Carotid dysfunction in senescent female mice is mediated by increased α1A-adrenoceptor activity and COX-derived vasoconstrictor prostanoids

Abstract

α-Adrenergic receptors are crucial regulators of vascular hemodynamics and essential pharmacological targets for cardiovascular diseases. With aging, there is an increase in sympathetic activation, which could contribute to the progression of aging-associated cardiovascular dysfunction, including stroke. Nevertheless, there is little information directly associating adrenergic receptor dysfunction in the blood vessels of aged females. This study determined the role of a-adrenergic receptors in carotid dysfunction of senescent female mice (accelerated-senescence prone, SAMP8), compared with a nonsenescent (accelerated-senescence prone, SAMR1). Vasoconstriction to phenylephrine (Phe) was markedly increased in common carotid artery of SAMP8 [area under the curve (AUC), 527 ± 53] compared with SAMR1 (AUC, 334 ± 30, P = 0.006). There were no changes in vascular responses to the vasoconstrictor agent U46619 or the vasodilators acetylcholine (ACh) and sodium nitroprusside (NPS). Hyperactivity to Phe in female SAMP8 was reduced by cyclooxygenase-1 and cyclooxygenase-2 inhibition and associated with augmented ratio of TXA2/PGI2 release (SAMR1, 1.1 ± 0.1 vs. SAMP8, 2.1 ± 0.3, P = 0.007). However, no changes in cyclooxygenase expression were seen in SAMP8 carotids. Selective α_1A-receptor antagonism markedly reduced maximal contraction, whereas α_1D antagonism induced a minor shift in Phe contraction in SAMP8 carotids. Ligand binding analysis revealed a threefold increase of α-adrenergic receptor density in smooth muscle cells (VSMCs) of SAMP8 vs. SAMR1. Phe rapidly increased intracellular calcium (Ca_i²⁺) in VSMCs via the α_1A-receptor, with a higher peak in VSMCs from SAMP8. In conclusion, senescence intensifies vasoconstriction mediated by α_1A-adrenergic signaling in the carotid of female mice by mechanisms involving increased Ca_i²⁺ and release of cyclooxygenase-derived prostanoids.NEW & NOTEWORTHY The present study provides evidence that senescence induces hyperreactivity of α₁-adrenoceptor-mediated contraction of the common carotid. Impairment of α₁-adrenoceptor responses is linked to increased Ca²⁺ influx and release of COX-derived vasoconstrictor prostanoids, contributing to carotid dysfunction in the murine model of female senescence (SAMP8). Increased reactivity of the common carotid artery during senescence may lead to morphological and functional changes in arteries of the cerebral microcirculation and contribute to cognitive decline in females. Because the elderly population is growing, elucidating the mechanisms of aging- and sex-associated vascular dysfunction is critical to better direct pharmacological and lifestyle interventions to prevent cardiovascular risk in both sexes.

Keywords: cerebrovascular function; common carotid; menopause; senescence-accelerated mice; α-adrenergic receptor.

Graphical abstract

Figure 1.

Senescence induced changes in adrenergic vasocontraction. Cumulative concentration-response curves to phenylephrine (Phe; A), U46619 (B), acetylcholine (ACh; C), and sodium nitroprusside (SNP; D) in endothelium-intact common carotid artery from 8-mo-old SAMR1 and SAMP8 females. Insets: graphs are means ± SE of the area under the curve calculated from each concentration-response curve (n = 6–11 mice/group). Statistical significance was calculated by the extra sum-of-squares F (fit of concentration-response curves) and t test with Welch’s correction (AUC). P values and comparisons are expressed on top of box and whiskers plots and by the curves. Significance is considered when P < 0.05. AUC, area under the crurve.

Figure 2.

