bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet-derived growth factor isoforms decrease insulin-like growth factor I gene expression in rat vascular smooth muscle cells and selectively stimulate the biosynthesis of insulin-like growth factor binding protein 4.
Authors: Giannella-Neto D , Kamyar A , Sharifi B , Pirola C J , Kupfer J , Rosenfeld R G , Forrester J S , Fagin J A
Published: June 2012

Platelet-derived growth factor (PDGF) is believed to be a critical mediator of vascular smooth muscle cell (SMC) proliferation. Because insulin-like growth factor (IGF) I (IGF-I) functions as a progression factor for the mitogenic effects of PDGF, we hypothesized that IGF-I gene expression and the production of IGF binding proteins (IGFBPs) by cultured rat aortic SMCs might be regulated by one or more of the three isoforms of PDGF: PDGF-AA, -BB, and -AB. IGF-I gene expression was highly dependent on cell density: IGF-I mRNA transcripts decreased markedly as a function of cell confluence. IGF-I mRNA content was inhibited to a similar degree by PDGF-AA, -BB, and -AB through a mechanism requiring protein synthesis. The inhibition was readily apparent at 4 hours, reaching approximately 25% of control levels after 24 hours. Radioimmunoassayable IGF-I was only barely detectable in SMC-conditioned serum-free medium and not significantly modulated by PDGF. Western ligand blot revealed that vascular SMCs release 30-kd and 24-kd IGFBP into serum-free conditioned medium. PDGF isoforms did not significantly alter release of the 30-kd IGFBP but evoked a fivefold to sixfold increase in the 24-kd IGFBP. The 24-kd IGFBP was found to comigrate with IGFBP-4, a recently identified binding protein that inhibits IGF action. The 30-kd protein was not merely a glycosylated form of IGFBP-4, because it was not sensitive to N-glycanase digestion. PDGF-AA, -BB, and -AB markedly induced expression of IGFBP-4 mRNA in a time- and concentration-dependent fashion. Vascular SMCs also express IGFBP-2 mRNA, but its abundance was not induced by PDGF. In conclusion, PDGF evokes a complex pattern of regulation of genes in the IGF/IGFBP system. By inhibiting IGF-I production and specifically inducing biosynthesis of the inhibitory binding protein IGFBP-4, PDGF may set in motion mechanisms to limit the final magnitude of the mitogenic response.
Ionic currents in single smooth muscle cells of the canine renal artery.
Authors: Gelband C H , Hume J R
Published: June 2012

Membrane currents from single smooth muscle cells enzymatically isolated from canine renal artery were recorded using the patch-clamp technique in the whole-cell and cell-attached configurations. These cells exhibited a mean resting potential, input resistance, membrane time constant, and cell capacitance of -51.8 +/- 2.1 mV, 5.2 +/- 0.98 G omega, 116.2 +/- 16.4 msec, and 29.1 +/- 2.0 pF, respectively. Inward current, when elicited from a holding potential of -80 mV, activated near -50 mV, reached a maximum near 0 mV and was sensitive to the dihydropyridine agonist Bay K 8644 and dihydropyridine antagonist nisoldipine. Two components of macroscopic outward current were identified from voltage-step and ramp depolarizations. The predominant charge carrier of the net outward current was identified as K+ by tail-current experiments (reversal potential, -61.0 +/- 0.8 mV in 10.8 mM [K+]o 0 mM [K+]i). The first component was a small, low-noise, voltage- and time-dependent current that activated between -40 and -30 mV (IK(dr)), and the second component was a larger, noisier, voltage- and time-dependent current that activated at potentials positive to +10 mV (IK(Ca)). Both IK(dr) and IK(Ca) displayed little inactivation during long (4-second) voltage steps. IK(Ca) and IK(dr) could be pharmacologically separated by using various Ca2+ and K+ channel blockers. IK(Ca) was substantially inhibited by external NiCl2 (500 microM), CdCl2 (300 microM), EGTA (5 mM), tetraethylammonium (Ki at +60 mV, 307 microM), and charybdotoxin (100 nM) but was insensitive to 4-aminopyridine (0.1-10 mM). IK(dr) was inhibited by 4-aminopyridine (Ki at +10 mV, 723 microM) and tetraethylammonium (Ki at +10 mV, 908 microM) but was insensitive to external NiCl2 (500 microM), CdCl2 (300 microM), EGTA (5 mM), and charybdotoxin (100 nM). Two types of single K+ channels were identified in cell-attached patches. The most abundant K+ channel that was recorded exhibited voltage-dependent activation, was blocked by external tetraethylammonium (250 microM), and had a large single-channel conductance (232 +/- 12 pS with 150 mM K+ in the patch pipette, 130 +/- 17 pS with 5.4 mM K+ in the patch pipette). The second channel was also voltage dependent, was blocked by 4-aminopyridine (5 mM), and exhibited a smaller single-channel conductance (104 +/- 8 pS with 150 mM K+ in the patch pipette, 57 +/- 6 pS with 5.4 mM K+ in the patch pipette). These results suggest that depolarization of canine renal artery cells opens dihydropyridine-sensitive Ca2+ channels and at least two K+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)
Rat carotid neointimal smooth muscle cells reexpress a developmentally regulated mRNA phenotype during repair of arterial injury.
Authors: Majesky M W , Giachelli C M , Reidy M A , Schwartz S M
Published: June 2012

