bq-123 and chelerythrine

Interstitial cells of Cajal (ICCs) are pacemaker cells that activate the periodic spontaneous depolarization (pacemaker potentials) responsible for the production of slow waves in gastrointestinal smooth muscle. Under current clamping, ICCs had a mean resting membrane potential of -58 ± 3 mV and externally applied ET produced membrane depolarization in a dosedependent manner. These effects were reduced by intracellular GDP beta S. A comparison of the concentration-dependent membrane depolarizations on pacemaker potentials to ET-1, ET-2 and ET-3 showed a rank order of potency ET-1≥ET-2≥ET-3 in cultured murine small intestinal ICCs. The pretreatment with Ca(2+)-free solution and thapsigargin, a Ca(2+)-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker potentials and suppressed the ET-1 induced membrane depolarizations. Chelerythrine and calphostin C, protein kinase C inhibitors or naproxen, an inhibitor of cyclooxygenase, did not block the ET-1 induced effects on pacemaker potentials. Pretreatment with BQ-123 (ET(A )receptor antagonist) or BQ-788 (ET(B )receptor antagonist) blocked the ET-1 induced effects on pacemaker potentials in cultured murine small intestinal ICCs. However, pretreatment with BQ-788 selectively did not block the ET-1 induced effects on pacemaker potentials in cultured murine large intestinal ICCs. Also, only externally applied selective ET(B )receptor agonist, IRL 1620 did not show any influence on pacemaker potentials in cultured murine large intestine ICCs. RT-PCR results indicated the presence of the ET(A )and ET(B )receptor in ICCs. These results suggested that ET-1 modulates pacemaker potentials through ET(A )and ET(B )receptor activation in murine small intestinal ICCs and ET(A )receptor activation in murine large intestinal ICCs by external Ca(2+) influx and internal Ca(2+) release via protein kinase C or cyclooxygenase-independent mechanism. Therefore, the ICCs are targets for ET and their interaction can affect intestinal motility.

Topics: Animals; Benzophenanthridines; Calcium; Calcium-Transporting ATPases; Cell Membrane; Cells, Cultured; Endothelin-1; Endothelin-2; Endothelin-3; Interstitial Cells of Cajal; Intestine, Large; Intestine, Small; Membrane Potentials; Mice; Mice, Inbred BALB C; Naproxen; Oligopeptides; Patch-Clamp Techniques; Peptides, Cyclic; Piperidines; Prostaglandin-Endoperoxide Synthases; Protein Kinase C; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Thapsigargin

A bioassay-guided fractionation of the 80 % ethanolic extract from Bocconia frutescens L. roots, showing a dose-dependent inhibitory effect towards both [(3)H]-angiotensin II and [(3)H]-BQ-123 binding to the human angiotensin II AT 1 and endothelin 1 ET(A) receptors, led to an alkaloidal subfraction as the only responsible fraction for the activity of the whole extract. Among the alkaloids present in this fraction sanguinarine and chelerythrine were significant inhibitors of [(3)H]-angiotensin II binding (hAT 1 receptor), with IC(50) values within the micromolar range. On the contrary, the [(3)H]-BQ-123 binding (ET(A) receptor) was only weakly inhibited. Moreover, other members of the isoquinoline alkaloid family such as chelidonine and some protoberberine alkaloids exhibited no affinity for the two receptors. The present work shows the possible structure-activity relationship for these benzophenanthridine alkaloids on a screening bioassay using both stably transfected Chinese hamster ovary (CHO) and the human neuroblastoma SK-N-MC cells. Furthermore, the ability of these compounds to block AT(1) and/or ET(A) receptors may provide some justification for the traditional use of Bocconia frutescens L. to control hypertension.

Topics: Alkaloids; Angiotensin II; Animals; Benzophenanthridines; Binding, Competitive; CHO Cells; Cricetinae; Dose-Response Relationship, Drug; Humans; Inhibitory Concentration 50; Isoquinolines; Ligands; Molecular Structure; Papaveraceae; Peptides, Cyclic; Phenanthridines; Plant Extracts; Plant Roots; Quantitative Structure-Activity Relationship; Receptor, Angiotensin, Type 1; Receptor, Endothelin A; Receptors, Angiotensin; Receptors, Endothelin; Tritium; Tumor Cells, Cultured

Cerebral arteries develop stretch-induced myogenic tone, which plays an important role in the regulation of blood flow to the brain. Although the effect of oxidized LDL (Ox-LDL) on many aspects of the vascular endothelial and smooth muscle cell function have been extensively investigated, its influence on myogenic activity has not been studied.. The effect of Ox-LDL on the myogenic tone that develops in the perfused rabbit posterior cerebral artery at intramural pressures between 40 and 90 mm Hg was examined.. Ox-LDL (10 microg/mL) significantly enhanced myogenic tone by 21.4+/-6.1% to 28.5+/-1.8% at 60 to 90 mm Hg pressure (P<0.05) but had no influence on norepinephrine- (0.5 to 1 micromol/L) and KCl (20 mmol/L)-induced constriction. Ox-LDL was effective whether the artery was exposed to it from the intraluminal or the extraluminal surface. Lysophosphatidylcholine (10 micromol/L), a lipid component of Ox-LDL, had an equivalent potentiating effect. Native LDL (100 microg/mL) was inactive. The myogenic tone-potentiating effect of Ox-LDL was abolished by endothelium removal but was not influenced by the NO synthase inhibitor N(G)-nitro-L-nitro-arginine methyl ester (50 micromol/L). This effect was reversed by the endothelin-1 (ET-1) antagonist BQ-123 (1 micromol/L). This concentration blocked 1 to 3 nmol/L ET-1-induced constriction without altering constriction induced by 40 mmol/L KCl. The potentiating effect was suppressed by the specific protein kinase C inhibitor chelerythrine (1 micromol/L).. Ox-LDL enhances myogenic tone through the release of ET-1 from the endothelium of the rabbit posterior cerebral artery.

