Arteriolar myogenic vasoconstriction occurs when increased stretch or membrane tension leads to smooth muscle cell depolarization and opening of voltage-gated Ca2+ channels. To prevent positive feedback and excessive pressure-induced vasoconstriction, studies in cerebral artery smooth muscle have suggested that activation of large conductance, Ca 2+ -activated K + channels (BK Ca ) provides an opposing hyperpolarizing influence reducing Ca 2+ channel activity. We have hypothesized that this mechanism may not equally apply to all vascular beds. To establish the existence of such heterogeneity in vascular reactivity, studies were performed on rat vascular smooth muscle (VSM) cells from cremaster muscle arterioles and cerebral arteries. Whole cell K + currents were determined at pipette [Ca 2+ ] of 100 nm or 5 μm in the presence and absence of the BK Ca inhibitor, iberiotoxin (IBTX; 0.1 μm). Similar outward current densities were observed for the two cell preparations at the lower pipette Ca 2+ levels. At 5 μm Ca 2+ , cremaster VSM showed a significantly (P < 0.05) lower current density compared to cerebral VSM (34.5 ± 1.9 vs 45.5 ± 1.7 pA pF −1 at +70 mV). Studies with IBTX suggested that the differences in K + conductance at 5 μm intracellular [Ca 2+ ] were largely due to activity of BK Ca . 17β-Oestradiol (1 μm), reported to potentiate BK Ca current via the channel's β-subunit, caused a greater effect on whole cell K + currents in cerebral vessel smooth muscle cells (SMCs) compared to those of cremaster muscle. In contrast, the α-subunit-selective BK Ca opener, NS-1619 (20 μm), exerted a similar effect in both preparations. Spontaneously transient outward currents (STOCs) were more apparent (frequency and amplitude) and occurred at more negative membrane potentials in cerebral compared to cremaster SMCs. Also consistent with decreased STOC activity in cremaster SMCs was an absence of detectable Ca 2+ sparks (0 of 76 cells) compared to that in cerebral SMCs (76 of 105 cells). Quantitative PCR showed decreased mRNA expression for the β1 subunit and a decrease in the β 1: α ratio in cremaster arterioles compared to cerebral vessels. Similarly, cremaster arterioles showed a decrease in total BK Ca protein and the β 1: α-subunit ratio. The data support vascular heterogeneity with respect to the activity of BK Ca in terms of both β-subunit regulation and interaction with SR-mediated Ca 2+ signalling.

In several species and in many vascular beds, ultrastructural studies describe close contact sites between the endothelium and smooth muscle of <∼20nm. Such sites are thought to facilitate the local action of signaling molecules and/or the passage of current, as metabolic and electrical coupling conduits between the arterial endothelium and smooth muscle. These sites have the potential for bidirectional communication between the endothelium and smooth muscle, as a key pathway for coordinating vascular function. The aim of this brief review is to summarize the literature on the ultrastructural anatomy and distribution of key components of MECC sites in arteries. In addition to their traditional role of facilitating electrical coupling between the two cell layers, data on the role of MECC sites in arteries, as signaling microdomains involving a spatial localization of channels, receptors and calcium stores are highlighted. Diversity in the density and specific characteristics of MECC sites as signaling microdomains suggests considerable potential for functional diversity within and between arteries in health and disease.

Diet-induced obesity is associated with changes in gastrointestinal function and induction of a mild inflammatory state. Serotonin (5-HT) containing enterochromaffin (EC) cells within the intestine respond to nutrients and are altered by inflammation. Thus, our aim was to characterize the uptake and release of 5-HT from EC cells of the rat ileum in a physiologically relevant model of diet-induced obesity. In chow-fed (CF) and Western diet-fed (WD) rats electrochemical methods were used to measure compression evoked (peak) and steady state (SS) 5-HT levels with fluoxetine used to block the serotonin reuptake transporter (SERT). The levels of mRNA for tryptophan hydroxylase 1 (TPH1) and SERT were determined by quantitative PCR, while EC cell numbers were determined immunohistochemically. In WD rats, the levels of 5-HT were significantly increased (SS: 19.2 ± 3.7 μm; peak: 73.5 ± 14.1 μm) compared with CF rats (SS: 12.3 ± 1.8 μm; peak: 32.2 ± 7.2 μm), while SERT-dependent uptake of 5-HT was reduced (peak WD: 108% of control versus peak CF: 212% control). In WD rats, there was a significant increase in TPH1 mRNA, a decrease in SERT mRNA and protein, and an increase in EC cells. In conclusion, our data show that foods typical of a Western diet are associated with an increased 5-HT availability in the rat ileum. Increased 5-HT availability is driven by the up-regulation of 5-HT synthesis genes, decreased re-uptake of 5-HT, and increased numbers and/or 5-HT content of EC cells which are likely to cause altered intestinal motility and sensation in vivo.

