bluuebunny/crossref_metadata_2025 huggingface dataset:
Heat produced by rabbit papillary muscle during anoxia and reoxygenation.
Authors: Dietrich D L , Elzinga G
Published: June 2012

Resting heat rate was measured in superfused rabbit papillary muscles at 20 degrees C during 40 minutes of anoxia and subsequent reoxygenation. To reveal the nature of the reactions underlying energy output under such conditions, the data obtained were compared with values predicted from data on chemical change. Before and after the anoxic period, muscles were stimulated at 0.2 Hz, during which time the contraction-related heat rate was measured. During anoxia, muscles were kept at rest or stimulated at 1 Hz. Stimulation was switched off intermittently to determine resting heat rate. Before anoxia, resting heat rate was 8.7 +/- 1.1 (mean +/- SEM) mW.g dry wt-1. During anoxia, it decreased to 38% and 50% of the preanoxic level in resting and stimulated muscles, respectively (P < .05). In resting muscles, heat rate increased with reoxygenation in approximately 10 to 15 minutes to 1.3 times the preanoxic level, whereas this was 3.7 times in stimulated muscles. Resting heat rate returned within 65 (resting muscles) or 150 (stimulated muscles) minutes to the baseline. The ratio of force- and contraction-related heat rate, ie, the economy of contraction, was not different before and after anoxia. We estimated that the heat produced by muscles during anoxia was not different from the heat to be expected from the hydrolysis of creatine phosphate, the breakdown of nucleotides, and the formation of lactate. The overshoot in resting heat during reoxygenation of resting muscles could be accounted for by the resynthesis of the energy store. The much larger overshoot in resting heat of stimulated muscles was due to the contracture. The finding that the economy of contraction was not altered by anoxia and reoxygenation suggests that both sarcoplasmic reticulum Ca(2+)-ATPase and myofibrillar ATPase are depressed by anoxia and that the enhancement of cytosolic calcium transients with reoxygenation, reported in other studies on papillary muscle, results from reduced binding of calcium rather than from enhanced release.
Molecular basis of cardiac troponin T isoform heterogeneity in rabbit heart.
Authors: Greig A , Hirschberg Y , Anderson P A , Hainsworth C , Malouf N N , Oakeley A E , Kay B K
Published: June 2012

In the rabbit heart, multiple isoforms of cardiac troponin T (cTnT1 through cTnT5, from largest in size to smallest), a protein essential for calcium-regulated myofibrillar ATPase activity, have been identified, and a correlation has been found between these isoforms and myofilament sensitivity to calcium. We have sought to establish the molecular basis of this diversity. Restriction-digest analysis of genomic DNA has indicated that the rabbit cTnT gene is a single-copy gene. cTnT cDNA clones were isolated from cDNA libraries, yielding a consensus sequence for the protein. Newborn rabbit heart cDNAs, obtained using the reverse-transcriptase polymerase chain reaction (RT-PCR), were amplified using primers derived from this cDNA. Three full-length cDNAs that differed by the inclusion or exclusion of three short nucleotide sequences within the cDNAs were obtained. Amplification in the 5' half of the cDNAs confirmed that multiple cTnT products arose because of the variable inclusion of an 18- and a 30-nt sequence. The 30-nt sequence has homology with previously described alternatively spliced exons in rat and chicken cTnT, whereas the 18-nt sequence has not been described previously. RT-PCR in the 3' half of the cDNAs confirmed an additional region of heterogeneity: the presence, in part or in full, or absence of a 9-nt region, which matches the alternatively spliced exon 12 described for rat cTnT. In vitro transcription and translation of four cDNA clones containing both the 18- and 30-nt sequences, the 30-nt sequence, the 18-nt sequence, or neither generated protein isoforms that comigrated with cTnT1, cTnT2, cTnT3, and cTnT4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Presence of chloride-formate exchange in vascular smooth muscle and cardiac cells.
Authors: Soleimani M , Howard R L
Published: June 2012

