Gene deletion of dopamine beta-hydroxylase and alpha1-adrenoceptors demonstrates involvement of catecholamines in vascular remodeling

In vitro studies have shown that stimulation of alpha1-adrenoceptors (ARs) directly induces proliferation, hypertrophy, and migration of arterial smooth muscle cells and adventitial fibroblasts. In vivo studies confirmed these findings and showed that catecholamine trophic activity becomes excessive after experimental balloon injury and contributes to neointimal growth, adventitial thickening, and lumen loss. However, past studies have been limited by selectivity of pharmacological agents. The aim of this study, in which mice devoid of norepinephrine and epinephrine synthesis [dopamine beta-hydroxylase (DBH-/-)] or deficient in alpha1-AR subtypes expressed in murine carotid (alpha1B-AR-/- and alpha1D-AR-/-) were used, was to test the hypothesis that catecholamines contribute to wall hypertrophy after injury. At 3 wk after injury of wild-type mice, lumen area and carotid circumference increased significantly, and hypertrophy of media and adventitia was in excess of that needed to restore circumferential wall stress to normal. In DBH-/- and alpha1B-AR-/- mice, increases in lumen area, circumference, and hypertrophy of the media and adventitia were reduced by 50-91%, resulting in restoration of wall tension to nearly normal (DBH-/-) or normal (alpha1B-AR-/-). In contrast, in alpha1D-AR-/- mice, increases in lumen area, circumference, and wall hypertrophy were unaffected and wall thickening remained in excess of that required to return tension to normal. When examined 5 days after injury, proliferation and leukocyte infiltration were inhibited in DBH-/- mice. These studies suggest that the trophic effects of catecholamines are mediated primarily by alpha1B-ARs in mouse carotid and contribute to hypertrophic growth after vascular injury.     

Adrenergic catecholamine trophic activity contributes to flow-mediated arterial remodeling

Stimulation of alpha1-adrenoceptors (ARs) induces proliferation, hypertrophy, and migration of vascular smooth muscle cells and adventitial fibroblasts in cell and organ culture. In vivo studies have confirmed this direct trophic action and found that endogenous catecholamines contribute to neointimal formation and wall hypertrophy induced by mechanical injury. In murine carotid artery, these effects are mediated by alpha 1B-ARs, whereas alpha 1D-ARs mediate contraction and alpha 1A-ARs are not expressed. Herein, we examined whether catecholamines also contribute to arterial wall growth in a noninjury model, i.e., flow-mediated remodeling. In wild-type mice or mice deficient in norepinephrine and epinephrine synthesis [dopamine beta-hydroxylase knockout (DBH-KO)], all distal branches of the left carotid artery (LC) except the thyroid artery were ligated to reduce flow in the LC and increase flow in the right carotid artery (RC). Twenty-one days later, negative hypertrophic remodeling of the LC [i.e., -20% (decrease) in lumen area, -2% in circumference of the external elastic lamina (CEEL), +98% (increase) in thickness of the intima media, and +71% in thickness for adventitia; P < 0.01 vs. sham ligation] and positive eutrophic remodeling of the RC [+23% in lumen area, +11% in CEEL; P < 0.01 vs. sham ligation] were inhibited in DBH-KO mice [LC: +10% intima media and +3% adventitia; RC: +9% lumen area and +3% CEEL]. This inhibition was associated with reduced proliferation in the RC and reduced apoptosis and leukocyte accumulation in the RC and LC when examined 5 days after ligation. Carotid remodeling in alpha 1D-AR-knockout mice evidenced little or no inhibition, which suggests dependence on alpha 1B-ARs. These findings suggest that catecholamine-induced trophic activity contributes to both flow-mediated negative remodeling and adaptive positive arterial remodeling.     