The spatial localization of TRPC3 and associated channels, receptors, and calcium stores are integral for myoendothelial microdomain function. TRPC3 facilitates endothelial SK(Ca) and IK(Ca) activation, as key components of EDH-mediated vasodilator activity and for regulating mesenteric artery tone.

Preeclampsia is a systemic vascular disorder of pregnancy and is associated with increased sensitivity to angiotensin II (AngII) and hypertension. The cause of preeclampsia remains unknown. We identified the role of regulator of G protein (heterotrimeric guanine nucleotide-binding protein) signaling 5 (RGS5) in blood pressure regulation during pregnancy and preeclampsia. RGS5 expression in human myometrial vessels is markedly suppressed in gestational hypertension and/or preeclampsia. In pregnant RGS5-deficient mice, reduced vascular RGS5 expression causes gestational hypertension by enhancing vascular sensitivity to AngII. Further challenge by increasing AngII results in preeclampsia-like symptoms, namely, more severe hypertension, proteinuria, placental pathology, and reduced birth weight. In pregnant heterozygote null mice, treatment with peroxisome proliferator-activated receptor (PPAR) agonists normalizes vascular function and blood pressure through effects on RGS5. These findings highlight a key role of RGS5 at the interface between AngII and PPAR signaling. Because preeclampsia is refractory to current standard therapies, our study opens an unrecognized and urgently needed opportunity for treatment of gestational hypertension and preeclampsia.

Myoendothelial microdomain signaling via localized calciumactivated potassium channel (K Ca ) and gap junction connexins (Cx) is critical for endothelium-dependent vasodilation in rat mesenteric artery. The present study determines the relative contribution of NO and gap junction-K Ca mediated microdomain signaling to endothelium-dependent vasodilation in human mesenteric artery. The hypothesis tested was that such activity is due to NO and localized K Ca and Cx activity. In mesenteric arteries from intestinal surgery patients, endotheliumdependent vasodilation was characterized using pressure myography with pharmacological intervention. Vessel morphology was examined using immunohistochemical and ultrastructural techniques. In vessel segments at 80 mm Hg, the intermediate (I)K Ca blocker 1-[(2-chlorophenyl)diphenyl-methyl]-1H-pyrazole (TRAM-34; 1 M) inhibited bradykinin (0.1 nM-3 M)-induced vasodilation, whereas the small (S) K Ca blocker apamin (50 and 100 nM) had no effect. Direct IK Ca activation with 1-ethyl-2-benzimidazolinone (1-EBIO; 10 -300 M) induced vasodilation, whereas cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methylpyrimidin-4-yl]-amine (1-30 M), the SK Ca activator, failed to dilate arteries, whereas dilation induced by 1-EBIO (10 -100 M) was blocked by TRAM-34. Bradykinin-mediated vasodilation was attenuated by putative gap junction block with carbenoxolone (100 M), with remaining dilation blocked by N-nitro L-arginine methyl ester (100 M) and [1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one] (10 M), NO synthase and soluble guanylate cyclase blockers, respectively. In human mesenteric artery, myoendothelial gap junction and IK Ca activity are consistent with Cx37 and IK Ca microdomain expression and distribution. Data suggest that endothelium-dependent vasodilation is primarily mediated by NO, IK Ca , and gap junction Cx37 in this vessel. Myoendothelial microdomain signaling sites are present in human mesenteric artery and are likely to contribute to endothelium-dependent vasodilation via a mechanism that is conserved between species.