The presence of chloride-formate anion exchange in vascular smooth muscle cells (VSMCs) and cardiac myocytes was investigated. Imposing an outward chloride gradient in sarcolemmal microsomes isolated from canine aorta stimulated [14C]formate uptake compared with the absence of a chloride gradient (24.3 +/- 2.33 versus 9.8 +/- 1.41 pmol/mg protein for 30 seconds, P < .03) and induced transient uphill [14C]formate uptake. The chloride-formate exchange was significantly inhibited in the presence of 1 mmol/L 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or furosemide (57% and 61%, respectively). Incubation of rat cultured VSMCs in a medium containing [14C]formate resulted in uptake of formate that was significantly DIDS and furosemide sensitive (79.34 +/- 2.47, 43.03 +/- 2.37, and 44.65 +/- 1.68 pmol/mg protein for 4 minutes in control, DIDS, and furosemide groups, respectively). Preincubation of the VSMCs in chloride-free medium significantly reduced the DIDS-sensitive (36.31 versus 16.85 pmol/mg protein for 4 minutes, P < .001) and furosemide-sensitive (34.72 versus 8.78 pmol/mg protein for 4 minutes, P < .001) [14C]formate uptake. These results are compatible with the presence of chloride-formate exchange in VSMCs. Influx of [14C]formate into sarcolemmal vesicles isolated from canine heart was significantly higher in the presence of an outward chloride gradient than in its absence (18.1 +/- 2.3 versus 9.6 +/- 1.7 pmol/mg protein for 30 seconds, P < .03). The chloride-formate exchange was significantly inhibited in the presence of 1 mmol/L DIDS or furosemide (41% and 52%, respectively). We conclude that the distribution of chloride-formate exchange may be more universal than previously suggested.(ABSTRACT TRUNCATED AT 250 WORDS)
Ventricular mechanoreflex and chemoreflex alterations in chronic heart failure.
Authors: Brändle M , Wang W , Zucker I H
Published: June 2012

Cardiac and arterial baroreflex control of the circulation is abnormal in both human and experimental heart failure. Ventricular vagal afferents mediate mechanical and chemical reflexes, which result in bradycardia and hypotension. The aim of the present study was to evaluate the changes that occur in ventricular mechanoreflexes and chemoreflexes in a conscious canine model of chronic heart failure. Dogs were instrumented for the measurement of left ventricular pressure, left atrial pressure, arterial pressure, and heart rate. Vascular occluders were placed on the ascending thoracic aorta, on the descending thoracic aorta, and on the thoracic inferior vena cava. A chronic left circumflex coronary artery catheter was also implanted. Finally, a pacing lead was secured to the left ventricular free wall. After recovery from surgery (10 to 14 days), the dogs were subjected to complete arterial baroreceptor denervation. The responses to vascular occlusions and intracoronary administration of prostacyclin (PGI2) were carried out before and after heart failure was induced by chronic cardiac pacing at 250 beats per minute. PGI2 was used as a chemical stimulus for ventricular afferents; ascending aortic occlusion was used as a mechanical stimulus. Before chronic pacing, ascending aortic occlusion resulted in a decrease in heart rate of 36.1 +/- 12.3 beats per minute (mean +/- SD, P < .001). After heart failure was induced, the heart rate response to ascending aortic occlusion was almost completely abolished. The slope of the linear relation between pulse interval and left ventricular end-diastolic pressure was reduced by 90.5% from a control value of 11.3 +/- 6.9 ms/mm Hg after heart failure had been induced.(ABSTRACT TRUNCATED AT 250 WORDS)
The mechanism of coronary collateral vasoconstriction in response to cyclooxygenase blockade.
Authors: Altman J D , Bache R J
Published: June 2012

The present study was performed to examine the mechanism by which cyclooxygenase blockade produces vasoconstriction in well-developed coronary collateral vessels. Eight dogs were studied 4 to 6 months after occlusion of the left anterior descending coronary artery (LAD) had been performed to stimulate collateral vessel growth. At the time of study, the LAD was cannulated at the site of occlusion for measurement of retrograde blood flow as an index of collateral blood flow. Levels of 6-ketoprostaglandin F1 alpha were 32 +/- 13% higher in blood diverted from the collateral-dependent LAD than in aortic blood (P < .05); the increase in this stable product of prostacyclin metabolism indicated production of prostacyclin across the coronary collateral system. Administration of arachidonic acid into the left main coronary artery to reach collateral vessels entering the LAD resulted in a 21 +/- 6% increase in retrograde flow (P < .01), demonstrating cyclooxygenase activity with production of vasodilator prostaglandins in the collateral system. Ibuprofen (10 mg/kg IV) caused a 55 +/- 7% decrease in retrograde flow (P < .03), suggesting that cyclooxygenase blockade inhibited tonic production of vasodilator prostaglandins in the collateral system. In contrast, neither thromboxane synthase inhibition with dazmegrel nor thromboxane receptor blockade with SQ 30741 caused a significant change in collateral flow, thus failing to support thromboxane-induced collateral constriction. After cyclooxygenase blockade, prostacyclin infused into the left main coronary artery was able to restore retrograde flow to the preibuprofen level.(ABSTRACT TRUNCATED AT 250 WORDS)
Myofilament Ca2+ sensitivity in intact versus skinned rat ventricular muscle.
Authors: Gao W D , Backx P H , Azan-Backx M , Marban E
Published: June 2012

