Calcium ion (Ca2+) influx through voltage-gated Ca2+ channels is important for the regulation of vascular tone. Activation of L-type Ca2+ channels initiates muscle contraction; however, the role of T-type Ca2+ channels (T-channels) is not clear. We show that mice deficient in the alpha1H T-type Ca2+ channel (alpha(1)3.2-null) have constitutively constricted coronary arterioles and focal myocardial fibrosis. Coronary arteries isolated from alpha(1)3.2-null arteries showed normal contractile responses, but reduced relaxation in response to acetylcholine and nitroprusside. Furthermore, acute blockade of T-channels with Ni2+ prevented relaxation of wild-type coronary arteries. Thus, Ca2+ influx through alpha1H T-type Ca2+ channels is essential for normal relaxation of coronary arteries.

The M5 muscarinic receptor is the most recent member of the muscarinic acetylcholine receptor family (M1-M5) to be cloned. At present, the physiological relevance of this receptor subtype remains unknown, primarily because of its low expression levels and the lack of M 5 receptor-selective ligands. To circumvent these difficulties, we used gene targeting technology to generate M 5 receptor-deficient mice (M5R ؊/؊ mice). M5R ؊/؊ mice did not differ from their wild-type littermates in various behavioral and pharmacologic tests. However, in vitro neurotransmitter release experiments showed that M 5 receptors play a role in facilitating muscarinic agonist-induced dopamine release in the striatum. Because M 5 receptor mRNA has been detected in several blood vessels, we also investigated whether the lack of M 5 receptors led to changes in vascular tone by using several in vivo and in vitro vascular preparations. Strikingly, acetylcholine, a powerful dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in M5R ؊/؊ mice. This effect was specific for cerebral blood vessels, because acetylcholine-mediated dilation of extracerebral arteries remained fully intact in M5R ؊/؊ mice. Our findings provide direct evidence that M 5 muscarinic receptors are physiologically relevant. Because it has been suggested that impaired cholinergic dilation of cerebral blood vessels may play a role in the pathophysiology of Alzheimer's disease and focal cerebral ischemia, cerebrovascular M 5 receptors may represent an attractive therapeutic target. M olecular cloning studies have revealed the existence of five molecularly distinct muscarinic acetylcholine receptor subtypes (M 1 -M 5 ) (1, 2). During the past decade, considerable progress has been made in delineating the physiological roles of the M 1 -M 4 muscarinic receptors (3, 4). In contrast, the physiological relevance of the M 5 receptor subtype, which is the most recent member of the muscarinic receptor family to be cloned (5, 6), remains unknown at present (7,8). However, expression of the cloned M 5 muscarinic receptor gene in cultured mammalian cells has shown that the encoded receptor protein is functional and efficiently couples to G proteins of the G q family, similar to the M 1 and M 3 receptor subtypes (5-8).Immunoprecipitation and in situ mRNA hybridization studies have demonstrated the presence of M 5 receptor protein͞mRNA in different areas of the brain including hippocampus, hypothalamus, and distinct midbrain regions (substantia nigra pars compacta and ventral tegmental area) (9-11). However, M 5 receptors are expressed at very low levels, representing less than 2% of the total muscarinic receptor population (M 1 -M 5 ) expressed in the brain (9). Interestingly, the use of highly sensitive reverse transcriptase-PCR (RT-PCR) techniques suggests that M 5 receptors are expressed in all major brain regions (12). More recently, M 5 receptors also have been detected in several peripheral tissues or cells including peripheral blood l...

We examined the vascular structure and endothelium-dependent relaxation in two genetic models of hypercholesterolemia: apolipoprotein E (apoE)-knockout mice and combined apoE/LDL receptor-double-knockout mice. Intimal area was increased markedly in proximal segments of thoracic aortas from apoE/LDL receptor-knockout mice [0.13 +/- 0.03 (mean +/- SE) mm2] compared with normal (C57BL/6J) mice (0.002 +/- 0.002 mm2, P < .05). Despite intimal thickening, the vascular lumen was not smaller in the aortas of apoE/LDL receptor-knockout mice (0.52 +/- 0.03 mm2) than in normal mice (0.50 +/- 0.03 mm2). In apoE-deficient mice, intimal thickening was minimal or absent, even though the concentration of plasma cholesterol was only modestly less than that in the double-knockout mouse (14.9 +/- 1.1 vs 18.0 +/- 1.2 mmol/L, respectively, P < .05). Relaxation of the aorta was examined in vitro in vascular rings precontracted with U46619. In normal mice, acetylcholine produced relaxation, which was markedly attenuated by the nitric oxide synthase inhibitor NG-nitro-L-arginine (100 microM). Relaxation to acetylcholine and the calcium ionophore A23187 was normal in apoE-deficient mice (in which lesions were minimal) but greatly impaired in the proximal segments of thoracic aortas of apoE/LDL receptor-deficient mice, which contained atherosclerotic lesions. Vasorelaxation to nitroprusside was similar in normal and apoE-knockout mice, with modest but statistically significant impairment in atherosclerotic segments of apoE/LDL receptor-knockout mice. In distal segments of the thoracic aorta of apoE/LDL receptor-deficient mice, atherosclerotic lesions were minimal or absent, and the endothelium-dependent relaxation to acetylcholine and calcium ionophore was normal. Thus, in apoE/LDL receptor-knockout mice (a genetic model of hyperlipidemia), there is vascular remodeling with preservation of the aortic lumen despite marked intimal thickening, with impairment of endothelium-dependent relaxation to receptor- and nonreceptor-mediated agonists. Atherosclerosis may be accelerated in the apoE/LDL receptor-double-knockout mouse compared with the apoE-knockout strain alone. We speculate that other factors, such as the absence of LDL receptors, may contribute to the differences in the extent of atherosclerosis in these two models of hyperlipidemia.