1 Agonists increase endothelial cell intracellular Ca 2+ , in part, by capacitative entry, which is triggered by the ®lling state of intracellular Ca 2+ stores. It has been suggested that depletion of endoplasmic reticulum (ER) Ca 2+ stores either leads to a physical coupling between the ER and a plasma membrane channel, or results in production of an intracellular messenger which a ects the gating of membrane channels. As an axis involving the IP 3 receptor has been implicated in a physical coupling mechanism the aim of this study was to examine the e ects of the putative IP 3 receptor antagonists/modulators, 2 aminoethoxydiphenyl borate (2APB) and xestospongin C, on endothelial cell Ca 2+ entry. 2 Studies were conducted in fura 2 loaded cultured bovine aortic endothelial cells and endothelial cells isolated from rat heart. 3 2APB (30 ± 300 mM) inhibited Ca 2+ entry induced by both agonists (ATP 1 mM, bradykinin 0.1 mM) and receptor-independent mechanisms (thapsigargin 1 mM, ionomycin 0.5 and 5 mM). 2APB did not diminish endothelial cell ATP-induced production of IP 3 nor e ect in vitro binding of [ 3 H]-IP 3 to an adrenal cortex binding protein. Capacitative Ca 2+ entry was also blocked by disruption of the actin cytoskeleton with cytochalasin (100 nM) while the initial Ca 2+ release phase was una ected. 4 Similarly to 2APB, xestospongin C (3 ± 10 mM) inhibited ATP-induced Ca 2+ release and capacitative Ca 2+ entry. Further, xestospongin C inhibited capacitative Ca 2+ entry induced by thapsigargin (1 mM) and ionomycin (0.5 mM). 5 The data are consistent with a mechanism of capacitative Ca 2+ entry in vascular endothelial cells which requires (a) IP 3 receptor binding and/or an event distal to the activation of the ER receptor and (b) a spatial relationship, dictated by the cytoskeleton, between Ca 2+ release and entry pathways.

Ca2+ entry mechanisms underlying spontaneous arteriolar tone and acute myogenic reactivity remain uncertain. These studies aimed to compare the effects of nifedipine and the putative T‐channel blocker, mibefradil, on arteriolar myogenic responsiveness and intracellular Ca2+ (Ca2+i). First order cremaster muscle arterioles (1A) were isolated from rats, cannulated, pressurized to 70 mmHg in the absence of intraluminal flow, and mechanical responses studied by video microscopy. The Ca2+i was measured using fluorescence imaging of Fura 2 loaded arterioles. Both nifedipine and mibefradil showed dose‐dependent inhibition of spontaneous myogenic tone (at 70 mmHg; pEC50 7.04±0.17 vs 6.65±0.20 respectively, n=6 for both, n.s.) and KCl‐induced vasoconstriction (at 70 mmHg; pEC50 6.93±0.38 vs 6.45±0.27 respectively, n=6 for both, n.s.). In arterioles maintained at 50 mmHg, nifedipine (10−7 and 10−5 M) caused a concentration dependent reduction in Ca2+i, however, mibefradil (10−7 and 10−5 M) had no effect. Furthermore nifedipine significantly attenuated the increase in Ca2+i associated with an acute pressure step (50–120 mmHg) whereas mibefradil was considerably less effective. Mibefradil (10−7 M) significantly attenuated contractile responses to 60 mM KCl without altering the KCl‐induced increase in Ca2+i, in contrast to nifedipine (10−7 M) which reduced both Ca2+i and contraction. Membrane potential of arterioles with spontaneous myogenic tone (70 mmHg) was −41.5±1.0 mV. Nifedipine (10−7 or 10−5 M) had no effect on membrane potential, however mibefradil (10−5 M) caused significant depolarization. In summary, both mibefradil and nifedipine inhibit arteriolar spontaneous tone and acute myogenic reactivity. While there may be overlap in the mechanisms by which these agents inhibit tone, differences in effects on membrane potential and intracellular Ca2+ levels suggest mibefradil exhibits actions other than blockade of Ca2+ entry in skeletal muscle arterioles. British Journal of Pharmacology (2000) 131, 1065–1072; doi: