bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeted inhibition of sarcoplasmic reticulum CaMKII activity results in alterations of Ca2+homeostasis and cardiac contractility
Authors: Ji Yong , Zhao Wen , Li Bailing , Desantiago Jaime , Picht Eckard , Kaetzel Marcia A. , Schultz Jo El J. , Kranias Evangelia G. , Bers Donald M. , Dedman John R.
Published: September 2005

Transgenic (TG) mice expressing a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitory peptide targeted to the cardiac myocyte longitudinal sarcoplasmic reticulum (LSR) display reduced phospholamban phosphorylation at Thr17and develop dilated myopathy when stressed by gestation and parturition (Ji Y, Li B, Reed TD, Lorenz JN, Kaetzel MA, and Dedman JR. J Biol Chem 278: 25063–25071, 2003). In the present study, these animals (TG) are evaluated for the effect of inhibition of sarcoplasmic reticulum (SR) CaMKII activity on the contractile characteristics and Ca2+cycling of myocytes. Analysis of isolated work-performing hearts demonstrated moderate decreases in the maximal rates of contraction and relaxation (±dP/d t) in TG mice. The response of the TG hearts to increases in load is reduced. The TG hearts respond to isoproterenol (Iso) in a dose-dependent manner; the contractile properties were reduced in parallel to wild-type hearts. Assessment of isolated cardiomyocytes from TG mice revealed 40–47% decrease in the maximal rates of myocyte shortening and relengthening under both basal and Iso-stimulated conditions. Although twitch Ca2+transient amplitudes were not significantly altered, the rate of twitch intracellular Ca2+concentration decline was reduced by ∼47% in TG myocytes, indicating decreased SR Ca2+uptake function. Caffeine-induced Ca2+transients indicated unaltered SR Ca2+content and Na+/Ca2+exchange function. Phosphorylation assays revealed an ∼30% decrease in the phosphorylation of ryanodine receptor Ser2809. Iso stimulation increased the phosphorylation of both phospholamban Ser16and the ryanodine receptor Ser2809but not phospholamban Thr17in TG mice. This study demonstrates that inhibition of SR CaMKII activity at the LSR results in alterations in cardiac contractility and Ca2+handling in TG hearts.
Age-related changes in cardiac structure and function in Fischer 344 × Brown Norway hybrid rats
Authors: Hacker Timothy A. , McKiernan Susan H. , Douglas Pamela S. , Wanagat Jonathan , Aiken Judd M.
Published: September 2005

The effects of aging on cardiovascular function and cardiac structure were determined in a rat model recommended for gerontological studies. A cross-sectional analysis assessed cardiac changes in male Fischer 344 × Brown Norway F1 hybrid rats (FBN) from adulthood to the very aged ( n = 6 per 12-, 18-, 21-, 24-, 27-, 30-, 33-, 36-, and 39-mo-old group). Rats underwent echocardiographic and hemodynamic analyses to determine standard values for left ventricular (LV) mass, LV wall thickness, LV chamber diameter, heart rate, LV fractional shortening, mitral inflow velocity, LV relaxation time, and aortic/LV pressures. Histological analyses were used to assess LV fibrotic infiltration and cardiomyocyte volume density over time. Aged rats had an increased LV mass-to-body weight ratio and deteriorated systolic function. LV systolic pressure declined with age. Histological analysis demonstrated a gradual increase in fibrosis and a decrease in cardiomyocyte volume density with age. We conclude that, although significant physiological and morphological changes occurred in heart function and structure between 12 and 39 mo of age, these changes did not likely contribute to mortality. We report reference values for cardiac function and structure in adult FBN male rats through very old age at 3-mo intervals.
Type VI collagen induces cardiac myofibroblast differentiation: implications for postinfarction remodeling
Authors: Naugle Jennifer E. , Olson Erik R. , Zhang Xiaojin , Mase Sharon E. , Pilati Charles F. , Maron Michael B. , Folkesson Hans G. , Horne Walter I. , Doane Kathleen J. , Meszaros J. Gary
Published: September 2005

Cardiac fibroblast (CF) proliferation and differentiation into hypersecretory myofibroblasts can lead to excessive extracellular matrix (ECM) production and cardiac fibrosis. In turn, the ECM produced can potentially activate CFs via distinct feedback mechanisms. To assess how specific ECM components influence CF activation, isolated CFs were plated on specific collagen substrates (type I, III, and VI collagens) before functional assays were carried out. The type VI collagen substrate potently induced myofibroblast differentiation but had little effect on CF proliferation. Conversely, the type I and III collagen substrates did not affect differentiation but caused significant induction of proliferation (type I, 240.7 ± 10.3%, and type III, 271.7 ± 21.8% of basal). Type I collagen activated ERK1/2, whereas type III collagen did not. Treatment of CFs with angiotensin II, a potent mitogen of CFs, enhanced the growth observed on types I and III collagen but not on the type VI collagen substrate. Using an in vivo model of myocardial infarction (MI), we measured changes in type VI collagen expression and myofibroblast differentiation after post-MI remodeling. Concurrent elevations in type VI collagen and myofibroblast content were evident in the infarcted myocardium 20-wk post-MI. Overall, types I and III collagen stimulate CF proliferation, whereas type VI collagen plays a potentially novel role in cardiac remodeling through facilitation of myofibroblast differentiation.
Cyclooxygenase-2 induction by bradykinin in aortic vascular smooth muscle cells
Authors: Rodriguez Jorge A. , De la Cerda Paula , Collyer Eileen , Decap Valerie , Vio Carlos P. , Velarde Victoria
Published: September 2005

Vascular smooth muscle cell proliferation and migration play an important role in the pathophysiology of several vascular diseases, including atherosclerosis. Prostaglandins that have been implicated in this process are synthesized by two isoforms of cyclooxygenase (COX), with the expression of the regulated COX-2 isoform increased in atherosclerotic plaques. Bradykinin (BK), a vasoactive peptide increased in inflammation, induces the formation of prostaglandins through specific receptor activation. We hypothesized that BK plays an important role in the regulation of COX-2, contributing to the increase in production of prostaglandins in vascular smooth muscle cells. Herein we examined the signaling pathways that participate in the BK regulation of COX-2 protein levels in primary cultured aortic vascular smooth muscle cells. We observed an increase in COX-2 protein levels induced by BK that was maximal at 24 h. This increase was blocked by a B2 kinin receptor antagonist but not a B1 receptor antagonist, suggesting that the B2 receptor is involved in this pathway. In addition, we conclude that the activation of mitogen-activated protein kinases p42/p44, protein kinase C, and nitric oxide synthase is necessary for the increase in COX-2 levels induced by BK because either of the specific inhibitors for these enzymes blocked the effect of BK. Using a similar approach, we further demonstrated that reactive oxygen species and cAMP were not mediators on this pathway. These results suggest that BK activates several intracellular pathways that act in combination to increase COX-2 protein levels. This study suggests a role for BK on the evolution of the atheromatous plaque by virtue of controlling the levels of COX-2.
Aortic stiffness affects the coronary blood flow response to percutaneous coronary intervention
Authors: Leung Michael C. H. , Meredith Ian T. , Cameron James D.
Published: September 2005

We examined the hypothesis that a stiff aorta is associated with reduced coronary blood flow (CBF) and CBF response to percutaneous coronary intervention (PCI). Aortic mechanical properties are thought to affect CBF, with increased stiffness associated with decreased coronary perfusion. Animal studies are conflicting, and human evidence is lacking. Even less is known about the effects of aortic stiffness on the CBF response to successful PCI. In 18 subjects undergoing elective PCI, a Doppler velocity guidewire was positioned proximal to a severe coronary stenosis to measure resting and adenosine-induced hyperemic CBF before and after PCI. Stenosis severity was assessed with Doppler velocity and pressure guidewires. Aortic mechanical indexes measured included central pulse-wave velocity (cPWV) and central pulse pressure (cPP). PCI was successful in all subjects (diameter stenosis: 88 ± 9% to 2 ± 7%; coronary flow velocity reserve: 1.8 ± 0.6 to 3.0 ± 0.8; fractional flow reserve: 0.57 ± 0.19 to 0.92 ± 0.06; all P < 0.001). With the adjustment for age and gender, resting and hyperemic CBF were inversely related to cPWV irrespective of the presence of stenosis (resting: before PCI, r2 = 0.452, P < 0.01; after PCI, r2 = 0.261, P = 0.043; hyperemic: before PCI r2 = 0.503, P = 0.005; after PCI r2 = 0.500, P = 0.002), whereas they were related to cPP in absence of stenosis (resting: r2 = 0.368, P = 0.022; hyperemic: r2 = 0.370, P = 0.016). Hyperemic CBF response ( P = 0.005) and hyperemic CBF improvement from PCI ( P = 0.025) were less marked in a stiff aorta than a compliant aorta. A stiff aorta is associated with a reduction in CBF, a lower hyperemic CBF response, and may reduce the improvement in hyperemic CBF after successful PCI.
Regulation of potassium channels in coronary smooth muscle by adenoviral expression of cytochromeP-450 epoxygenase
Authors: Campbell William B. , Holmes Blythe B. , Falck John R. , Capdevila Jorge H. , Gauthier Kathryn M.
Published: September 2005

Epoxyeicosatrienoic acids (EETs) are endothelium-derived cytochrome P-450 (CYP) metabolites of arachidonic acid that relax vascular smooth muscle by large-conductance calcium-activated potassium (BKCa) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase BKCachannel activity. Bovine coronary SMCs were transduced with adenovirus coding the CYP Bacillus megaterium -3 (F87V) (CYP BM-3) epoxygenase that metabolizes arachidonic acid exclusively to 14( S),15( R)-EET. Adenovirus containing the cytomegalovirus promoter- Escherichia coli β-galactosidase was used as a control. With the use of an anti-CYP BM-3 (F87V) antibody, a 124-kDa immunoreactive protein was detected only in CYP BM-3-transduced cells. Protein expression increased with increasing amounts of virus. When CYP BM-3-transduced cells were incubated with [14C]arachidonic acid, HPLC analysis detected 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) and 14,15-EET. The identity of 14,15-EET and 14,15-DHET was confirmed by mass spectrometry. In CYP BM-3-transduced cells, methacholine (10−5M) increased 14,15-EET release twofold and BKCachannel activity fourfold in cell-attached patches. Methacholine-induced increases in BKCachannel activity were blocked by the CYP inhibitor 17-octadecynoic acid (10−5M). 14( S),15( R)-EET was more potent than 14( R),15( S)-EET in relaxing bovine coronary arteries and activating BKCachannels. Thus CYP BM-3 adenoviral transduction confers SMCs with epoxygenase activity. These cells acquire the capacity to respond to the vasodilator agonist by synthesizing 14( S),15( R)-EET from endogenous arachidonic acid to activate BKCachannels. These studies indicate that 14( S),15( R)-EET is a sufficient endogenous activator of BKCachannels in coronary SMCs.
Scaling laws of vascular trees: of form and function
Authors: Kassab Ghassan S.
Published: September 2005

