Adenovirus-mediated leptin expression normalises hypertension associated with diet-induced obesity.

In our previous study, moderate increases in plasma leptin levels achieved via administration of recombinant adenovirus containing the rat leptin cDNA were shown to correct the abnormal metabolic profile in rats with diet-induced obesity, suggesting that these animals had developed resistance to the metabolic effects of leptin, which could be reversed by leptin gene over-expression. However, the effect of this therapeutic strategy on blood pressure was not investigated. The present study aimed to determine whether a moderate increase of endogenous plasma leptin levels affected arterial blood pressure in rats with diet-induced obesity and hypertension.

Modification of cardiovascular ion channels by gene therapy.

Delivery of genes to the heart and vasculature for therapeutic purposes is an exciting strategy that is approaching clinical reality. Abnormalities of expression or function of ion channels is central to many cardiovascular diseases and gene delivery to modify ion channels is an appealing alternative to traditional therapy with small-molecule drugs. Potential therapeutic targets include hypertrophy and heart failure, atrioventricular node modification in atrial fibrillation, ventricular tachycardia and hypertension.

Delivery of ion channel genes to treat cardiovascular diseases.

Modifying ion channel expression and function in the heart and vasculature are potentially useful, novel approaches to managing cardiac hypertrophy, atrial fibrillation and hypertension. Calcium channels play a pivotal role in the heart and vasculature in controlling muscle contraction as well as other aspects of calcium-dependent signaling. The present investigation reports development of mutated L-type calcium channel beta subunits that are delivered by an adenoviral vector to vascular smooth muscle tissue.

Design of mutant beta2 subunits as decoy molecules to reduce the expression of functional Ca2+ channels in cardiac cells.

Calcium influx through long-lasting ("L-type") Ca(2+) channels (Ca(V)) drives excitation-contraction in the normal heart. Dysregulation of this process contributes to Ca(2+) overload, and interventions that reduce expression of the pore-forming alpha(1) subunit may alleviate cytosolic Ca(2+) excess. As a molecular approach to disrupt the assembly of Ca(V)1.

Vascular calcium channels and high blood pressure: pathophysiology and therapeutic implications.

Long-lasting Ca(2+) (Ca(L)) channels of the Ca(v)1.2 gene family are heteromultimeric structures that are minimally composed of a pore-forming alpha(1C) subunit and regulatory beta and alpha(2)delta subunits in vascular smooth muscle cells. The Ca(L) channels are the primary pathways for voltage-gated Ca(2+) influx that trigger excitation-contraction coupling in small resistance vessels.

High glucose inhibits apoptosis induced by serum deprivation in vascular smooth muscle cells via upregulation of Bcl-2 and Bcl-xl.

Apoptosis plays a critical role in normal vascular development and atherosclerosis. To test the hypothesis that diabetic vasculopathy may be due in part to altered apoptosis pathways, we investigated the effects of high glucose treatment on serum withdrawal-induced apoptosis, expression of Bcl-2 family members, and inhibitor of apoptosis protein (IAP)-1 in vascular smooth muscle cells (VSMCs). Treatment with a high concentration of glucose (22 mmol/l) significantly attenuated apoptosis in response to serum withdrawal in cultured rat VSMCs compared with cells treated with a normal glucose concentration (5.

In vivo role of endothelin-converting enzyme-1 as examined by adenovirus-mediated overexpression in rats.

We previously reported that adenovirus-mediated overexpression of endothelin-1 (ET-1) elevates systemic blood pressure in rats. In this model, plasma big ET-1: ET-1 ratios were almost 30, whereas they were only 5 in the control group, suggesting that endothelin-converting enzyme (ECE) may be a rate-limiting step in the production of ET-1 under these conditions. To further investigate the role of ECE in vivo, we prepared recombinant adenovirus strains carrying a soluble, secretory form of bovine ECE-1 cDNA (Ad.

Systemic hypertension induced by hepatic overexpression of human preproendothelin-1 in rats.

Endothelin-1 (ET-1) has been implicated in the regulation of vascular tone in various pathological conditions. To examine the effect of in vivo overexpression of the peptide in rats, we prepared recombinant adenovirus stocks encoding the human preproET-1 cDNA (Ad.ET-1) or Escherichia coli lacZ (Ad.

Pharmacology of endothelins: vascular preparations for studying ETA and ETB receptors.

Three rabbit vessels, the carotid and pulmonary arteries and the jugular vein were investigated to identify vascular monoreceptor systems (either ETA or ETB) to be used in structure-activity studies on endothelins and their antagonists. The RbCA has been found to behave as a monoreceptor ETA preparation, since it shows much greater sensitivity to ET-1 than to ET-3 and is insensitive to IRL 1620. The contractile response of the RbCA to ET-1 is reduced in the presence of BQ-123 but is not influenced by BQ-788.