Topics: Alkaloids; Animals; Benzophenanthridines; Blood Pressure; Cerebrovascular Circulation; Endothelin Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Enzyme Inhibitors; Lipoproteins, LDL; Lysophosphatidylcholines; Male; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Norepinephrine; Oxidation-Reduction; Peptides, Cyclic; Phenanthridines; Posterior Cerebral Artery; Potassium Chloride; Protein Kinase C; Rabbits; Vasoconstrictor Agents; Vasodilator Agents; Vasomotor System

Myocardial stretch is a well-known stimulus that leads to hypertrophy. Little is known, however, about the intracellular pathways involved in the transmission of myocardial stretch to the cytoplasm and nucleus. Studies in neonatal cardiomyocytes demonstrated stretch-induced release of angiotensin II (Ang II). Because intracellular alkalinization is a signal to cell growth and Ang II stimulates the Na+/H+ exchanger (NHE), we studied the relationship between myocardial stretch and intracellular pH (pHi). Experiments were performed in cat papillary muscles fixed by the ventricular end to a force transducer. Muscles were paced at 0.2 Hz and superfused with HEPES-buffered solution. pHi was measured by epifluorescence with the acetoxymethyl ester form of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM). Each muscle was progressively stretched to reach maximal developed force (Lmax) and maintained in a length that was approximately 92% Lmax (Li). During the "stretch protocol," muscles were quickly stretched to Lmax for 10 minutes and then released to Li; pHi significantly increased during stretch and came back to the previous value when the muscle was released to Li. The increase in pHi was eliminated by (1) specific inhibition of the NHE (EIPA, 5 micromol/L), (2) AT1-receptor blockade (losartan, 10 micromol/L), (3) inhibition of protein kinase C (PKC) (chelerythrine, 5 micromol/L), (4) blockade of endothelin (ET) receptors with a nonselective (PD 142,893, 50 nmol/L) or a selective ETA antagonist (BQ-123, 300 nmol/L). The increase in pHi by exogenous Ang II (500 nmol/L) was also reduced by both ET-receptor antagonists. Our results indicate that after myocardial stretch, pHi increases because of stimulation of NHE activity. This involves an autocrine-paracrine mechanism in which protein kinase C, Ang II, and ET play crucial roles.

Topics: Alkalies; Alkaloids; Amiloride; Angiotensin II; Animals; Anti-Arrhythmia Agents; Autocrine Communication; Benzophenanthridines; Cats; Endothelin Receptor Antagonists; Endothelins; Enzyme Inhibitors; Heart; Hydrogen-Ion Concentration; Losartan; Muscle Contraction; Muscle Fibers, Skeletal; Oligopeptides; Papillary Muscles; Paracrine Communication; Peptides, Cyclic; Phenanthridines; Protein Kinase C; Sodium-Hydrogen Exchangers

Protection from ischaemic preconditioning (IP) is dependent on activation of protein kinase C (PKC), and preconditionings protection can be mimicked by stimulation of various membrane receptors which are known to activate PKC. It is well known that KATP channel activation is cardioprotective. We tested the hypothesis that preischaemic treatment with endothelin-1 (ET-1) can protect against infarction by a PKC-dependent mechanism and by activating KATP channels.. Buffer-perfused isolated rat hearts were subjected to 30 min regional ischaemia and 120 min reperfusion. Risk zone was determined by fluorescent particles, and infarct size by TTC staining.. Treatment with ET-1 in a dose of 1 nM prior to ischaemia significantly reduced infarct size in % of the risk zone compared to the control group (infarct size: 14.1 +/- 2.6 vs. 41.9 +/- 3.4%), while ET-1 0.1 nM did not protect (infarct size: 40.9 +/- 3%). AS the protective dose of ET-1 resulted in a significant reduction of coronary flow, a control group with a similar preischaemic flow-reduction was included (infarct size: 48.1 +/- 4.2%). Both the nonselective ETA/ETB receptor antagonist bosentan (1 microM) and the ET(A)-receptor-selective antagonist BQ 123 (2 microM) abolished protection from ET-1 (infarct size: 43.3 +/- 3.5 and 41.3 +/- 3.3%, respectively), as did the PKC inhibitor chelerythrine (2 microM) (infarct size: 41.1 +/- 5.2%) and the KATP blocker 5-hydroxydecanoate (infarct size: 41.7 +/- 2.9%). None of the ET receptor antagonists bosentan and BQ-123 influenced infarct size alone (infarct size: 42.7 +/- 2.5 and 41.3 +/- 3.3%, respectively). IP, similarly to ET-1, reduced infarct size (infarct size: 6.1 +/- 1.4%), but the nonselective ET receptor antagonist bosentan did not interfere with preconditioning's protection (infarct size: 13.2 +/- 4.3%).. ET-1 treatment prior to ischaemia can protect against infarction via ETA receptors by a PKC-dependent mechanism and by activating KATP channels, but ET does not mediate IP in the isolated rat heart.

Topics: Alkaloids; Animals; Anti-Arrhythmia Agents; Benzophenanthridines; Bosentan; Coronary Circulation; Decanoic Acids; Endothelin Receptor Antagonists; Endothelins; Hydroxy Acids; Ischemic Preconditioning, Myocardial; Male; Myocardial Infarction; Myocardium; Peptides, Cyclic; Perfusion; Phenanthridines; Potassium Channel Blockers; Potassium Channels; Protein Kinase C; Rats; Rats, Wistar; Sulfonamides