bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamics of normal and ischemic canine papillary muscles.
Authors: Rayhill S C , Daughters G T , Castro L J , Niczyporuk M A , Moon M R , Ingels N B , Stadius M L , Derby G C , Bolger A F , Miller D C
Published: June 2012

This investigation was designed to elucidate the dynamics of the left ventricular (LV) papillary muscles. Miniature tantalum myocardial markers were placed on the tip and base of each papillary muscle in six dogs. Markers were also implanted into the LV myocardium to define two orthogonal equatorial diameters and the long-axis dimension. Two weeks later, after recovery from thoracotomy, markers were visualized by biplane fluoroscopy, and video images were recorded during control conditions, after autonomic blockade, after inotropic stimulation with calcium, after methoxamine infusion (to increase afterload), and after blood volume augmentation (to increase preload). Two days later, radiographic recordings were made before and after occlusion of the left circumflex coronary artery. Computer-aided analysis of the video recordings was used to determine three-dimensional coordinates of the markers. It was found that before circumflex coronary occlusion, the dynamics of both papillary muscles closely mimicked the dynamics of the LV as a whole. The papillary muscles shortened during ejection and lengthened during diastole. Their lengths changed minimally during the isovolumic periods, and this behavior was not altered by any of the interventions except coronary occlusion. During circumflex coronary artery occlusion, the ischemic posterior papillary muscle lengthened during isovolumic contraction and most of ejection and shortened only when LV pressure began to fall. Hence, we believe that previous studies demonstrating papillary muscle lengthening during isovolumic contraction and shortening during isovolumic relaxation may have been confounded by coexistent myocardial ischemia or stunning.
T-type Ca2+ channels are abnormal in genetically determined cardiomyopathic hamster hearts.
Authors: Sen L , Smith T W
Published: June 2012

Although there is substantial evidence of abnormal Ca2+ homeostasis in heart cells of the cardiomyopathic Syrian hamster (Bio 14.6 strain), the mechanism by which these myocytes become Ca(2+)-overloaded is not known. To elucidate the role of voltage-sensitive Ca2+ channels in the pathogenesis of myopathy, whole-cell Ca2+ currents were measured in myopathic and normal control cardiac myocytes. These studies demonstrate the presence of two voltage-sensitive Ca2+ channel types in ventricular myocytes isolated from 200- to 300-day-old cardiomyopathic and age-matched normal hamsters. The two Ca2+ channel types were identified by their unitary conductance properties and pharmacologic sensitivities. Both L-type and T-type Ca2+ channels were present in cardiomyopathic and normal cells. Current density through L-type Ca2+ channels was the same in cardiomyopathic and normal control myocytes. However, the mean current density of T-type Ca2+ channels in cardiomyopathic cells was significantly higher than in normal cells (myopathic, 12.3 +/- 1.8 pA/pF; normal, 5.8 +/- 1.1 pA/pF; n = 8; P < .01). The T-type Ca2+ current in cardiomyopathic myocytes was activated and inactivated at more negative potentials than in cells from normal hamster hearts. These findings demonstrate no abnormality of the dihydropyridine-sensitive voltage-dependent L-type Ca2+ channel. In contrast, the observed abnormalities in T-type Ca2+ channel function in cardiomyopathic hamster myocytes suggest that this alteration may be related to the pathogenesis of Ca2+ overload and the arrhythmias in this genetically determined form of cardiomyopathy.
Capillary perfusion of the rat brain cortex. An in vivo confocal microscopy study.
Authors: Villringer A , Them A , Lindauer U , Einhäupl K , Dirnagl U
Published: June 2012

Confocal laser-scanning microscopy was used to visualize subsurface cerebral microvessels labeled with intravascular fluorescein in a closed cranial window model of the anesthetized rat. In noninvasive optical sections up to 250 microns beneath the brain surface, plasma perfusion and blood cell perfusion of individual capillaries were studied. Under resting conditions, in all cerebral capillaries the presence of plasma flow as demonstrated by the appearance of an intravenously injected fluorescent tracer within 20 seconds after injection. Plasma flow was verified even in capillaries that contained stationary erythrocytes or leukocytes; 91.1% of the capillaries contained flowing blood cells, 5.2% contained stationary blood cells, and no blood cells were seen in 3.6%. Mean blood cell velocity was 498.3 +/- 443.9 microns/s, and the mean blood cell supply rate was 35.75 +/- 28.01 cells per second. When capillaries were continuously observed for 1 minute, "on" and "off" periods of blood cell flow were noted. During hypercapnia (increase of PCO2 from 33.25 to 50.26 mm Hg), mean blood cell flux increased from 38.6 +/- 17.2 to 55.5 +/- 12.2 per second (P < .005, paired t test of mean values in six animals), and blood cell velocity increased from 519.5 +/- 254.8 to 828.5 +/- 460.8 microns/s (P = .074, paired t test of mean values in six animals). Homogeneity of blood cell flux increased as indicated by the coefficient of variation decreasing from 44.6% to 22.0%, and the portion of poorly perfused capillaries (blood cell flux, < 40 per second) decreased from 59.2% to 22.4%.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of angiotensin II and enalapril on transfer of low-density lipoprotein into aortic intima in rabbits.
Authors: Nielsen L B , Stender S , Kjeldsen K , Nordestgaard B G
Published: June 2012