Different pressor and bronchoconstrictor properties of human big-endothelin-1, 2 (1-38) and 3 in ketamine/xylazine-anaesthetized guinea-pigs.

1. In the present study, the precursors of endothelin-1, endothelin-2 and endothelin-3 were tested for their pressor and bronchoconstrictor properties in the anaesthetized guinea-pig. In addition, the effects of big-endothelin-1 and endothelin-1 were assessed under urethane or ketamine/xylazine anaesthesia.

Block of endothelin-1-induced release of thromboxane A2 from the guinea pig lung and nitric oxide from the rabbit kidney by a selective ETB receptor antagonist, BQ-788.

1. The present study characterizes the receptors responsible for endothelin-1-induced release of thromboxane A2 from the guinea pig lung and of endothelium-derived nitric oxide from the rabbit perfused kidney, by the use of the selective ETA receptor antagonist, BQ-123, and a novel selective ETB receptor antagonist, BQ-788. 2.

Characterization of receptors for endothelins in the perfused arterial and venous mesenteric vasculatures of the rat.

1. Endothelin-1 and -3 induced marked arterial and venous constrictions in the perfused mesenteric vasculature of the rat with endothelin-3 being at least 20 times less active than endothelin-1, on both arterial and venous sides of the vasculature. 2.

Increased plasma levels of endothelin during anaphylactic shock in the guinea-pig.

Intravenous injection of ovalbumin into actively and passively sensitized guinea-pig resulted in acute circulatory collapse. The plasma level of immunoreactive endothelin rose from 22 +/- 2 to 40 +/- 7 fmol/ml (n = 12, P < 0.01) and 29 +/- 5 fmol/ml (n = 12, P < 0.

Endothelin-1 induces vasoconstriction and prostacyclin release via the activation of endothelin ETA receptors in the perfused rabbit kidney.

Endothelin-1 (0.005 and 0.01 nmol) induced a dose-dependent increase in perfusion pressure in the perfused rabbit kidney.

Pharmacologic evidence for the specificity of the phosphoramidon-sensitive endothelin-converting enzyme for big endothelin-1.

The pharmacology of human big endothelin-1 (big ET-1) and big ET-3 was compared in five pharmacologic models: perfused rat and guinea pig lungs, perfused rabbit kidney, and in the rat and the guinea pig in vivo (blood pressure monitoring). In these models, big ET-1 consistently induced concentration- or dose-dependent pharmacologic effects sensitive to phosphoramidon (vasopressor or prostanoid-releasing effects). In contrast, big ET-3, dissolved in either phosphate-buffered saline (pH 7.

ETA-dependent pressor effects and release of prostacyclin induced by endothelins in pulmonary and renal vasculature.

BQ-123, a selective endothelin A (ETA) receptor antagonist, was used to study the receptors involved in the ET-1-induced release of prostacyclin (PGI2) in perfused rat lung and rabbit kidney and the pressor effects of ET-1 in rabbit renal vasculature. In perfused rat lung, infusion of ET-1 (5 nM) significantly increased the release of PGI2, which was markedly reduced after a 15-min infusion of BQ-123 (1 microM). In rabbit kidney, the PGI2 release induced by ET-1 (10 nM) was abolished by a 15-min pretreatment with BQ-123 (1 microM).

Nonadrenergic and noncholinergic arterial dilatation and venoconstriction are mediated by calcitonin gene-related peptide1 and neurokinin-1 receptors, respectively, in the mesenteric vasculature of the rat after perivascular nerve stimulation.

In the present study, selective calcitonin gene-related peptide (CGRP) and neurokinin (NK) agonists and antagonists were used to characterize the receptors mediating the nerve-induced arterial vasodilation and venous vasoconstriction in the rat mesenteric vasculature. In guanethidine-pretreated preparations, perivascular nerve stimulation (PNS) induced a frequency-dependent vasodilation in the arterial vasculature (precontracted with methoxamine), yet only induced an atropine-resistant contraction in the venous mesenteric vasculature (precontracted with U46619) of the rat. hCGRP alpha induced a marked dose-dependent relaxation of the arterial side, whereas only a slight vasodilation was seen at a high dose on the precontracted venous side.

Phosphoramidon-sensitive effects of big endothelins in the perfused rabbit kidney.

