Katrina Grosfils

Amperometric determination of choline released from rat submandibular gland acinar cells using a choline oxidase biosensor.

A choline (CHO) biosensor based on the determination of H(2)O(2) generated at the electrode surface by the enzyme choline oxidase (CHOx) was developed. The biosensor consisted of CHOx retained onto a horseradish peroxidase (HRP) immobilized solid carbon paste electrode (sCPE). The HRPsCPE contained the molecule phenothiazine as redox mediator and CHOx was physically retained on the electrode surface using a dialysis membrane. Several parameters have been studied such as, mediator amount, influence of applied potential, etc. The CHO measurements were performed in 0.1 M phosphate buffer, pH 7.4. Amperometric detection of CHO was realized at an applied potential of 0.0 mV vs Ag/AgCl. The response is linear over the concentration range 5.0x10(-7)-7.0x10(-5) M, with a detection limit of 1.0x10(-7) M. This biosensor was used to detect choline released from phosphatidylcholine (PC) by phospholipase D (PLD) in isolated rat salivary gland cells stimulated by a purinergic agonist (ATP).

Ramipril Prevents Endothelial Dysfunction Induced by Oxidized Low-Density Lipoproteins A Bradykinin-Dependent Mechanism

We wished to determine whether the acute toxic effects of oxidized LDL are attenuated in aortas isolated from rats chronically treated with an angiotensin-converting enzyme (ACE) inhibitor. In aortic rings incubated with human oxidized LDL (300 microg/mL), the endothelium-dependent relaxations to acetylcholine were attenuated, but not those to A23187 and to nitroprusside. This toxic effect of oxidized LDL was completely prevented in preparations coincubated with oxidized LDL and the nitric oxide (NO) precursor L-arginine (0.3 mmol/L). In aortas isolated from rats orally treated for 6 weeks with 10 mg/kg ramipril (group 1) or 1 mg/kg ramipril (group 2), this toxic effect of oxidized LDL was also markedly attenuated. In contrast, in aortas isolated from rats cotreated with ramipril (10 mg/kg) for 6 weeks and subcutaneous injections of Hoe 140 (a B2 kinin antagonist), 500 microg/kg per day for the last 2 weeks (group 3) or from rats orally treated for 6 weeks with losartan (an AT1-type angiotensin II receptor antagonist), 20 mg/kg (group 4), the inhibitory effect of oxidized LDL on acetylcholine-induced relaxations was similar to that observed in the control group (group 5). Moreover, long-term treatment with ramipril increased relaxations to acetylcholine in groups 1 and 2 and also relaxations to A23187 and aortic cGMP content in group 1, suggesting an enhanced NO availability. Thus, the protective effect of long-term ACE inhibition against the acute vascular toxicity of oxidized LDL is bradykinin dependent and seems to involve a facilitation of NO release via endothelial B2 kinin receptors.

Chronic Angiotensin-Converting Enzyme Inhibition and Endothelial Function of Rat Aorta

To determine whether chronic angiotensin-converting enzyme (ACE) inhibition produces functional changes in the aorta normotensive rats, four groups of rats were studied in parallel for 6 weeks. Group 1 orally received ramipril and beta 2-kinin antagonist HOE140 500 micrograms/kg per day s.c. by injection for the remaining 2 weeks; group 3, hydralazine 100 mg/kg per day PO for 6 weeks; group 4 (control), subcutaneous injections of saline solution during the last 2 of 6 weeks. In aorta isolated from group 1 the relaxations induced by bradykinin, acetylcholine, and histamine were significantly potentiated compared with those of group 4. In group 3, despite a decrease in systolic blood pressure similar to that induced by ramipril treatment, the responses to these three endothelium-dependent vasodilators were not different from those of group 4. In group 2, bradykinin-induced relaxations were completely abolished whereas acetylcholine-induced and histamine-induced relaxations were to those of group 4. The inhibitory effect of the endothelium on serotonin-induced contractions was significantly increased in preparations of group 1 compared with those of groups 2 through 4. Indirect measurements of nitric oxide formation such as contractions evoked by NG-monomethyl-L-arginine (L-NMMA) and aortic cGMP content were also significantly enhanced in preparations from group 1 compared with those of groups 2 through 4. Moreover, because the relaxations to nitroglycerin and nitroprusside were similar in groups 1, 2, and 4 an alteration of the guanylate cyclase activity by ramipril treatment is quite unlikely. Thus long-term treatment with ramipril potentiates the endothelium-dependent responses in the rat aorta by enhancing nitric oxide availability.

Regulation by thapsigargin and carbachol of the intracellular calcium concentration in rat submandibular glands.

1. Carbachol and thapsigargin both increased the intracellular calcium concentration in rat submandibular cells in the presence and in the absence of extracellular calcium. Depletion of intracellular calcium pools with thapsigargin prevented the response to carbachol. 2. The two agents also increased the influx of calcium. The muscarinic agonist stimulated the efflux of calcium outside the cell. 3. From these results it is concluded that submandibular cells possess several intracellular calcium pools sensitive to thapsigargin, among which some are sensitive to IP3. Depletion of these pools increase the uptake of extracellular calcium.

Inhibitory effect of caffeine on the response to carbachol in rat pancreatic acini

1. Caffeine did not affect by itself the amylase release, nor intracellular calcium concentration in rat pancreatic acini. 2. Concentration of caffeine higher than 1 mM inhibited the amylase released in response to the muscarinic agonist carbamylcholine. This inhibition was also seen on the intracellular calcium elevation: 20 mM caffeine inhibited by 80% the calcium elevation in response to 100 microM carbachol. 3. Caffeine also prevented the specific binding of [3H]-N-methylscopolamine ([3H]-NMS) on rat pancreatic muscarinic receptors. 4. We conclude that caffeine behaves as a muscarinic antagonist.

Mepacrine inhibits amylase secretion mediated by a guanylnucleotide binding protein.

1. In intact rat pancreatic acini, 10 nM bombesin, 100 nM phorbol ester TPA and 10 mM fluoroaluminate increased amylase secretion 5-, 3- and 4-fold respectively. 2. Mepacrine dose-dependently inhibited the response to bombesin and fluoroaluminate but did not affect the response to TPA. 3. In permeabilized acini, mepacrine inhibited the secretory response to GTP gamma S without modifying the response to TPA.