Smooth muscle cells (SMCs) cultured from the neointima of injured rat carotid arteries have a different shape and organization in vitro than SMCs from the uninjured media. The morphology of neointimal SMCs from adult rats strongly resembles that of a subset of medial SMCs from 12-day-old rat pups. In the present study, we show that adult carotid neointimal SMCs in vitro express the platelet-derived growth factor (PDGF)-B gene but have little or no PDGF alpha-receptor mRNA. In contrast, medial SMCs from contralateral uninjured carotids, grown and passaged under identical conditions, contain abundant PDGF alpha-receptor mRNA but little or no PDGF-B mRNA. Transcript levels for PDGF-A or PDGF beta-receptor were not different in neointimal versus medial SMC cultures. The PDGF mRNA phenotype of adult neointimal SMCs strongly resembles that of an aortic medial SMC subset from newborn rat pups. Although intriguing, the differences in SMC phenotypes we observed in cell culture may depend on unique conditions in vitro and do not necessarily mean that analogous SMC diversity also exists in vivo. To address this question, we constructed and screened a SMC cDNA library for additional molecular markers of the common "pup-intimal" SMC phenotype. Two cDNA clones were identified whose cognate mRNA levels were developmentally regulated in rat aorta in vivo and were present at high levels in the adult carotid neointima formed 2 weeks after balloon catheter injury. Importantly, elevated levels of these two cognate mRNAs in carotid neointima compared with underlying media were maintained in cultures of neointimal versus medial SMCs in vitro. DNA sequence analysis indicated that the cDNA clones encoded rat tropoelastin and alpha 1 procollagen (type I). These results provide further evidence that neointima formation in the adult rat carotid artery depends on reexpression of an SMC phenotype or subpopulation with special properties characteristic of earlier stages of artery wall development. Our studies to date indicate that two of these special properties are paracrine growth factor production and extracellular matrix synthesis.
Decreased collagen gene expression and absence of fibrosis in thyroid hormone-induced myocardial hypertrophy. Response of cardiac fibroblasts to thyroid hormone in vitro.
Authors: Yao J , Eghbali M
Published: June 2012

The regulatory effects of thyroid hormone on biosynthesis of myocardial proteins that originate from cardiac myocytes are well established. Little is known, however, of regulatory effects of thyroid hormone on interstitial proteins. In this study we examined the effects of thyroid hormone on collagen gene expression in thyroid hormone-induced myocardial hypertrophy and the response of cardiac fibroblasts to thyroid hormone in culture. Adult male Sprague-Dawley rats were treated intraperitoneally with L-thyroxin (10 micrograms/100 g body wt) for 2 hours or 1, 2, 3, 6, 12, or 14 days. Northern blot analysis of RNA from total ventricular tissue showed that after 2 hours of treatment, the abundance of mRNA for pro alpha 2(I) collagen decreased by 53% (p less than 0.05) and reached the lowest level (60% decrease, p less than 0.02) at day 1, remained diminished at day 3, and then gradually returned toward normal levels. After transient transfection of chimeric DNA containing collagen type I promoter-chloramphenicol acetyl transferase (CAT) gene into the thyroxin-treated cardiac fibroblasts, the level of CAT activity decreased significantly. Treatment of cardiac fibroblasts in culture (10 nM L-thyroxin) resulted in a 33% (p less than 0.005) decrease in the abundance of mRNA for pro alpha 2(I) collagen. The stability of the mRNA for pro alpha 2(I) collagen in cardiac fibroblasts, as measured by mRNA half-life, was slightly (16.6%) decreased by thyroid-hormone treatment. Collagen synthesis as shown by immunofluorescent staining of intracellular collagen in cultured fibroblasts and in frozen sections of myocardium was also diminished.(ABSTRACT TRUNCATED AT 250 WORDS)
Measurement of endothelial permeability to 125I-low density lipoproteins in rabbit arteries by use of en face preparations.
Authors: Truskey G A , Roberts W L , Herrmann R A , Malinauskas R A
Published: June 2012

A procedure of en face quantitative autoradiography of the endothelium (Hautchen preparations) was developed to examine regional variations in 125I-low density lipoprotein (125I-LDL) permeability in the arterial wall in vivo. Endothelial preparations from fixed arterial tissue and calibration standards consisting of known concentrations of 125I-albumin were dipped in nuclear emulsion, exposed for 1-3 months, developed, and stained with hematoxylin. Digital image analysis was used to analyze dark-field images of autoradiographs. Background grain densities on cold endothelial preparations were 30-100% higher than on glass, but the variability in grain densities on the two different surfaces was similar. Regression slopes of grain density versus concentration for calibration standards were the same for sections placed on cold tissue or glass. For 1-5-microns-thick calibration standards of the same concentration, the grain density was proportional to the total amount of radioactivity per unit area. The results indicated that errors arising from nonuniformities in preparation thickness were minimal, and permeabilities and intimal concentrations could be determined. Rabbits were killed 10 minutes after injection of 125I-LDL, and endothelial preparations were made. For regions of uniformly low grain density in the rabbit aorta, the 125I-LDL permeability was 1.9 +/- 0.8 x 10(-8) cm/sec, and the effective diffusion coefficient was 5.4 +/- 3.1 x 10(-10) cm2/sec. Errors in the estimated permeability arising from nonuniformities in tissue thickness were the same as the reported experimental variability. Analysis of elevated regions of permeability suggested that 125I-LDL was binding to the extracellular matrix. Approximately 25% of the sites of elevated grain density were associated with mitotic endothelial cells, and such regions had higher permeabilities than sites associated with nonmitotic cells. Around intercostal arteries, sites of highest permeability were distal and lateral to the vessels and occurred where lesions first develop in hypercholesterolemic rabbits.
Endothelin increases myofilament Ca2+ sensitivity in alpha-toxin-permeabilized rabbit mesenteric artery.
Authors: Nishimura J , Moreland S , Ahn H Y , Kawase T , Moreland R S , van Breemen C
Published: June 2012