The Ca2+ sensitivity of myofilaments was compared before and after skinning in the same rat trabeculae at a diastolic sarcomere length of 2.2 to 2.3 microns. Trabeculae from rat right ventricle were loaded with fura-2 salt by iontophoretic microinjection, and [Ca2+]i was determined from the epifluorescence at 510 nm when excited at 340 and 380 nm. Steady-state activation was achieved by stimulating the muscle at 10 Hz after 10 to 20 minutes of application of ryanodine (5 mumol/L). The muscles were then skinned with Triton X-100 (1%) for 15 to 25 minutes and subsequently activated with solutions containing varied [Ca2+]. The intact force-[Ca2+] relation was highly cooperative (Hill coefficient, 4.87 +/- 0.35; n = 10), with a low [Ca2+]i required for half-maximal activation (K1/2) (0.62 +/- 0.03 mumol/L). After skinning, the Hill coefficient fell to 2.72 and the K 1/2 shifted rightward to 2.2 mumol/L in the presence of 1.2 mmol/L free Mg2+. Because of uncertainty regarding the appropriate [Mg2+], we measured [Mg2+]i at 0.72 +/- 0.06 mmol/L (n = 11) with Mg-fura-2 salt. When activating solutions were modified to contain [Mg2+] = 0.5 mmol/L, the force-[Ca2+] relation was shifted to the left (K 1/2 = 0.93 +/- 0.1, n = 10) with a Hill coefficient of 3.75 +/- 0.37, but the changes were not sufficient to superimpose with the intact force-[Ca2+] relation (P < .05 versus intact). These results suggest that, despite the significant effect of Mg2+ on the force-[Ca2+] relation in skinned muscles, the Ca2+ responsiveness of the myofilaments is still altered by skinning.(ABSTRACT TRUNCATED AT 250 WORDS)
Endothelium-mediated and direct actions of acetylcholine on rabbit aortic baroreceptors.
Authors: Munch P A
Published: June 2012

Effects of exogenous acetylcholine on single-unit aortic baroreceptors were studied using a rabbit in vitro aortic arch/aortic nerve preparation. The arch was perfused with Krebs-Henseleit solution at constant pressure while simultaneously recording arch diameter and baroreceptor discharge frequency. Administration of acetylcholine over a wide range of concentrations (10(-9) to 10(-4) mol/L) evoked multiple dose-dependent changes in baroreceptor activity. "Low" concentrations (< 10(-6) mol/L) consistently dilated arch and increased baroreceptor discharge (n = 11). These responses were prevented by prerelaxing smooth muscle with sodium nitroprusside (10(-7) mol/L, n = 6) and were augmented by precontracting smooth muscle with norepinephrine (0.5 x 10(-7) mol/L, n = 11). Thus, the baroreceptor responses were dependent quantitatively on the level of preexisting vasoactive tone. The responses also were antagonized by atropine (10(-7) mol/L, n = 6), which inhibits release of endothelium-derived relaxing factor (EDRF), and by N-monomethyl-L-arginine (10(-5) mol/L, n = 6), a specific blocker of nitric oxide formation (primary relaxing factor responsible for acetylcholine vasorelaxation). Hexamethonium was ineffective (n = 6). These results indicate that "low-dose" responses to acetylcholine were mediated solely by release of EDRF. In contrast, "high" concentrations of acetylcholine (> 10(-6) mol/L) constricted arch and reduced baroreceptor discharge in most units (66%). These responses were blocked by atropine, but not by hexamethonium, and were attributed to direct contraction of the smooth muscle. In the remaining baroreceptors (33%), discharge increased irrespective of changes in diameter or preexisting vasoactive tone, but not in the presence of hexamethonium. Therefore, some baroreceptors were stimulated chemically by acetylcholine (or an intermediary substance), probably through activation of nicotinic receptors on the sensory endings. The effects of acetylcholine on the baroreceptor pressure-frequency curve also were examined by injecting slow pressure ramps before and during acetylcholine treatment. Low concentrations that relaxed smooth muscle shifted the curve to lower pressures; high concentrations that contracted smooth muscle shifted the curve to higher pressures. Last, when acetylcholine was given with the diameter rather than the pressure held constant, the baroreceptor reciprocal responses to smooth muscle relaxation and contraction were directionally reversed but remained consistent with the changes in wall tension.(ABSTRACT TRUNCATED AT 400 WORDS)
Mechanisms of the endothelial toxicity of cyclosporin A. Role of nitric oxide, cGMP, and Ca2+.
Authors: Gallego M J , García Villalón A L , López Farre A J , García J L , Garrón M P , Casado S , Hernando L , Caramelo C A
Published: June 2012