To assess the mechanism behind a possible atherosclerosis-promoting effect of angiotensin II, the influence of angiotensin II, noradrenaline, and enalapril on transfer of low-density lipoprotein (LDL) into the arterial wall was investigated in conscious rabbits. Intravascular infusion of angiotensin II (1.4 micrograms/kg per minute) initially increased the mean blood pressure from 70 to 80 mm Hg to 125 to 150 mm Hg; this effect was transient, and the blood pressure returned to baseline values within 2 hours, despite continuous infusion of angiotensin II. The normalized influx of LDL into the aortic intima, determined after in vivo exposure to 125I-LDL for 1 hour, was 88 +/- 17 (n = 6), 12 +/- 12 (n = 5), and 28 +/- 6 (n = 5) nL/cm2 per hour (mean +/- SEM) during angiotensin II infusion at high blood pressure, during angiotensin II infusion after the blood pressure had been normalized, and during continuous saline infusions, respectively (P < .05 for high blood pressure versus low blood pressure and saline). When noradrenaline was used to increase blood pressure to a level similar to that induced by angiotensin II, the normalized influx of LDL in noradrenaline-treated rabbits was also increased markedly. Production of endogenous angiotensin II was inhibited with enalapril (2.9 mg/kg per day). Compared with placebo rabbits, enalapril-treated rabbits had a 92% lower plasma angiotensin-converting enzyme activity and a 23% lower blood pressure. The normalized influx of LDL, however, was similar in the two groups at 18 +/- 2 (n = 10) and 20 +/- 3 (n = 10) nL/cm2 per hour, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Nitric oxide influences neuronal activity in the nucleus tractus solitarius of rat brainstem slices.
Authors: Tagawa T , Imaizumi T , Harada S , Endo T , Shiramoto M , Hirooka Y , Takeshita A
Published: June 2012

Nitric oxide (NO) is shown to be synthesized in the central nervous system as well as in vascular endothelial cells. However, the physiological role of NO in cardiovascular regulation in the central nervous system remains unclear. The present study examines whether NO plays a role in the regulation of neuronal activity in the nucleus tractus solitarius (NTS). Single-unit extracellular recordings were obtained from NTS neurons in slices (400 microns) of the rat brainstem, which had spontaneous discharges at a frequency of 0.5 to 3 spikes per second. Eighty-one neurons were tested for sensitivity to L-arginine, which is the physiological precursor of NO. L-Arginine (10(-7) to 10(-4) mol/L) increased neuronal activity dose dependently in 33 (40.7%) of 81 neurons tested, but D-arginine (10(-5) mol/L) did not. The neurons that responded to L-arginine responded to glutamate as well. NG-Monomethyl-L-arginine (10(-5) to 3 x 10(-5) mol/L), an inhibitor of the formation of NO, dose-dependently blocked increases in the neuronal activity evoked with L-arginine (10(-5) mol/L). Hemoglobin (1.5 mg/L), a trapper of NO, and methylene blue (10(-5) mol/L), an inhibitor of guanylate cyclase, also blocked increases in the neuronal activity evoked with L-arginine (10(-5) mol/L). Sodium nitroprusside (SNP, 10(-5) to 10(-4) mol/L), which spontaneously produces NO, increased the neuronal activity in the neurons that responded to L-arginine. SNP did not alter the neuronal activity of the neurons that did not respond to L-arginine.(ABSTRACT TRUNCATED AT 250 WORDS)
Endothelial cell storage and release of endothelin as a cardioregulatory mechanism.
Authors: McClellan G , Weisberg A , Rose D , Winegrad S
Published: June 2012

Coronary vascular endothelial cells release substances into the coronary circulation that modify the contractile system of cardiac myocytes, and cardiac myocytes may release factors that modulate the secretion of cardioregulatory substances by endothelial cells. This regulatory loop is sensitive to the rate of coronary flow and tissue oxygen tension. In the present study, coronary venous effluent from isolated perfused hearts and the contents of the coronary vascular endothelial cells have been collected, the latter by disrupting the cells with coronary perfusion at high pressure. The relative amounts of upregulating and downregulating factors in both collections have been estimated by assaying their effects on the contractility of isolated cardiac trabeculas. The amount of upregulating factor stored in the endothelial cells is sensitive to the rate of coronary flow just before disruption of the cells. The quantity of endothelin in the coronary venous effluent and in the vascular endothelial cell contents was measured by radioimmunoassay and compared with the degree of upregulation of contractility produced by the two types of solutions. Upregulation was never produced in the absence of endothelin. The extent of the increase in contractility that was observed with endothelial cell contents correlated with the concentration of endothelin and was approximately the same as the increase in contractility from similar concentrations of endothelin added to standard Krebs' solution. The amount of the increase in contractility from coronary effluent could be accounted for by the concentration of endothelin in the effluent with the additional presence of some downregulating factor as well. The endothelin antagonist BQ123 inhibited the upregulation from coronary perfusate. It appears that endothelin alone can account for all of the upregulation of contractility produced by the vascular endothelial cells. Coronary flow, probably through shear forces, seems to regulate the production of endothelin possibly from an inactive precursor. Tissue oxygen tension appears to modulate the rate of release of the endothelin from endothelial cells though substances released by cardiac myocytes or other cells in the tissue. The downregulating factor is stored to a much smaller extent.
Inhibition of smooth muscle cell proliferation by visible light-activated psoralen.
Authors: Sumpio B E , Li G , Deckelbaum L I , Gasparro F P
Published: June 2012

