Left ventricular geometry, tissue composition, and residual stress in High Fat Diet Dahl-Salt sensitive rats.

BACKGROUND: Hypertension drives myocardial remodeling, leading to changes in structure, composition and mechanical behavior, including residual stress, which are linked to heart disease progression in a gender-specific manner. Emerging therapies are also targeting constituent-specific pathological features. All previous studies, however, have characterized remodeling in the intact tissue, rather than isolated tissue constituents, and did not include sex as a biological variable. OBJECTIVE: In this study we first identified the contribution of collagen fiber network and myocytes to the myocardial residual stress/strain in Dahl-Salt sensitive rats fed with high fat diet. Then, we quantified the effect of hypertension on the remodeling of the left ventricle (LV), as well as the existence of sex-specific remodeling features. METHODS: We performed mechanical tests (opening angle, ring-test) and histological analysis on isolated constituents and intact tissue of the LV. Based on the measurements from the tests, we performed a stress analysis to evaluate the residual stress distribution. Statistical analysis was performed to identify the effects of constituent isolation, elevated blood pressure, and sex of the animal on the output of both experimental measures and modeling results. RESULTS: Hypertension leads to reduced residual stress/strain intact tissue, isolated collagen fibers, and isolated myocytes in male and female rats. Collagen remains the largest contributor to myocardial residual stress in both normotensive and hypertensive animals. We identified sex-differences in both hypertensive and normotensive animals. CONCLUSIONS: We observed both constituent- and sex-specific remodeling features in the LV of an animal model of hypertension.

Interleukin-10 does not contribute to the anti-contractile nature of PVAT in health.

Perivascular adipose tissue (PVAT) is protective and reduces contraction of blood vessels in health. PVAT is composed of adipocytes, multiple types of immune cells and stromal cells. Interleukin (IL)-10, an anti-inflammatory cytokine usually produced by T cells, B cells and macrophages, was identified as one of the highly expressed (mRNA) cytokines in the mesenteric PVAT of healthy rats. One report suggested that exogenous IL-10 causes relaxation of mouse mesenteric arteries, also suggesting that IL-10 maybe a potential anti-contractile factor. Hence, we hypothesized that PVAT-derived IL-10 causes vasorelaxation and/or reduces vasoconstriction, thus contributing to the anti-contractile nature of PVAT in health. Mesenteric arteries from rats and mice expressed the receptor for IL-10 (in tunica intima and media) as determined by immunohistochemistry. Mesenteric resistance arteries for rats and superior mesenteric artery for mice were used for isometric contractility studies. Increasing concentrations [0.4-100 ng/mL] of recombinant rat/mouse (rr/mr) IL-10 or vehicle was directly added to half-maximally constricted (phenylephrine, PE) vessels (without PVAT, with endothelium). IL-10 did not cause a direct vasorelaxation. Further, the ability of rrIL-10 to cause a rightward or downward shift of a vasoconstriction-response curve was tested in the rat. The vessels were incubated with rrIL-10 [100 ng/mL or 10 ng/mL] or vehicle for 1.5 h in the tissue bath followed by a cumulative PE [10-8-10-4 M] or U46619 [10-10-10-5 M] response curve. The maximal contractions and EC50 values were similar in IL-10 incubated vessels vs vehicle. Thus, acute exposure of exogenous IL-10 did not reduce local vasoconstriction. To further test if endogenous IL-10 from PVAT was anti-contractile, superior mesenteric arteries from IL-10 WT and KO mice, with and without PVAT, were subjected to increasing concentrations of PE. The anti-contractile nature of PVAT was preserved with both short-term and prolonged depletion (using younger and older mice, respectively) of endogenous IL-10 in males and females. Contrary to our hypothesis, PVAT-derived IL-10 neither caused vasorelaxation nor reduced local vasoconstriction directly/indirectly. Therefore, IL-10 does not contribute to the anti-contractile nature of PVAT in healthy rodents.

Perivascular adipose tissue contains functional catecholamines.

The sympathetic nervous system and its neurotransmitter effectors are undeniably important to blood pressure control. We made the novel discovery that perivascular adipose tissue (PVAT) contains significant concentrations of catecholamines. We hypothesized that PVAT contains sufficient releasable catecholamines to affect vascular function. HPLC, isometric contractility, immunohistochemistry, whole animal approaches and pharmacology were used to test this hypothesis. In normal rat thoracic aorta and superior mesenteric artery, the indirect sympathomimetic tyramine caused a concentration-dependent contraction that was dependent on the presence of PVAT. Tyramine stimulated release of NA, dopamine (DA) and the tryptamine serotonin (5-HT) from PVAT isolated from both arteries. In both arteries, tyramine-induced concentration-dependent contraction was rightward-shifted and reduced by the noradrenaline transporter inhibitor nisoxetine (1 µM), the vesicular monoamine transporter tetrabenazine (10 µM) and abolished by the α adrenoreceptor antagonist prazosin (100 nM). Inhibitors of the DA and 5-HT transporter did not alter tyramine-induced, PVAT-dependent contraction. Removal of the celiac ganglion as a neuronal source of catecholamines for superior mesenteric artery PVAT did not significantly reduce the maximum or shift the concentration dependent contraction to tyramine. Electrical field stimulation of the isolated aorta was not affected by the presence of PVAT. These data suggest that PVAT components that are independent of sympathetic nerves can release NA in a tyramine-sensitive manner to result in arterial contraction. Because PVAT is intimately apposed to the artery, this raises the possibility of local control of arterial function by PVAT catecholamines.