The branching pattern and vascular geometry of biological tree structure are complex. Here we show that the design of all vascular trees for which there exist morphometric data in the literature (e.g., coronary, pulmonary; vessels of various skeletal muscles, mesentery, omentum, and conjunctiva) obeys a set of scaling laws that are based on the hypothesis that the cost of construction of the tree structure and operation of fluid conduction is minimized. The laws consist of scaling relationships between 1) length and vascular volume of the tree, 2) lumen diameter and blood flow rate in each branch, and 3) diameter and length of vessel branches. The exponent of the diameter-flow rate relation is not necessarily equal to 3.0 as required by Murray's law but depends on the ratio of metabolic to viscous power dissipation of the tree of interest. The major significance of the present analysis is to show that the design of various vascular trees of different organs and species can be deduced on the basis of the minimum energy hypothesis and conservation of energy under steady-state conditions. The present study reveals the similarity of nature's scaling laws that dictate the design of various vascular trees and the underlying physical and physiological principles.
Differential sympathetic outflow and vasoconstriction responses at kidney and skeletal muscles during fictive locomotion
Authors: Koba Satoshi , Yoshida Takayoshi , Hayashi Naoyuki
Published: September 2005

We compared sympathetic and circulatory responses between kidney and skeletal muscles during fictive locomotion evoked by electrical stimulation of the mesencephalic locomotor region (MLR) in decerebrate and paralyzed rats ( n = 8). Stimulation of the MLR for 30 s at 40-μA current intensity significantly increased arterial pressure (+38 ± 6 mmHg), triceps surae muscle blood flow (+17 ± 3%), and both renal and lumbar sympathetic nerve activities (RSNA +113 ± 16%, LSNA +31 ± 7%). The stimulation also significantly decreased renal cortical blood flow (−18 ± 6%) and both renal cortical and triceps surae muscle vascular conductances (RCVC −38 ± 5%, TSMVC −17 ± 3%). The sympathetic and vascular conductance changes were significantly dependent on current intensity for stimulation at 20, 30, and 40 μA. The changes in LSNA and TSMVC were significantly less than those in RSNA and RCVC, respectively, at all current intensities. At the early stage of stimulation (0–10 s), decreases in RCVC and TSMVC were significantly correlated with increases in RSNA and LSNA, respectively. These data demonstrate that fictive locomotion induces less vasoconstriction in skeletal muscles than in kidney because of less sympathetic activation. This suggests that a neural mechanism mediated by central command contributes to blood flow distribution by evoking differential sympathetic outflow during exercise.
Enhanced neurally evoked responses and inhibition of norepinephrine reuptake in rat mesenteric arteries after spinal transection
Authors: Brock James A. , Yeoh Melanie , McLachlan Elspeth M.
Published: September 2005

In patients with high thoracic spinal lesions that remove most of the central drive to splanchnic preganglionic neurons, visceral or nociceptive stimuli below the lesion can provoke large increases in blood pressure (autonomic dysreflexia). We have examined the effects of T4 spinal transection on isometric contractions of mesenteric arteries isolated from spinalized rats. Nerve-evoked contractions involved synergistic roles for norepinephrine and ATP. At 7 wk after spinal transection, responses to perivascular stimulation at 1–5 Hz were enhanced fivefold, whereas the α1-adrenoceptor antagonist prazosin (10 nM) produced a twofold larger reduction in contraction (to 20 pulses at 10 Hz) than in unoperated controls. In contrast, the reduction in nerve-evoked contractions by the P2-purinoceptor antagonist suramin (0.1 mM) and the responses to the P2-purinoceptor agonist α,β-methylene ATP or to high K+ concentration did not greatly differ between groups, indicating that arteries from spinalized rats were not generally hyperreactive. Sensitivity to the α1-adrenoceptor agonist phenylephrine was enhanced in arteries from spinalized rats, and the difference from controls was abolished by the norepinephrine uptake blocker desmethylimipramine. Sensitivity to the α1-adrenoceptor agonist methoxamine, which is not a substrate for the neuronal norepinephrine transporter, was similar among the groups. Thus the increased neurally evoked response after spinal transection appeared to be due to a reduction in neuronal uptake of released norepinephrine, a mechanism that did not explain the enhanced response of tail arteries after spinal transection that we previously reported. The findings provide further support for potentiated neurovascular responses contributing to the genesis of autonomic dysreflexia.
Contractile effects of adenosine A1 and A2A receptors in isolated murine hearts
Authors: Tikh Eugene I. , Fenton Richard A. , Dobson James G.
Published: September 2005

The adenosine A1 receptor (A1R) inhibits β-adrenergic-induced contractile effects (antiadrenergic action), and the adenosine A2A receptor (A2AR) both opposes the A1R action and enhances contractility in the heart. This study investigated the A1R and A2AR function in β-adrenergic-stimulated, isolated wild-type and A2AR knockout murine hearts. Constant flow and pressure perfused preparations were employed, and the maximal rate of left ventricular pressure (LVP) development (+dp/d tmax) was used as an index of cardiac function. A1R activation with 2-chloro- N6-cyclopentyladenosine (CCPA) resulted in a 27% reduction in contractile response to the β-adrenergic agonist isoproterenol (ISO). Stimulation of A2AR with 2- P(2-carboxyethyl)phenethyl-amino-5′- N-ethylcarboxyamidoadenosine (CGS-21680) attenuated this antiadrenergic effect, resulting in a partial (constant flow preparation) or complete (constant pressure preparation) restoration of the ISO contractile response. These effects of A2AR were absent in knockout hearts. Up to 63% of the A2AR influence was estimated to be mediated through its inhibition of the A1R antiadrenergic effect, with the remainder being the direct contractile effect. Further experiments examined the effects of A2AR activation and associated vasodilation with low-flow ischemia in the absence of β-adrenergic stimulation. A2AR activation reduced by 5% the depression of contractile function caused by the flow reduction and also increased contractile performance over a wide range of perfusion flows. This effect was prevented by the A2AR antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385). It is concluded that in the murine heart, A1R and A2AR modulate the response to β-adrenergic stimulation with A2AR, attenuating the effects of A1R and also increasing contractility directly. In addition, A2AR supports myocardial contractility in a setting of low-flow ischemia.
Vascular dysfunction produced by hyperhomocysteinemia is more severe in the presence of low folate
Authors: Symons J. David , Rutledge John C. , Simonsen U. , Pattathu Roshny A.
Published: September 2005

