In response to the specific question about relaxation to acetylcholine sometimes we find that, depending on the specific vessel, other muscarinic agonists are better including carbachol and bradykinin so these may be worth a try. Also with regard to oxygenation, buffer, temperature and pH - which solution are you using in the bath? Are you measuring temperature, oxygen tension and pH? What gas are you bubbling with?

Thank you

You mean that we used carbachol and bradykinin are better than Acetylecholine?

because we used Panlab 4 channel organ bath we didnot have problem with temperature, but we have problem with pH and oxygenation

we have two bottle one 100% Oxygen other 100% CO2, we used only Oxygen continuously but between 3 minute just only for one time we open CO2 100% mixing with Oxygen 100%, because we didnt have carbogen (95% O2 and 5% CO2)

therefore we have problem with Oxygenation and pH

thanks for reply

It depends on the blood vessel you are using but these are an alternative option to acetylcholine for studying endothelium dependent relaxation. Are you submaximally preconstricting with a vasoconstrictor before adding the acetylcholine? With regard to the gases I have always used Carbogen as 5% CO2 is required to maintain the buffer I use (Krebs ) at pH7.4. Therefore it is highly likely that your pH will vary if you are not using a premixed gas 95% O2 and 5%CO2.

By which pathway Higher concentrations of acetylcholine can have direct constrictor actions on rat aorta.

Acetylcholine would act on the M3 receptor subtype in smooth muscle cells activating a Gq coupled receptor leading to release of IP3 and DAG with subsequent release of calcium from the SR and vasoconstriction.

Thank you. I know the mechanism of M3 receptor stimulation by ACh but I have problem how can ACh with high concentration lead to contraction?

Thank you

I always use Carbogen as 5% CO2 and maintain the Krebs at pH7.4. I did not think you can mixt the gases during the experiment everyday in the same standart.

Also, it might be helpfull, to you, I prepare my Krebs usually in the same day, and check its pH before adding to reservoir several times per experiment.

I am happy with phenylephrine and Ach combination.

Please check the tissue bath temparature with conventional lab. thermomether, (put it inside the bath) Does electronic recorder says the right about the heat?

I agree that you need to take great care in tissue preparation, to avoid damage to the endothelium. Also, you will need to precontract using EC50-EC70 concentration of phenylephrine. If you are using a bicarbonate buffer then you need to bubble with 95% O2, 5% CO2 gas (carbogen) mixture. If you must use 100% oxygen because of non-availability of carbogen, then you will need to change your buffer to either TRIS or HEPES. Check the literature for these. Good kuck!

I'm used to work with PSS buffer, which has a bicarbonate concentration at 25 mM. It's possible to maintain a very stable pH with a gas mixture containing 10% CO2 in oxygen. But you need to have two sensitive flow meters for each gas.