Cyclosporin A (CyA) is an efficient immunosuppressive agent, which, however, causes functional and structural alterations in endothelial cells. The aim of the present study was to examine the mechanisms of CyA-induced endothelial disfunction. CyA administration (Wistar rats, 25 mg/kg per day for 15 days) induced a significant inhibition of endothelium-dependent relaxation to acetylcholine on isolated femoral arteries. No changes with CyA were detected in the relaxation response to the endothelium-independent agent (sodium nitroprusside) or the endothelium-dependent receptor-independent agent (Ca2+ ionophore). The addition of L-arginine (10(-5) mol/L) shifted to the left the acetylcholine-mediated vasorelaxing response in CyA-treated segments, an effect that was accompanied by a marked increase of cGMP. 45Ca2+ uptake was higher in CyA-treated segments with respect to control segments but became normalized after incubation with L-arginine or sodium nitroprusside. De-endothelialization or incubation with the L-arginine competitive analogue N omega-nitro-L-arginine (NwNLA) increased 45Ca2+ uptake in control segments but not in CyA-treated segments. In conclusion, in isolated rat arteries, chronic CyA therapy affects endothelial function by uncoupling the acetylcholine-mediated relaxation and interfering with an endothelium-mediated pathway that regulates 45Ca2+ uptake by a mechanism reversed by an L-arginine-dependent cGMP generation.
Significance of inwardly directed transmembrane current in determination of local myocardial electrical activation during ventricular fibrillation.
Authors: Witkowski F X , Plonsey R , Penkoske P A , Kavanagh K M
Published: June 2012

Ventricular fibrillation (VF) is the principle cardiac rhythm disorder responsible for sudden cardiac death in humans. The accurate determination of local cardiac activation during VF is essential for its mechanistic elucidation. This has been hampered by the rapidly changing and markedly heterogeneous electrophysiological nature of VF. These difficulties are manifested when attempting to differentiate true propagating electrical activity from electrotonic signals and when identifying local activation from complex and possibly fractionated electrograms. The purpose of this investigation was to test the hypothesis that the presence of a balanced inwardly and outwardly directed transmembrane charge, obtained from the ratio of the inward to outward area under the cardiac transmembrane current curve (-/+ Im area), could reliably differentiate propagating from electrotonic deflections during VF. To test this hypothesis, we applied a recently described technique for the in vivo estimation of the transmembrane current (Im) during cardiac activation. A 17-element orthogonal epicardial electrode array was combined with an immediately adjacent optical fiber array to record electrical and optically coupled transmembrane potential signals during VF. Recordings were obtained during electrically induced VF in six dogs to determine the Im associated with activation and the time course of repolarization, as well as unipolar electrograms and bipolar electrograms recorded at multiple center-to-center interelectrode distances from 0.2 to 3 mm. Propagating local activations were associated with the presence of an easily identified inwardly directed Im, with a balanced inward and outward charge (-/+ Im area approximately 1.0). Electrotonic wave-forms lacked this inward Im (-/+ Im area approximately 0.0). Normal Na(+)-mediated inward currents were directly demonstrated to be responsible for some activations during VF.
Expression and insulin-like growth factor-dependent proteolysis of insulin-like growth factor-binding protein-4 are regulated by cell confluence in vascular smooth muscle cells.
Authors: Kamyar A , Pirola C J , Wang H M , Sharifi B , Mohan S , Forrester J S , Fagin J A
Published: June 2012

Insulin-like growth factor (IGF)-I is markedly induced after balloon injury in the rat aorta, where it may serve to mediate vascular repair. Because the bioavailability of IGF-I is modulated by IGF-binding proteins (IGFBPs), we examined the regulation of IGFBPs by IGFs in primary cultures of rat aortic smooth muscle cells (SMCs). Serum-deprived SMC-conditioned medium contains IGFBPs of 38 to 45 kD (only in confluent cultures), 30 kD (possibly IGFBP-2), 28 kD, and 24 kD (IGFBP-4), the latter being the most abundant. IGF-I and IGF-II but not insulin evoked a marked decrease of IGFBP-4 as early as 4 hours after treatment. IGFBP-4 mRNA abundance, however, was entirely unaffected by IGF-I for up to 48 hours. IGF-I analogues with high affinity for the IGF-I receptor and weak affinity for IGFBP paradoxically evoked a small increase in IGFBP-4, probably through a general increase in protein synthesis. IGF-I only minimally decreased IGFBP-4 content in medium of sparse cultures, whereas it completely abolished IGFBP-4 content in conditioned medium of superconfluent SMCs. IGF-I also evoked a concentration-dependent increase in the abundance of IGFBP-3 in confluent, but not sparse, SMCs without affecting IGFBP-3 mRNA. Addition of IGF-I to cell-free medium conditioned by confluent, but not by sparsely cultured, SMCs led to rapid degradation of IGFBP-4. Interestingly, IGFBP-4 mRNA was markedly induced in confluent relative to sparsely grown SMCs in an IGF-I independent fashion. Thus, both biosynthesis and IGF-dependent proteolysis of IGFBP-4 are increased in confluent SMCs. Proteolysis was maximal at 37 degrees C and was abrogated by EDTA and by benzamidine. Phenylmethylsulfonyl fluoride and the plasmin inhibitor bdellin had minor inhibitory activity, whereas aprotinin, angiotensin-converting enzyme inhibitors, and N-ethylmaleimide were without effect. The protease does not affect the structure of IGF-I as determined by reverse-phase high-performance liquid chromatography and size-exclusion chromatography of 125I-IGF-I incubated for up to 24 hours with SMC-conditioned medium containing IGFBP-4. In summary, SMCs elaborate a cation-dependent protease in a confluence-dependent fashion, which degrades bound IGFBP-4 and likely releases free structurally intact IGF-I, presumably to interact with the cell surface receptor and/or other IGFBPs.
Input impedance of the lower abdominal aorta in chronically instrumented fetal sheep.
Authors: Schröder H , Cetin E , Hüneke B , Carstensen M
Published: June 2012