Earlier we reported that dietary folate depletion causes hyperhomocysteinemia (HHcy) and arterial dysfunction in rats (Symons JD, Mullick AE, Ensunsa JL, Ma AA, and Rutledge JC. Arterioscler Thromb Vasc Biol 22: 772–780, 2002). Both HHcy and low folate (LF) are risk factors for cardiovascular disease. Therefore, the dysfunction we observed could have resulted from HHcy, LF, and/or their combination (HHcy + LF). We tested the hypothesis that HHcy-induced vascular dysfunction is more severe in the presence of LF. Four groups of rats consumed diets for ∼10 wk that produced plasma homocysteine (μM) and liver folate (μg folate/g liver) concentrations, respectively, of 7 ± 1 and 15 ± 1 (Control; Con; n = 16), 17 ± 2 and 15 ± 2 (HHcy; n = 17), 10 ± 1 and 8 ± 1 (LF; n = 14), and 21 ± 2 and 8 ± 1 (HHcy + LF; n = 18). We observed that maximal ACh-evoked vasorelaxation was greatest in aortas and mesenteric arteries from Con rats vs. all groups. While the extent of dysfunction was similar between LF and HHcy animals, it was less severe compared with arteries from HHcy + LF rats. Maximal ACh-evoked vasorelaxation in coronary arteries was not different between Con and LF rats, but both were greater than HHcy + LF animals. In segments of aortas, 1) ACh-evoked vasorelaxation was similar among groups after incubation with the nonenzymatic intracellular O[Formula: see text] scavenger Tiron, 2) vascular O[Formula: see text] estimated using dihydroethidium staining was greatest in HHcy + LF vs. all groups, and 3) tension development in response to nitric oxide (NO) synthase inhibition was greatest in Con vs. all other groups. We conclude that HHcy + LF evokes greater dysfunction than either HHcy alone (aortas, mesentery) or LF alone (aortas, mesentery, coronary), likely by producing more O[Formula: see text] within the vasculature and thereby reducing NO bioavailability.
Adrenergic origin of very low-frequency blood pressure oscillations in the unanesthetized rat
Authors: Radaelli Alberto , Castiglioni Paolo , Centola Marco , Cesana Francesca , Balestri Giulia , Ferrari Alberto U. , Di Rienzo Marco
Published: September 2005

Spectral analysis of cardiovascular signals has been extensively used to investigate circulatory homeostatic mechanisms. However, the nature of very low-frequency (VLF) fluctuations remains unclear. Because we previously observed enhanced VLF fluctuations in blood pressure (BP) in the sympathectomized rat (a model characterized by markedly increased plasma epinephrine levels), the aims of our study were to assess whether the genesis of VLF fluctuations in BP depends on circulating catecholamines and to determine which adrenergic receptor(s) and which membrane ion channel(s) are involved. We used continuous intra-arterial BP recordings from unanesthetized unrestrained rats to compute the power of VLF fluctuations in BP in the intact condition, during acute ganglionic blockade with hexamethonium, and after restoration of BP levels by infusion (in addition to hexamethonium) of adrenergic agonists (epinephrine, norepinephrine, and clonidine) or nonadrenergic vasoconstrictors (vasopressin). Effects of infusion of specific adrenergic receptor blockers (propranolol, prazosin, and yohimbine) with hexamethonium and catecholamines and infusion of various membrane ion channel blockers on VLF fluctuations in BP were also evaluated. Our results are as follows. 1) Ganglionic blockade drastically reduced BP levels and VLF fluctuations. 2) All vasoconstrictors restored BP levels, but only adrenergic vasoconstrictors generated striking VLF fluctuations in BP. 3) Catecholamine-induced fluctuations were abolished by α2-, but not α1- or β-, adrenergic receptor blockade and by Ba2+-sensitive K+channel or L-type Ca2+channel, but not by other ion channel, blockers. We conclude that, in the conscious, unrestrained ganglion-blocked rat, catecholamine infusion generates VLF fluctuations in BP through stimulation of α2-receptors and activation of Ba2+-sensitive K+channels. These fluctuations may have (patho)physiological relevance under conditions of disrupted circulatory homeostasis.
Persistent splanchnic hyperemia during upright tilt in postural tachycardia syndrome
Authors: Stewart Julian M. , Medow Marvin S. , Glover June L. , Montgomery Leslie D.
Published: September 2005