Ryanodine receptors are uncoupled from contraction in rat vena cava.

Ryanodine receptors (RyR) are Ca(2+)-sensitive ion channels in the sarcoplasmic reticulum (SR) membrane, and are important effectors of SR Ca(2+) release and smooth muscle excitation-contraction coupling. While the relationship between RyR activation and contraction is well characterized in arteries, little is known about the role of RyR in excitation-contraction coupling in veins. We hypothesized that RyR are present and directly coupled to contraction in rat aorta (RA) and vena cava (RVC). RA and RVC expressed mRNA for all 3 RyR subtypes, and immunofluorescence showed RyR protein was present in RA and RVC smooth muscle cells. RA and RVC rings contracted when Ca(2+) was re-introduced after stores depletion with thapsigargin (1µM), indicating both tissues contained intracellular Ca(2+) stores. To assess RyR function, contraction was then measured in RA and RVC exposed to the RyR activator caffeine (20mM). In RA, caffeine caused contraction that was attenuated by the RyR antagonists ryanodine (10µM) and tetracaine (100µM). However, caffeine (20mM) did not contract RVC. We next measured contraction and intracellular Ca(2+) (Ca(2+)(i)) simultaneously in RA and RVC exposed to caffeine. While caffeine increased Ca(2+)(i) and contracted RA, it had no significant effect on Ca(2+)(i) or contraction in RVC. These data suggest that ryanodine receptors, while present in both RA and RVC, are inactive and uncoupled from Ca(2+) release and contraction in RVC.

The existence of a local 5-hydroxytryptaminergic system in peripheral arteries.

BACKGROUND AND PURPOSE: 5-HT is a vasoconstrictor exhibiting enhanced effects in systemic arteries from subjects with cardiovascular disease. The effect of endogenous 5-HT on arteries is controversial, because the concentration of free circulating 5-HT is low and a 5-hydroxytryptaminergic system has not been identified in peripheral arteries. We hypothesized that a local 5-hydroxytryptaminergic system (including 5-HT synthesis, metabolism, uptake and release) with physiological function exists in peripheral arteries. EXPERIMENTAL APPROACH: The presence of key components of a 5-hydroxytryptaminergic system in rat aorta and superior mesenteric artery was examined using western blot analyses, immunohistochemistry and immunocytochemistry. The function of the rate-limiting enzyme in 5-HT biosynthesis, tryptophan hydroxylase (TPH), and 5-HT transporter was tested by measuring enzyme activity and 5-HT uptake, respectively. Isometric contraction of arterial strips was used to demonstrate the function of released endogenous 5-HT in arterial tissues. KEY RESULTS: mRNA for TPH-1 was present in arteries, with low levels of TPH protein and TPH activity. Expression and function of MAO A (5-HT metabolizing enzyme) was supported by immunohistochemistry, western analyses and the elevation of concentrations of 5-hydroxyindoleacetic acid (5-HT metabolite) after exposure to exogenous 5-HT. The 5-HT transporter was localized to the plasma membrane of freshly isolated aortic smooth muscle cells. Peripheral arteries actively took up 5-HT in a time-dependent and 5-HT transporter-dependent manner. The 5-HT transporter substrate, (+)-fenfluramine, released endogenous 5-HT from peripheral arteries, which potentiated noradrenaline-induced arterial contraction. CONCLUSIONS AND IMPLICATIONS: This study revealed the existence of a local 5-hydroxytryptaminergic system in peripheral arteries.

Arterial epidermal growth factor receptor expression in deoxycorticosterone acetate-salt hypertension.