This study was designed to investigate the mechanism of endothelin-1 (ET-1) contractions in Staphylococcus alpha-toxin-permeabilized vascular smooth muscle. Rabbit small mesenteric arteries permeabilized with alpha-toxin were mounted for isometric or isotonic force recording or were processed for determination of myosin light chain (MLC) phosphorylation levels. Addition of 100 nM ET-1 plus 10 microM GTP significantly enhanced myofilament Ca2+ sensitivity as compared with the addition of Ca2+ alone (EC50, 0.47 microM Ca2+ for Ca2+ alone and 0.13 microM Ca2+ for ET-1 plus (GTP). This enhanced sensitivity was reversed by GDP beta S. ET-1-induced contractions were relaxed at a constant [Ca2+] by the addition of 30 microM cAMP or cGMP, demonstrating a direct effect of the cyclic nucleotides on contractile regulation. Inhibition of protein kinase C activity by 100 nM staurosporine relaxed ET-1 plus GTP-induced contractions, and pretreatment with 40 microM chelerythrine inhibited the ET-1 plus GTP increase in force. At 0.32 microM Ca2+, steady-state levels of shortening velocity were not increased by ET-1 plus GTP, although steady-state levels of MLC phosphorylation were significantly enhanced. The ET-1-induced increase in MLC phosphorylation was not altered by changes in [Ca2+], whereas the shortening velocity was Ca2+ dependent, suggesting that the increase MLC phosphorylation level may be the result of protein kinase C, rather than MLC kinase, activation. These results are consistent with the hypothesis that ET-1 increases myofilament Ca2+ sensitivity by a G protein-dependent pathway and subsequent activation of protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)
Multiple connexins confer distinct regulatory and conductance properties of gap junctions in developing heart.
Authors: Veenstra R D , Wang H Z , Westphale E M , Beyer E C
Published: June 2012

Multiple gap junction proteins (connexins) and channels have been identified in developing and adult heart. Functional expression of the three connexins found in chick heart (connexin42, connexin43, and connexin45) by stable transfection of communication-deficient neuro2A (N2A) cells revealed that all three connexin cDNAs are capable of forming physiologically distinct gap junctions that differ in their transjunctional voltage dependence and unitary channel conductances. The transjunctional voltage dependences of connexin45 and connexin42 closely resembled those of 4-day and 18-day embryonic chick heart gap junctions, respectively. The multiple channel conductances between 80 and 240 pS, including the predominant 160 pS channel, observed in embryonic chick heart were also common to connexin42. The expression of multiple gap junction channels with distinct conductance and regulatory properties within a given tissue may account for developmental changes in intercellular communication.
Prostaglandin D2 relaxes bovine coronary arteries by endothelium-dependent nitric oxide-mediated cGMP formation.
Authors: Braun M , Schrör K
Published: June 2012

This study investigates the vasomotor activities of prostaglandin (PG) D2 in bovine coronary arteries in relation to endothelial function. Isolated segments of bovine coronary arteries with intact endothelium were concentration-dependently relaxed by PGD2 (0.01-1 microM), a reaction that was blocked by a selective PGD receptor antagonist (BW A868C). There was a tight correlation between PGD2- and acetylcholine-induced relaxations (r = 0.894, n = 96, p < 0.001). Removal of endothelium abolished the PGD2-induced relaxation and unmasked a contractile activity of the compound. Inhibition of endogenous PGI2 formation by indomethacin did not modify these responses, whereas inhibition of endogenous nitric oxide generation by NG-nitro-L-arginine and NG-monomethyl L-arginine (10 or 100 microM) or scavenging of released nitric oxide by oxyhemoglobin (3 microM) considerably (> 50%) antagonized the PGD2-induced relaxation. The vessel relaxation by PGD2 was associated with a threefold to fourfold increase in vascular cGMP. A considerable reduction in vascular cGMP was measured after removal of the endothelium (by 53%) and inhibition of endogenous nitric oxide generation by NG-nitro-L-arginine (by 70%). This also resulted in a complete inhibition of PGD2-induced cGMP accumulation. Similar results were obtained with the stable PGD2 mimetic ZK 110.841, suggesting that these biological activities of PGD2 were due to the compound itself and not caused by any PGD2 metabolite. A slight but significant increase in cAMP was observed in arteries with intact endothelium as well as after removal of endothelium. Because the relaxing effect of PGD2 was strictly endothelium dependent, the observed relaxation cannot be explained by cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)
Activation of ATP-sensitive K+ channels by cromakalim. Effects on cellular K+ loss and cardiac function in ischemic and reperfused mammalian ventricle.
Authors: Venkatesh N , Stuart J S , Lamp S T , Alexander L D , Weiss J N
Published: June 2012