The present study was designed to evaluate the effect of 8-methoxypsoralen (8-MOP) activated with visible light (419 nm) on the suppression of smooth muscle cell (SMC) proliferation in vitro. We hypothesize that if visible light (VL) instead of UVA is used to photoactivate 8-MOP, cytotoxic 8-MOP-DNA cross-link formation can be minimized. Bovine aorta SMCs (2 x 10(4)/cm2) were incubated with 8-MOP (1 micrograms/mL) for 30 minutes (in the dark) and exposed to a range of VL (2 to 69 J/cm2) to determine the dose of VL that inhibits SMC proliferation with minimal toxicity. The results show that 8-MOP in combination with 2 to 12 J/cm2 VL reversibly inhibited SMC proliferation for up to 5 days after treatment. SMC viability was confirmed by trypan blue exclusion. 8-MOP in combination with 23- or 69-J/cm2 VL irreversibly inhibited SMC proliferation. In cell cycle studies, 12-J/cm2 VL was used to activate 8-MOP. A phase-specific G2 blockade that correlated temporally with recovery of SMC replication was observed. Photoadduct repair studies showed that cell proliferation rates recovered when 60% of the adducts had been removed. These results demonstrate for the first time the possibility of using VL to activate 8-MOP to inhibit cell proliferation and suggest that 8-MOP/VL photochemotherapy can be used to control SMC growth.
Role of Na(+)-H+ exchange in mediating effects of endothelin-1 on normal and ischemic/reperfused hearts.
Authors: Khandoudi N , Ho J , Karmazyn M
Published: June 2012

Endothelin (ET) has been shown to be elevated under conditions of cardiac pathology and to produce diverse cardiac effects, including coronary constriction and a positive inotropic influence. We characterized the concentration- and time-dependent effects of the most potent of the ET isoforms, ET-1 (0.4, 2, and 4 nmol/L), on myocardial contractility and coronary resistance and assessed its effects on the ischemic and reperfused heart. Because ET-1 has been shown to activate the Na(+)-H+ exchanger in cardiac myocytes, we determined the contribution of the antiport by examining the effects of ET-1 in the presence of the Na(+)-H+ exchange inhibitor methylisobutyl amiloride (MIA). At all three concentrations, ET-1 produced an initial positive inotropic effect that was reversed with continued perfusion, the degree of the reversal being dependent on ET-1 concentration. With 0.4 nmol/L, contractility returned to pre-ET-1 values, whereas after 75 minutes of perfusion with 4 nmol/L ET-1, contractility was depressed by 75%. At all concentrations, ET-1 produced a coronary-constricting effect, whereas an elevation in resting tension was observed only with 4 nmol/L ET-1. MIA significantly prevented the positive inotropic effect of ET-1 but had no effect on loss in function or elevation in resting tension produced by 4 nmol/L ET-1. Furthermore, MIA partially, but not significantly, attenuated the constricting effects of all ET-1 concentrations. In the ischemic heart, 0.4 nmol/L ET-1 appeared to delay the loss in contractility produced by cessation of flow, although the effect was not significant. Higher concentrations of ET-1 were without effect on ischemia-induced contractile depression, although their presence produced a marked elevation in resting tension during ischemia that was attenuated by MIA. Recovery in contractility was reduced by all concentrations of ET-1, although the effects of the lowest concentration were associated primarily with defective relaxation. The depressant effects of ET-1 either in normal or ischemic/reperfused hearts were irreversible. The inhibitory effects of ET-1 on contractile recovery were associated with diminished tissue glycogen and elevated lactate levels. High-energy phosphates after reperfusion were depressed in hearts treated with 4 nmol/L ET-1. The attenuation in contractile recovery and alterations in metabolite content were prevented by MIA. These results provide evidence that ET-1 produces complex effects on heart function that are likely mediated via different mechanisms and demonstrate its ability to aggravate ischemic and reperfusion injury through a mechanism possibly involving Na(+)-H+ exchange activation.
Acute changes in myosin heavy chain synthesis rate in pressure versus volume overload.
Authors: Imamura T , McDermott P J , Kent R L , Nagatsu M , Cooper G , Carabello B A
Published: June 2012

The left ventricular hypertrophy that develops with the volume overload of mitral regurgitation is relatively less than that which develops with the pressure overload of aortic stenosis even when both lesions are severe. The hypertrophy that develops must be the sum of changes in the rate of myocardial protein synthesis and degradation. In the present canine study, we explored early changes in the synthesis rate of myosin heavy chain in response to severe acute pressure overload versus that of the severe acute volume overload of mitral regurgitation. We tested the hypothesis that in acute overload, the rate of protein synthesis would increase less in the volume-overload model than in the pressure-overload model, a potential partial mechanism for the discrepancy in the eventual total amount of hypertrophy that develops in these two lesions. Acute pressure overload was produced by inflating a balloon in the descending aorta, and acute volume overload was produced by using our closed-chest mitral chordal rupture technique. In both models, the hemodynamic lesion that was created was severe. In eight dogs with pressure overload, the average gradient across the balloon was 119.8 +/- 6.1 mm Hg. In six dogs with volume overload, the average regurgitant fraction was 0.67 +/- 0.06. Six other dogs served as controls. The average rate of myosin heavy chain synthesis in control dogs was 2.7 +/- 0.2% per day, virtually identical to the rate we found in the severe volume-overload model. In contrast, the rate was increased in the pressure-overload model by 30% to 3.5 +/- 0.3% per day (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)
Cardiolipin-protein complexes and initiation of complement activation after coronary artery occlusion.
Authors: Rossen R D , Michael L H , Hawkins H K , Youker K , Dreyer W J , Baughn R E , Entman M L
Published: June 2012