Epidermal growth factor (EGF) causes contraction in arteries from deoxycorticosterone acetate (DOCA)-salt hypertensive rats but not in normotensive sham rats. We hypothesized that an increase in the number of EGF receptors (EGFRs) in arteries from DOCA-salt rats enables the observed contraction to EGF to occur. DOCA-salt rats had a systolic blood pressure >170 mm Hg, whereas all sham rats had a systolic blood pressure <125 mm Hg. Thoracic aorta were removed for measurement of isometric force, EGFR mRNA levels, and EGFR protein levels. EGF caused a significant contraction in endothelium-denuded aorta from DOCA-salt rats (38+/-7% of maximal phenylephrine-induced [10 micromol/L] contraction) compared with aorta from sham rats (4+/-2%). The EGFR tyrosine kinase-specific inhibitors 4,5-dianilinophthalimide (10 micromol/L) and AG1478 (250 nmol/L) reduced contraction in aorta from DOCA-salt by 85+/-14% and 65+/-10%, respectively. EGFR mRNA in DOCA-salt aorta was increased 4.2-fold compared with that in sham aorta. However, Western analyses of membrane-enriched and whole-tissue lysate of aorta from sham and DOCA-salt revealed no statistical difference in the density of EGFR protein between sham and DOCA-salt aorta. These data refute our hypothesis and suggest that a change downstream of EGFR is responsible for enabling EGF-induced contraction in hypertension.

Enhanced contraction to 5-hydroxytryptamine is not due to "unmasking" of 5-hydroxytryptamine(1b) receptors in the mesenteric artery of the deoxycorticosterone acetate-salt rat.

5-Hydroxytryptamine(1B) (5-HT(1B)) receptors have been implicated in mediating arterial contraction to 5-HT. Additionally, the 5-HT(1B) receptor has been reported to be "unmasked" by depolarizing stimuli. We hypothesized that 5-HT(1B) receptors in arteries from hypertensive animals, arteries reported to have a depolarized resting membrane potential in smooth muscle cells, are unmasked and participate in the supersensitivity observed to 5-HT in hypertension. We used the isolated tissue bath apparatus and examined the response of superior mesenteric arteries from normotensive sham and hypertensive deoxycorticosterone acetate (DOCA)-salt rats. The 5-HT(1B) agonists CP93129 and sumatriptan (10(-9) to 10(-5) mol/L) caused a maximal contraction (50+/-12% of phenylephrine [10(-5) mol/L] contraction) in arteries from DOCA-salt rats; no contraction was observed in arteries from normotensive rats. The 5-HT(1B) receptor antagonist GR55562 (100 nmol/L) inhibited both the 5-HT- (4-fold rightward shift) and CP93129-induced (11-fold rightward shift) contractions in mesenteric arteries from hypertensive DOCA-salt rats. In other experiments, arteries from normotensive rats were incubated with 15 mmol/L KCl, as a depolarizing stimulus, and then exposed to 5-HT and CP93129. In the presence of KCl, a small leftward shift to 5-HT was observed. However, the presence of a depolarizing stimulus was unable to produce changes in the 5-HT maximal response to resemble that of arteries from DOCA-salt rats, nor was contraction to CP93129 observed. These data support the conclusions that 5-HT(1B) receptors mediate contraction in mesenteric arteries from hypertensive rats and that this enhanced response to 5-HT is not due to membrane depolarization alone.

Inability of serotonin to activate the c-Jun N-terminal kinase and p38 kinase pathways in rat aortic vascular smooth muscle cells.

BACKGROUND: Serotonin (5-HT, 5-hydroxytryptamine) activates the Extracellular Signal-Regulated Kinase (ERK)/ Mitogen-Activated Protein Kinase (MAPK) pathways, in vascular smooth muscle cells. Parallel MAPK pathways, the c-Jun N-terminal Kinase (JNK) and p38 pathway, are activated by stimulators of the ERK/MAPK pathway. We hypothesized that 5-HT would activate the JNK and p38 pathways in rat vascular smooth muscle cells. RESULTS: Results were determined using standard Western analysis and phosphospecific JNK and p38 antibodies. No significant activation by 5-HT (10(-9) - 10(-5) M; 30 min) of the JNK or p38 pathways, as measured by protein phosphorylation, was observed in any of these experiments. These experiments were repeated in the presence of the serine/threonine phosphatase inhibitor okadaic acid (1 uM) and the tyrosine phosphatase inhibitor sodium orthovanadate (1 uM) to maximize any observable signal. Even under these optimized conditions, no activation of the JNK or p38 pathways by 5-HT was observed. Time course experiments (5-HT 10(-5) M; 5 min, 15 min, 30 min and 60 min) showed no significant activation of JNK after incubation with 5-HT at any time point. However, we detected strong activation of JNK p54 and p46 (5- and 7 fold increases in bands p54 and p46, respectively over control levels) by anisomycin (500 ng/ml, 30 min). Similarly, a JNK activity assay failed to reveal activation of JNK by 5-HT, in contrast to the strong stimulation by anisomycin. CONCLUSION: Collectively, these data support the conclusion that 5-HT does not activate the JNK or p38 pathways in rat vascular smooth muscle cells.

Activation of Erk mitogen-activated protein kinase proteins by vascular serotonin receptors.