Pharmacological modulation of [K+]o accumulation and action potential changes during acute myocardial ischemia is under evaluation as a promising new antiarrhythmic and cardioprotective strategy during myocardial ischemia and reperfusion. We studied the effects of cromakalim, a K+ channel opener that activates ATP-sensitive K+ channels, in isolated arterially perfused rabbit interventricular septa subjected to ischemia and reperfusion and, through use of the patch clamp technique, in inside-out membrane patches excised from guinea pig ventricular myocytes. During aerobic perfusion, 5 microM cromakalim shortened action potential duration (APD) from 217 +/- 7 to 201 +/- 10 msec, had no effect on [K+]o, and reduced tension by 17 +/- 3% (n = 11). During ischemia, pretreatment with 5 microM cromakalim resulted in 1) more rapid APD shortening (71 +/- 9 versus 166 +/- 7 msec at 10 minutes and 63 +/- 12 versus 122 +/- 8 msec at 30 minutes), 2) similar [K+]o accumulation after 10 minutes (8.9 +/- 0.3 versus 9.6 +/- 0.5 mM) but a trend toward increased [K+]o accumulation after 30 minutes (11.0 +/- 1.7 versus 9.6 +/- 1.0 mM), and 3) similar times for tension to decline to 50% of control (2.14 +/- 0.16 versus 2.14 +/- 0.19 minutes) but shorter time to fall to 20% of control (4.34 +/- 0.33 versus 4.90 +/- 0.22 minutes; p = 0.003). After 60 minutes of reperfusion following 30 minutes of ischemia, recovery of function was similar, with a trend toward better recovery of developed tension (to 58 +/- 9% versus 39 +/- 10% of control; p = 0.18) and tissue ATP levels in cromakalim-treated hearts but no differences in APD or rest tension. Thus, 5 microM cromakalim had mild effects in normal heart but greatly accelerated APD shortening during ischemia without markedly increasing [K+]o accumulation, possibly because the more rapid APD shortening reduced the time-averaged driving force for K+ efflux through ATP-sensitive K+ channels. A significant cardioprotective effect during 30 minutes of ischemia plus 60 minutes of reperfusion could not be demonstrated in this model. In excised membrane patches studied at room temperature, the ability of cromakalim to activate ATP-sensitive K+ channels was significantly potentiated by 100 microM but not 15 microM cytosolic ADP, suggesting that in addition to the modest fall in cytosolic ATP during early ischemia, the rapid increases in cytosolic ADP may further sensitize cardiac ATP-sensitive K+ channels to activation by cromakalim.(ABSTRACT TRUNCATED AT 400 WORDS)
Age-related changes in fibronectin expression in spontaneously hypertensive, Wistar-Kyoto, and Wistar rat hearts.
Authors: Mamuya W , Chobanian A , Brecher P
Published: June 2012

The effects of age and blood pressure on fibronectin expression in the rat heart were studied in the normotensive Wistar and Wistar-Kyoto (WKY) strains and in the spontaneously hypertensive rat (SHR). Fibronectin mRNA expression decreased threefold between 10 and 40 weeks of age in Wistar hearts, with changes of similar magnitude occurring between 6 and 24 weeks in WKY rats. In contrast, no decrease in fibronectin mRNA was observed in SHR hearts during this time span. These results are in contrast to changes observed previously in the aorta, where an increase in fibronectin mRNA occurred with age in all three rat strains. Ribonuclease protection analysis showed a small age-specific increase in the relative content of EIIIA+ fibronectin mRNA isoforms in hearts from Wistar rats, whereas no change was found in the relative amount of either EIIIA or EIIIB isoforms in SHR hearts. Changes similar to those observed for fibronectin mRNA, although of different magnitudes, were observed in mRNA levels for collagen alpha 1(III) and beta 1 integrin. In Wistar hearts, collagen alpha 1(III) mRNA levels decreased fivefold to sixfold between 10 and 40 weeks of age, whereas a twofold to threefold decrease in beta 1 integrin was observed in WKY hearts between 6 and 24 weeks of age. Western blot analysis revealed a positive correlation between fibronectin mRNA and protein for age-dependent changes in ventricular tissue but not in the atria, suggesting that the regulation of fibronectin expression during the changes common to both aging and hypertrophy could involve both transcriptional and posttranscriptional mechanisms.
Age-related alterations in the phosphorylation of sarcoplasmic reticulum and myofibrillar proteins and diminished contractile response to isoproterenol in intact rat ventricle.
Authors: Jiang M T , Moffat M P , Narayanan N
Published: June 2012

Previous studies have shown that the inotropic response of the heart to beta-adrenergic stimulation declines with aging. This alteration has been attributed partly to an age-related impairment in the activation of the beta-adrenoceptor-G protein-adenylate cyclase complex. To further understand the mechanisms underlying the age-related deficit, the present study compared beta-adrenergic-mediated contractile response, cAMP accumulation, and phosphorylation of sarcoplasmic reticulum and myofibrillar proteins in isolated perfused hearts from adult (6-8 months) and aged (28-30 months) Fischer 344 rats. In isometrically contracting, electrically paced (240 beats per minute) hearts perfused at constant flow rate (9 ml/min per gram ventricle), the baseline contractile performance differed significantly between adult and aged hearts. Thus, contraction duration was prolonged (approximately 15%, p < 0.001) in the aged relative to the adult heart, and this was due to increases in time to peak tension and relaxation time. Further, developed peak tension, normalized per gram ventricular wet weight, was significantly lower (approximately 20%, p < 0.05) in the aged compared with the adult heart. In these isolated perfused heart preparations, beta-adrenergic stimulation with isoproterenol (ISO, 0.001-1 microM) evoked concentration-dependent positive inotropic and lusitropic responses, both of which were significantly lower (15-20%, p < 0.05-0.001) in the aged compared with the adult heart. These age-related differences were manifested as relatively smaller ISO-induced increases in 1) developed peak tension, 2) maximum rate of tension development (+dT/dt), and 3) maximum rate of relaxation (-dT/dt) in the aged compared with the adult heart. The ISO-induced abbreviation of time to half relaxation was also less marked in the aged heart. Under similar experimental conditions, ISO (0.1 microM)-induced increase in tissue cAMP content was also lower (approximately 18%, p < 0.05) in the aged heart. ISO (0.1 microM)-induced phosphorylation of the sarcoplasmic reticulum protein phospholamban and myofibrillar protein troponin I was significantly diminished (approximately 38% and 25% decline, respectively, for phospholamban and troponin I; p < 0.05-0.001) in the aged compared with the adult heart. No significant age-related difference was, however, evident in ISO-induced phosphorylation of C protein of myofibrils. These data suggest that age-related decrements in beta-adrenergic-mediated cAMP accumulation and phosphorylation of phospholamban and troponin I contribute to the diminished contractile responses of the aged heart to beta-adrenergic stimulation.
Effects of lysolipids and oxidatively modified low density lipoprotein on endothelium-dependent relaxation of rabbit aorta.
Authors: Mangin E L , Kugiyama K , Nguy J H , Kerns S A , Henry P D
Published: June 2012

