Complexity of aortic rings constriction induced by endothelin to anticipate pharmacological effects on blood pressure

Complexity of aortic rings constriction induced by endothelin to anticipate pharmacological effects on blood pressure

Printemps R, Didier H, Le Grand B, Letzelter C, PhysioStim, Zone Industriellede Brénas, 81440 Lautrec, France.

Rat aortic ring Endothelin

Isolated aortic rings are frequently used to anticipate putative effects on blood pressure. However, the choice of the vasoconstrictive agent for the ex-vivo experiments might be taken carefully into account. Therefore, the aim of this study was to investigate whether blocking functional endothelin receptor activity could be a candidate for pharmacological evaluation of the aorta reactivity. In vitro, the effect of endothelin was determined by measurement of isometric contractions in isolated rat aortic arteries. Endothelium intact (E+) and de-endothelialized (E-) segments were examined after pre-incubated with a well-known endothelin ETa/ETb receptor antagonist: bosentan (from 1 to 10 µM). Relaxation of precontracted rings with endothelin was investigated with a cholinomimetic: carbachol (from 0.01 to 10 µM). Application of endothelin from 0.1 to 30 nM induced a concentration-dependent contraction of the rings in endothelium intact (E+) and de-endothelialized (E-) segments with an EC50 value respectively of 9.4 nM and of 5.1 nM. In de-endothelialized (E-) segments, bosentan induced a shift of the concentration dependent curve toward the elevated concentration of endothelin. For example, the EC50 value in the presence of bosentan 10 µM was 61.5 nM vs 5.1 nM without bosentan. This parallel shift of the curve clearly suggested a competitive antagonism against the ET receptor. Finally, carbachol induced a concentration dependent relaxation of endothelin contraction in endothelium intact (E+) segment with an EC50 of 0.3µM whereas it was devoid of significant effect in de-endothelialized (E-) segments. In conclusion, the present study indicates the existence of functional endothelin receptor activity in rat aorta artery, which is different in the endothelium (presumably ETb) and the smooth muscle layer (presumably a combination of ETa/ETb). This endothelin receptor activity could be blocked by bosentan confirming the involvement of ETa/ETb in the smooth muscle layer. Therefore, the heterogenous activity of endothelin receptors between endothelium and smooth muscle layer renders complex the interpretation of constrictive activity of endothelin with aorta. Thus, the experimentations with aortic rings to anticipate the effect of compound on endothelin induced regulation of blood pressure might be used carefully.


Cardiac toxicity comparison of Roundup® and glyphosate

Cardiac toxicity comparison of Roundup® and glyphosate on human Induced Pluripotent Stem cells derived cardiomyocytes

Printemps R, GuilbotS, Le Grand M, PhysioStim, Zone Industriellede Brénas, 81440 Lautrec, France.

Although pesticides are known to be key actors of modern agriculture,they are source of controversies due to their suspected hazardous health effects.
Roundup®, one of the most popularized pesticide, is an herbicide formulation based on an active ingredient, glyphosate, and adjuvants. The broad use of Roundup® through they ears in agricultural practices as well as home garden care led to large exposure of humans and mammalians to glyphosate. Evaluation of the safety and potential health risks from exposure to pesticides remains a major challenge.
Roundup® and glyphosate were compared using human cell based model to evaluate their impacts on cardiovascular function after exposition.

Materials and Methods

  • iCell® Cardiomyocytes2 were supplied by Cellular Dynamics (a Fujifilm company) and seeded on 48 well plate following manufacturers instruction and analyzed using the xCELLigence RTCA cardio ECR platform (simultaneous measurement of MEA and impedance).
  • 4 days after seeding, iCell2 cardiomyocytes were exposed during 24h to 5 different concentrations of Roundup® or glyphosate(from 0.01 to 100μM) prepared from stock solutions diluted in distilled water(final concentration of distilled water was 0.3%).
  • Roundup® solution used during this study is composed of 400g/L of glyphosate. Concentration of Roundup® presented here after correspond to the concentration of glyphosate present in the solution (Roundup® 10μM correspond to 10μM of glyphosate)
  • Data are expressed as Mean ± SD. Each concentration of compound was administrated in 4 wells.
  • The following parameters were monitored: Cell Index (CI), Amplitude of contraction, Beat rate (BR),Beating period(BP),Individual Beating Duration(IBD),Field Potential Duration(FPD) and FPD corrected by Fridericia(FPDc),Spike amplitude,Beating Rhythm Irregularity (BRI).

Results

Roundup®

Glyphosate

  • No effect of glyphosate from 0.01 μM to 100 μM on viability, contractility and electrical activity.
  • Roundup® up to 10 μM had no effect on viability and electrical activity, only as light decrease of amplitude of contraction was recorded (-11%) after 6 hand sustained within 24h at 10μM.
  • iPSC-CMs stopped beating during Roundup® 100μM.

conclusion

  • The absence of effects of glyphosate on cardiac function indicates that the cardiotoxicity observed with Roundup®could be attributed to Roundup®’sadjuvants.
  • This predictive and sensitive assay can be very useful for cardiotoxicity assessment of pesticides using a human cell based model. This work demonstrates that the evaluation of pesticides’ adjuvants is necessary to better address the safety of pesticides which can also be enlarged to a broader class of molecules.

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