We previously demonstrated that 5-hydroxytryptamine 2A (5-HT 2A ) receptor-mediated rat arterial contraction was dependent on activation of tyrosine kinases, including mitogen-activated protein kinase (MAPK) kinase. In the current study, we examined arterial smooth muscle for the presence of serotonin (5-hydroxytryptamine, 5-HT) 5-HT 1B, 5-HT 1D, 5-HT 1F, 5-HT 2A, 5-HT 2B, and 5-HT 7 receptor mRNA and hypothesized that, if present, activation of these receptors would stimulate the extracellular signal-regulated kinase (Erk) MAPK pathway and an Erk MAPK-dependent contraction. RT-PCR analyses of rat aortic smooth muscle cells, cultured and fresh, indicated the presence of 5-HT 1B, 5-HT 1D, 5-HT 1F, 5-HT 2A, 5-HT 2B, and 5-HT 7 receptor mRNA. The 5-HT 1B agonists RU24969 and CGS12066B, 5-HT 1B/1D/1F receptor agonist sumatriptan, and 5-HT 2B receptor agonist BW723C86 (10(-9) - 10(-4) M ) did not contract the aorta, nor did the 5-HT 7 receptor antagonist LY215840 leftward shift 5-HT-induced contraction. The 5-HT 1E/1F receptor agonist BRL54443 induced contraction, but this was abolished by the 5-HT 2A/2C receptor antagonist ketanserin (10 nM ); contraction was not observed with a different 5-HT 1F receptor agonist, LY344864. 5-HT and alpha-methyl-5-HT produced a concentration-dependent increase in Erk MAPK activity in cultured aortic smooth muscle cells and in aorta contracted with these agonists. All other agonists were inactive; a high concentration of BRL54443 (10 microM ) stimulated Erk MAPK activation (150% basal). Thus, while mRNA and possibly protein for multiple 5-HT receptors are present in aortic smooth muscle, only the 5-HT 2A receptor plays a significant role in directly modulating contractility and activating the Erk MAPK pathway.

Mineralocorticoids upregulate arterial contraction to epidermal growth factor.

The present studies test the hypothesis that contraction to EGF is dependent on mineralocorticoids and/or an elevation in systolic blood pressure (SBP). Endothelium-denuded thoracic aortas from sham normotensive, N(omega)-nitro-L-arginine (L-NNA) hypertensive, Wistar-Kyoto (WKY), and spontaneously hypertensive rats (SHR) were used in isolated tissue-bath experiments. Maximal contraction to epidermal growth factor [EGF; percentage of phenylephrine (PE; 10 umol/l)-induced contraction] was greater in strips from L-NNA (32 +/- 5%) and SHR (53 +/- 8%) rats compared with sham and WKY rats (17 +/- 1 and 12 +/- 4%, respectively). Wistar-Furth rats became only mildly hypertensive when given DOCA salt (134 +/- 6 mmHg) compared with Wistar rats (176 +/- 9 mmHg), but aortas from both strains had a similarly enhanced contraction to EGF (approximately 9 times the maximal contraction of sham aorta). Furthermore, in vitro incubation of aortas from Wistar and Wistar-Furth rats with aldosterone (10 nmol/l) increased EGF-receptor mRNA expression by >50%. These data indicate that arterial contraction to EGF may occur independent of hypertension and be stimulated by mineralocorticoids.

Characterization of the serotonin receptor mediating contraction in the mouse thoracic aorta and signal pathway coupling.

The purpose of this study was to characterize pharmacologically the 5-HT receptor(s) mediating contraction in the mouse aorta and the pathways these receptors are coupled with to mediate contraction. We hypothesized that a 5-HT2A receptor, as in the rat, mediates contraction by activating L-type calcium channels, phospholipase C (PLC), and tyrosine kinase(s). Endothelium-denuded aortic strips were placed in a tissue bath for measurement of isometric contractile force. 5-HT, the 5-HT2A receptor agonist alpha-methyl-5-HT, and partial 5-HT2A receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (+/--DOI) caused the most potent and efficacious contraction. The 5-HT(1E/1F) receptor agonist BRL 54443 also induced contraction (-log EC(50) = 6.52); however, the 5-HT2A receptor antagonist ketanserin antagonized this contraction. Our hypothesis was further supported by the finding that antagonists with affinity for the 5-HT2A receptor, ketanserin, 1-(1-naphthyl)piperazine, spiperone, and LY53857, reduced 5-HT-induced contraction. A correlation of 0.927 was found between literature-derived compound binding affinities for the agonists and antagonists at the 5-HT2A receptor of the rat and the data generated in our experiments (-log EC(50) and pK(B) values). The L-type calcium channel blockers nifedipine and nitrendipine, PLC inhibitor 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate, and tyrosine kinase inhibitors genistein and PD 098,059 all shifted and/or reduced maximum contraction to 5-HT. We conclude that contraction to 5-HT in the mouse aorta is mediated primarily by a 5-HT2A receptor and is coupled to L-type calcium channels, PLC, and tyrosine kinases.