Specific rabbit anti-cardiolipin (anti-CL) antibodies were used to investigate the hypothesis that cardiolipin, associated with mitochondrial membrane proteins, binds C1 and facilitates activation of the complement cascade following reperfusion of ischemic myocardium. By immunoelectron microscopy, anti-CL localized to subsarcolemmal mitochondria, emerging through breaks in membranes of damaged cardiac myocytes. Anti-CL reacted with > 15 mitochondrial constituents, most of which comigrated with the proteins that bind C1q in transblots of subsarcolemmal mitochondria, fractionated by polyacrylamide gel electrophoresis under reducing conditions in the presence of sodium dodecyl sulfate. A subset of the C1q-binding proteins > 24 to 37 kDa served as stable sites for assembly of C3, C5, and C9. Cardiac lymph, collected during the first hour after reperfusion of ischemic myocardium, contained proteins of diverse size that reacted with both anti-CL and C1q. Cardiac lymph, collected before occlusion and 4 to 5 hours after reperfusion, in comparison, had few if any C1q or anti-CL reactive proteins. Treatment with phospholipase suppressed the C1q-binding activity and anti-CL reactivity of the proteins in reperfusion lymph and those with similar properties in mitochondrial extracts. Our data suggest that during ischemia, mitochondria, extruded through breaks in the sarcolemma, unfold and release membrane fragments in which cardiolipin and protein are intimately associated. By binding C1 and supplying sites for the assembly of later-acting complement components, these fragments provide the means to disseminate the complement-mediated inflammatory response to ischemic injury.
Effects of clonidine on the reflex cardiovascular responses and release of substance P during muscle contraction.
Authors: Ally A , Meintjes A F , Mitchell J H , Wilson L B
Published: June 2012

The effects of microdialyzing clonidine into the L-7 dorsal horn on the cardiovascular responses, renal sympathetic nerve activity (RSNA), and release of substance P (SP) evoked by static contraction of the triceps surae muscle were studied using anesthetized cats. A microdialysis probe was inserted into the spinal cord ipsilateral to the muscle being contracted or stretched. Contraction, evoked by stimulation of the distal ends of the cut L-7 and S-1 ventral roots for 1 minute, increased mean arterial pressure (MAP), heart rate (HR), and RSNA by 48 +/- 6 mm Hg, 18 +/- 2 beats per minute, and 66 +/- 5%, respectively. Passive stretch of the same muscle for 1 minute also increased MAP, HR, and RSNA by 51 +/- 6 mm Hg, 17 +/- 2 beats per minute, and 50 +/- 3%, respectively. Microdialysis of clonidine (380 mumol/L) blunted the contraction-evoked responses: MAP, HR, and RSNA increased by 19 +/- 4 mm Hg, 7 +/- 1 beats per minute, and 24 +/- 5%, respectively. The increases elicited by passive stretch were also attenuated (MAP, 22 +/- 4 mm Hg; HR, 6 +/- 1 beats per minute; and RSNA, 15 +/- 4%). This attenuation by clonidine was dose dependent (3.8 mumol/L, 38 mumol/L, 380 mumol/L, and 3.8 mmol/L). Preadministration of the alpha 2-adrenergic antagonist yohimbine (3 mmol/L) blocked the effect of clonidine (380 mumol/L) on the cardiovascular and RSNA responses to muscle contraction. Clonidine (380 mumol/L) did not alter the release of SP in the dorsal horn during contraction (before clonidine, 0.380 +/- 0.018 fmol/100 microL; after clonidine, 0.356 +/- 0.012 fmol/100 microL).(ABSTRACT TRUNCATED AT 250 WORDS)
Cyclic ADP-ribose competes with ATP for the adenine nucleotide binding site on the cardiac ryanodine receptor Ca(2+)-release channel.
Authors: Sitsapesan R , McGarry S J , Williams A J
Published: June 2012

We have investigated the mechanism of action of the putative second messenger, cyclic ADP-ribose (cADPR), on the cardiac ryanodine-sensitive Ca(2+)-release channel. Current fluctuations through single Ca(2+)-release channels have been monitored after incorporation into planar phospholipid bilayers. We demonstrate that activation of the channel by cADPR is dependent on activating levels of cytosolic Ca2+ and lifetime analysis indicates that the mechanism of action may be sensitization of the channel to Ca2+. In the absence of ATP, cADPR activates the channel in a concentration-dependent manner in the presence of 10 mumol/L cytosolic Ca2+. However, in the presence of ATP, cADPR tends to decrease open probability, indicating that cADPR may be acting at the adenine nucleotide binding site. In addition, we demonstrate that the precursor of cADPR, beta-NAD+, and the breakdown product, ADP-ribose, also activate the channel. As cADPR will have to compete with much higher concentrations of beta-NAD+, ADP-ribose, and ATP, we suggest that cADPR does not act as a direct endogenous trigger for the opening of the cardiac Ca(2+)-release channel.
Pituitary-specific transcription factor (Pit-1) binding site in the human renin gene 5'-flanking DNA stimulates promoter activity in placental cell primary cultures and pituitary lactosomatotropic cell lines.
Authors: Sun J , Oddoux C , Gilbert M T , Yan Y , Lazarus A , Campbell W G , Catanzaro D F
Published: June 2012

Renin gene expression is limited to a number of specific tissues, including the kidney, adrenal glands, reproductive organs (of particular relevance to this study, the placenta), and the pituitary gland. In the present study, we investigated the human renin (hRen) 5'-flanking DNA sequences required to drive the expression of a luciferase reporter gene in placental and pituitary cells and in two cell lines, 293 and JEG-3, which have been proposed as model systems with which to study transcriptional regulation of renin genes. The activities of specific sequences in the hRen 5'-flanking DNA sequences in human placental cell primary cultures were very similar to those that we previously reported in pituitary cells, suggesting the involvement of common promoter elements and related transcription factors. Accordingly, the binding site for the pituitary-specific transcription factor (Pit-1) was the major determinant of renin promoter activity in both pituitary and placental cells. Gel mobility shift analysis showed a placental nuclear factor with a gel mobility different from that of Pit-1. However, Northern blot analysis failed to demonstrate abundant Pit-1-related mRNAs in renin-expressing cultures of chorionic and decidual cells, suggesting that the placental factor is not closely related to Pit-1. Although a factor from 293 cells also bound to the Pit-1 site, it had gel mobility shift characteristics different from Pit-1 and the placental factor. Moreover, the low promoter activity in 293 cells was independent of this site or, indeed, of sequences upstream from the TATA box. In JEG-3 cells, renin 5'-flanking DNA sequences showed virtually no transcriptional activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Increased protein kinase C and isozyme redistribution in pressure-overload cardiac hypertrophy in the rat.
Authors: Gu X , Bishop S P
Published: June 2012