Previous investigations have allowed for stratification of patients with postural tachycardia syndrome (POTS) on the basis of peripheral blood flow. One such subset, comprising “normal-flow POTS” patients, is characterized by normal peripheral resistance and blood volume in the supine position but thoracic hypovolemia and splanchnic blood pooling in the upright position. We studied 32 consecutive 14- to 22-yr-old POTS patients comprising 13 with low-flow POTS, 14 with normal-flow POTS, and 5 with high-flow POTS and 12 comparably aged healthy volunteers. We measured changes in impedance plethysmographic (IPG) indexes of blood volume and blood flow within thoracic, splanchnic, pelvic (upper leg), and lower leg regional circulations in the supine posture and during incremental tilt to 20°, 35°, and 70°. We validated IPG measures of thoracic and splanchnic blood flow against indocyanine green dye-dilution measurements. We validated IPG leg blood flow against venous occlusion plethysmography. Control subjects developed progressive vasoconstriction with incremental tilt. Splanchnic blood flow was increased in the supine position in normal-flow POTS, despite marked peripheral vasoconstriction, and did not change during incremental tilt, producing progressive splanchnic hypervolemia. Absolute hypovolemia was present in low-flow POTS, all supine flows and volumes were reduced, there was no vasoconstriction with tilt in all segments, and segmental volumes tended to increase uniformly throughout tilt. Lower body (pelvic and leg) flows were increased in high-flow POTS at all angles, with consequent lower body hypervolemia during tilt. Our main finding is selective and maintained orthostatic splanchnic vasodilation in normal-flow POTS, despite marked peripheral vasoconstriction in these same patients. Local splanchnic vasoregulatory factors may counteract vasoconstriction and venoconstriction in these patients. Lower body vasoconstriction in high-flow POTS was abnormal, and vasoconstriction in low-flow POTS was sustained at initially elevated supine levels.
The direct physiological effects of mitoKATPopening on heart mitochondria
Authors: Costa Alexandre D. T. , Quinlan Casey L. , Andrukhiv Anastasia , West Ian C. , Jabůrek Martin , Garlid Keith D.
Published: September 2005

The mitochondrial ATP-sensitive K+channel (mitoKATP) has been assigned multiple roles in cell physiology and in cardioprotection. Each of these roles must arise from basic consequences of mitoKATPopening that should be observable at the level of the mitochondrion. MitoKATPopening has been proposed to have three direct effects on mitochondrial physiology: an increase in steady-state matrix volume, respiratory stimulation (uncoupling), and matrix alkalinization. Here, we examine the evidence for these hypotheses through experiments on isolated rat heart mitochondria. Using perturbation techniques, we show that matrix volume is the consequence of a steady-state balance between K+influx, caused either by mitoKATPopening or valinomycin, and K+efflux caused by the mitochondrial K+/H+antiporter. We show that increasing K+influx with valinomycin uncouples respiration like a classical uncoupler with the important difference that uncoupling via K+cycling soon causes rupture of the outer mitochondrial membrane and release of cytochrome c. By loading the potassium binding fluorescent indicator into the matrix, we show directly that K+influx is increased by diazoxide and inhibited by ATP and 5-HD. By loading the fluorescent probe BCECF into the matrix, we show directly that increasing K+influx with either valinomycin or diazoxide causes matrix alkalinization. Finally, by comparing the effects of mitoKATPopeners and blockers with those of valinomycin, we show that four independent assays of mitoKATPactivity yield quantitatively identical results for mitoKATP-mediated K+transport. These results provide decisive support for the hypothesis that mitochondria contain an ATP-sensitive K+channel and establish the physiological consequences of mitoKATPopening for mitochondria.
Role of calcitonin gene-related peptide in the postischemic anti-inflammatory effects of antecedent ethanol ingestion
Authors: Kamada Kazuhiro , Gaskin F. Spencer , Yamaguchi Taiji , Carter Patsy , Yoshikawa Toshikazu , Yusof Mozow , Korthuis Ronald J.
Published: September 2005

The aim of this study was to determine the role of calcitonin gene-related peptide (CGRP) in the postischemic anti-inflammatory effects of antecedent ethanol ingestion. Ethanol was administered to wild-type C57BL/6 mice on day 1 as a bolus by gavage at a dose that produces a peak plasma ethanol of 45 mg/dl 30 min after administration. Twenty-four hours later ( day 2), the superior mesenteric artery was occluded for 45 min followed by 70 min of reperfusion (I/R). Intravital fluorescence microscopy was used to quantify the numbers of rolling (LR) and adherent (LA) leukocytes labeled with carboxyfluorescein diacetate succinimidyl ester in postcapillary venules of the small intestine. I/R increased LR and LA, effects that were prevented by antecedent ethanol. The postischemic anti-inflammatory effects of ethanol consumption were abolished by administration of a specific CGRP receptor antagonist [CGRP-(8–37)] or after sensory nerve neurotransmitter depletion using capsaicin administered 4 days before ethanol ingestion, which initially induces rapid release of CGRP from sensory nerves, thereby depleting stored neuropeptide. Administration of exogenous CGRP or induction of endogenous CGRP release by treatment with capsaicin 24 h before I/R mimicked the postischemic anti-inflammatory effects of antecedent ethanol ingestion. Preconditioning with capsaicin 24 h before I/R was prevented by coincident treatment with CGRP-(8–37), while exogenous CGRP induced an anti-inflammatory phenotype in mice depleted of CGRP by capsaicin administration 4 days earlier. Our results indicate that the effect of antecedent ethanol ingestion to prevent postischemic LR and LA is initiated by a CGRP-dependent mechanism.
Inhibition of p38 reduces myocardial infarction injury in the mouse but not pig after ischemia-reperfusion
Authors: Kaiser Robert A. , Lyons Jefferson M. , Duffy Jodie Y. , Wagner Connie J. , McLean Kelly M. , O'Neill Timothy P. , Pearl Jeffrey M. , Molkentin Jeffery D.
Published: September 2005