Exposure of isolated arteries to oxidatively modified low density lipoprotein (LDL) has been reported to suppress endothelium-dependent relaxation (EDR). To determine whether lipid degradation products in oxidized LDL contribute to impaired relaxation, we have tested the responsiveness of isolated rabbit aortas to endothelium-dependent relaxants (acetylcholine, ATP, and calcium ionophore A23187) and nitroglycerin before and after 2-hour incubations with selected lipids and LDL preparations. Concentrations (10 microM) of lecithin, phosphatidylserine, lysophosphatidylserine, sphingomyelin, phosphatidic acid, palmitate, arachidonate, and auto-oxidized arachidonate had no effect on EDR. Concentrations (10 microM) of lysolecithin, lyso-platelet activating factor, and sphingosine significantly suppressed endothelium-dependent relaxation. Native LDL (100 micrograms/ml incubation buffer) containing only small amounts of lysophosphatidylcholine exerted no effect on EDR. In contrast, LDL preparations oxidatively modified by exposure to cultured endothelial cells or copper inhibited EDR. When modified LDL was depleted of its lysolecithin by treatment with a selective phospholipase B (lysolecithinase), the inhibitory effects were attenuated. In contrast, native LDL accumulating lysolecithin under the influence of a phospholipase A2 (lecithinase) exerted inhibitory effects mimicking those of oxidized LDL. Lipids and lipoproteins had no effect on the responsiveness to nitroglycerin, an endothelium-independent vasodilator. We conclude that lysolecithin in oxidatively modified LDL contributes importantly to its vasomotor effects.
Substrate-induced changes in the lipid content of ischemic and reperfused myocardium. Its relation to hemodynamic recovery.
Authors: de Groot M J , Coumans W A , Willemsen P H , van der Vusse G J
Published: June 2012

To investigate the effect of lactate, pyruvate, and glucose on the endogenous levels of lipids in the normoxic, ischemic, and reperfused myocardium, isolated working rat hearts were exposed to various grades of ischemic insult (15, 30, or 45 minutes). Glucose was present as the basal substrate in the perfusion medium, and lactate (5 mM) or pyruvate (5 mM) was added as the cosubstrate. Lipid metabolism was evaluated by fatty acid accumulation, triacylglycerol turnover, and phospholipid homeostasis. Exogenous lactate significantly increased fatty acid content above preischemic levels after 45 minutes of ischemia. In glucose-perfused hearts, fatty acid levels were even slightly higher than in lactate-perfused hearts, whereas pyruvate-perfused hearts demonstrated less accumulation of fatty acids. By reperfusion, fatty acid levels in glucose-perfused hearts returned to control values. In lactate- and pyruvate-perfused hearts, fatty acid accumulation was further enhanced by reperfusion. When the fatty acid content exceeded 400 nmol/g dry wt during reperfusion, hemodynamic function was impaired, whereas fatty acid levels below 400 nmol/g dry wt did not correlate with hemodynamic recovery. The total triacylglycerol content did not change during ischemia and reperfusion. However, accumulation of glycerol was remarkable during the first 15 minutes of ischemia in all hearts, and release of glycerol by reperfusion was considerable in lactate-perfused hearts after 30 minutes of ischemia and in all groups of hearts after 45 minutes of ischemia. Release of glycerol in association with maintained levels of triacylglycerols suggests turnover of the triacylglycerol pool. The rate of triacylglycerol cycling correlated poorly with hemodynamic recovery. Accumulation of arachidonic acid revealed disturbances in phospholipid turnover. Arachidonic acid accumulation during reperfusion demonstrated a strong relation with impairment of cardiac function. Hence, derangements in phospholipid homeostasis during reperfusion might be involved in myocardial damage, which is influenced by the substrates available.
Impairment by damage of the pterygopalatine ganglion of nitroxidergic vasodilator nerve function in canine cerebral and retinal arteries.
Authors: Toda N , Ayajiki K , Yoshida K , Kimura H , Okamura T
Published: June 2012

Histochemical study revealed that transcutaneous injection of ethanol into the vicinity of the pterygopalatine ganglion greatly decreased the positive staining for NADPH diaphorase activity after 1 week in the ipsilateral ganglion of a dog and abolished the staining of perivascular nerves in the middle and posterior cerebral arteries. Transmural electrical stimulation or nicotine produced a relaxation in middle and posterior cerebral arteries isolated from the side with the nontreated ganglion (control side), whereas the relaxation was abolished or reversed to a contraction in the arteries from the side with the ethanol-treated ganglion. Nitric oxide-induced relaxations did not differ in the arteries from both sides. The response to nerve stimulation of the control arteries was suppressed by treatment with NG-nitro-L-arginine (L-NA), an inhibitor of nitric oxide synthase, and the inhibition was reversed by L-arginine. Nicotine produced a contraction followed by a relaxation in central retinal arterial strips obtained from the control side; the relaxation was abolished and the contraction was potentiated in the arteries from the treated side. The nicotine-induced relaxation was abolished by L-NA, and the contraction was suppressed by phentolamine. On the other hand, the nicotine-induced relaxation in superficial temporal arteries, susceptible to L-NA, was not attenuated by treatment with ethanol. The findings obtained so far support our hypothesis that nitric oxide released from the vasodilator nerve acts as a transmitter to produce arterial smooth muscle relaxation and suggest that the nerve fibers to the cerebral and retinal arteries arise from the pterygopalatine ganglion.
NG-nitro L-arginine methyl ester and other alkyl esters of arginine are muscarinic receptor antagonists.
Authors: Buxton I L , Cheek D J , Eckman D , Westfall D P , Sanders K M , Keef K D
Published: June 2012