Effects of salt intake and angiotensin II on vascular reactivity to endothelin-1.

Hypertension produced by chronic infusion of angiotensin II (Ang II) is significantly blunted by blockade of endothelin-1 (ET-1) ET(A) subtype receptors. Furthermore, this model is salt-sensitive, and the antihypertensive response to ET(A) receptor blockade is more pronounced in animals on high salt intake. The goal of these experiments was to evaluate the effect of salt intake and Ang II on vascular reactivity to ET-1. In superior mesenteric arteries from normal male rats, studied in a standard muscle bath, incubation for 1 h with a subcontractile concentration of Ang II (10(-10) M) did not affect concentration-response curves to ET-1. Pressor responses in vivo to 2-h infusions of Ang II (5 ng/min) in rats maintained on normal or high salt intake were abolished by pretreatment with the ET(A) receptor antagonist ABT-627. The antagonist had no effect on pressor responses to phenylephrine (PE). In other experiments, rats maintained on either high or normal salt intake received continuous infusion of Ang II (5 ng/min i.v.) for 7 days, and then their superior mesenteric arteries were tested in the muscle bath. The maximum contractile response to ET-1 in arteries from Ang II-infused rats on normal salt intake was larger than in arteries from rats not receiving Ang II. Conversely, maximum responses to ET-1 in arteries from Ang II-infused rats on high salt intake were depressed compared with controls. No differences in vascular reactivity to PE were found. Thus, chronic in vivo exposure to Ang II results in specific salt-dependent changes in vascular reactivity to ET-1.

Insulin-like growth factor inhibits vascular contraction to 5-hydroxytryptamine: involvement of tyrosine phosphatase.

This study tests the hypothesis that insulin-like growth factor 1 (IGF-1)-induced vasodilation is due to the stimulation of tyrosine phosphatase. Rat aortic segments (endothelium intact) were placed in muscle baths for force measurement. Segments were contracted to serotonin [5-hydroxytyptamine (5-HT), 10(-7)-10(-5) M] before and after incubation with IGF-1 (10-100 nM; 90 min). IGF-1 caused a 20% inhibition of 5-HT-induced contractions. This inhibition was reversed by the tyrosine phosphatase inhibitors sodium orthovanadate and molybdate. Orthovanadate did not alter inhibitory properties of the calcium channel antagonist verapamil, suggesting that the phosphatase inhibitors were relatively specific. IGF-1-induced inhibition was not altered by blockade of nitric oxide synthase. Western blot analysis confirmed that the 5-HT-induced stimulation of tyrosine phosphorylation of the 42-kDa extracellular signal-regulated mitogen-activated protein kinase protein was reduced by IGF-1 (52% inhibition), an inhibition that was attenuated by orthovanadate. These data are consistent with the hypothesis that the vasodilator activity of IGF-1 is mediated by the activation of a tyrosine phosphatase.

5-Hydroxytryptamine-induced potentiation of endothelin-1- and norepinephrine-induced contraction is mitogen-activated protein kinase pathway dependent.

5-Hydroxytryptamine (5-HT)-induced arterial contraction depends on activation of the tyrosine kinase-dependent extracellular signal-regulated mitogen-activated protein kinase (Erk MAPK) pathway. The importance of 5-HT in the control of peripheral resistance has been questioned because circulating free levels of 5-HT are low (in the nanomolar range). We tested the hypothesis that physiologically relevant concentrations of 5-HT potentiate arterial contraction in response to agonists proved to have importance in blood pressure maintenance (norepinephrine [NE] and endothelin-1 [ET-1]) in a tyrosine kinase- and an Erk MAPK-dependent manner. Strips of endothelium-denuded rat tail artery were used for the measurement of isometric force. The general tyrosine kinase inhibitor genistein (5 micromol/L) and the inhibitor of MAPK/Erk kinase activation PD098059 (10 micromol/L) shifted concentration-response curves to 5-HT (1x10(-9) to 3x10(-4) mol/L) rightward but did not shift concentration-response curves to NE or ET-1. In separate experiments, 5-HT (10 nmol/L) potentiated contraction in response to NE (20 nmol/L) by approximately 200% to 300% and to ET-1 (0.3 and 1 nmol/L) by 640% and 180%, respectively. Genistein and PD098059 significantly (66% to 100%) reduced 5-HT-induced potentiation of both NE (20 nmol/L)- and ET-1 (0.3 and 1 nmol/L)-induced contraction. Thus, these data support the ability of low physiological concentrations of 5-HT to amplify arterial responses to hormones with bona fide effects on blood pressure in the novel manner of depending on a tyrosine kinase/Erk MAPK pathway. Although these findings were generated in large arteries, we speculate that they may be applicable to vascular functioning in the deoxycorticosterone acetate salt model of hypertension in which all 3 hormones, 5-HT, NE, and ET-1, have been implicated as causal factors.