The MAPK family member p38 is activated in the heart after ischemia-reperfusion (I/R) injury. However, the cardioprotective vs. proapoptotic effects associated with p38 activation in the heart after I/R injury remain unresolved. Another issue to consider is that the majority of past studies have employed the rodent as a model for assessing p38's role in cardiac injury vs. protection, while the potential regulatory role in a large animal model is even more uncertain. Here we performed a parallel study in the mouse and pig to directly compare the extent of cardiac injury after I/R at baseline or with the selective p38 inhibitor SB-239063. Infusion of SB-239063 5 min before ischemia in the mouse prevented ischemia-induced p38 activation, resulting in a 25% reduction of infarct size compared with vehicle-treated animals (27.9 ± 2.9% vs. 37.5 ± 2.7%). In the pig, SB-239063 similarly inhibited myocardial p38 activation, but there was no corresponding effect on the degree of infarction injury (43.6 ± 4.0% vs. 41.4 ± 4.3%). These data suggest a difference in myocardial responsiveness to I/R between the small animal mouse model and the large animal pig model, such that p38 activation in the mouse contributes to acute cellular injury and death, while the same activation in pig has no causative effect on these parameters.
Contribution of CFTR to apical-basolateral fluid transport in cultured human alveolar epithelial type II cells
Authors: Fang Xiaohui , Song Yuanlin , Hirsch Jan , Galietta Luis J. V. , Pedemonte Nicoletta , Zemans Rachel L. , Dolganov Gregory , Verkman A. S. , Matthay Michael A.
Published: September 2005

Previous studies in intact lung suggest that CFTR may play a role in cAMP-regulated fluid transport from the distal air spaces of the lung. However, the potential contribution of different epithelial cells (alveolar epithelial type I, type II, or bronchial epithelial cells) to CFTR-regulated fluid transport is unknown. In this study we determined whether the CFTR gene is expressed in human lung alveolar epithelial type II (AT II) cells and whether the CFTR chloride channel contributes to cAMP-regulated fluid transport in cultured human AT II cells. Human AT II cells were isolated and cultured on collagen I-coated Transwell membranes for 120–144 h with an air-liquid interface. The cultured cells retained typical AT II-like features based on morphologic studies. Net basal fluid transport was 0.9 ± 0.1 μl·cm−2·h−1and increased to 1.35 ± 0.11 μl·cm−2·h−1(mean ± SE, n = 18, P < 0.05) by stimulation with cAMP agonists. The CFTR inhibitor, CFTRinh-172, inhibited cAMP stimulated but not basal fluid transport. In short-circuit current ( Isc) studies with an apical-to-basolateral transepithelial Cl−gradient, apical application of CFTRinh-172 reversed the forskolin-induced decrease in Isc. Real time RT-PCR demonstrated CFTR transcript expression in human AT II cells at a level similar to that in airway epithelial cells. We conclude that CFTR is expressed in cultured human AT II cells and may contribute to cAMP-regulated apical-basolateral fluid transport.
VDUP1: a potential mediator of expansion-induced lung growth and epithelial cell differentiation in the ovine fetus
Authors: Filby C. E. , Hooper S. B. , Sozo F. , Zahra V. A. , Flecknoe S. J. , Wallace M. J.
Published: September 2005

The degree of fetal lung expansion is a critical determinant of fetal lung growth and alveolar epithelial cell (AEC) differentiation, although the mechanisms involved are unknown. As VDUP1 (vitamin D3-upregulated protein 1) can modulate cell proliferation, can induce cell differentiation, and is highly expressed in the lung, we have investigated the effects of fetal lung expansion on VDUP1 expression and its relationship to expansion-induced fetal lung growth and AEC differentiation in fetal sheep. Alterations in fetal lung expansion caused profound changes in VDUP1 mRNA levels in lung tissue. Increased fetal lung expansion significantly reduced VDUP1 mRNA levels from 100 ± 8% in control fetuses to 37 ± 4, 46 ± 4, and 45 ± 9% of control values at 2, 4, and 10 days of increased fetal lung expansion, respectively. Reduced fetal lung expansion increased VDUP1 mRNA levels from 100 ± 16% in control fetuses to 162 ± 16% of control values after 7 days. VDUP1 was localized to airway epithelium in small bronchioles, AECs, and some mesenchymal cells. Its expression was inversely correlated with cell proliferation during normal lung development ( R2= 0.972, P < 0.002) as well as in response to alterations in fetal lung expansion ( R2= 0.956, P < 0.001) and was positively correlated with SP-B expression during normal lung development ( R2= 0.803, P < 0.0001) and following altered lung expansion ( R2= 0.817, P < 0.001). We suggest that VDUP1 may be an important mediator of expansion-induced lung cell proliferation and AEC differentiation in the developing lung.
Soluble guanylyl cyclase expression is reduced in allergic asthma
Authors: Papapetropoulos Andreas , Simoes Davina C. M. , Xanthou Georgia , Roussos Charis , Gratziou Christina
Published: September 2005