Protein kinase C (PKC) activity and isozyme distribution were evaluated during development of pressure-overload-induced cardiac hypertrophy. Three-week-old rats were loosely banded on the ascending aorta (left ventricular hypertrophy [LVH] group). Two weeks later, when left ventricular mass was 50% greater than in the sham-operated control group and cardiac mass was still rapidly increasing beyond that of normal growth, PKC activity and [3H]phorbol 12,13-dibutyrate (PDBu) binding capacity were determined. In LVH, PKC activity was 119 +/- 14%, 158 +/- 17%, and 152 +/- 9% of the control value in cytosol, membrane, and nuclear-cytoskeletal fractions, respectively (n = 9 or 10). [3H]PDBu binding assay revealed increased PKC concentration in LVH cytosolic (control, 0.51 +/- 0.06 pmol/L per milligram; LVH, 0.78 +/- 0.09 pmol/L per milligram; n = 5; P < .05) and membrane fractions (control, 1.33 +/- 0.15; LVH, 2.32 +/- 0.39; n = 5; P < .05). Scatchard analysis indicated no difference in Kd values between control and LVH groups. Immunoblot analysis using PKC isoform-specific antibodies showed that both Ca(2+)-dependent (alpha and beta) and Ca(2+)-independent (delta, epsilon, and zeta) isoforms were present in the left ventricle. Compared with the control value, there was increased concentration in the membrane and nuclear-cytoskeletal fractions for beta 1,2 and epsilon and in the cytosol for beta 1,2. PKC-delta could be detected only in the nuclear-cytoskeletal fraction and was not changed in LVH. PKC-alpha and -zeta were present in all three fractions but were not altered in LVH. These data indicate that PKC activity and concentration increase during development of LVH induced by pressure overload. The increased PKC isozymes were mainly limited to PKC-beta 1,2 and PKC-epsilon, and the increase was present mainly in the membrane and nuclear-cytoskeletal fractions.
Localization of atrial natriuretic peptide in caveolae of in situ atrial myocytes.
Authors: Page E , Upshaw-Earley J , Goings G E
Published: June 2012

The plasma membrane-associated non-clathrin-coated vesicles called caveolae are multifunctional organelles thought to be implicated in the sequestration and transport of small molecules (potocytosis) as well as in the binding of Ca2+ ions, signal transduction, and processing of hormonal and mechanosensitive signals. We have previously suggested that the apparent contiguity of caveolar and atrial granule membranes observed in electron micrographs of in situ mouse atrial myocytes might reflect externalization of atrial natriuretic peptide through caveolae. Using Tokuyasu's classic technique, we now show by immunoelectron microscopy of glutaraldehyde-fixed and cryosectioned mouse and rat atria that antibody against atrial natriuretic peptide prohormone is present within caveolae of in situ atrial myocytes. We confirm this intracaveolar localization by stereoimaging colloidal gold-labeled antibody to the prohormone in electron micrographs of glutaraldehyde/osmium tetroxide-fixed positively stained atrial thin sections. Because profiles of caveolae were rarely immunolabeled with antibody against atrial peptide unless there was a profile of an immunolabeled atrial granule nearby in the subjacent cytoplasm, we concluded that the intracaveolar hormone was derived predominantly from a direct interaction of atrial granules with caveolae. Perturbations that markedly increase the rate of natriuretic peptide secretion via the regulated pathway, including atrial stretch, contractions, and increased external Ca2+ concentration, failed to alter caveolar immunostaining. These results suggest that atrial peptide can pass from atrial granules into caveolae by transiently open pathways between the interiors of granules and caveolae. The results are interpreted as suggesting the presence of a second pathway for externalization of atrial natriuretic peptide through caveolae in addition to the classic pathway for regulated atrial peptide secretion at noncaveolar plasmalemma.
Control of cardiac Ca2+ levels. Inhibitory actions of sphingosine on Ca2+ transients and L-type Ca2+ channel conductance.
Authors: McDonough P M , Yasui K , Betto R , Salviati G , Glembotski C C , Palade P T , Sabbadini R A
Published: June 2012

The naturally occurring second messenger sphingosine (SPH) was examined for its ability to influence cardiac myocyte Ca2+ regulation. SPH inhibited intracellular Ca2+ transients in adult and neonatal rat ventricular myocytes. The inhibition was steeply dose dependent, with complete blockage of the Ca2+ transients occurring in the 20- to 25-mumol/L range. Whole-cell patch clamping revealed substantial inhibition of the L-type Ca2+ channel current (ICa) by SPH. The ability of SPH to block both the Ca2+ transients and ICa was not dependent on protein kinases, since the general protein kinase inhibitor H7 failed to prevent the actions of SPH. The specificity of the effect of SPH was determined in experiments showing that SPH analogues did not produce comparable effects. Neither the naturally occurring ceramide, N-stearoyl SPH, nor the cell-permeant ceramide, N-acetyl SPH, had SPH-like actions on the Ca2+ transients or L-type channel conductances. Caffeine-induced Ca2+ transients were also inhibited by the actions of SPH on cardiac sarcoplamic reticulum Ca2+ release, and the threshold for caffeine-induced Ca2+ release was raised. We conclude that SPH inhibits excitation-contraction coupling in cardiac myocytes by reducing the amount of entering "trigger Ca2+" for Ca(2+)-induced Ca2+ release and by simultaneously raising the threshold of the ryanodine receptor for Ca(2+)-induced Ca2+ release. Consequently, we propose that sphingolipids produced by the sphingomyelin signal transduction pathway could be physiologically relevant regulators of cardiac [Ca2+]i and therefore cardiac contractility.
Microvascular Responses to Inhibition of Nitric Oxide Production
Authors: Kurose Iwao , Wolf Robert , Grisham Matthew B. , Aw Tak Yee , Specian Robert D. , Granger D. Neil
Published: June 2012