Systemic alpha 1A-adrenoceptor antagonist inhibits neointimal growth after balloon injury of rat carotid artery

Previous in vitro and in vivo studies have shown that norepinephrine, acting through alpha(1A)-adrenoceptors, stimulates hypertrophy, proliferation, and migration of vascular smooth muscle cells and adventitial fibroblasts and may contribute to neointimal growth, lumen loss, and inward remodeling caused by iatrogenic wall injury and vascular disease. Our present aim was to determine whether intravenous administration of the alpha(1A)-adrenoceptor antagonist KMD-3213, at dosages without systemic hemodynamic effects, inhibits wall growth after injury. Inhibition of alpha(1A)-adrenoceptors with 12.8 and 32 microg/kg KMD-3213 had no effect on arterial pressure or renal and hindquarter resistances in anesthetized rats. A second group then received carotid balloon injury and continuous intravenous KMD-3213 at 4 and 10 microg x kg(-1) x h(-1) for 2 wk. Mean, systolic, and diastolic arterial pressures and heart rate of conscious unrestrained rats were unaffected. KMD-3213 reduced neointima growth by approximately 30 and 46% at the two doses (P < 0.01). These data support the novel hypothesis that a direct alpha(1A)-adrenoceptor-dependent trophic action of catecholamines is augmented by injury and may contribute significantly to hypertrophic vascular disease.     

Enhanced alpha1-adrenergic trophic activity in pulmonary artery of hypoxic pulmonary hypertensive rats

Mechanisms that induce the excessive proliferation of vascular wall cells in hypoxic pulmonary hypertension (PH) are not fully understood. Alveolar hypoxia causes sympathoexcitation, and norepinephrine can stimulate alpha(1)-adrenoceptor (alpha(1)-AR)-dependent hypertrophy/hyperplasia of smooth muscle cells and adventitial fibroblasts. Adrenergic trophic activity is augmented in systemic arteries by injury and altered shear stress, which are key pathogenic stimuli in hypoxic PH, and contributes to neointimal formation and flow-mediated hypertrophic remodeling. Here we examined whether norepinephrine stimulates growth of the pulmonary artery (PA) and whether this is augmented in PH. PA from normoxic and hypoxic rats [9 days of 0.1 fraction of inspired O(2) (Fi(O(2)))] was studied in organ culture, where wall tension, Po(2), and Pco(2) were maintained at values present in normal and hypoxic PH rats. Norepinephrine treatment for 72 h increased DNA and protein content modestly in normoxic PA (+10%, P < 0.05). In hypoxic PA, these effects were augmented threefold (P < 0.05), and protein synthesis was increased 34-fold (P < 0.05). Inferior thoracic vena cava from normoxic or hypoxic rats was unaffected. Norepinephrine-induced growth in hypoxic PA was dose dependent, had efficacy greater than or equal to endothelin-1, required the presence of wall tension, and was inhibited by alpha(1A)-AR antagonist. In hypoxic pulmonary vasculature, alpha(1A)-AR was downregulated the least among alpha(1)-AR subtypes. These data demonstrate that norepinephrine has trophic activity in the PA that is augmented by PH. If evident in vivo in the pulmonary vasculature, adrenergic-induced growth may contribute to the vascular hyperplasia that participates in hypoxic PH.     

Inhibition of in-stent restenosis by oral copper chelation in porcine coronary arteries

Stress-induced release of IL-1alpha and fibroblast growth factor-1 is dependent on intracellular copper and is a major driver of neointimal hyperplasia. Therefore, we assessed the effect of tetrathiomolybdate (TTM), a clinically proven copper chelator, on in-stent restenosis. Nine pigs were treated with TTM (5 mg/kg po) twice daily for 2 wk before stent implantation and for 4 wk thereafter, and nine pigs served as controls. In-stent restenosis was assessed by quantitative coronary angiography (QCA), intravascular ultrasound (IVUS), and histomorphometry. Serum ceruloplasmin activity was used as a surrogate marker of copper bioavailability. In TTM-treated animals, ceruloplasmin dropped 70 +/- 10% below baseline levels. Baseline characteristics were comparable in TTM-treated and control animals. At 4-wk follow-up, all parameters relevant to in-stent restenosis were significantly reduced in TTM-treated animals: minimal lumen diameter by QCA was 2.03 +/- 0.57 and 1.47 +/- 0.45 mm in TTM-treated and control animals, respectively (P < 0.05), percent stenosis diameter was 39% less in TTM-treated animals (27.1 +/- 16.6% vs. 44.5 +/- 16.1%, P < 0.05), minimal lumen area by IVUS was 60% larger in TTM-treated animals (4.27 +/- 1.56 vs. 2.67 +/- 1.19 mm(2), P < 0.05), and neointimal volume by histomorphometry was 37% less in TTM-treated animals (34.9 +/- 11.5 vs. 55.2 +/- 19.6 mm(3), P < 0.05). We conclude that systemic copper chelation with a clinically approved chelator significantly inhibits in-stent restenosis.     