Impact of senescence on endothelium-derived factors and phenylephrine (Phe) contraction. Cumulative concentration-response curves to Phe in endothelium-intact common carotid artery from 8-mo-old SAMR1 and SAMP8 females. The role of NO, ${\text{O}}_2^{-}$, and prostanoids in Phe-induced contraction was assessed by treatment with l-NAME (A and B), Tempol (C and D), and indomethacin (E and F). Insets: graphs are means ± SE of the area under the curve calculated from each concentration-response curve (n = 6–12 mice/group). G: delta difference in the AUC of basal vs. treated arteries. Statistical significance was calculated by the extra sum-of-squares F (fit of concentration-response curves) and t test with Welch’s correction (AUC). P values and comparisons are expressed on top of box and whiskers plots and by the curves. Significance is considered when P < 0.05. AUC, area under the crurve.

Figure 3.

Impact of senescence on COX-mediated signaling pathway. Cumulative concentration-response curves to phenylephrine (Phe) in endothelium-intact common carotid artery from 8-mo-old SAMR1 and SAMP8 females. The role of COX-1- and COX-2-derived prostanoids to Phe-induced contraction was assessed by treatment with selective COX-1 (A and C) or COX-2 (B and D) inhibitors. Insets: graphs are means ± SE of the area under the curve calculated from each concentration-response curve (n = 6–11 mice/group). The levels of prostacyclin (E), thromboxane A₂ (F), and the ratio of release (G) were determined after concentration-response curves to Phe. The expression of mRNA of COX-1 (H) and COX-2 (I) was determined in the common carotid of SAMR1 and SAMP8. Statistical significance was calculated by the extra sum-of-squares F (fit of concentration-response curves) and t test with Welch’s correction (AUC). P values and comparisons are expressed on top of box and whiskers plots and by the curves. Significance is considered when P < 0.05. AUC, area under the crurve.

Figure 4.

Role of senescence on a₁-adrenergic receptor-mediated contraction. Cumulative concentration-response curves to phenylephrine (Phe) in endothelium-intact common carotid artery from 8-mo-old SAMR1 and SAMP8 females. The contribution of specific α-adrenergic receptors to Phe-induced contraction was determined by pretreatment of arteries with α_1A-adrenergic antagonist (5-methyl-urapidil, n = 8) and α_1D-adrenergic antagonist (BMY 7378, n = 8). The antagonist concentrations of 5-methyl-urapidil (A) and BMY 7378 (B) to be used in the studies concertation were tested in the common carotid of SAMR1 and then tested as a single concentration (10⁻⁷ M) in the common carotid of SAMR1 (C and D) and SAMP8 (E and F). The density of α₁-adrenergic receptors was determined in the primary culture of VSMCs from SAMR1 and SAMP8 (G) and normalized by the amount of protein (H). Statistical significance was calculated by the extra sum-of-squares F (fit of concentration-response curves) and t test with Welch’s correction (receptor density and EC₅₀). P values and comparisons are expressed on top of box and whiskers plots and by the curves. Significance is considered when P < 0.05. VSMCs, smooth muscle cells.

Figure 5.

Senescence increases intracellular free calcium via the ɑ_1A-adrenergic receptor. A: schematic representation of protocol for determining intracellular free calcium (Ca_i²⁺) in response to Phe (10⁻⁷ M) in VSMCs of SAMR1 and SAMP8 female mice. B: time-course of Ca_i²⁺ in response to Phe stimuli (10⁻⁷ M) in VSMCs of SAMR1 and SAMP8, and in response to ionomycin, as a positive control. C and D: time course of Ca_i²⁺ in the absence and the presence of ɑ_1A antibody in VSMCs of SMAR1 (C) and SAMP8 (D). E: box and whiskers plots show the differences in the AUC of Ca_i²⁺ time course in all groups (n = 4 individual sample/group). Statistical significance was calculated by Brown Forsythe and Welch ANOVA, followed by Dunnett’s T3 post hoc analysis. P values and comparisons are expressed on top of box and whiskers plots. Significance is considered when P < 0.05. A was created using a licensed version of BioRender. AUC, area under the crurve; VSMCs, smooth muscle cells.