Abstract
The objective of this study was to assess the potential contribution of hydrogen peroxide (H
2
O
2
) to the leukocyte–endothelial cell adhesion and increased microvascular permeability observed in rat mesenteric venules after inhibition of nitric oxide synthesis with
N
G
-nitro-
l
-arginine methyl ester (L-NAME). Leukocyte adherence and emigration and leakage of fluorescein isothiocyanate–labeled albumin were monitored in postcapillary venules before and after exposure of the tissue to L-NAME. H
2
O
2
production in mesenteric tissue was monitored by using dihydrorhodamine 123 (DHR), the H
2
O
2
-sensitive fluorochrome. L-NAME elicited a rapid increase in both the rate of albumin extravasation and oxidation of DHR, which was followed by an increased adherence and emigration of leukocytes in postcapillary venules. Treatment with either catalase or dimethylthiourea attenuated the L-NAME–induced oxidative stress, albumin leakage, and leukocyte–endothelial cell adhesion. Oxidation of DHR was enhanced in animals treated with either 3-amino-1,2,4-triazole (ATZ), an inhibitor of endogenous catalase, or a combination of ATZ and maleic acid diethyl ester, which depletes intracellular glutathione. Animals receiving a CD11/CD18-specific antibody to prevent leukocyte adhesion/emigration exhibited a reduced oxidation of DHR in response to L-NAME. These findings indicate that most of the H
2
O
2
(and secondarily derived oxidants) generated in mesenteric tissue exposed to an inhibitor of nitric oxide production is due to accumulation of activated leukocytes.
Characteristics of the Delayed Rectifier Current (I
Kr
and I
Ks
) in Canine Ventricular Epicardial, Midmyocardial, and Endocardial Myocytes
Authors: Liu Da-Wei , Antzelevitch Charles
Published: June 2012

Abstract
Recent studies have described regional differences in the electrophysiology and pharmacology of ventricular myocardium in canine, feline, rat, guinea pig, and human hearts. In this study, we use standard microelectrode and whole-cell patch-clamp techniques to examine the characteristics of the action potential and the delayed rectifier K
+
current (I
K
) in epicardial, M region (deep subepicardial to midmyocardial), and endocardial cells isolated from the canine left ventricle. Cells from the M region displayed much longer action potential durations (APDs) at slow rates. At a basic cycle length of 4 s, APD measured at 90% repolarization was 358±16 (mean±SEM), 262±12, and 287±11 ms in cells from the M region, epicardium, and endocardium, respectively. Steady state APD-rate relations were steeper in cells from the M region. In complete Tyrode’s solution, I
K
was smaller in myocytes from the M region when compared with those isolated from the epicardium or endocardium. Further characterization of I
K
was conducted in a Na
+
-, K
+
-, and Ca
2+
-free bath solution to isolate the slowly activating component of the delayed rectifier (I
Ks
) from the rapidly activating component (I
Kr
). I
Ks
was significantly smaller in M cells than in epicardial and endocardial cells. With repolarization to −20 mV, I
Ks
tail current density was 1.99±0.30 pA/pF (mean±SEM) in epicardial cells, 1.83±0.18 pA/pF in endocardial cells, and 0.92±0.14 pA/pF in M cells. Voltage dependence and time course of activation and deactivation of I
Ks
were similar in the three cell types. The relative contribution of I
Kr
and I
Ks
among the three cell types was examined by using 6 mmol/L [K
+
]
o
Tyrode’s solution with and without E-4031, a highly selective blocker of I
Kr
. An E-4031–sensitive current was observed in the presence but not in the absence of extracellular K
+
. This rapidly activating component showed characteristics similar to those of I
Kr
as described in rabbit and cat ventricular cells. Deactivation of I
Kr
was significantly slower than that of I
Ks
. I
Kr
(E-4031–sensitive component) tail current density was similar in the three cell types, whereas I
Ks
(E-4031–insensitive component) tail current density was significantly smaller in the M cells. Our results suggest that the distinctive phase-3 repolarization features of M cells are due in part to a lesser contribution of I
Ks
and that this distinction may also explain why M cells are the main targets for agents that prolong APD in ventricular myocardium. These findings may advance our understanding of the ionic basis for the electrocardiographic T wave, U wave, and long QT intervals as well as our understanding of factors contributing to the development of cardiac arrhythmias.
Recombinant Apolipoprotein A-I
Milano
Dimer Inhibits Carotid Intimal Thickening Induced by Perivascular Manipulation in Rabbits
Authors: Soma Maurizio R. , Donetti Elena , Parolini Cinzia , Sirtori Cesare R. , Fumagalli Remo , Franceschini Guido
Published: June 2012