Analogues of L-arginine with modifications at the terminal guanidino nitrogen and/or the carboxyl terminus of the molecule have been widely used for their ability to inhibit the production of nitric oxide and are thought to be competitive antagonists of nitric oxide synthase. The present studies were designed to test the possibility that these agents are also muscarinic receptor antagonists. Acetylcholine produced concentration-dependent contraction of endothelium-denuded rabbit coronary artery as well as isolated strips of canine colonic smooth muscle. The arginine analogue NG-nitro L-arginine methyl ester (L-NAME, 100 microM) but not NG-monomethyl L-arginine (L-NMMA, 100 microM) significantly shifted these contractile relations to the right, an effect that was not reversed by addition of 1 mM L-arginine. In radioligand binding studies using the muscarinic radioligand [3H]quinuclidinyl benzilate and tissues known to contain differing contributions of M1, M2, and M3 muscarinic receptors, addition of increasing concentrations of L-NAME resulted in a monophasic competition of binding with affinities (Ki) ranging from 68 microM in endothelium to 317 microM in whole aorta. Addition of the hydrolysis-resistant guanosine 5'-triphosphate analogue GTP gamma S (100 microM) had no effect on L-NAME competition of [3H]quinuclidinyl benzilate binding. Addition of L-NAME in radioligand binding competition studies using the agonist carbachol did not result in an alteration of the receptor's affinity for agonist, confirming the competitive nature of the interaction of L-NAME with the muscarinic receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
Nitric oxide mediates vasodilatation in response to activation of N-methyl-D-aspartate receptors in brain.
Authors: Faraci F M , Breese K R
Published: June 2012

Neurons release nitric oxide (NO) in response to activation of receptors for the excitatory amino acid N-methyl-D-aspartate (NMDA). We examined the hypothesis that activation of receptors for NMDA produces dilatation of the cerebral microcirculation that is mediated by NO. Diameters of cerebral arterioles were measured using a closed cranial window in anesthetized rabbits. Under control conditions, topical NMDA produced concentration-related dilatation of pial arterioles. Dilatation in response to NMDA was inhibited selectively by MK-801 (an NMDA receptor antagonist) and tetrodotoxin, suggesting that responses to NMDA were receptor mediated and dependent on neuronal activation. Increases in arteriolar diameter in response to NMDA were not affected by L-arginine but were inhibited by NG-nitro-L-arginine, suggesting that the vasodilatation was mediated by NO. Dilatation of cerebral arterioles in response to NMDA was not inhibited by indomethacin, suggesting that cyclooxygenase products do not mediate the response. Using isolated cerebral arteries, we also examined whether NMDA elicited direct cerebral vascular effects. In intact arteries studied in vitro, NMDA had no effect on vascular tone, suggesting that cerebral arteries lack receptors for NMDA. These findings suggest that NO generated in response to activation of receptors for NMDA in vivo is neuronally derived and not due to a direct vascular effect. Thus, NO may mediate increases in local blood flow during increases in neuronal activity in response to excitatory amino acids.
Characterization and localization of endothelin receptor subtypes in the human atrioventricular conducting system and myocardium.
Authors: Molenaar P , O'Reilly G , Sharkey A , Kuc R E , Harding D P , Plumpton C , Gresham G A , Davenport A P
Published: June 2012

The characterization and localization of endothelin A (ETA) and endothelin B (ETB) receptors have been determined in tissue sections of the human atrioventricular conducting system, surrounding regions of atrial and ventricular myocardium, and the left ventricular free wall by use of radioligand binding, polymerase chain reaction, and in situ hybridization. Selective ETA (BQ123) and ETB (BQ3020) compounds in conjunction with [125I]endothelin-1 revealed the presence of ETA and ETB receptors in the left ventricular free wall (BQ123: 57 +/- 5% ETA, 43 +/- 2% ETB, n = 3; BQ3020: 67 +/- 3% ETA, 33 +/- 3% ETB, n = 3). Autoradiography using [125I]endothelin-1 in the absence or presence of BQ3020, BQ123, or endothelin-1 showed ETA and ETB receptors localized to atrial and ventricular myocardium, the atrioventricular conducting system, and endocardial cells. There was a higher proportion of ETB receptors in the atrioventricular node and the penetrating and branching bundles of His than in the surrounding interventricular and interatrial septa (p < 0.0001). There was a lower density of ETB receptors in the interventricular septum compared with the interatrial septum and the atrioventricular conducting system (p = 0.009) and a lower density of ETA receptors in the atrioventricular conducting system compared with interatrial and interventricular septa (p = 0.008). Isolated right atrial myocytes showed a higher proportion of ETA receptors (91 +/- 12%, n = 3). Amplification of left ventricular free wall cDNA by polymerase chain reaction revealed the presence of ETA and ETB receptor mRNA. mRNA for both subtypes was detected in isolated atrial myocytes. In situ hybridization showed ETA and ETB receptor mRNA localization to atrial and ventricular myocardium, the atrioventricular conducting system, and endocardial cells. These studies demonstrate the presence of ETA and ETB receptors in human myocardium and the atrioventricular conducting system.
Spiral waves of excitation underlie reentrant activity in isolated cardiac muscle.
Authors: Pertsov A M , Davidenko J M , Salomonsz R , Baxter W T , Jalife J
Published: June 2012