Interaction of the beta adrenergic receptor antagonist bucindolol with serotonergic receptors.

Bucindolol is a nonselective beta-adrenergic receptor antagonist that has additional vasodilating properties. Because some beta-adrenergic receptor antagonists such as cyanopindolol are used as 5-HT1A/5-HT1B receptor antagonists, we tested the hypothesis that bucindolol can interact with 5-HT receptors. Both in vitro and in vivo methods were used to examine the interaction of bucindolol with 5-HT receptors relevant to the cardiovascular system-the 5-HT1A, 5-HT1D, 5-HT2A, and 5-HT2B receptors-and with alpha1-adrenergic receptors. In binding studies, bucindolol displayed high affinity for the 5-HT1A receptor (Ki, 11 nM), modest affinity for the 5-HT2A receptor (Ki, 382 nM), and no measurable affinity for the 5-HT1D receptor; binding affinity for the 5-HT2B receptor was not studied. Bucindolol also displayed significant binding affinity (Ki, 69 nM) for the alpha1-adrenergic receptors. Alpha1-Adrenergic receptor antagonist activity was confirmed by the ability of bucindolol (1 mg/kg) to act as a competitive antagonist against 0.01-30 microg/kg phenylephrine-induced pressor responses in conscious rats. In conscious permanently instrumented rats, bucindolol (0.1-3.0 mg/kg, i.v.) did not cause bradycardia similar to that elicited by the 5-HT1A-receptor agonist 8-OH-DPAT (3-300 microg/kg, i.v.), nor did bucindolol (1 mg/kg) block the 8-OH-DPAT-induced bradycardia. Bucindolol (10(-9)-10(-5) M) did not cause relaxation in the PGF2alpha-contracted, endothelium-intact porcine coronary artery, nor did bucindolol (10(-5) M) block 5-HT-induced coronary artery relaxation, indicating that bucindolol does not have significant interactions at the 5-HT1D receptor. Bucindolol also displayed no agonist activity at the 5-HT2A and 5-HT2B receptor (endothelium-denuded rat thoracic aorta and rat stomach fundus, respectively), but did act as a weak 5-HT2A-receptor antagonist (-log K(B) [M] = 5.4+/-0.1) and 5-HT2B-receptor antagonist (-log K(B) [M] = 7.8+/-0.1). Thus, these data suggest that bucindolol lacks the ability to activate the 5-HT1A, 5-HT1D, 5-HT2A, and 5-HT2B receptor, but can block alpha1-adrenergic receptors and act as a weak 5-HT2A- and 5-HT2B-receptor antagonist. The relevance of these serotoninergic effects as it pertains to the mechanism of bucindolol-induced vasodilation is unknown.

Mechanisms of 5-hydroxytryptamine(2A) receptor activation of the mitogen-activated protein kinase pathway in vascular smooth muscle.

5-Hydroxytryptamine (5-HT) activates the extracellular signal-regulated kinase (Erk) mitogen-activated protein kinases (MAPKs) in the vasculature, resulting in contraction. The mechanisms by which this occurs are unclear. G protein-coupled receptors can activate Erk MAPK pathways through a variety of mechanisms, including stimulation of Src, phosphoinositide-3 kinase (PI-3-K), protein kinase C (PKC), or the epidermal growth factor (EGF) receptor tyrosine kinase. We hypothesize that 5-HT uses one or more of these pathways. In isolated strips of rat aorta, the MAPK/Erk kinase inhibitor U0126 (50 microM), Src inhibitor PP1 (0.5 microM), PKC inhibitors calphostin C (1 microM) and chelerythrine (10 microM), and the PI-3-K inhibitor LY294002 (1-20 microM) reduced 5-HT-induced contraction. The EGF receptor tyrosine kinase inhibitor AG1478 (0.25-1 microM) was without effect. Thus, 5-HT activates PKC, Src, and possibly PI-3-K to result in contraction. In rat aortic myocytes, 5-HT (1 microM) activated Erk MAPK proteins 2- to 3-fold over basal values; activation was reduced by U0126, PP1, and LY294002 and unaffected by calphostin C or chelerythrine, wortmannin, or AG1478. The lack of effect of EGF receptor tyrosine kinase and PI-3-K inhibitors was confirmed in that the EGF receptor immunoprecipitated from 5-HT-exposed cells did not display an increase in autophosphorylation, nor did 5-HT significantly increase activation of Akt/protein kinase B, a downstream substrate for PI-3-K. These data suggest that the rat aortic 5-HT(2A) receptor uses Src but not PKC, PI-3-K, or the EGF receptor tyrosine kinase in stimulating Erk MAPK activation.

