The endothelin (ET) family consists of three 21-amino acid peptides designated ET-1, 2]. The biological actions of the ETs are primarily mediated by two distinct, G-protein-coupled receptor subtypes designated ET A and ET B [2]. Endothelin A receptors have a higher affinity for ET-1 and ET-2 than ET-3 and the ET B receptor binds all three isoforms with equal affinity. Translation of preproET mRNA generates preproET, which is converted to big ET and finally cleaved by ET converting enzyme (ECE) to facilitate production of biologically active peptide. Endothelin-1 released by vascular endothelial cells exerts an autocrine influence by promoting vasodilatation, subsequent to activation of ET B receptors located on endothelial cells. It also exerts a paracrine effect on adjacent vascular smooth muscle cells (VSMC) in evoking vasoconstrictor and mitogenic actions by activation of both ET A and ET B receptors. The primary target of ET-1 is the vasculature where it evokes transient vasodilatation mediated by endothelial ET B receptors, followed by slow-onset and sustained contraction mediated by ET A and ET B receptors located on VSMC. The functional response to ET-1 varies throughout different tissues and vascular beds due to differences in distribution and expression of these two receptor subtypes. Endothelin-1 Diabetologia (1999) AbstractSince the discovery of endothelin-1 as the most potent endothelial-derived vasoconstrictor/mitogenic peptide a decade ago, considerable evidence has implicated this peptide in various cardiovascular disease states, including diabetes mellitus. Plasma and tissue concentrations of endothelin-1 as well as responses to the peptide are changed in various forms of the disease in humans and animals. Endothelin activity is also altered in atherosclerotic and ischaemic disease, nephropathy, retinopathy, erectile dysfunction, and neuropathy, many of the well-known complications of diabetes. Striking new evidence shows that antagonists of the endothelin system might beneficially affect and potentially overcome some of these complications. Despite this, lack of direct proof of causation makes this peptide's role in the disease uncertain. This review examines the current state of thought on the role of endothelin in diabetes and in the complications of the disease as well as the likely roles of altered metabolic variables in modulating endothelin-1 concentrations and its activity. It is concluded that although alterations in endothelin-1 release and action are clearly associated with the diabetic state, further studies using inhibitors of the endothelin system are warranted to determine its precise role in the complications of the disease. [Diabetologia (1999

These data suggest that vascular tissue from the metabolically dysregulated obese Zucker rat exhibits attenuated endothelin-1 peptide production and elevated endothelin receptor levels. Since elevated insulin levels have been linked to increased endothelin receptor expression, it is plausible that hyperinsulinemia upregulates endothelin receptors contributing to elevated vasoconstrictor responses to endothelin-1 in this model of obesity and hypertension.

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While insulin is known to promote vascular smooth muscle (VSM) relaxation, it also enhances endothelin-1 (ET-1) secretion and action in conditions such as NIDDM and hypertension. We examined the effect of insulin pretreatment on intracellular free calcium ([Ca2+]i) responses to ET-1 in cultured aortic smooth muscle cells (ASMCs) isolated from Sprague-Dawley (SD) rats and measured ET(A) receptor characteristics and ET-1-evoked tension responses in aorta obtained from insulin-resistant, hyperinsulinemic Zucker-obese (ZO) and control Zucker-lean (ZL) rats. Pretreatment of rat ASMCs with insulin (10 nmol/l for 24 h) failed to affect basal [Ca2+]i levels but led to a significant increase in peak [Ca2+]i response (1.7-fold; P < 0.01) to ET-1. The responses to IRL-1620 (an ET(B) selective agonist), ANG II, and vasopressin remained unaffected. ET-1-evoked peak [Ca2+]i responses were significantly attenuated by the inclusion of the ET(A) antagonist, BQ123, in both groups. The ET(B) antagonist, BQ788, abolished [Ca2+]i responses to IRL-1620 but failed to affect the exaggerated [Ca2+]i responses to ET-1. Saturation binding studies revealed a twofold increase (P < 0.01) in maximal number of binding sites labeled by 125I-labeled ET-1 in insulin-pretreated cells and no significant differences in sites labeled by 125I-labeled IRL-1620 between control and treatment groups. Northern blot analysis revealed an increase in ET(A) mRNA levels after insulin pretreatment for 20 h, an effect that was blocked by genistein, actinomycin D, and cycloheximide. Maximal tension development to ET-1 was significantly greater (P < 0.01), and microsomal binding studies using [3H]BQ-123 revealed a twofold higher number of ET(A) specific binding sites (P < 0.01) in aorta from ZO rats compared with that of ZL rats. These data suggest that insulin exaggerates ET-1-evoked peak [Ca2+]i responses via increased vascular ET(A) receptor expression, which may contribute to enhanced vasoconstriction observed in hyperinsulinemic states.