The mechanism of reentrant ventricular tachycardia was studied in computer simulations and in thin (approximately 20 x 20 x 0.5-mm) slices of dog and sheep ventricular epicardial muscle. A two-dimensional matrix consisting of 96 x 96 electrically coupled cells modeled by the FitzHugh-Nagumo equations was used to analyze the dynamics of self-sustaining reentrant activity in the form of elliptical spiral waves induced by premature stimulation. In homogeneous anisotropic media, spirals are stationary and may last indefinitely. However, the presence of small parameter gradients may lead to drifting and eventual termination of the spiral at the boundary of the medium. On the other hand, spirals may anchor and rotate around small discontinuities within the matrix. Similar results were obtained experimentally in 10 preparations whose electrical activity was monitored by means of a potentiometric dye and high-resolution optical mapping techniques; premature stimulation triggered reproducible episodes of sustained or nonsustained reentrant tachycardia in the form of spiral waves. As a rule, the spirals were elongated, with the major hemiaxis parallel to the longitudinal axis of the cells. The period of rotation (183 +/- 68 msec [mean +/- SD]) was longer than the refractory period (131 +/- 38 msec) and appeared to be determined by the size of the spiral's core, which was measured using a newly devised "frame-stack" plot. Drifting of spiral waves was also observed experimentally. Drift velocity was 9.8% of the velocity of wave propagation. In some cases, the core became stationary by anchoring to small arteries or other heterogeneities, and the spiral rotated rhythmically for prolonged periods of time. Yet, when drift occurred, spatiotemporal variations in the excitation period were manifested as a result of a Doppler effect, with the excitation period ahead of the core being 20 +/- 6% shorter than the excitation period behind the core. As a result of these coexisting frequencies, a pseudoelectrocardiogram of the activity in the presence of a drifting spiral wave exhibited "QRS complexes" with an undulating axis, which resembled those observed in patients with torsade de pointes. The overall results show that spiral wave activity is a property of cardiac muscle and suggest that such activity may be the common mechanism of a number of monomorphic and polymorphic tachycardias.
Ionic bases for electrophysiological distinctions among epicardial, midmyocardial, and endocardial myocytes from the free wall of the canine left ventricle.
Authors: Liu D W , Gintant G A , Antzelevitch C
Published: June 2012

Recent studies from our laboratory involving syncytial preparations have delineated electrophysiological distinctions between epicardium, endocardium, and a unique population of cells in the deep subepicardial to midmyocardial layers (M region) of the canine ventricle. In the present study, we used standard microelectrode, single microelectrode switch voltage-clamp, and whole-cell patch-clamp techniques to examine transmembrane action potentials, steady-state current-voltage relations, and the 4-aminopyridine-sensitive transient outward current (Ito1) in myocytes enzymatically dissociated from discrete layers of the free wall of the canine left ventricle. Action potential characteristics of myocytes isolated from the epicardium, M region, and endocardium were very similar to those previously observed in syncytial preparations isolated from the respective regions of the ventricular wall. A prominent spike and dome was apparent in myocytes from epicardium and the M region but not in myocytes from endocardium. Action potential duration-rate relations were considerably more pronounced in cells isolated from the M region. Current-voltage relations recorded from cells of epicardial, M region, and endocardial origin all displayed an N-shaped configuration with a prominent negative slope-conductance region. The magnitude of the inward rectifier K+ current (IK1) was 392 +/- 86, 289 +/- 65, and 348 +/- 115 pA in epicardial, M region, and endocardial myocytes, respectively, when defined as steady-state current blocked by 10 mM Cs+. Similar levels were obtained when IK1 was defined as the steady-state difference current measured in the presence (6 mM) and absence of extracellular K+. Ito1 was significantly greater in epicardial and M region myocytes than in endocardial myocytes. At a test potential of +70 mV (holding potential, -80 mV), Ito1 amplitude was 4,203 +/- 2,370, 3,638 +/- 1,135, and 714 +/- 286 pA in epicardial, M region, and endocardial cells, respectively. No significant differences were observed in the voltage dependence of inactivation of Ito1 in the three cell types. The time course of reactivation of Ito1 was slower in cells from the M region compared with either epicardial or endocardial cells. Our data suggest that prominent heterogeneity exists in the electrophysiology of cells spanning the canine ventricular wall and that differences in the intensity of the transient outward current contribute importantly, but not exclusively, to this heterogeneity. These findings should advance our understanding of basic heart function and the ionic bases for the electrocardiographic J wave, T wave, U wave, and long QT intervals as well as improve our understanding of some of the complex factors contributing to the development of cardiac arrhythmias.
Altered vascular responses to platelets from hypercholesterolemic humans.
Authors: Kaul S , Waack B J , Padgett R C , Brooks R M , Heistad D D
Published: June 2012