Different alpha-adrenoceptors mediate migration of vascular smooth muscle cells and adventitial fibroblasts in vitro

Norepinephrine directly induces growth of the vascular wall, which may involve not only proliferation of smooth muscle cells (SMCs) and adventitial fibroblasts (AFBs) but also augmentation of their migration. To test this hypothesis, growth-arrested SMCs and AFBs from rat aorta were exposed to norepinephrine. Norepinephrine caused dose-dependent migration of both cell types that was dependent on chemotaxis. In contrast, platelet-derived growth factor (PDGF)-BB, used as a positive control, stimulated both chemotaxis and chemokinesis. Only alpha(1D)-adrenoceptors (AR) and alpha(2)-AR antagonists inhibited norepinephrine migration of SMCs, whereas norepinephrine migration of AFBs was only inhibited by alpha(1A)-AR and alpha(1B)-AR antagonists; beta-AR blockade was without effect. Norepinephrine and PDGF-BB were additive for AFB, but not SMC, migration. Stimulation of migration was reversed at high norepinephrine concentrations (10 microM); this inhibition was mediated by alpha(2)- and beta-ARs in AFBs but not in SMCs. Thus norepinephrine induces migration of SMCs and AFBs via different alpha-ARs. This action may participate in wall remodeling and norepinephrine potentiation of injury-induced intimal lesion growth.     

Catecholamines augment collateral vessel growth and angiogenesis in hindlimb ischemia

Catecholamine stimulation of alpha1-adrenoceptors exerts growth factor-like activity, mediated by generation of reactive oxygen species, on arterial smooth muscle cells and adventitial fibroblasts and contributes to hypertrophy and hyperplasia in models of vascular injury and disease. Adrenergic trophic activity also contributes to flow-mediated positive arterial remodeling by augmenting proliferation and leukocyte accumulation. To further examine this concept, we studied whether catecholamines contribute to collateral growth and angiogenesis in hindlimb insufficiency. Support for this hypothesis includes the above-mentioned studies, evidence that ischemia augments norepinephrine release from sympathetic nerves, and proposed involvement of reactive oxygen species in angiogenesis and collateral growth. Mice deficient in catecholamine synthesis [by gene deletion of dopamine beta-hydroxylase (DBH-/-)] were studied. At 3 wk after femoral artery ligation, increases in adductor muscle perfusion were similar in DBH-/- and wild-type mice, whereas recovery of plantar perfusion and calf microsphere flow were attenuated, although not significantly. Preexisting collaterals in adductor of wild-type mice showed increases in lumen diameter (60%) and medial and adventitial thickness (57 and 119%, P < 0.05 here and below). Lumen diameter increased similarly in DBH-/- mice (52%); however, increases in medial and adventitial thicknesses were reduced (30 and 65%). Leukocyte accumulation in the adventitia/periadventitia of collaterals was 39% less in DBH-/- mice. Increased density of alpha-smooth muscle actin-positive vessels in wild-type adductor (45%) was inhibited in DBH-/- mice (2%). Although both groups experienced similar atrophy in the gastrocnemius (approximately 22%), the increase in capillary-to-muscle fiber ratio in wild-type mice (21%) was inhibited in DBH-/- mice (7%). These data suggest that catecholamines may contribute to collateral growth and angiogenesis in tissue ischemia.     

Alpha1-adrenoceptor-dependent vascular hypertrophy and remodeling in murine hypoxic pulmonary hypertension