Studies of the coronary circulation have divided vascular resistances into three large components: large vessels, small resistance vessels, and veins. Studies of the epicardial microcirculation in the beating heart using stroboscopic illumination have suggested that resistance is more precisely controlled in different segments of the circulation. Measurements nents (conductance vessels, resistance vessels, and veins) functions in a homogeneous manner. Recent advances in research technology have made it possible to study the three major components of coronary circulation in greater detail than has been possible. The results of these investigations indicate that there is substantial heterogeneity of function within each of the three major components that regulate myocardial perfusion.The purpose of this review is to describe advances in understanding the role of small coronary arterial resistance vessels and the regulation of myocardial perfusion. The interested reader is referred to recent reviews on the large conduit coronary vessels' and the coronary veins.2 Historical AspectsThe first direct visualization of coronary arterial microvessels in the beating heart was reported by Martini

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 ...

The purposes of this study were to determine if coronary dilation secondary to an increase in myocardial oxygen consumption (MVO2) affects the microcirculation in a homogeneous or heterogeneous manner and to determine if comparable degrees of coronary dilation produced by increasing MVO2 or exogenous (intravenous adenosine) or endogenous (intravenous dipyridamole) adenosine have similar effects in the coronary microcirculation. The epimyocardial coronary microcirculation was observed through an intravital microscope by stroboscopic epi-illumination in anesthetized open-chest dogs. Aortic pressure and heart rate were controlled by an aortic snare and atrioventricular sequential pacing, respectively, during experimental procedures. In group 1 (n = 15), coronary arterial microvessel diameters were measured under control condition and during rapid pacing at 300 beats/min, which doubled MVO2. Increases in MVO2 caused heterogeneous vasodilation in coronary arterial microvessels (40-380 microns). There was an inverse relation between control diameter and percent increase in diameter. In group 2 (n = 15) or group 3 (n = 10), adenosine or dipyridamole was infused intravenously to increase myocardial perfusion to the same level as that obtained with rapid pacing. Adenosine and dipyridamole did not change MVO2. Adenosine and dipyridamole also caused heterogeneous vasodilation, but the effects of adenosine and dipyridamole were restricted to arterial microvessels smaller than 150 microns. From these results, we conclude that increases in MVO2 produce widespread but heterogeneous vasodilation, that is, greater dilation in smaller arterial microvessels. Comparable increases in coronary flow produced by increasing MVO2 or endogenous and exogenous adenosine do not produce identical changes in the distribution of coronary microvascular resistance.

Previous studies have demonstrated that responses to endothelium-dependent vasodilators are absent in the aortas from mice deficient in expression of endothelial nitric oxide synthase (eNOS -/- mice), whereas responses in the cerebral microcirculation are preserved. We tested the hypothesis that in the absence of eNOS, other vasodilator pathways compensate to preserve endothelium-dependent relaxation in the coronary circulation. Diameters of isolated, pressurized coronary arteries from eNOS -/-, eNOS heterozygous (+/-), and wild-type mice (eNOS +/+ and C57BL/6J) were measured by video microscopy. ACh (an endothelium-dependent agonist) produced vasodilation in wild-type mice. This response was normal in eNOS +/- mice and was largely preserved in eNOS -/- mice. Responses to nitroprusside were also similar in arteries from eNOS +/+, eNOS +/-, and eNOS -/- mice. Dilation to ACh was inhibited by N(G)-nitro-L-arginine, an inhibitor of NOS in control and eNOS -/- mice. In contrast, trifluoromethylphenylimidazole, an inhibitor of neuronal NOS (nNOS), decreased ACh-induced dilation in arteries from eNOS-deficient mice but had no effect on responses in wild-type mice. Indomethacin, an inhibitor of cyclooxygenase, decreased vasodilation to ACh in eNOS-deficient, but not wild-type, mice. Thus, in the absence of eNOS, dilation of coronary arteries to ACh is preserved by other vasodilator mechanisms.

The mechanisms that control the timing of labor have yet to be fully characterized. In a previous study, the overexpression of small conductance calcium-activated K(+) channel isoform 3 in transgenic mice, Kcnn3(tm1Jpad)/Kcnn3(tm1Jpad) (also known as SK3(T/T)), led to compromised parturition, which indicates that KCNN3 (also known as SK3) plays an important role in the delivery process. Based on these findings, we hypothesized that SK3 channel expression must be downregulated late in pregnancy to enable the uterus to produce the forceful contractions required for parturition. Thus, we investigated the effects of SK3 channel expression on gestation and parturition, comparing SK3(T/T) mice to wild type (WT) mice. Here, we show in WT mice that SK3 transcript and protein are significantly reduced during pregnancy. We also found the force produced by uterine strips from Pregnancy Day 19 (P19) SK3(T/T) mice was significantly less than that measured in WT or SK3 knockout control (SK3(DOX)) uterine strips, and this effect was reversed by application of the SK3 channel inhibitor apamin. Moreover, two treatments that induce labor in mice failed to result in complete delivery in SK3(T/T) mice within 48 h after injection. Thus, stimuli that initiate parturition under normal circumstances are insufficient to coordinate the uterine contractions needed for the completion of delivery when SK3 channel activity is in excess. Our data indicate that SK3 channels must be downregulated for the gravid uterus to generate labor contractions sufficient for delivery in both term and preterm mice.