Five fetal sheep (gestational age, 118 to 125 days) were instrumented to measure impedances of the lower abdominal aorta. Four days after surgery, flow and pressure pulses were recorded during control conditions and during infusion of norepinephrine (0.3 to 3 micrograms.min-1.kg-1 body weight) or angiotensin II (0.2 to 3 micrograms.min-1.kg-1). The protocol was repeated after injection of hexamethonium (10 mg.kg-1). Moduli and phases of impedances for the first ten harmonics were calculated by fast Fourier transformation. During control, input resistance was 4300 +/- 940 dyne.s.cm-5 (mean +/- SD) at a mean blood flow of 13.3 +/- 2.4 cm3.s-1 and pressure of 54,960 +/- 6980 dyne.cm-2. Moduli fell to 50% of input resistance between 2 and 3 Hz and, declining continuously, reached a minimum of 20% near 10 to 12 Hz, then increased slightly to 30% at about 30 Hz. At the first three harmonics, flow was always leading pressure. Infusion of angiotensin or norepinephrine increased impedance moduli significantly. Resistance increase was largest with angiotensin (19,800 +/- 11,370 dyne.s.cm-5), but no difference was detectable between angiotensin and norepinephrine when related to the same increase of input resistance. The position of the minimum seemed to be unchanged at high resistance values, but relative impedance moduli were smaller than during control, and low-frequency phases were significantly more negative. An analog of inertance, compliance, and resistance to steady flow was used to simulate impedances, and the effect of resistance increases on flow waveforms in the fetal abdominal aorta was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)
Blockade of cardiac inflammation in Mg2+ deficiency by substance P receptor inhibition.
Authors: Weglicki W B , Mak I T , Phillips T M
Published: June 2012

In previous work we reported the elevation of circulating inflammatory cytokines in rodents maintained on a Mg(2+)-deficient diet. Within the first week of Mg2+ deficiency, significant elevation of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) occurs. The present study was designed to assess the effects of SP receptor blockade by CP-96,945 and its inactive enantiomer CP-96,344 on tissue cytokine levels and in vivo oxidative indexes. CP-96,345 had no significant effect on circulating levels of SP or CGRP; however, at the tissue level, a significant decrease (P < .01) in myocardial accumulation of SP occurred; the inactive enantiomer was only slightly effective. In addition, CP-96,345 significantly reduced (by 53%) the accumulation of tumor necrosis factor-alpha (TNF-alpha) (but not interleukin-1 and interleukin-6) within the lesions; the effect of the enantiomer was insignificant. We conclude that treatment with CP-96,345 inhibits SP and TNF-alpha tissue levels in cardiac lesions, indicating a linkage between this neuropeptide and TNF-alpha. Both SP and TNF-alpha can trigger free radical production; plasma thiobarbituric acid-reactive materials were elevated 2.5-fold and red blood cell reduced glutathione was reduced 55% during Mg2+ deficiency. In the presence of CP-96,345, both indexes of in vivo oxidation were significantly attenuated; the enantiomer was ineffective. These latter observations point to a neuropeptide/TNF-alpha/free radical-triggered mechanism that may be the major pathway of systemic oxidative injury inducing the cardiomyopathic lesions seen during Mg2+ deficiency.
Anisotropy of water diffusion in the myocardium of the rat.
Authors: Garrido L , Wedeen V J , Kwong K K , Spencer U M , Kantor H L
Published: June 2012

