Antiproliferative effect induced by novel imidazoline S43126 in PC12 cells is mediated by ROS, stress activated MAPKs and caspases.

BACKGROUND: Some imidazoline compounds have pleiotropic effects including cell death in vitro. We examined the antiproliferative action of a novel imidazoline compound S43126, and the role of the I1-imidazoline receptor, ROS, MAPKs and caspases in S43126-induced cell death. METHODS: PC 12 cells were treated with various concentrations of S43126 in the presence or absence of several ligands, and the effects on cell proliferation, ROS levels, and apoptosis were evaluated using Trypan Blue, Alamar Blue, Western blot and microscopy. RESULTS: We showed that S43126 reduced PC12 cell proliferation by greater than 50%, increased cell death by greater than 40% and increased apoptotic body formation. These effects were reversed by I1R-antagonist, efaroxan. S43126 also increased intracellular ROS levels by greater than 2.5-fold relative to vehicle-treated control. These effects were significantly inhibited by N-acetyl-cysteine. In addition, pharmacologic inhibitors of ERK, JNK and p38 MAPK, significantly reduced S43126-induced antiproliferative activity. Caspases 3, 8 and 9 were all activated in a time-dependent manner by S43126. Pan caspase inhibitor z-VAD-fmk, ameliorated the effects of S43126 on cell death and cell proliferation. CONCLUSION: Our data showed that the effects of S43126 on PC12 cell death were partly mediated by ROS production, MAPK and caspase activation. These results further indicate an emerging role for I1R in apoptotic processes.

Pharmacological properties of the central antihypertensive agent, moxonidine.

The sympathetic nervous system plays a central role in the pathophysiology not only of hypertension and other cardiovascular diseases but also metabolic disorders including disturbances of glucose and lipid homeostasis. A centrally acting sympathetic agent is therefore attractive not only for lowering blood pressure, but also intervening with multiple disease processes. Older agents such as clonidine and guanabenz have numerous side effects, including sedation and dry mouth that limit their acceptability to patients. Moxonidine and the related agent rilmenidine have greatly reduced side effects, because they have reduced activity at the α(2) -adrenergic receptors that mediate these undesirable actions. Instead, moxonidine and rilmenidine act primarily through a novel cellular site, termed the I(1) -imidazoline receptor. The molecular biology of the I(1) -imidazoline receptor protein has recently been described, and the cell signaling pathways linked to this protein have been characterized. Moxonidine has unique effects on a number of cell types through this unusual cellular site of action. There are multiple therapeutic implications of these cellular actions, especially for metabolic syndrome and its associated derangements in glucose and lipid metabolism. Finally, the clinical trials that seemed to identify an unfavorable outcome in severe heart failure are dissected and critiqued. We conclude that moxonidine and future successors to this agent could be of great value in treating multiple chronic diseases.

Novel I1-Imidazoline Agonist S43126 Augment Insulin Secretion in Min6 Cells.

The I1-imidazoline receptor is a novel drug target for hypertension and insulin resistance which are major disorders associated with Type II diabetes. In the present study, we examined the effects of a novel imidazoline agonist S43126 on calcium fluxes and insulin secretion from Min6 ß-cells. We also examined the effects of S43126 on the induction of IRAS, and phosphorylation of components in the I1-imidazoline signaling pathways, namely ERK and PKB. Min6 ß-cells were treated with varying doses of S43126 [10-8M to 10-5M] for various time (5-60mins). S43126 at higher dose [10-5M] stimulated insulin secretion under elevated glucose concentration compared to basal. In addition, insulin secretion and Ca2+ influx mediated by S43126 [10-5M] were decreased following co-treatment with efaroxan (I1-antagonist) and nifedipine (L-type voltage-gated Ca2+-channel blocker) at various times (5-60mins). Furthermore, S43126 at [10-5M] increased Ca2+ oscillation, [Ca2+] and 45Ca2+ uptake in a time and dose-dependent manner. Moreover, Western blot analysis of treated samples showed that S43126 caused an increased protein expression of IRAS as well as phosphorylation of both ERK1/2 and PKB in a concentration-dependent manner. We conclude that S43126 exerts its insulinotropic effect in a glucose dependent manner by a mechanism involving L-type calcium channels and imidazoline I1-receptors.

Novel I1-imidazoline S43126 enhance insulin action in PC12 cells.