Soluble guanylyl cyclase (sGC) is an enzyme highly expressed in the lung that generates cGMP contributing to airway smooth muscle relaxation. To determine whether the bronchoconstriction observed in asthma is accompanied by changes in sGC expression, we used a well-established murine model of allergic asthma. Histological and biochemical analyses confirmed the presence of inflammation in the lungs of mice sensitized and challenged with ovalbumin (OVA). Moreover, mice sensitized and challenged with OVA exhibited airway hyperreactivity to methacholine inhalation. Steady-state mRNA levels for all sGC subunits (α1, α2, and β1) were reduced in the lungs of mice with allergic asthma by 60–80%, as estimated by real-time PCR. These changes in mRNA were paralleled by changes at the protein level: α1, α2, and β1 expression was reduced by 50–80% as determined by Western blotting. Reduced α1 and β1 expression in bronchial smooth muscle cells was demonstrated by immunohistochemistry. To study if sGC inhibition mimics the airway hyperreactivity seen in asthma, we treated naïve mice with a selective sGC inhibitor. Indeed, in mice receiving ODQ the methacholine dose response was shifted to the left. We conclude that sGC expression is reduced in experimental asthma contributing to the observed airway hyperreactivity.
Distribution of epithelial sodium channels and mineralocorticoid receptors in cardiovascular regulatory centers in rat brain
Authors: Amin Md Shahrier , Wang Hong-Wei , Reza Erona , Whitman Stewart C. , Tuana Balwant S. , Leenen Frans H. H.
Published: September 2005

Epithelial sodium channels (ENaC) are important for regulating sodium transport across epithelia. Functional studies indicate that neural mechanisms acting through mineralocorticoid receptors (MR) and sodium channels (presumably ENaC) are crucial to the development of sympathoexcitation and hypertension in experimental models of salt-sensitive hypertension. However, expression and localization of the ENaC in cardiovascular regulatory centers of the brain have not yet been studied. RT-PCR and immunohistochemistry were performed to study ENaC and MR expression at the mRNA and protein levels, respectively. Both mRNA and protein for α-, β-, and γ-ENaC subunits and MR were found to be expressed in the rat brain. All three ENaC subunits and MR were present in the supraoptic nucleus, magnocellular paraventricular nucleus, hippocampus, choroid plexus, ependyma, and brain blood vessels, suggesting the presence of multimeric channels and possible regulation by mineralocorticoids. In most cortical areas, thalamus, amygdala, and suprachiasmatic nucleus, notable expression of γ-ENaC was undetectable, whereas α- and β-ENaC were abundantly expressed pointing to the possibility of a heterogeneous population of channels. The findings suggest that stoichiometrically different populations of ENaC may be present in both epithelial and neural components in the brain, which may contribute to regulation of cerebrospinal fluid and interstitial Na+ concentration as well as neuronal excitation.
Enhanced role for RhoA-associated kinase in adrenergic-mediated vasoconstriction in gracilis arteries from obese Zucker rats
Authors: Naik Jay S. , Xiang Lusha , Hester Robert L.
Published: September 2005

Obesity, insulin resistance, dyslipidemia, and hypertension are components of the pathophysiological state known as metabolic syndrome. Adrenergic vasoconstriction is mediated through increases in cytosolic Ca2+ and the myofilaments' sensitivity to Ca2+. In many pathophysiological states, there is an enhanced role for Rho kinase (ROK)-mediated increases in Ca2+ sensitivity of the contractile apparatus. Thus we hypothesized that there is a greater role for ROK-mediated increases in Ca2+ sensitivity in α1-adrenergic vasoconstriction in arteries from obese Zucker (OZ) rats. Therefore, small gracilis muscle arteries from 11- to 12-wk-old and 16- to 18-wk-old lean and OZ rats were isolated, cannulated, and pressurized to 75 mmHg. For some experiments, vessels were loaded with fura 2-AM. Changes in luminal diameter and vessel wall Ca2+ concentration ([Ca2+]) were measured in response to phenylephrine (PE), the thromboxane mimetic U-46619, and KCl. α1-Adrenergic vasoconstriction was similar between 11- to 12-wk-old lean and obese animals and greater in older obese animals compared with controls. PE-induced increases in vascular smooth muscle cell [Ca2+] were blunted in OZ animals compared with lean controls in both age groups of animals. KCl and U-46619 elicited similar vasoconstriction and vascular smooth muscle cell [Ca2+] in both groups. ROK inhibition attenuated PE vasoconstriction to a greater degree in arteries from 11- to 12-wk-old OZ rats compared with lean animals; ROK inhibition in arteries from older rats right shifted both concentration-response curves to the same point. Total RhoA and ROKα protein expressions were similar between groups. These results suggest an enhanced role for the ROK pathway in α1-adrenergic vasoconstriction in metabolic syndrome.
Angiotensin II in dorsomedial hypothalamus modulates cardiovascular arousal caused by stress but not feeding in rabbits
Authors: De Matteo Robert , Head Geoffrey A. , Mayorov Dmitry N.
Published: September 2005

The dorsomedial hypothalamus (DMH) is critically implicated in the cardiovascular response to emotional stress. This study aimed to determine whether the DMH is also important in cardiovascular arousal associated with appetitive feeding behavior and, if so, whether locally released angiotensin II and glutamate are important in this arousal. Emotional (air-jet) stress and feeding elicited similar tachycardic (+51 and +45 beats/min, respectively) and pressor (+16 and +9 mmHg, respectively) responses in conscious rabbits. Bilateral microinjection of GABAA agonist muscimol (500 pmol) into the DMH, but not nearby hypothalamic regions, attenuated pressor and tachycardic responses to air-jet by 56–63% and evoked anorexia. Conversely, stimulation of the DMH with the glutamate analog kainic acid (250 pmol) elicited hypertension (+25 mmHg) and tachycardia (+114 beats/min) and activated feeding behavior. Local microinjection of a glutamate receptor antagonist, kynurenic acid (10 nmol), decreased pressor responses to stress and eating by 46 and 72%, respectively, without affecting feeding behavior. Bilateral microinjection of a selective AT1-receptor antagonist, candesartan (500 pmol), into the DMH, but not nearby sites, attenuated pressor and tachycardic stress responses by 31 and 33%, respectively. Candesartan did not alter feeding behavior or circulatory response to feeding. These results indicate that, in addition to its role in mediating stress responses, the DMH may be important in regulating cardiovascular arousal associated with feeding. Local glutamatergic inputs appear to regulate cardiovascular response to both stress and feeding. Conversely, angiotensin II, acting via AT1 receptors, may selectively modulate, in the DMH, cardiovascular response to stress, but not feeding.
Cathepsin B-mediated yolk protein degradation during killifish oocyte maturation is blocked by an H+-ATPase inhibitor: effects on the hydration mechanism
Authors: Raldúa Demetrio , Fabra Mercedes , Bozzo María G. , Weber Ekkehard , Cerdà Joan
Published: September 2005