Pulsed field gradient nuclear magnetic resonance methods combined with nuclear magnetic resonance imaging were used to determine the water diffusion anisotropy in perfused rat hearts at 37 degrees C. It was found that the observed diffusion coefficient D(app) (apparent diffusion coefficient) depends on the orientation of the applied gradient g. When g is parallel to the epicardial surface, the observed diffusivity is D(app) parallel = 1.8 +/- 0.4 x 10(-9) m2.s-1, whereas when g is perpendicular to it, diffusivity is D(app) perpendicular = 2.5 +/- 0.5 x 10(-9) m2.s-1. To better characterize this directional dependence, images of the second-order diffusion tensor D of the myocardium were obtained. These data demonstrate several essential features of cardiac myoarchitecture, including the helicity of fiber orientation with respect to the ventricular axis and the variation of fiber pitch angle with transmural depth. Diffusion anisotropy may be quantified in a coordinate-independent manner by the eigenvalues of the diffusion tensor. In the myocardial midwall, these eigenvalues were E1 = 3.29 +/- 0.57, E2 = 2.01 +/- 0.42, and E3 = 0.77 +/- 0.58 x 10(-9) m2.s-1 (mean +/- SD). These data suggest that myocardial water diffusion is essentially unrestricted parallel to the myofibers. They further show that failure to measure the complete diffusion tensor may lead to substantial underestimates of diffusion anisotropy in the myocardium.
ADP inhibits the sliding velocity of fluorescent actin filaments on cardiac and skeletal myosins.
Authors: Yamashita H , Sata M , Sugiura S , Momomura S , Serizawa T , Iizuka M
Published: June 2012

We studied the effect of MgADP on the mechanical interaction of actomyosin in cardiac and skeletal muscles using an in vitro motility assay. The sliding velocities of fluorescently labeled actin filaments on rat cardiac and skeletal myosins were measured at various MgATP and MgADP concentrations. The filament velocity depended on MgATP concentration according to classic Michaelis-Menten kinetics with apparent Michaelis constants (Km) of 43 and 137 mumol/L and maximum velocity of 5.6 and 8.6 microns/s for cardiac and skeletal myosins, respectively. The presence of 2 mmol/L MgADP decreased the filament velocity and shifted the substrate concentration dependence of the velocity toward higher MgATP concentrations, yielding the inhibition constants of 194 and 478 mumol/L for cardiac and skeletal myosins, respectively. The activation energies determined by the temperature dependence of the velocity were 61 and 83 kJ/mol for rat V1 and rabbit cardiac myosins, which were similar to those of the dissociation rate constant of actomyosin-ADP complex reported in a solution study. The inhibition of the velocity by MgADP can be explained by the crossbridge scheme in which MgADP competes with MgATP for the substrate site on myosin molecules. In cardiac myosin, addition of a concentration of MgADP as low as 25 mumol/L significantly inhibited the velocity in the presence of 2 mmol/L MgATP, suggesting that increased intracellular MgADP may reduce the rate of crossbridge detachment, resulting in a decreased ATP consumption and an increased economy of force production under ischemic conditions. The present results support the idea that MgADP may be a physiologically important modulator of contraction in cardiac muscle.
Preparation, characterization, and evaluation of a monoclonal antibody against the rabbit platelet glycoprotein IIb/IIIa in an experimental angioplasty model.
Authors: Azrin M A , Ling F S , Chen Q , Pawashe A , Migliaccio F , Homer R , Todd M , Ezekowitz M D
Published: June 2012

The deposition of platelets at the site of balloon angioplasty is thought to play a major role in the pathogenesis of restenosis. The antibody AZ-1, which binds to the rabbit platelet glycoprotein IIb/IIIa receptor and inhibits platelet function both in vitro and in vivo, was produced and tested in an experimental model of angioplasty. Atherosclerosis was induced by desiccation injury of the femoral artery, followed by a 28-day diet with 2% cholesterol and 6% peanut oil. Rabbits were randomized to receive an infusion of saline, a single infusion of 0.5 mg/kg of AZ-1, or an infusion of 0.6 mg/kg AZ-1 before angioplasty. The latter group received a second infusion of 0.6 mg/kg 72 hours later. Functional platelet inhibition was demonstrated by prolongation of the bleeding time in all treated animals. Angiography was performed at baseline, immediately after a standardized angioplasty, and again 28 days after angioplasty on a total of 42 vessels. There were no significant differences between the antibody-treated group and the control group in the mean angiographic minimum luminal diameter at any of the time points. There was also no difference in the initial improvement after angioplasty (acute gain), in the decrease in luminal diameter from immediately after angioplasty to 28 days after angioplasty (late loss), or in the overall improvement from before angioplasty to 28 days after angioplasty. Quantitative histological analysis confirmed the lack of a beneficial effect of AZ-1. There were no significant differences in the area of the intima, the media, or the combined intima and media between the antibody-treated groups and the control group. Thus, potent platelet inhibition for up to 6 days after balloon angioplasty using a monoclonal antibody that inhibits platelet aggregation did not reduce the response to vascular injury after balloon angioplasty in this rabbit model of experimental atherosclerosis.
Histamine induces K+, Ca2+, and Cl- currents in human vascular endothelial cells. Role of ionic currents in stimulation of nitric oxide biosynthesis.
Authors: Groschner K , Graier W F , Kukovetz W R
Published: June 2012