Excessive proliferation of vascular wall cells underlies the development of elevated vascular resistance in hypoxic pulmonary hypertension (PH), but the responsible mechanisms remain unclear. Growth-promoting effects of catecholamines may contribute. Hypoxemia causes sympathoexcitation, and prolonged stimulation of alpha(1)-adrenoceptors (alpha(1)-ARs) induces hypertrophy and hyperplasia of arterial smooth muscle cells and adventitial fibroblasts. Catecholamine trophic actions in arteries are enhanced when other conditions favoring growth or remodeling are present, e.g., injury or altered shear stress, in isolated pulmonary arteries from rats with hypoxic PH. The present study examined the hypothesis that catecholamines contribute to pulmonary vascular remodeling in vivo in hypoxic PH. Mice genetically deficient in norepinephrine and epinephrine production [dopamine beta-hydroxylase(-/-) (DBH(-/-))] or alpha(1)-ARs were examined for alterations in PH, cardiac hypertrophy, and vascular remodeling after 21 days exposure to normobaric 0.1 inspired oxygen fraction (Fi(O(2))). A decrease in the lumen area and an increase in the wall thickness of arteries were strongly inhibited in knockout mice (order of extent of inhibition: DBH(-/-) = alpha(1D)-AR(-/-) > alpha(1B)-AR(-/-)). Distal muscularization of small arterioles was also reduced (DBH(-/-) > alpha(1D)-AR(-/-) > alpha(1B)-AR(-/-) mice). Despite these reductions, increases in right ventricular pressure and hypertrophy were not attenuated in DBH(-/-) and alpha(1B)-AR(-/-) mice. However, hematocrit increased more in these mice, possibly as a consequence of impaired cardiovascular activation that occurs during reduction of Fi(O(2)). In contrast, in alpha(1D)-AR(-/-) mice, where hematocrit increased the same as in wild-type mice, right ventricular pressure was reduced. These data suggest that catecholamine stimulation of alpha(1B)- and alpha(1D)-ARs contributes significantly to vascular remodeling in hypoxic PH.     

Characterization of noradrenergic transmission at the dorsal motor nucleus of the vagus involved in reflex control of fundus tone

Quantitative analysis of innervation to dorsal motor nucleus of the vagus (DMV) fundus-projecting neurons indicates that approximately 17% of input neurons are noradrenergic. To determine whether this small percentage of neurons innervating DMV output to the stomach is physiologically relevant, we evaluated the role of norepinephrine at the DMV in mediating a vagovagal reflex controlling the fundus. A strain gauge was sutured onto the fundus of isoflurane-anesthetized rats to monitor changes in tone evoked by esophageal distension (ED). ED produced a decrease in fundus tone of 0.31 +/- 0.02 g (P < 0.05), which could be reproduced after a 30-min interval between distensions. Bilateral cervical vagotomy and/or pretreatment with intravenous atropine methylbromide prevented the reflex-induced fundus relaxation. In contrast, intravenous N(G)-nitro-L-arginine methyl ester had no effect. Bilateral microinjection of alpha2-adrenoreceptor antagonists (yohimbine and RS-79948) into the DMV also prevented the response. Before microinjection of alpha2-adrenoreceptor antagonists, ED decreased fundus tone by 0.33 +/- 0.05 g (P < 0.05). After antagonist microinjection, ED decreased fundus tone by only 0.05 +/- 0.06 g (P > 0.05). Bilateral microinjection of prazosin into the DMV had no effect on the response. Microinjection of norepinephrine into the DMV mimicked the effect of ED and was also prevented by prior microinjection of an alpha2-adrenoreceptor antagonist. Our results indicate that noradrenergic innervation of DMV fundus-projecting neurons is physiologically important and suggest that norepinephrine released at the DMV acts on alpha2-adrenoreceptors to inhibit activity in a cholinergic-cholinergic excitatory pathway to the fundus.     

Endothelin-1 expression in vascular adventitial fibroblasts

Endothelial cells are a major source of endothelin (ET)-1, but the possibility that vascular adventitial fibroblasts generate ET-1 has not been explored. We hypothesized that aortic adventitial fibroblasts have the ability to produce ET-1, which may contribute to extracellular matrix synthesis. Vascular adventitial fibroblasts were isolated from mouse aorta and incubated with various concentrations of angiotensin II (ANG II). mRNA levels of preproET-1 and type I procollagen were detected with relative RT-PCR. ET-1 levels in culture medium were measured with ELISA. Protein levels of procollagen were detected with Western blotting. ANG II (10 and 100 nM, 1 microM) induced a time- and concentration-dependent increase in preproET-1 mRNA levels (P < 0.05). Induction of preproET-1 mRNA was accompanied by release of immunoreactive peptide ET-1 (P < 0.05). ANG II-evoked increases in preproET-1 mRNA expression and ET-1 release were blocked by losartan (100 microM), an AT1 receptor antagonist, but not PD-123319 (100 microM), an AT2 receptor antagonist. To further confirm our findings, we cloned and then sequenced vascular fibroblast preproET-1 bidirectionally with T7 and M13 reverse sequencing primers. Their nucleotide sequences were identical to preproET-1 cDNA from mouse vascular endothelial cells (accession no. AB081657). Moreover, ANG II-induced type I procollagen mRNA and protein expression were inhibited by BQ-123 (10 microM), an ET(A) receptor inhibitor, but not BQ-788 (10 microM), an ET(B) receptor inhibitor, suggesting a significant role of adventitial ET-1 in regulation of extracellular matrix synthesis. The results demonstrate that vascular adventitial fibroblasts are able to synthesize and release ET-1 in response to ANG II.     