In teleost oocytes, yolk proteins (YPs) derived from the yolk precursors vitellogenins are partially cleaved into free amino acids and small peptides during meiotic maturation before ovulation. This process increases the osmotic pressure of the oocyte that drives its hydration, which is essential for the production of buoyant eggs by marine teleosts (pelagophil species). However, this mechanism also occurs in marine species that produce benthic eggs (benthophil), such as the killifish ( Fundulus heteroclitus), in which oocyte hydration is driven by K+. Both in pelagophil and benthophil teleosts, the enzymatic machinery underlying the maturation-associated proteolysis of YPs is poorly understood. In this study, lysosomal cysteine proteinases potentially involved in YP processing, cathepsins L, B, and F (CatL, CatB, and CatF, respectively), were immunolocalized in acidic yolk globules of vitellogenic oocytes from the killifish. During oocyte maturation in vitro induced with the maturation-inducing steroid (MIS), CatF disappeared from yolk organelles and CatL became inactivated, whereas CatB proenzyme was processed into active enzyme. Consequently, CatB enzyme activity and hydrolysis of major YPs were enhanced. Follicle-enclosed oocytes incubated with the MIS in the presence of bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, underwent maturation in vitro, but acidification of yolk globules, activation of CatB, and proteolysis of YPs were prevented. In addition, MIS plus bafilomycin A1-treated oocytes accumulated less K+than those stimulated with MIS alone; hence, oocyte hydration was reduced. These results suggest that CatB is the major protease involved in yolk processing during the maturation of killifish oocytes, whose activation requires acidic conditions maintained by a vacuolar-type H+-ATPase. Also, the data indicate a link between ion translocation and YP proteolysis, suggesting that both events may be equally important physiological mechanisms for oocyte hydration in benthophil teleosts.
Differential effects of heptanoate and hexanoate on myocardial citric acid cycle intermediates following ischemia-reperfusion
Authors: Okere Isidore C. , McElfresh Tracy A. , Brunengraber Daniel Z. , Martini Wenjun , Sterk Joseph P. , Huang Hazel , Chandler Margaret P. , Brunengraber Henri , Stanley William C.
Published: September 2005

In the normal heart, there is loss of citric acid cycle (CAC) intermediates that is matched by the entry of intermediates from outside the cycle, a process termed anaplerosis. Previous in vitro studies suggest that supplementation with anaplerotic substrates improves cardiac function during myocardial ischemia and/or reperfusion. The present investigation assessed whether treatment with the anaplerotic medium-chain fatty acid heptanoate improves contractile function during ischemia and reperfusion. The left anterior descending coronary artery of anesthetized pigs was subjected to 60 min of 60% flow reduction and 30 min of reperfusion. Three treatment groups were studied: saline control, heptanoate (0.4 mM), or hexanoate as a negative control (0.4 mM). Treatment was initiated after 30 min of ischemia and continued through reperfusion. Myocardial CAC intermediate content was not affected by ischemia-reperfusion; however, treatment with heptanoate resulted in a more than twofold increase in fumarate and malate, with no change in citrate and succinate, while treatment with hexanoate did not increase fumarate or malate but increased succinate by 1.8-fold. There were no differences among groups in lactate exchange, glucose oxidation, oxygen consumption, and contractile power. In conclusion, despite a significant increase in the content of carbon-4 CAC intermediates, treatment with heptanoate did not result in improved mechanical function of the heart in this model of reversible ischemia-reperfusion. This suggests that reduced anaplerosis and CAC dysfunction do not play a major role in contractile and metabolic derangements observed with a 60% decrease in coronary flow followed by reperfusion.
Adenosine A2A receptors mediate GABAergic inhibition of respiration in immature rats
Authors: Mayer Catherine A. , Haxhiu Musa A. , Martin Richard J. , Wilson Christopher G.
Published: September 2005

Adenosine is a known inhibitor of respiratory output during early life. In this study we investigated the developmental changes in adenosine A2A-receptor activation on respiratory timing, as well as the relationship between adenosine and GABA. The specific adenosine A2A-receptor agonist CGS-21680 (CGS) or vehicle control was injected into the fourth ventricle of 14-day ( n = 9), 21-day ( n = 9), and adult ( n = 5) urethane-anesthetized rats while diaphragm electromyogram was monitored as an index of respiratory neural output. CGS injection resulted in a decrease in frequency and/or apnea in all 14-day-old rats and in 66% of 21-day-old rats. There was no effect of CGS injection on respiratory timing in adult rats. Prior injection of the GABAA-receptor blocker bicuculline at 14 and 21 days eliminated the CGS-induced decrease in frequency and apnea. We conclude from these studies that the inhibitory effect of A2A-receptor activation on respiratory drive is age dependent and is mediated via GABAergic inputs to the inspiratory timing neural circuitry. These findings demonstrate an important mechanism by which xanthine therapy alleviates apnea of prematurity.