We have reported previously that liver X receptor-␣ (LXR␣) can mediate a novel cAMP-dependent increase in renin and c-myc gene transcription by binding as a monomer to a unique regulatory element termed the cAMP-negative response element (CNRE). To determine whether this novel action of LXR␣ has global implications on gene regulation, we employed expression profiling to identify other genes regulated by this unique mechanism. Here we report the existence of a set of known and unknown transcripts regulated in parallel with renin. Querying the Celera Mouse Genome Assembly revealed that a majority of these genes contained the consensus CNRE. We have confirmed the functionality of these CNREs by competition for LXR␣ binding via electrophoretic mobility shift assays (EMSA) and by the use of CNRE decoy molecules documenting the abolishment of the cAMP-mediated gene induction. Taken together, these results demonstrate that the interaction between cAMP-activated LXR␣ and the CNRE enhancer element is responsible for widespread changes in gene expression and identify a set of LXR␣/cAMP-regulated genes that may have important biological implications.The transcription factor liver X receptor-␣ (LXR␣), 1 a member of the nuclear hormone receptor superfamily, regulates the expression of genes involved in cholesterol homeostasis and bile acid synthesis (1, 2). The best known mechanism of LXR␣-mediated transcriptional activation occurs through interactions with compounds such as oxysterols (22-cho) or retinoic acid (9cRA) and results in heterodimerization to other transcription factors including retinoid X receptor ␣ and peroxisome proliferator-activated receptor ␥ (3). Transcription of target genes such as the cholesterol 7␣-hydroxylase gene occurs through a classical DR4/LXRE (5Ј-GGTTTAAATAAGTTCA-3Ј) response element (4).The aspartyl protease renin is synthesized in the kidney and secreted into the plasma. It is the rate-limiting enzyme in angiotensin biosynthesis and thus plays an important role in blood pressure and volume regulation. Renin gene expression and secretion are mediated in part by intracellular levels of cAMP in kidney juxtaglomerular (JG) cells (5). Previously, we have identified a cAMP-responsive element distinct from the classical cAMP-responsive element in the promoter region of the mouse renin gene and have termed this element CNRE (5Ј-TACCTAACTTGGCTCACAGGCAGAATTTATC-3Ј) (6). Homologues of this element found at positions Ϫ619 to Ϫ588 of the mouse Ren-1 D gene have been found in the mouse Ren-1 C and Ren-2 genes as well as in the rat and human renin genes (7). Furthermore, using a yeast one-hybrid screening approach, we demonstrated that LXR␣ bound to this sequence, whereas functional studies using promoter/reporter gene chimeric constructs revealed that LXR␣ increased basal levels of renin expression and mediated the cAMP-dependent induction of mouse and human renin gene expression (8). Interestingly, the previously reported LXR␣ ligands such as 22-cho had no effect on renin gene expression nor on the expre...

Type I (insulin-dependent) diabetes mellitus is associated with metabolic and cardiovascular abnormalities as exemplified by the streptozotocin (STZ) induced diabetic rat. Along with profound hyperglycaemia and reduced plasma insulin concentrations, STZ rats shaw altered responses to vasoconstrictor stimuli, and over time, develop complications characteristic of Type I diabetes [1]. Endothelin has been implicated in various disorders such as renal disease, atherosclerosis, diabetes mellitus and hypertension [2±6]. The STZ diabetic rat has been shown to exhibit either attenuated or enhanced vascular responses to various agonists, including endothelin (ET) [7±17]. In addition, some studies have shown plasma ET concentrations to be increased in both STZ rats and in patients with Type I diabetes [4, 15, 17±19] and other studies demonstrated decreased or unchanged plasma ET concentrations in diabetic models compared with non-diabetic control subjects [20±22]. The rea- Diabetologia (1998 Insulin and vanadate restore decreased plasma endothelin concentrations and exaggerated vascular responses to normal in the streptozotocin diabetic rat

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The presence of arginine vasopressin (AVP) V1 receptors on neonatal rat cardiomyocytes (NRCs) linked to processes capable of elevating intracellular free calcium ([Ca2+]i) is now firmly established. This study examined the sources and signaling involved in [Ca2+]i elevations evoked by AVP in NRCs. AVP promoted increases in both [Ca2+]i and 1,4,5-inositoltrisphosphate (IP3) levels in NRCs. The degree of [Ca2+]i elevation was less than that of angiotensin II, but greater than that of endothelin-1. Extracellular Mg2+ depletion led to diminution of the maximal [Ca2+]i response, with a rightward shift in the concentration-response curves to AVP. The phospholipase C inhibitors, D-609, NCDC, or U73122, and the IP3 receptor blocker, heparin, abolished the [Ca2+]i response to AVP. Neither cyclooxygenase inhibition with indomethacin nor PKC inhibition with staurosporine had any effect. Neither ryanodine nor caffeine, which deplete sarcoplasmic reticulum (SR) Ca2+ stores, nor ruthenium red, which inhibits both SR and mitochondrial Ca2+ stores, affected [Ca2+]i responses to AVP. The SR Ca2+ pump inhibitor, cyclopiazonic acid, abolished, and removal of extracellular Ca2+ attenuated, the response to AVP. These data indicate that activation of cardiac V1 receptors by AVP results in mobilization of Ca2+ from a distinct, non-SR, nonmitochondrial, intracellular Ca2+ pool that is Ca2+ pump replenished and IP3 sensitive. This process occurs secondary to phospholipase C (PLC)-mediated generation of IP3, requires the presence of Mg2+ and extracellular Ca2+, and occurs in a manner independent of PKC and cyclooxygenase activation. Such mechanisms of Ca2+ mobilization might indicate a distinct role for AVP in cardiac physiology and disease.