Adrenomedullin gene transfer induces neointimal apoptosis and inhibits neointimal hyperplasia in injured rat artery

Arterial wall injury leads to inflammatory reaction and release of growth factors that may mediate intimal regrowth. It is hypothesized that the neointimal cells may originate from adventitial myofibroblasts, medial smooth muscle cells, or differentiated bone marrow derived cells. Adrenomedullin (AM), an auto/paracrine cardiovascular peptide that is secreted from fibroblasts, endothelial cells, and vascular smooth muscle cells, may have a regulatory role in the intimal regeneration. In order to investigate the role of AM in neointimal growth, stimulation of stem cell migration, and apoptosis, we overexpressed AM with recombinant adenovirus in a rat arterial injury model. The intimae were significantly thinner in the arteries treated with AM adenovirus compared to the control group. Intima/media ratios were 0.48 +/- 0.18 and 1.01 +/- 0.20 (P < 0.05) in the AM group and the control group, respectively. In addition, a significantly higher apoptotic index of neointimal cells was seen in the AM gene transfer group compared to the control (2.78 +/- 0.5 vs. 0.57 +/- 0.20, P < 0.01). The neointimal cells stained positive for alpha-smooth muscle actin and negative for desmin suggesting possible myofibroblast origin. Very few c-Kit+ or MDR1+ cells were detected 2 weeks after the injury. We conclude that AM overexpression inhibits neointimal growth. The inhibition is associated with enhanced apoptosis of the neointimal cells which may be of myofibroblast origin.     

Analysis of responses to sympathetic nerve stimulation in the feline pulmonary vascular bed

The adrenergic receptor subtypes mediating the response to sympathetic nerve stimulation in the pulmonary vascular bed of the cat were investigated under conditions of controlled blood flow and constant left atrial pressure. The increase in lobar vascular resistance in response to sympathetic nerve stimulation was reduced by prazosin and to a lesser extent by yohimbine, the respective alpha 1- and alpha 2-adrenoceptor antagonists. Moreover, in animals pretreated with a beta-adrenoceptor antagonist to prevent an interaction between alpha- and beta 2-adrenoceptors, responses to nerve stimulation were reduced by prazosin, but yohimbine had no significant effect. On the other hand, in animals pretreated with a beta-adrenoceptor antagonist, yohimbine had an inhibitory effect on responses to tyramine and to norepinephrine. Propranolol had no significant effect on the response to nerve stimulation, whereas ICI 118551, a selective beta 2-adrenoceptor antagonist, enhanced responses to nerve stimulation and injected norepinephrine. The present data suggest that neuronally released norepinephrine increases pulmonary vascular resistance in the cat by acting mainly on alpha 1-adrenoceptors and to a lesser extent on postjunctional alpha 2-adrenoceptors but that this effect is counteracted by an action on presynaptic alpha 2-receptors. The present studies also suggest that neuronally released norepinephrine acts on beta 2-adrenoceptors and that the response to sympathetic nerve stimulation represents the net effect of the adrenergic transmitter on alpha 1-, alpha 2-, and beta 2-adrenoceptors in the pulmonary vascular bed.     

Prostacyclin synthesis elicited by cholinergic agonists is linked to activation of M2 alpha and M2 beta muscarinic receptors in the rabbit aorta