The nature of the membrane currents mediating agonist-induced Ca2+ entry and enhanced nitric oxide (NO) production in endothelial cells is still unclear. Using both perforated-patch and conventional whole-cell clamp technique, we have studied the membrane response associated with histamine stimulation of human vascular endothelial cells. In perforated-patch experiments, the initial histamine (10 mumol/L)-induced current reversed close to the K+ equilibrium potential and was blocked by tetrabutylammonium ions (TBA, 10 mmol/L). In addition, a TBA-insensitive current that developed slowly in the presence of histamine was recorded. This delayed histamine-induced current reversed close to neutral potential and was inhibited by SK&F 96365 (25 mumol/L), a putative blocker of receptor-operated Ca2+ channels. Similar histamine effects were observed in conventional whole-cell experiments using pipette solutions with low Ca(2+)-buffering capacity. Strong buffering of intracellular free Ca2+ suppressed the initial, but not the delayed, current response. The delayed component of histamine-induced current was substantially inhibited by the Cl- channel blocker N-phenylanthranilic acid (NPA, 100 mumol/L), and an eightfold change in the Cl- gradient shifted the reversal potential of this current by 30 mV. In Cl(-)-free solutions, histamine induced an SK&F 96365-sensitive NPA-resistant current, which, according to reversal potential measurements in 20 mmol/L extracellular Ca2+, corresponded to a cation conductance with 13- to 25-fold selectivity for Ca2+ over K+. Both SK&F 96365 and TBA strongly suppressed histamine-induced rises in intracellular free Ca2+ and cellular cGMP levels, whereas NPA did not. Our results provide the first demonstration that three distinct ionic conductances contribute to the histamine-induced membrane response of endothelial cells. It is suggested that histamine induces a Cl- conductance that is apparently not involved in Ca2+ homeostasis and regulation of NO biosynthesis, while, in parallel, joint activation of a rapidly induced K+ permeability and a slowly developing cation permeability mediate Ca2+ entry and stimulation of endothelial NO production.
Single amino acid substitutions within the ion permeation pathway alter single-channel conductance of the human L-type cardiac Ca2+ channel.
Authors: Yatani A , Bahinski A , Mikala G , Yamamoto S , Schwartz A
Published: June 2012

Voltage-dependent L-type Ca2+ channels select for Ca2+ and other divalent cations by high-affinity Ca2+ binding and ion-ion interactions in the permeation pathway. We have recently identified a series of highly conserved glutamate residues, located within the SS2 segments of each of the four repeats of the human heart Ca2+ channel alpha 1 subunit, as major determinants of ion selectivity of the channel. To further investigate the functional role of these glutamate residues in ion permeation, we have individually neutralized the glutamic acids in repeats II and IV by substitution with alanine or glutamine. Wild-type and mutant Ca2+ channels were expressed in Xenopus oocytes. Apparent affinity for external Ca2+ was assessed by measuring the block by Ca2+ of inward Li+ currents through the Ca2+ channels. Mutations reducing net negative charge at these positions resulted in a 10-fold reduction in the affinity of the channel for external Ca2+. Single-channel conductance was measured with either divalent or monovalent cations as the charge carriers. Substitution of glutamic acid at position 677 with either alanine or glutamine increased single-channel conductance, whereas the same substitutions at position 1387 resulted in a decrease in single-channel conductance with Ba2+ as the charge carrier. In contrast, the unitary current amplitude carried by Na+, in the absence of external divalent cations, was not altered by these mutations. The results suggest that these conserved glutamate residues participate in high-affinity Ca2+ binding within the pore. The different effects on Ba2+ permeation indicate an asymmetric arrangement of the glutamate residues between repeats II and IV.(ABSTRACT TRUNCATED AT 250 WORDS)
Altered pulmonary microvascular reactivity to norepinephrine in canine pacing-induced heart failure.
Authors: Townsley M I , Pitts V H , Ardell J L , Zhao Z , Johnson W H
Published: June 2012

Pulmonary hypertension in congestive heart failure causes medial hypertrophy in pulmonary vessels and thickening of the endothelial basement membrane. In this study, the functional consequences of such pulmonary vascular adaptations were evaluated. Heart failure was induced in dogs by rapid ventricular pacing (240 beats per minute) for 28 days, at which time left ventricular shortening fraction was decreased by 57% compared with that at baseline. Lung lobes from paced (n = 56) and control dogs (n = 68) were isolated and perfused with autologous blood. Total, arterial (Ra), and venous (Rv) vascular resistances were significantly increased and vascular capacitance decreased in lobes from paced animals compared with controls. Increments in Ra and Rv after intra-arterial boluses of norepinephrine were measured before and after sequential addition of the alpha 1- and alpha 2-receptor antagonists prazosin (16 mumol/L) and yohimbine (0.1 mumol/L) in the presence or absence of propranolol (5 mumol/L). Norepinephrine (1 to 40 micrograms) had little effect on Ra in the absence of propranolol, a pattern that persisted in control lobes after propranolol. However, when lobes from paced animals were pretreated with propranolol, norepinephrine increased Ra, Rv was increased after norepinephrine in control lobes, an effect that was enhanced in the paced group. In both groups, the increment in Rv was greater after propranolol. Irrespective of propranolol pretreatment, prazosin significantly attenuated, if not abolished, the response to norepinephrine. The enhancement in venous vascular reactivity in lobes from paced animals remained when venous pressure was elevated to 20 cm H2O. In control lobes under conditions of elevated tone or when endothelium-dependent relaxing factor was blocked, responses to norepinephrine did not mimic those observed in the paced group. Microvascular permeability, as measured by the capillary filtration coefficient, was not altered in the paced group. We conclude that the pulmonary adaptations to 4 weeks of rapid ventricular pacing include functional changes in pulmonary hemodynamics and vascular reactivity but not in microvascular permeability.
Extracellular matrix remodeling after balloon angioplasty injury in a rabbit model of restenosis.
Authors: Strauss B H , Chisholm R J , Keeley F W , Gotlieb A I , Logan R A , Armstrong P W
Published: June 2012