Abstract
Apolipoprotein A-I
Milano
(apoA-I
M
), a natural variant of apolipoprotein A-I (apoA-I), confers to the carriers a significant protection against vascular disease. The antiatherogenic activity of a recombinant disulfide-linked apoA-I
M
dimer (rA-I
M
/A-I
M
) was analyzed in vivo by evaluating its effect on neointimal formation induced by periarterial manipulation in 1% cholesterol–fed rabbits. A flexible collar was applied around the carotid artery 21 days after the beginning of the dietary regimen, and animals were killed 10 days later. Rabbits were injected five times with reconstituted high-density lipoprotein containing egg phosphatidylcholine (EPC) and rA-I
M
/A-I
M
(119 mg EPC+40 mg protein per dose) or with EPC liposomes (119 mg EPC per dose) beginning either 5 days before or at the day of collar positioning. Neither treatment affected plasma cholesterol levels. A significant intimal thickening was observed in control animals; the intima-to-media (I/M) ratio was 0.63±0.11 versus 0.03±0.05 for the sham-operated contralateral arteries. Neointimal formation was markedly inhibited in animals pretreated with rA-I
M
/A-I
M
before lesion induction (I/M, 0.26±0.19) but not in those in which treatment began the day of collar insertion (I/M, 0.74±0.14). EPC liposomes did not affect neointimal formation (I/M, 0.50±0.14 and 0.51±0.07 in the two treatment groups). Proliferation of smooth muscle cells, assessed by direct incorporation of bromo-2′-deoxyuridine (BrdU) into replicating DNA, was reduced by ≈30% and 75% in the intimal and medial tissues of rA-I
M
/A-I
M
–pretreated rabbits. Thus, a short-term treatment with rA-I
M
/A-I
M
inhibits smooth muscle cell proliferation and intimal thickening in rabbits, providing evidence for the potential use of rA-I
M
/A-I
M
in the treatment of atherosclerosis.
Rapamycin-FKBP Inhibits Cell Cycle Regulators of Proliferation in Vascular Smooth Muscle Cells
Authors: Marx Steven O. , Jayaraman Thottala , Go Loewe O. , Marks Andrew R.
Published: June 2012

Abstract
Multiple growth factors can stimulate quiescent vascular smooth muscle cells to exit from G0 and reenter the cell cycle. The macrolide antibiotic rapamycin, bound to its cytosolic receptor FKBP, is an immunosuppressant and a potent inhibitor of cellular proliferation. In the present study, the antiproliferative effects of rapamycin on human and rat vascular smooth muscle cells were examined and compared with the effects of a related immunosuppressant, FK520. In vascular smooth muscle cells, rapamycin, at concentrations as low as 1 ng/mL, inhibited DNA synthesis and cell growth. FK520, an analogue of the immunosuppressant FK506, is structurally related to rapamycin and binds to FKBP but did not inhibit vascular smooth muscle cell growth. Molar excesses of FK520 blocked the antiproliferative effects of rapamycin, indicating that the effects of rapamycin required binding to FKBP. Rapamycin-FKBP inhibited retinoblastoma protein phosphorylation at the G1/S transition. This inhibition of retinoblastoma protein phosphorylation was associated with a decrease in p33

cdk2

kinase activity. These observations suggest that rapamycin, but not FK520, inhibits vascular smooth muscle cell proliferation by reducing cell-cycle kinase activity.
Arterial Wall Mechanics in Conscious Dogs
Authors: Armentano Ricardo Luis , Barra Juan Gabriel , Levenson Jaime , Simon Alain , Pichel Ricardo Horacio
Published: June 2012

Abstract
To evaluate arterial physiopathology, complete arterial wall mechanical characterization is necessary. This study presents a model for determining the elastic response of elastin (ς
E
, where ς is stress), collagen (ς
C
), and smooth muscle (ς
SM
) fibers and viscous (ς
η
) and inertial (ς
M
) aortic wall behaviors. Our work assumes that the total stress developed by the wall to resist stretching is governed by the elastic modulus of elastin fibers (E
E
), the elastic modulus of collagen (E
C
) affected by the fraction of collagen fibers (f
C
) recruited to support wall stress, and the elastic modulus of the maximally contracted vascular smooth muscle (E
SM
) affected by an activation function (f
A
). We constructed the constitutive equation of the aortic wall on the basis of three different hookean materials and two nonlinear functions, f
A
and f
C
:

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[{\varsigma}{=}{\varsigma}_{E}{+}{\varsigma}_{C}{+}{\varsigma}_{SM}{+}{\varsigma}_{{\eta}}{+}{\varsigma}_{M}{=}E_{E}\ {\cdot}\ ({\varepsilon}{-}{\varepsilon}_{0E}){+}E_{C}\ {\cdot}\ f_{C}\ {\cdot}\ {\varepsilon}{+}E_{SM}\ {\cdot}\ f_{A}\ {\cdot}\ {\varepsilon}\] \end{document}

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[{+}{\eta}\ {\cdot}\ \frac{d{\varepsilon}}{dt}{+}M\ {\cdot}\ \frac{d^{2}{\varepsilon}}{dt^{2}}\] \end{document}

where ε is strain and ε
0E
is strain at zero stress. Stress-strain relations in the control state and during activation of smooth muscle (phenylephrine, 5 μg · kg
−1
· min
−1
IV) were obtained by transient occlusions of the descending aorta and the inferior vena cava in 15 conscious dogs by using descending thoracic aortic pressure (microtransducer) and diameter (sonomicrometry) measurements. The f
C
was not linear with strain, and at the onset of significant collagen participation in the elastic response (break point of the stress-strain relation), 6.02±2.6% collagen fibers were recruited at 23% of stretching of the unstressed diameter. The f
A
exhibited a skewed unimodal curve with a maximum level of activation at 28.3±7.9% of stretching. The aortic wall dynamic behavior was modified by activation increasing viscous (η) and inertial (M) moduli from the control to active state (viscous, 3.8±1.3×10
4
to 7.8±1.1×10
4
dyne · s · cm
−2
,
P
<.0005; inertial, 61±42 to 91±23 dyne · s
2
· cm
−2
,
P
<.05). Finally, the purely elastic stress-strain relation was assessed by subtracting the viscous and inertial behaviors.
Dye Tracers Define Differential Endothelial and Smooth Muscle Coupling Patterns Within the Arteriolar Wall
Authors: Little T. L. , Xia J. , Duling B. R.
Published: June 2012