5-HT2B-receptor antagonist LY-272015 is antihypertensive in DOCA-salt-hypertensive rats.

We previously demonstrated a change in the receptors mediating 5-hydroxytryptamine (5-HT)-induced contraction in arteries of deoxycorticosterone acetate (DOCA)-salt-hypertensive rats. Specifically, contraction to 5-HT is mediated primarily by 5-HT2A receptors in arteries from normotensive sham rats and by both 5-HT2A and 5-HT2B receptors in arteries from hypertensive rats. We hypothesized that the 5-HT2B receptor may play a role in maintaining the high blood pressure of DOCA-salt-hypertensive rats, and herein we provide data connecting in vitro and in vivo findings. The endothelium-denuded isolated superior mesenteric artery of DOCA-salt rats displayed a marked increase in maximum contraction to the newly available 5-HT2B-receptor agonist BW-723C86 compared with that of arteries from sham rats, confirming that the 5-HT2B receptor plays a greater role in 5-HT-induced contraction in arteries from DOCA-salt rats. In chronically instrumented rats, the 5-HT2B-receptor antagonist LY-272015 (0.3, 1.0, and 3.0 mg/kg iv at 30-min intervals) was given cumulatively 1 time/wk during 4 wk of continued DOCA-salt treatment. LY-272015 did not reduce blood pressure of the sham-treated rats at any time or dose. However, LY-272015 (1.0 and 3. 0 mg/kg) significantly reduced mean blood pressure in a subgroup of week 3 (-20 mmHg) and week 4 DOCA-salt (-40 mmHg) rats that had extremely high blood pressure (mean arterial blood pressure approximately 200 mmHg). Blockade of 5-HT2B receptors by in vivo administration of LY-272015 (3.0 mg/kg) was verified by observing reduced 5-HT-induced contraction in rat stomach fundus, the tissue from which the 5-HT2B receptor was originally cloned. These data support the novel hypothesis that 5-HT2B-receptor expression is induced during the development of DOCA-salt hypertension and contributes to the maintenance of severe blood pressure elevations.

Epidermal growth factor: a potent vasoconstrictor in experimental hypertension.

We have tested the hypothesis that growth factor signaling pathways are augmented in hypertension, a disease associated with vascular smooth muscle cell growth. Thoracic aorta was dissected from deoxycorticosterone acetate-salt (DOCA-salt) and one kidney, one clip (1K, 1C) hypertensive rats and from sham normotensive rats for use in isolated tissue bath experiments. Systolic blood pressure was significantly higher in DOCA-salt and 1K, 1C than in normotensive sham rats: 192 +/- 7, 185 +/- 10, and 117 +/- 4 mmHg, respectively. Although virtually no contraction to epidermal growth factor (EGF) was observed in endothelium-denuded sham rat aorta [1 +/- 1% phenylephrine (PE) (10 micromol/l)-induced contraction], the maximal EGF-induced contraction was 45 +/- 7% in endothelium-denuded aorta from DOCA-salt hypertensive rats and 39 +/- 7% in aorta from 1K, 1C rats. Although slightly attenuated, a contraction to EGF was still observed in endothelium-intact aortic strips from 28-day DOCA-salt hypertensive rats. We also conducted concentration-response curves to EGF on days 1, 3, 5, 7, 14, and 21 of DOCA-salt therapy. A significant contraction to EGF in aorta from DOCA-salt rats was observed on day 14, when DOCA-salt rats had significantly higher blood pressure than sham rats: 188 +/- 6 and 122 +/- 3 mmHg, respectively. Transforming growth factor-alpha, an agonist of the EGF receptor, contracted DOCA-salt rat aorta (30 +/- 7% PE-induced contraction) but not sham aorta (3 +/- 3%). The EGF receptor tyrosine kinase inhibitor 4,5-dianilinophthalimide (10 micromol/l), the mitogen-activated protein kinase kinase inhibitor PD-098059 (10 micromol/l), and the L-type voltage-gated calcium channel inhibitor diltiazem (1 mol/l), but not the cyclooxygenase inhibitor indomethacin (10 micromol/l), virtually abolished EGF-induced contraction (85, 98, and 99% reduction, respectively). These data support a striking difference in EGF signaling between normotensive and hypertensive animals. Furthermore, they provide evidence that growth factors should be considered vasoconstrictors as well as growth modulators in hypertension.

Is functional upregulation of the 5-HT2B receptor in deoxycorticosterone acetate salt-treated rats blood pressure dependent?