The enzymatic conversion of human big endothelins (1, 2, and 3) to their respective active metabolites (endothelin-1, -2, and -3) was investigated in the perfused rabbit kidney through the pressor- and eicosanoid-releasing properties of these peptides. Intra-arterial bolus injections of endothelin-1 and -2 (5-50 pmol), endothelin-3 (100-250 pmol), and big endothelin-1 and -2 (100-250 pmol) into the kidney produced dose-dependent increases of perfusion pressure, whereas big endothelin-3 was inactive at doses up to 1,000 pmol. Endothelin-1 and -2 (10 nM), endothelin-3 (100 nM), and big endothelin-1 and -2 (100 nM) are potent enhancers of prostacyclin release without inducing any release of thromboxane B2 in the perfused kidney.

Human big-endothelin-1 and endothelin-1 release prostacyclin via the activation of ET1 receptors in the rat perfused lung.

Although ET1 and ET2 binding sites were found in rat lung membranes, a selective ET1 receptor antagonist, BQ-123 (10 microM), did not displace [125I]-endothelin-1 ([125I]ET-1) from ET2 sites, illustrating the selectivity of the angatonist for ET1 receptors. In rat perfused lungs, BQ-123 (1 microM) markedly reduced the prostacyclin (PGI2) releasing properties of endothelin-1 (ET-1: 5 nM) and human big-ET-1 (100 nM) suggesting that both peptides induce the release of PGI2 via the selective activation of ET1 receptors.

Neurokinins produce selective venoconstriction via NK-3 receptors in the rat mesenteric vascular bed.

The vasoactive properties of the neurokinins (substance P (SP), neurokinin A (NKA), neurokinin B (NKB)) and some selective analogues were assessed in the arterial and venous mesenteric beds of the rat. Although both sides of the mesenteric vasculature displayed endothelium-dependent relaxation in response to acetylcholine (ACh) or bradykinin (BK) (1 and 10 nmol), SP and the selective NK-1 analogue, [Sar9,Met(O2)11]SP were inactive. Of the three selective neurokinin agonists used, [Sar9,Met(O2)11]SP (NK-1), [beta-Ala8]NKA-(4-10) (NK-2) and [MePhe7]NKB (NK-3), only the latter induced a dose-dependent pressor effect in the venous mesenteric vasculature.

Different pharmacological profiles of big-endothelin-3 and big-endothelin-1 in vivo and in vitro.

1. Human big-endothelin-1 (big-ET-1) and endothelin-1 (ET-1) are equipotent as pressor agents and produce a significant change in mean arterial blood pressure (MAP) in anaesthetized guinea-pigs (2 nmol kg-1: peak delta MAP: 23 +/- 6 mmHg and 26 +/- 5 mmHg, respectively). 2.

Presence of a phosphoramidon-sensitive endothelin-converting enzyme which converts big-endothelin-1, but not big-endothelin-3, in the rat vas deferens.

Endothelin-1 (ET-1) enhanced field stimulation-evoked (0.1 Hz), nerve-mediated contractions of the prostatic portion of the rat vas deferens. The human precursor of ET-1, big-endothelin (1-38) (big-ET-1) was only two-fold less potent than ET-1 (pD2 values: 7.

Structure-activity studies of bradykinin and related peptides. B2-receptor antagonists.

Thirty-seven compounds were tested as antagonists of kinin B2- and B1-receptors to identify the chemical changes required to obtain antagonism, improve antagonist affinity, and eliminate residual agonistic activities. Apparent affinity of antagonists was evaluated in terms of pA2 on the rabbit jugular vein, the dog carotid and renal arteries, the hamster urinary bladder, the guinea pig ileum, the rat vas deferens, the guinea pig trachea, and the rabbit aorta, using bradykinin and desArg9-bradykinin as B2- and B1-receptor activators. Replacement of Pro7 of bradykinin with D-Phe leads to antagonism; substitution of Pro3 by Hyp and extension of the peptide chain at the N-terminal with a D-Arg residue improves the affinity of antagonists; acetylation of N-terminal amine function reduces residual agonistic activity; these changes, combined with the replacement of Phe8 by Leu as in Ac-D-Arg[Hyp3,D-Phe7,Leu8]-bradykinin, led to potent full B2-receptor antagonists.

Human big endothelin releases prostacyclin in vivo and in vitro through a phosphoramidon-sensitive conversion to endothelin-1.

Human big endothelin (big ET) and endothelin-1 (ET-1) induce similar increases in left ventricular systolic pressure in the anesthetized rabbit. Unlike ET-1, human big ET does not induce an initial transient hypotension. Human big ET (3 nmol/kg) inhibits ADP-induced platelet aggregation ex vivo by 60% whereas ET-1 at 1 nmol/kg inhibits platelet aggregation by more than 80%.