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Keyword:cecal ligation and puncture, hyperresponsiveness, hyporeactivity, septic shock, vascular dysfunction https://mc06.manuscriptcentral.com/cjpp-pubs AbstractWe evaluated the effects of phenylephrine, norepinephrine, angiotensin II and vasopressin in mesenteric, renal, carotid and tail arteries, and in perfused mesenteric vascular bed from rats subjected to the cecal ligation and puncture (CLP) model of sepsis. Phenylephrine and angiotensin II were less efficacious in mesenteric arteries from the CLP 6 h and CLP 18 h groups than in preparations from non-septic animals, but no differences were found for norepinephrine and vasopressin between the preparations. In renal arteries, none of the vasoconstrictors had impaired activity in the CLP groups. Nonetheless, carotid arteries from the CLP 18 h group presented reduced reactivity to all vasoconstrictors tested, but only phenylephrine and norepinephrine had their effects reduced in carotid arteries from the CLP 6 h group. Despite the reduced responsiveness to phenylephrine, tail arteries from septic rats were hyperreactive to vasopressin and norepinephrine at 6 h and 18 h after the CLP surgery, respectively. The mesenteric vascular bed from CLP groups was hyporeactive to phenylephrine, norepinephrine and angiotensin II, but not to vasopressin. The vascular contractility in sepsis varies from the well-described refractoriness, to unaltered or even hyperresponsiveness to vasoconstrictors, depending on the vessel, the vasoactive agent, and the time period evaluated.

We have shown that arteries present differential responses to α1 adrenergic receptors (α1ARs) agonists in sepsis (Bernardelli et al., Can J Physiol Pharmacol, 94, 1227–36, 2016). Indeed, in animals subjected to the cecal ligation and perforation (CLP) model of sepsis, activation of α1ARs by norepinephrine (NE) results in a small degree of hyporesponsiveness, compared with the responses generated by phenylephrine (PE). It is well known that increased amounts of nitric oxide (NO) are produced in sepsis. NO plays a crucial role in septic vasoplegia, so we hypothesized that α1ARs agonists could differentially modulate the activity of NO synthases (NOS). Here, endothelium‐intact aortic rings from male Wistar rats subjected to CLP were studied in organ baths at 6 h after the septic insult. Control experiments were conducted with arteries from naïve animals. The effects of increasing concentrations (1 nM–300 mM) of PE and NE were evaluated in preparations previously incubated with L‐NAME (a non‐selective inhibitor of NOS; 100 μM), 1400W (a selective inhibitor of the inducible isoform of NOS; 10 μM), or S‐Methyl‐L‐thiocitrulline (a selective inhibitor of the neuronal isoform of NOS; 10 μM). We also investigated the influence of sodium nitroprusside (SNP; 0.1 μM, during 30 min) in the responses of control arteries to PE and NE. Aortic sections were also evaluated in experiments using the fluorescent BODIPY® FL prazosin (QAPB) (250 nM) and DAF‐FM (10 μM). Incubation with L‐NAME increased the contractile effects of PE and NE in both the control and CLP group. On the other hand, both 1400W and S‐methyl‐L‐thiocitrulline had no influence in the effects of PE and NE in control preparations, and increased the responses to PE (but not to NE) from 1.31 ± 0.06 g in control to 2.24 ± 0.11 and 2.21 ± 0.14 g, respectively, in the CLP group (p < 0.05). The fluorescence of QAPB in aortic sections did not differ between the groups, regardless of the previous incubation with PE or NE, suggesting that there were no significant differences in the density of α1ARs between the groups. Besides, neither PE nor NE were able to increase NO production, as detected by the NO sensitive fluorescence probe DAF‐FM. Interestingly, incubation with SNP reduced the maximal contractile effect of NE and PE by 35 and 65%, respectively (p < 0.05). These results suggest that neither an impaired interaction of PE and NE with α1ARs nor a differential modulation of NO production account for the distinct degree of vascular hyporeactivity observed. Nevertheless, our findings reveal remarkable differences between the intracellular signaling activated by PE and NE, regarding their sensitivity to NO. The better comprehension of the behavior of α1ARs can allow the development of ligands with enhanced efficacy for the management of vasoplegia and hypotension in sepsis. Support or Funding Information Financial Support: CNPq (Brazil), with a Ph.D. fellowship to Bernardelli, A. K. This study was approved by the Animal Ethics Committee of UFSC (6327180718).

Endothelial cells (ECs) are electrically coupled to arterial smooth muscle cells in the vascular wall and can modulate their contractility through direct control of membrane potential and via the production of several vasoactive substances. ECs express several cation channels which are known to regulate arterial contractility. In contrast, physiological functions of anion channels in ECs are unclear. ECs express TMEM16A, a Ca2+-activated Cl-channel. To examine physiological functions of TMEM16A channels in ECs, we generated an inducible, EC-specific TMEM16A knockout (TMEM16A ecKO) mouse. An increase in intracellular Ca2+ concentration ([Ca2+]i) or the application of acetylcholine (ACh) activated Cl‑ currents in fresh-isolated mesenteric artery ECs of control ( TMEM16Afl/fl) mice that were inhibited by tannic acid or benzbromarone, which are TMEM16A channel blockers. In contrast, an increase in [Ca2+]i or ACh did not activate Cl‑ currents in TMEM16A ecKO ECs. HC067047, a TRPV4 channel blocker, inhibited TMEM16A current activation by ACh in TMEM16Afl/fl ECs. GSK101, a TRPV4 channel activator, stimulated TMEM16A currents in TMEM16Afl/fl ECs, but not in TMEM16A ecKO ECs. Super resolution single-molecule localization microscopy demonstrated that surface clusters of TMEM16A and TRPV4 channels locate in nanoscale spatial proximity in the plasma membrane of ECs. ACh produced membrane hyperpolarization in pressurized TMEM16Afl/fl arteries that was attenuated in TMEM16A ecKO arteries. Vasodilation to ACh was smaller in pressurized TMEM16A ecKO arteries than in TMEM16Afl/fl arteries. TMEM16A knockout in ECs increased blood pressure in mice. These data indicate that ACh activates TRPV4-coupled TMEM16A channels in ECs to induce membrane hyperpolarization, vasodilation and a reduction in blood pressure. National Institutes of Health This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.