This study tests the hypothesis that the functional upregulation of the arterial 5-hydroxytryptamine (5-HT)2B receptor in arteries of deoxycorticosterone acetate (DOCA)-salt hypertensive rats depends on the development of high blood pressure. Wistar-Furth and Wistar rats were given sham or DOCA-salt treatment (200 mg/kg DOCA, SC; 1.0% NaCl and 0.2% KCI in drinking water). Systolic blood pressures (4 week; mm Hg) were: Wistar Sham (120+/-3), Wistar DOCA (176+/-6), Wistar-Furth Sham (112+/-3) and Wistar-Furth DOCA (136+/-4). Isolated mesenteric arteries from Wistar DOCA and Wistar-Furth DOCA rats displayed a three- to fivefold leftward shift in contraction to 5-HT that was insensitive to blockade by the 5-HT2A receptor antagonist ketanserin (10 nM) and a significantly increased maximal contraction to the 5-HT2B receptor agonist BW723C86 [Wistar DOCA = 90+/-17% phenylephrine contraction; Wistar Sham = 1+/-1%; Wistar-Furth DOCA = 33+/-8%; Wistar-Furth Sham = 0%]. Arteries from Sprague-Dawley rats receiving salt or DOCA alone displayed similar systolic blood pressures (151+/-11 mm Hg and 144+/-5 mm Hg, respectively), but only tissues from rats receiving DOCA displayed an increased contraction to BW723C86 (DOCA alone = 60.7+/-16% vs. sham = 13+/-5.3%). These data suggest that upregulation of the arterial 5-HT2B receptor is largely independent of an increase in blood pressure.

Dissociation of angiotensin II-stimulated activation of mitogen-activated protein kinase kinase from vascular contraction.

Angiotensin II (Ang II) is a potent pressor hormone, a stimulus for vascular smooth muscle hypertrophy and an activator of multiple tyrosine kinases. The physiological effects of Ang II are mediated through activation of AT1 and AT2 receptors, receptors that have been coupled to tyrosine kinase(s) and tyrosine phosphatases, respectively. Agonists of G protein-coupled receptors, of which Ang II is one, have recently been shown to stimulate smooth muscle contraction in part via activation tyrosine kinases. We tested the hypothesis that Ang II-induced contraction in the rat aorta was dependent on activation of tyrosine kinase(s) and specifically investigated the role of the tyrosine kinase mitogen-activated protein kinase kinase (MEK), a kinase important to the mitogen activated protein kinase (MAPK) pathway. Rat thoracic aortic strips denuded of endothelium and cultured aortic smooth muscle cells were used in isolated tissue baths for measurement of isometric contractile force and Western analyses of protein tyrosyl-phosphorylation. Ang II (0.1-100 nM)-induced contraction in the aorta was completely blocked by the AT1 receptor antagonist losartan (1 microM) but unaffected by the AT2 receptor antagonist PD123319 (100 nM) or tyrosine phosphatase inhibitor sodium orthovanadate (1 microM), indicating an AT1 receptor mediates aortic contraction to Ang II. Neither the tyrosine kinase inhibitor genistein (5 microM), inactive tyrosine kinase inhibitor daidzein (5 microM) nor MEK inhibitor PD098059 (10 microM) reduced Ang II-induced contraction; the concentrations of inhibitors used maximally reduced contraction stimulated by other agonists of G protein-coupled receptors such as serotonin. Moreover, Ang II-induced contraction was not altered by the combination of PD098059 and PD123319, indicating that it is unlikely AT2 receptor stimulation masks activation of the MAPK pathway through AT1 receptor activation. The nonflavone tyrosine kinase inhibitor tyrphostin B42 (30 microM) reduced Ang II-induced maximal contraction (to 11.2% control) but, unlike the other tyrosine kinase inhibitors, also reduced KCl-induced contraction (to 55.2% control), indicating a probable nonselectivity of tyrphostin B42. Ang IIinduced maximal contraction was reduced by the L-type voltage gated calcium channel antagonist nifedipine (50 nM), consistent with the activation of calcium channels by Ang II. In cultured rat aortic smooth muscle cells, Ang II (0.1-1000 nM) stimulated concentration-dependent tyrosyl-phosphorylation of the extracellular signal regulated kinase (Erk) mitogen activated protein kinases (maximal stimulation, fold basal: Erk-1 = 17-fold, Erk-2 = 3-fold), indicating that Ang II can activate MEK. Losartan (1 microM) abolished Ang II (10 nM)-induced Erk tyrosyl-phosphorylation and PD098059 (10 microM), which did not diminish Ang II-induced aortic contraction, reduced Ang II (10 nM)-stimulated phosphorylation of Erk-2 by 72%. Finally, Ang II (1 microM) increased tyrosyl-phosphorylation of the Erk proteins in isolated aorta exposed to Ang II for 5 min. Thus, while Ang II can stimulate both MEK activation and vascular contraction via interaction with AT1 receptors, stimulation of MEK does not appear to be important for Ang II-induced contraction. These findings dissociate the process of Ang II-stimulated Erk protein tyrosyl-phosphorylation from Ang II-induced contraction in the rat aorta.