The I(1)-imidazoline receptor is a novel target for drug development for hypertension and insulin resistance, major disorders associated with type 2 diabetes. In the present study, we examined the effects of a novel imidazoline agonist S43126, on phosphorylation of protein kinase B (PKB/Akt) and extracellular signal-regulated kinase (ERK1/2) in PC12 cells. We further examined the effects of S43126 on insulin stimulated PKB and ERK phosphorylation. PC12 cells were treated with varying doses of S43126 (10(-10) to 10(-6) M) or insulin (10(-10) to 10(-6) M) or combination treatment with insulin (10(-6) M) and varying doses of S43126 (10(-6) - 10(-11) M) for 10 min. Western blot analysis of treated samples showed that S43126 increased both ERK1/2 and PKB phosphorylation by 5 fold. Combination treatment with insulin (10(-6) M) and varying doses of S43126 (10(-6) - 10(-11) M) enhanced phosphorylation of PKB and ERK1/2 above the level of insulin alone, in a dose and time dependent manner. Treatment with siRNA against Nischarin (mouse homologue of I(1)-imidazoline receptor) reduced the phosphorylation of both ERK and PKB following combination treatments. These results indicate that S43126 has the potential to augment insulin action and should be further studied as a possible candidate drug for the treatment of insulin resistance states.

Centrally acting imidazolines stimulate vascular alpha 1A-adrenergic receptors in Rat-Tail Artery.

: 1. Centrally acting imidazoline antihypertensive agents clonidine and moxonidine also act peripherally to contract blood vessels. While these agents act at both I(1)-imidazoline and alpha 2 adrenergic receptors centrally, the receptor types by which they mediate contraction require further definition. We therefore characterized the receptor subtype by which these agents mediate contraction of proximal rat-tail artery. 2. Dose-response curves were determined for phenylephrine and for several imidazoline ligands, using endothelium denuded, isolated ring segments, of tail arteries from adult male Sprague-Dawley rats. Ring segments were mounted on a force transducer with platinum wires and immersed in a tissue bath containing Krebs solution, to which drugs could be added. Signals were digitized and recorded by a computer. 3. Tail artery contractions expressed as a percent of contraction to 106 mM potassium were phenylephrine (96%), moxonidine (88%), clonidine (52%), and UK14304 (30%). Neither rilmenidine nor harmane caused contraction. Contraction of tail artery to moxonidine or clonidine could be blocked by alpha 1 antagonist urapidil or prazosin, and also by alpha 1A subtype selective antagonist WB4101. Schild plots were generated and a calculated pA2 value of 9.2 for prazosin in the presence of clonidine confirms clonidine as an agonist at alpha 1A receptors in proximal segments of rat-tail artery. 4. Our work suggests that clonidine and moxonidine are promiscuous compounds at micromolar concentrations and that harmane and rilmenidine are more selective compounds for in vivo imidazoline research.

Moxonidine, an antihypertensive agent, is permissive to alpha1-adrenergic receptor pathway in the rat-tail artery.

To investigate whether alpha1-adrenergic receptors were involved in the contractile response of tail arteries to moxonidine, isolated ring segments of tail arteries from male adult Sprague-Dawley rats were studied. Moxonidine (EC50 = 1.3 microM) and the alpha1-agonist phenylephrine (EC50 = 2.5 microM) increased tension development in the rat-tail artery similarly. The response to moxonidine (1 microM) could be blocked by both alpha1-adrenoceptor blockers prazosin (IC50 = 1 nM), and urapidil (IC50 = 14 nM), and also by alpha2-adrenoceptor blockers, yohimbine (IC50 = 49 nM) and efaroxan (IC50 = 49 nM). Combination drug treatment (urapidil and yohimbine, or yohimbine and prazosin) was more effective in blocking the contractile response to moxonidine, than treatment with prazosin or urapidil alone. Comparison of pA2 values for prazosin in the presence of moxonidine (9.35) or phenylephrine (10.2) confirm that alpha1-adrenergic receptors are involved in the contractile response of rat-tail artery to moxonidine.

Apparent membrane pore-formation by Portuguese Man-of-war (Physalia physalis) venom in intact cultured cells.

Intracellular, ratiometric microfluorimetry with fura-2 reveals that low doses of Portuguese Man-of-war (Physalia physalis) venom cause a linear increase in intracellular calcium accumulation by cultured L-929 cells. The influx of calcium is preceded by a lag period that is relatively independent of venom concentration, except at very low concentrations. Electron micrographs of negatively stained preparations of membranes from venom-treated L-929 and GH(4)C(1) cells exhibit 10-80 nm diameter lesions. The number and diameter of these lesions correlate with venom concentration. The venom forms lesions in GH(4)C(1) cells at much lower concentrations than in L-929 cells. Osmotic protectants such as sucrose and polyethylene glycol (PEG), reduce the extent of lactate dehydrogenase (LDH) release from venom-treated cells with the higher molecular weight PEG causing a greater inhibition of LDH release than sucrose. These results imply that Man-of-war venom produces pore-like structures in the membranes of target cells, which leads to colloid osmotic swelling with subsequent release of intracellular proteins and cell lysis.