This study was conducted to investigate the subtypes of muscarinic receptors involved in the action of cholinergic agents on prostacyclin synthesis in the rabbit aorta. Prostacyclin production measured as 6-keto-PGF1 alpha was assessed after exposing the aortic rings to different cholinergic agents. Acetylcholine (ACh) (M1 and M2 agonist) (1-10 microM) and arecaidine proparagyl ester (APE) (M2 selective agonist) (1-10 microM) enhanced 6-keto-PGF1 alpha output in a concentration-dependent manner. A selective M1 receptor agonist, McN-A-343, at 1 microM-1 mM did not alter 6-keto-PGF1 alpha output. ACh- and APE induced increases in 6-keto-PGF1 alpha output were attenuated by the M1/M2 antagonist atropine (0.1 microM), M2 alpha antagonist (AF-DX 116), (0.1-1.0 microM), and by selective M2 beta antagonist, hexahydro-sila-difendiol (HHSiD) (0.1-1.0 microM), but not by the M1 antagonist pirenzepine (1.0 microM). 6-Keto-PGF1 alpha output elicited by ACh- or APE was not altered by the adrenergic receptor antagonists phentolamine and propranolol or by the nicotinic receptor blocker hexamethonium. Similarly, the arachidonic acid- or norepinephrine induced 6-keto-PGF1 alpha accumulation was not altered by these muscarinic receptor antagonists. Indomethacin, a cyclooxygenase inhibitor, prevented arachidonic acid, ACh- or APE induced 6-keto-PGF1 alpha output. Removal of the endothelium abolished the production of 6-keto-PGF1 alpha elicited by ACh, APE, bradykinin, and calcium ionophore A 23187, but not that induced by angiotensin II, K+ or norepinephrine. These data suggest that vascular prostaglandin generation elicited by cholinergic agonists is mediated via activation of M2 alpha and M2 beta but not M1 muscarinic receptors, which are most likely located on the endothelium.     

Mediation of Norepinephrine-Stimulated Cyclic AMP Accumulation by Adrenergic Receptors in Hypothalamic and Preoptic Area Slices: Effects of Estradiol

Adrenergic receptor agonists and antagonists were employed to establish (a) which receptor subtypes mediate the cyclic AMP response to norepinephrine in hypothalamic and preoptic area slices from gonadectomized female rats and (b) which receptor subtypes might be modulated by the steroid hormone estradiol. Slice cyclic AMP levels were elevated by the beta receptor agonist isoproterenol, but not by alpha 1 (phenylephrine, methoxamine) or alpha 2 (clonidine) agonists. However, the alpha agonist phenylephrine potentiated the effect of the beta agonist isoproterenol on slice cyclic AMP accumulation. In slices from rats given no hormone treatment, the beta antagonist propranolol inhibited norepinephrine-stimulated cyclic AMP production, while the alpha 1 antagonist prazosin was without effect. In contrast, the cyclic AMP response to norepinephrine in slices from estradiol-treated rats was blocked more effectively by prazosin than by propranolol. Estradiol treatment also attenuated the production of cyclic AMP by the beta agonist isoproterenol. The data suggest (a) that norepinephrine induction of cyclic AMP accumulation in hypothalamic and preoptic area slices is mediated by beta receptors and potentiated by alpha receptor activation and (b) that estradiol depresses beta and increases alpha 1 receptor function in slices from brain regions associated with reproductive physiology.     

Vasohibin prevents arterial neointimal formation through angiogenesis inhibition

Vasohibin is a VEGF-inducible angiogenesis inhibitor in vascular endothelium. Here we examined the presence of vasohibin in human arterial wall, and found it in endothelium of adventitial microvessels in atherosclerotic lesion. Adventitial angiogenesis is involved in the progression of neointimal formation. Even in the presence of endogenous angiogenesis inhibitors, pathological angiogenesis persists. However, the supplementation of exogenous angiogenesis inhibitors can prevent pathological angiogenesis. We evaluated the potential role of vasohibin in neointimal formation. Adenovirus-mediated human vasohibin gene transfer in mouse liver resulted in the release of vasohibin in plasma and exhibited anti-angiogenic effects at remote sites. This gene transfer inhibited adventitial angiogenesis, macrophage infiltration, and neointimal formation after cuff placement on mouse femoral artery. Vasohibin exhibited no direct effect on migration and proliferation of smooth muscle cells. Thus, vasohibin has an activity to prevent neointimal formation by inhibiting adventitial angiogenesis.     

Renal sympathetic nerve activity modulates afferent renal nerve activity by PGE2-dependent activation of alpha1- and alpha2-adrenoceptors on renal sensory nerve fibers