Activated platelets release potent vasoactive factors. Previous studies have focused on mechanisms by which vascular abnormalities lead to altered responses of atherosclerotic arteries. We tested the hypothesis that the activation of platelets from hypercholesterolemic humans produces abnormal vascular responses. Responses to intraluminal and abluminal activation of platelets from normal subjects and type II hypercholesterolemic patients (total cholesterol, 274 +/- 16 [mean +/- SEM] mg/dl) were examined in carotid arteries from normal rabbits perfused in vitro. Intraluminal activation of normal platelets produced pronounced dilatation of arteries preconstricted with phenylephrine. Vasodilator responses to intraluminal activation of platelets from hypercholesterolemic patients were greatly impaired. Vasodilator responses to platelets from hypercholesterolemic patients were not restored to normal by LY53,857 (10(-5) M), a 5-hydroxytryptamine2-serotonergic antagonist, by SQ29,548 (10(-5) M), a thromboxane A2/prostaglandin H2 receptor antagonist, or by apyrase (1.5 units/ml), an enzyme with ADPase activity. Abluminal activation of normal platelets produced modest constriction in quiescent arteries, and abluminal activation of platelets from hypercholesterolemic patients produced augmented vasoconstrictor responses. The major finding is that vasodilator responses to platelets from hypercholesterolemic patients are profoundly impaired, and vasoconstrictor responses to platelets from hypercholesterolemic patients are augmented. Mechanisms in addition to increased release of serotonin, thromboxane, and ADP appear to contribute to impaired vasodilator responses to hypercholesterolemic platelets. Thus, alteration of platelets by hypercholesterolemia, as well as altered vascular reactivity, may contribute to abnormal vascular responses in atherosclerosis.
Flow-related responses of intracellular inositol phosphate levels in cultured aortic endothelial cells.
Authors: Prasad A R , Logan S A , Nerem R M , Schwartz C J , Sprague E A
Published: June 2012

In vitro and in vivo evidence indicates that hemodynamic wall shear stress evokes a diversity of biological responses in vascular endothelial cells, ranging from cell shape changes to alterations in low density lipoprotein receptor expression. The signal transduction mechanisms by which the level of fluid mechanical shear stress is recognized by the endothelial cell and translated into these diverse biological responses remain to be elucidated. The present study focuses on the association between the onset of elevated shear stress and activation of the phosphoinositide signal transduction pathway, as measured by the intracellular release of inositol phosphates, in cultured bovine aortic endothelial cells (BAECs). BAECs were seeded, grown to confluence on large polyester sheets, and preincubated with 0.3 microCi/ml [3H]inositol for 24 hours before insertion in parallel-plate flow chambers for exposure to high shear stress (HS) at 30 dynes/cm2 or low shear stress (LS) at < 0.5 dyne/cm2 for periods ranging from 15 seconds to 24 hours. The induction of HS was associated with an early, transient but significant increase (142%, HS/LS x 100%) in inositol trisphosphate (IP3) measured at 15 seconds of shear stress exposure followed by a major peak in IP3 (189%) observed at 5 minutes after HS onset. After these initial increases, IP3 levels returned to near resting levels within 30 minutes of continued HS exposure and then continued to decline to significantly lower (75%) levels relative to LS-treated cells within 4 hours and remained lower throughout the remainder of the 24-hour HS exposure. LS-treated cells exhibited no significant changes in inositol phosphate levels throughout the 24-hour exposure periods. Exposure of BAECs to shear stress of 60 dynes/cm2 resulted in an approximately fourfold increase in IP3 levels (396%) measured at 5 minutes, almost double the levels measured in cells exposed to 30 dynes/cm2 for 5 minutes. Pretreatment of BAECs for 30 minutes with 5 mM neomycin, an inhibitor of phosphoinositide metabolism, before HS exposure inhibited both the early increases in inositol phosphates and subsequent cell elongation and alignment observed in untreated BAECs simultaneously exposed to HS without inhibiting protein synthesis. These results indicate that the exposure of cultured BAECs to elevated wall shear stress is associated with an early biphasic IP3 increase followed by a resetting of intracellular inositol phosphate concentrations to levels below that observed in static cultured BAECs. Furthermore, neomycin inhibition of this IP3 response to shear stress is associated with an inhibition of one of the major endothelial biological responses to shear stress, i.e., cell shape change and orientation.(ABSTRACT TRUNCATED AT 400 WORDS)
Inhibition of gap junctional conductance by long-chain acylcarnitines and their preferential accumulation in junctional sarcolemma during hypoxia.
Authors: Wu J , McHowat J , Saffitz J E , Yamada K A , Corr P B
Published: June 2012

Electrophysiological and biochemical sequelae of myocardial ischemia occur within minutes of the onset of myocardial ischemia in vivo. Both conduction delay and conduction block occur rapidly within the same time interval as the accumulation of long-chain acylcarnitines. In the present study, double whole-cell voltage-clamp procedures were used to assess the influence of long-chain acylcarnitines on gap junctional conductance in isolated pairs of canine ventricular myocytes. Long-chain acylcarnitine (5 microM) decreased gap junctional conductance from 153 to 48 nS in a time-dependent and reversible manner. Although the amplitude of junctional current was reduced by 68%, the current continued to demonstrate a linear current-voltage relation. The extent of endogenous accumulation of long-chain acylcarnitines in junctional regions of the sarcolemma was assessed in isolated myocytes in which endogenous free, short-chain, and long-chain acylcarnitine pools had been equilibrated with [3H]carnitine. Under normoxic conditions, long-chain acylcarnitines were not detectable in junctional sarcolemma of myocytes as assessed using electron microscopic autoradiography. Exposure of myocytes to hypoxia (PO2, < 15 mm Hg) for 10 minutes resulted in the preferential accumulation of endogenous long-chain acylcarnitines in junctional sarcolemma (173 +/- 5 x 10(5) molecules/microns 3), a concentration that was sevenfold greater than that found in nonjunctional sarcolemma. Therefore, endogenous long-chain acylcarnitines accumulate preferentially in junctional regions of the sarcolemma during short intervals of hypoxia. Exogenously supplied long-chain acylcarnitines can markedly decrease cellular coupling in a reversible manner, suggesting that this amphiphile may contribute to the marked slowing in conduction velocity in the ischemic heart in vivo, not only by suppressing the rapid Na+ inward current directly, as has been shown previously, but also by decreasing cellular coupling.