Role of Sex Differences and Effects of Endothelial NO Synthase Deficiency in Responses of Carotid Arteries to Serotonin

Abstract

Abstract-We examined the hypothesis that contraction of the carotid arteries to serotonin is normally inhibited by endothelial NO synthase (eNOS) and is enhanced in mice lacking the gene for eNOS. Because the influence of eNOS may vary with the sex of the mouse, we also tested whether responses to serotonin were dependent on sex. We studied carotid arteries in vitro from littermate control (eNOS ϩ/ϩ ) mice, heterozygous (eNOS ϩ/Ϫ ) mice, and homozygous eNOS-deficient (eNOS Ϫ/Ϫ ) mice (male and female). Contraction to serotonin was greater in male eNOS ϩ/ϩ mice than in female eNOS ϩ/ϩ mice. In male mice, contraction to serotonin increased by Ϸ40% and 2.5-fold in male eNOS ϩ/Ϫ and eNOS Ϫ/Ϫ mice, respectively. Contraction to serotonin was more than doubled in female eNOS ϩ/Ϫ mice and increased Ͼ5-fold in arteries from eNOS Ϫ/Ϫ mice. In contrast, maximum vasoconstriction to U46619 was similar in male and female eNOS ϩ/ϩ , eNOS ϩ/Ϫ , and eNOS Ϫ/Ϫ mice. Relaxation to acetylcholine was not different in male and female eNOS ϩ/ϩ or eNOS ϩ/Ϫ mice but was absent in eNOS Ϫ/Ϫ mice. These findings suggest that the contraction of carotid arteries to serotonin is influenced by the sex of the animal. eNOS deficiency in gene-targeted mice is associated with enhanced contraction to serotonin, particularly in female mice, providing direct evidence that eNOS is a major determinant of vascular effects of serotonin. The results with eNOS ϩ/Ϫ mice suggest a "gene-dosing" effect for vascular responses to serotonin. Key Words: NO synthase Ⅲ acetylcholine Ⅲ thromboxane Ⅲ mice Ⅲ genetically altered mice T he endothelium can modulate vascular function by the production and release of a variety of vasodilator and vasoconstrictor agents. 1 NO released from the endothelium has been identified as a major endothelium-derived relaxing factor 2 that primarily produces relaxation of vascular muscle by the activation of soluble guanylate cyclase and cGMPdependent protein kinase I. 3 In addition, studies that used pharmacological approaches and gene-targeted mice suggest that NO is the primary mediator of endothelium-dependent relaxation in several blood vessels, including coronary, carotid, and cerebral arteries. 4 -9 In addition to mediating vasorelaxation, a second major role for endothelium is to inhibit responses of vascular muscle to vasoconstrictors. For example, contraction of coronary and cerebral arteries to serotonin is augmented after the removal of endothelium. 10,11 Mechanisms that may account for increased vasoconstrictor responses to serotonin after endothelial removal may include deficiency of NO or other endothelium-derived relaxing factors that oppose the direct contractile effect of serotonin on vascular muscle. Inhibitors of NO synthase (NOS) increase vasoconstriction and/or decrease vasodilation to serotonin, 11-13 but limitations related to the specificity of these pharmacological agents may complicate the interpretation of the studies. This is particularly true if one is attempting to define the role of specific isoforms of ...

Structure of Cerebral Arterioles in Mice Deficient in Expression of the Gene for Endothelial Nitric Oxide Synthase

“…We found previously that responses of carotid artery to acetylcholine, nitroprusside, and serotonin are altered in heterozygous eNOS-deficient mice, although to a lesser degree than in homozygous eNOS-deficient mice. 25,26 Based on this finding, we anticipated that some degree of cerebral arteriolar hypertrophy might develop in heterozygous, as well as homozygous, eNOS-deficient mice, although possibly to a lesser extent than in the homozygous group. Cross-sectional area of the vessel wall, however, was virtually the same in cerebral arterioles of heterozygous eNOS-deficient mice and wild-type mice.…”

“…We included the study of eNOS ϩ/Ϫ mice, because it has been observed previously that vascular responses to some stimuli are altered in eNOS ϩ/Ϫ mice. 25,26 In addition, experts in the field of genetics encourage the study of heterozygous deficient mice because, depending on the results, they may encourage the study of genetic polymorphisms in humans. 27 Genotyping of the animals was performed by Southern blotting or polymerase chain reaction from tail biopsy specimens as previously described.…”

“…27 Genotyping of the animals was performed by Southern blotting or polymerase chain reaction from tail biopsy specimens as previously described. 25,26,28,29 To determine whether effects of eNOS deficiency on cerebral arterioles resulted from increases in arterial pressure, separate groups of eNOS ϩ/ϩ and eNOS Ϫ/Ϫ mice (obtained from Jackson Laboratories) underwent unilateral carotid ligation at 4 weeks of age. Mice were anesthetized with Avertin (0.2 to 0.3 mL, intraperitoneally), shaved, and prepared with a 70% alcohol solution, and a ligature was tied tightly around the left common carotid artery.…”

Ceramide-Induced Impairment of Endothelial Function Is Prevented by CuZn Superoxide Dismutase Overexpression

“…These findings suggest that relaxation of the carotid artery to ACh in nontransgenic mice is normally mediated by NO and is consistent with our previous findings in eNOS-deficient mice. 28 In vessels treated with ceramide (1 mol/L), relaxation in response to higher concentrations of ACh was inhibited by Ϸ25%. For example, relaxation to 100 mol/L ACh was 92Ϯ6% and 71Ϯ6% in vehicle-treated and ceramide-treated (1 mol/L) vessels, respectively ( Figure 3A).…”

Mechanisms Responsible for Serotonin Vascular Reactivity Sex Differences in the Internal Mammary Artery

“…Nuno et al33 and Lamping and Faraci34 previously revealed male hypersensitivity to serotonin but not thromboxane A 2 in mice. However, no sex difference to serotonin responses was observed in studies using porcine vessels 35, 36.…”