KATP activity that was suppressed by 10 μM ATP in the bath. In voltage-clamp recording, PMA enhanced tolbutamide-sensitive membrane currents elicited by repetitive ramp pulses from -90 to -50 mV in a concentration-dependent manner, and this potentiation could not be prevented by pre-treatment of cell with BIM. 4α-phorbol 12,13-didecanoate (4α-PDD), a non-PKC-activating phorbol ester, mimicked the effect of PMA on both current-clamp and voltage-clamp recording configurations. With either 5.5 or 16.6 mM glucose in the extracellular solution, PMA (80 nM) increased insulin secretion from rat islets. However, in islets pretreated with BIM (1 μM), PMA did not increase, but rather reduced insulin secretion.
In rat pancreatic β-cells, PMA modulates insulin secretion through a mixed mechanism: increases insulin secretion by activation of PKC, and meanwhile decrease insulin secretion by impairing β-cell excitability in a PKC-independent manner. The enhancement of KATP activity by reducing sensitivity of KATP to ATP seems to underlie the PMA-induced impairment of β-cells electrical excitation in response to glucose stimulation.
PMCID: PMC55693  PMID: 11560763
12.  The effect of GABA receptor ligands in experimental spina bifida occulta 
BMC Pharmacology  2001;1:2.
The pathophysiology behind spina bifida and other neural tube defects (NTDs) is unclear. Folic acid is one variable, but other factors remain. Studies suggest that substances active at the GABA receptor may produce NTDs. To test this hypothesis pregnant rats were exposed to either the GABA a agonist muscimol (1, 2 or 4 mg/kg), the GABA a antagonist bicuculline (.5, 1, or 2 mg/kg), the GABA b agonist baclofen (15, 30, 60 mg/kg), or the GABA b antagonist hydroxysaclofen (1, 3, or 5 mg/kg) during neural tube formation. Normal saline was used as a control and valproic acid (600 mg/kg) as a positive control. The embryos were analyzed for the presence of a spina bifida like NTD.
After drug administration the pregnancies were allowed to proceed to the 21st day of gestation. Then embryos were removed and skeletons staining and cleared. Vertebral arch closure was measured. Results indicate that the GABAa receptor agonist muscimol, the GABAa receptor antagonist bicuculline, and the GABAb agonist baclofen produced NTDs characterized by widening of the vertebral arch. Oppositely the GABAb antagonist hydroxysaclofen produced narrowing of the vertebral arches.
The findings indicate that GABA a or b ligands are capable of altering neural formation. GABA may play a greater than appreciated role in neural tube formation and may be important in NTDs. The narrowing of the vertebral arch produced by the GABA b antagonist hydroxysalcofen suggests that GABA b receptor may play an undefined role in neural tube closure that differs from the GABA a receptor.
PMCID: PMC48147  PMID: 11532198
13.  Flow cytometry analysis of the effect of allopurinol and the dinitroaniline compound (Chloralin) on the viability and proliferation of Leishmania infantum promastigotes 
BMC Pharmacology  2001;1:1.
Leishmaniasis is a major parasitic disease in the tropical regions. However, Leishmania infantum has recently emerged as a very important cause of opportunistic infections for individuals positive for human immunodeficiency virus (HIV). However, there is a lack of in vitro tests for assessing the effect of anti-parasitic drugs on the viability and proliferation of Leishmania infantum. The aim of this study is to assess the efficacy of anti-parasitic drugs like allopurinol and Chloralin on the viability and proliferation of L. infantum promastigotes by utilizing two complementary flow cytometric approaches after exposure of the promastigotes to various concentrations of the drugs.
The density of the cultures in the presence and absence of allopurinol was determined by haemocytometer enumeration. The two flow cytometric approaches used to monitor the drug effect were: (i) a quantitative method to measure cell division using 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining and (ii) evaluation of cell viability by dual-staining with the membrane-permeable nuclear stain, SBRY-14 and propidium iodide. It was found that concentrations of allopurinol above 50 μg/ml yielded a clear decrease in the proliferation rate of the promastigotes. However, the viability results showed that about 46.8% of the promastigotes incubated in the presence of 800 μg/ml of allopurinol were still alive after 96 hours. In sharp contrast, more than 90% of promastigotes treated with Chloralin 10 μM (2.7 μg/ml) were dead after 48 hours of treatment. These flow cytometric findings suggest that allopurinol has a leishmaniostatic effect while the dinitroaniline compound (Chloralin) has a leishmaniocidal effect against promastigotes.
The flow cytometric data on proliferation and viability were consistent with results obtained from haemocytometer counts and allowed us to develop a model for assessing in vitro the effects of medicaments like allopurinol and chloralin on L. infantum promastigotes on a cellular level.
PMCID: PMC30939  PMID: 11299045

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