Increasing efferent renal sympathetic nerve activity (ERSNA) increases afferent renal nerve activity (ARNA). To test whether the ERSNA-induced increases in ARNA involved norepinephrine activating alpha-adrenoceptors on the renal sensory nerves, we examined the effects of renal pelvic administration of the alpha(1)- and alpha(2)-adrenoceptor antagonists prazosin and rauwolscine on the ARNA responses to reflex increases in ERSNA (placing the rat's tail in 49 degrees C water) and renal pelvic perfusion with norepinephrine in anesthetized rats. Hot tail increased ERSNA and ARNA, 6,930 +/- 900 and 4,870 +/- 670%.s (area under the curve ARNA vs. time). Renal pelvic perfusion with norepinephrine increased ARNA 1,870 +/- 210%.s. Immunohistochemical studies showed that the sympathetic and sensory nerves were closely related in the pelvic wall. Renal pelvic perfusion with prazosin blocked and rauwolscine enhanced the ARNA responses to reflex increases in ERSNA and norepinephrine. Studies in a denervated renal pelvic wall preparation showed that norepinephrine increased substance P release, from 8 +/- 1 to 16 +/- 1 pg/min, and PGE(2) release, from 77 +/- 11 to 161 +/- 23 pg/min, suggesting a role for PGE(2) in the norepinephrine-induced activation of renal sensory nerves. Prazosin and indomethacin reduced and rauwolscine enhanced the norepinephrine-induced increases in substance P and PGE(2). PGE(2) enhanced the norepinephrine-induced activation of renal sensory nerves by stimulation of EP4 receptors. Interaction between ERSNA and ARNA is modulated by norepinephrine, which increases and decreases the activation of the renal sensory nerves by stimulating alpha(1)- and alpha(2)-adrenoceptors, respectively, on the renal pelvic sensory nerve fibers. Norepinephrine-induced activation of the sensory nerves is dependent on renal pelvic synthesis/release of PGE(2).     

Tetrahydrobiopterin deficiency exaggerates intimal hyperplasia after vascular injury

Decreased levels of tetrahydrobiopterin (BH4), an absolute cofactor for nitric oxide synthase (NOS), lead to uncoupling of NOS into a superoxide v. nitric oxide producing enzyme, and it is this uncoupling that links it to the development of vascular disease. However, the effects of in vivo deficiency of BH4 on neointimal formation after vascular injury have not been previously investigated. Hph-1 mice, which display 90% deficiency in guanine triphosphate cyclohydrolase I, the rate limiting enzyme in BH4 synthesis, were used. Hph-1 and wild-type mice, treated with either vehicle or BH4 (n = 15 per group), were subjected to wire-induced femoral artery injury, and NOS expression and activity, inflammation, cell proliferation, superoxide production, and neointimal formation were assessed. The major form of NOS expressed over vessel wall after vascular injury was endothelial NOS. Hph-1 mice exhibited lower NOS activity (2.8 +/- 0.3 vs. 4.5 +/- 0.4 pmol/min/mg protein, P < 0.01), and higher aortic superoxide content (5.2 +/- 2.0 x 10(5) cpm vs. 1.6 +/- 0.7 x 10(5) cpm, P < 0.01) compared with wild-type controls, indicating uncoupling of NOS. Treatment of hph-1 mice with BH4 significantly increased NOS activity (from 2.8 +/- 0.3 to 4.1 +/- 0.4 pmol.min(-1).mg protein(-1), P < 0.05), and attenuated superoxide production (from 5.2 +/- 2.0 x 10(5) cpm to 0.8 +/- 0.7 x 10(5) cpm, P < 0.05). Hph-1 mice also had higher inflammatory reactions and more cell proliferation after vascular injury. Furthermore, hph-1 mice responded by a marked increase in neointimal formation at 4 wk after vascular injury, compared with wild-type controls (intima:media ratio: 4.5 +/- 0.5 vs. wild-type 0.7 +/- 0.1, P < 0.001). Treatment of hph-1 mice with BH4 prevented vascular injury-induced increase in neointimal formation (intima:media ratio: 1.4 +/- 0.1 vs. hph-1, P < 0.001). Treatment had no effect on wild-type controls. In summary, we describe, for the first time, that in vivo BH4 deficiency facilitates neointimal formation after vascular injury. Modulation of BH4 bioavailability is an important therapeutic target for restenosis.     