Abstract
Dye tracers were chosen, based on net charge, chemical structure, and reactive groups, to test for the existence of and to provide novel insight into channel selectivities of junctional pathways connecting smooth muscle and endothelial cells of the arteriolar wall. Dyes were injected into individual smooth muscle or endothelial cells of hamster cheek pouch arterioles using microiontophoresis. Coupling, independent of tracer net charge, was seen both within and between cell layers. Endothelial cells were well coupled by all of the tested dyes. Smooth muscle junctions appeared less effective in dye transfer than endothelial junctions. Lucifer yellow was confirmed to be a poor tracer of smooth muscle gap junctions, and remarkably this dye and other related sulfate-containing molecules interfered with dye movement through smooth muscle but not endothelial junctions. Myoendothelial junctions showed a striking polarity of dye movement, with dye transfer from endothelial to smooth muscle cells but little or no transfer in the reverse direction. Because the dyes have size and charge characteristics similar to those of known cellular second messengers, these findings have important implications for cell-cell signaling in the vessel wall.
Nitric Oxide Synthesis by Cultured Endothelial Cells Is Modulated by Flow Conditions
Authors: Noris Marina , Morigi Marina , Donadelli Roberta , Aiello Sistiana , Foppolo Marco , Todeschini Marta , Orisio Silvia , Remuzzi Giuseppe , Remuzzi Andrea
Published: June 2012

Abstract
In the present study, we examined the hypothesis that dynamic characteristics of flow modulate the production of vasoactive mediators, namely nitric oxide (NO) and endothelin-1 (ET-1), by human umbilical vein endothelial cells (HUVECs). Cells were exposed for 6 hours in a cone-and-plate apparatus to different types of flow: steady laminar, with shear stresses of 2, 8, and 12 dyne/cm
2
; pulsatile laminar, with shear stress from 8.2 to 16.6 dyne/cm
2
and a frequency of 2 Hz; periodic laminar, with square wave cycles of 15 minutes and shear stress from 2 to 8 dyne/cm
2
; and turbulent, with shear stress of 8 dyne/cm
2
on average. A second culture dish was kept in a normal incubator as a static control for each experiment. Laminar flow induced synthesis of NO by HUVECs that was dependent on shear-stress magnitude. Laminar shear stress at 8 dyne/cm
2
also upregulated the level of NO synthase mRNA. As observed with steady laminar flow, pulsatile flow also induced an increase in NO release by endothelial cells. When HUVECs were subjected to step-change increases of laminar shear, a further increase of NO synthesis was observed, compared with steady laminar shear of the same magnitude. Turbulent flow did not upregulate NO synthase mRNA or increase NO release. Both laminar and turbulent shear stress reduced, although not significantly, ET-1 mRNA and ET-1 production compared with the static condition. These results indicate that local blood flow conditions modulate the production of vasoactive substances by endothelial cells. This may affect vascular cell functions such as nonthrombogenicity, regulation of blood flow, and vascular tone.
Characterization of Bradykinin B
2
Receptors in Adult Myocardium and Neonatal Rat Cardiomyocytes
Authors: Minshall Richard D. , Nakamura Fumiaki , Becker Robert P. , Rabito Sara F.
Published: June 2012

Abstract
Specific [
125
I-Tyr
8
]bradykinin (BK) binding was observed on myocardial membranes from adult guinea pigs, dogs, rats, and rabbits that was displaced by unlabeled BK with an IC
50
between 0.1 and 30 nmol/L. In the adult guinea pig ventricular myocardium, which displays both high- and low-affinity binding, guanosine 5′-
O
-(3-thiotriphosphate) (GTPγS; 100 μmol/L) eliminated high-affinity binding and reduced total specific [2,3-prolyl-3,4-
3
H(
N
)]BK ([
3
H]BK) binding by >60%. Agonist competition binding to rat myocardial membranes was characterized as being of one affinity for BK in the nanomolar range, and it was not altered by GTPγS. Saturation binding studies with [
125
I-Tyr
8
]BK and [
3
H]BK, performed on cultured neonatal rat cardiac myocytes, revealed a single class of BK binding sites with a
K
d
and B
max
of 0.24±0.04 nmol/L and 18.4±1.1 fmol/mg protein, respectively (≈1500 receptors per cell). In competitive binding assays, unlabeled BK, Hoe 140 (a specific BK B
2
receptor antagonist), and des-Arg
9
,[Leu
8
]BK (a BK B
1
receptor antagonist) displaced [
125
I-Tyr
8
]BK with an IC
50
of 4.3, 0.041, and 307 nmol/L, respectively. In the presence of 100 μmol/L GTPγS, [
3
H]BK binding to myocyte membranes was reduced by 40%, but the IC
50
did not change. Cardiac fibroblasts, evaluated in parallel to the myocytes, contain a single class of [
3
H]BK binding sites (248±72 fmol/mg) with a 130-fold lower relative affinity (32.4±11.3 nmol/L) than that determined in rat cardiomyocytes. BK stimulated the inositol 1,4,5-trisphosphate (IP
3
) production by cardiomyocytes, which reached a maximum after 20 seconds of stimulation and increased from a baseline of 138.4±23.2 pmol/mg protein to 1020.7±75.9 pmol/mg with 1 μmol/L BK (EC
50
=15.3 nmol/L). The effect was significantly blocked by 1 μmol/L Hoe 140. The IP
3
response by cardiomyocytes was fourfold greater and sixfold more sensitive than that by cardiac fibroblasts (EC
50
=92.3 nmol/L). These data suggest the presence of high-affinity BK B
2
receptors on cardiomyocytes, which are functionally coupled via a G protein to the production of IP
3
.