Cerebrovascular smooth muscle culture. II. Characterization of adrenergic receptors linked to adenylate cyclase

Cultured and propagated smooth muscle cells contain adenylate cyclase (AC) responsive to catecholamines and their analogues. Isoproterenol and zinterol were the most effective stimulants of AC activity with EC50 = 8.5 X 10(-8)M. They were followed by epinephrine, phenylephrine and norepinephrine (EC50 = 7.5 X 10(-7)M, 6.5 X 10(-6)M and 4 X 10(-6)M, respectively). When the selective antagonists for beta 1 and beta 2 receptors (beta 1-type practolol and atenolol, beta 1/beta 2-type propranolol and beta 2-type butoxamine) were tested against isoproterenol, epinephrine and norepinephrine stimulation of AC activity, the beta 1 in contrast to beta 2 antagonists were found ineffective. The alpha-blockers (phentolamine alpha 1/alpha 2-type antagonists) and yohimbine (alpha 2-type antagonist) alone or in the presence of propranolol did not significantly inhibit the catecholamine-induced enhancement of cAMP formation. On the other hand, prazosine (alpha 1-type antagonist) blocked the stimulatory effect of epinephrine and norepinephrine on AC system. Similarly, the alpha 2-agonist, clonidine, did not affect the catecholamines' stimulated AC activity while alpha 1 agonist, phenylephrine, induced an additive enhancement of norepinephrine production of cAMP. The findings of beta-2- and alpha-1-type adrenergic receptors in the cultured cerebrovascular smooth muscle provide additional support for the implicated involvement of adrenergic innervation in the regulation of cerebral blood flow and/or systemic blood pressure.     

Adventitial delivery of dominant-negative p67phox attenuates neointimal hyperplasia of the rat carotid artery

Several essential components of NADPH oxidase, including p22phox, gp91phox (nox2) and its homologs nox1 and nox4, p47phox, p67phox, and rac1, are present in the vasculature. We previously reported that p67phox is essential for adventitial fibroblast NADPH oxidase O2- production. Thus we postulated that inhibition of adventitial p67phox activity would attenuate angioplasty-induced hyperplasia. To test this hypothesis, we treated the adventitia of carotid arteries with a control adenovirus (Ad-control), a virus expressing dominant-negative p67phox (Ad-p67dn), or a virus expressing a competitive peptide (gp91ds) targeting the p47phox-gp91phox interaction (Ad-gp91ds). Common carotid arteries (CCAs) from male Sprague-Dawley rats were transfected with Ad-control, Ad-p67dn, or Ad-gp91ds in pluronic gel. After 2 days, a 2-F (Fogarty) catheter was used to injure CCAs in vivo. After 14 days, CCAs were perfusion-fixed and analyzed. In 13 experiments, digital morphometry suggested a reduction of neointimal hyperplasia with Ad-p67dn compared with Ad-control; however, the reduction did not reach statistical significance (P = 0.058). In contrast, a significant reduction was achieved with Ad-gp91ds (P = 0.006). No changes in medial area or remodeling were observed with either treatment. Moreover, adventitial fibroblast proliferation in vitro was inhibited by Ad-gp91ds but not by Ad-p67dn, despite confirmation that Ad-p67dn inhibits NADPH oxidase in fibroblasts. These data appear to suggest that a multicomponent vascular NADPH oxidase plays a role in neointimal hyperplasia. However, inhibition of p47phox may be more effective than inhibition of p67phox at attenuating neointimal growth.     

Regulation of adenylate cyclase of neuroblastoma x glioma hybrid cells by alpha-adrenergic receptors. I. Inhibition of adenylate cyclase mediated by alpha receptors.

(-)-Norepinephrine and other catecholamines inhibit basal and prostaglandin E1-stimulated adenylate cyclase activities by 35 to 60% in homogenates of NG108-15 neuroblastoma x gloma hybrid cells and markedly reduce adenosine 3'35:'-monophosphate levels of intact cells, but do not affect guanosine 3':5'-monophosphate levels. The specificity of the NG108-15 receptor for ligands is that of an alpha receptor, possibly a presynaptic alpha 2 receptor. The inhibition of adenylate cyclase by norepinephrine is reversed by alpha receptor antagonists such as dihydroergotamine or phentolamine, but not by the beta receptor antagonist propranolol. The effect of norepinephrine on adenylate cyclase activity initially is dependent on GTP; half-maximal inhibition of enzyme activity by norepinephrine is obtained with 0.2 micron GTP. The inhibition of adenylate cyclase activity by norepinephrine is reduced by 10 mM NaF and is abolished by 0.05 mM guanyl-5'-yl imidodiphosphate. Inhibitions of NG108-15 adenylate cyclase mediated by alpha receptors, opiate receptors, and muscarinic acetylcholine receptors are not additive; this suggests that the three species of receptors can be functionally coupled to the same adenylate cyclase molecules or molecules regulating the enzyme.