Remodeling of the vessel wall after balloon angioplasty injury is incompletely understood, and in particular, the role of extracellular matrix synthesis in restenosis has received little attention. The objective of the present study was to determine the sequence of changes in collagen, elastin, and proteoglycan synthesis and content after balloon injury and to relate these changes to growth of the intimal lesions and extent of cell proliferation. In a double-injury non-cholesterol-fed model, right iliac arterial lesions in 43 rabbits were treated with balloon angioplasty, and the rabbits were killed at five time points ranging from immediate to 12 weeks. Vessel wall collagen and elastin content and synthesis were measured after incubation with 14C-proline and separation with a cyanogen bromide extraction procedure. Sulfated glycosaminoglycan synthesis was measured after incubation with [35S]sulfate, papain digestion, and ethanol precipitation. Continuous in vivo infusion of bromodeoxyuridine (96 hours) was used to assess cell proliferation. The intimal area significantly increased from 0.27 +/- 0.08 to 0.73 +/- 0.11 mm2 between 0 and 12 weeks. Intimal and medial cell proliferation were modest and peaked at 1 week (labeling indexes of 4.8% and 3.0%, respectively) and then markedly declined by 2 weeks. Significant increases in collagen, elastin, and proteoglycan synthesis, up to 4 to 10 times above control nondamaged contralateral iliac arteries, were noted at 1, 2, and 4 weeks. These increases in synthesis were accompanied by significant increases in collagen and elastin content (by approximately 35%) that coincided with the temporal increase in cross-sectional area.(ABSTRACT TRUNCATED AT 250 WORDS)
Localization of muscarinic receptor mRNAs in rat heart and intrinsic cardiac ganglia by in situ hybridization.
Authors: Hoover D B , Baisden R H , Xi-Moy S X
Published: June 2012

Although the heart is considered a relatively pure source of m2 muscarinic receptors, the possible expression of other muscarinic receptor genes at discrete sites within the myocardium or by intrinsic cardiac ganglia had not been evaluated. Accordingly, the present study used in situ hybridization histochemistry with 35S-labeled oligonucleotide probes to address this issue. Initial experiments demonstrated that the localization of m2 mRNA was similar to that reported for muscarinic receptors labeled with the nonselective muscarinic antagonist quinuclidinyl benzilate; however, there were two important exceptions. The conducting system contained less message than expected, whereas the intrinsic cardiac ganglia contained more. The mismatch between muscarinic receptor and m2 mRNA densities in the conducting system could not be explained by the local expression of other muscarinic receptor genes, since m1, m3, and m4 mRNAs were not detected at this or any other site within the myocardium. However, the presence of a high density of prejunctional muscarinic receptors in the conducting system would be consistent with such a mismatch. Surprisingly, the intrinsic cardiac ganglia contained more than four times as much m2 mRNA as found in the atria. This level of message may be necessary for the production of prejunctional receptors on cholinergic nerve fibers within the heart and receptors localized to the ganglion cell bodies. The ganglia also contained smaller amounts of m1 and m4 mRNAs. These observations suggest that prejunctional muscarinic receptors could have a prominent role in regulating cholinergic neurotransmission in the conducting system and that multiple muscarinic receptors are present in the intrinsic cardiac ganglia.
Perfusion pressure and volume status determine the microvascular response of the rat kidney to NG-monomethyl-L-arginine.
Authors: Lockhart J C , Larson T S , Knox F G
Published: June 2012

This study investigated the role of volume status and perfusion pressure on the hemodynamic response of cortical and medullary renal capillaries to systemic inhibition of nitric oxide. NG-Monomethyl-L-arginine (L-NMMA) was infused intravenously (15-mg/kg bolus and 500-micrograms.min-1.kg-1 infusion), and blood flow in cortical capillaries (QCC) and in descending (QDVR) and ascending vasa recta (QAVR) was measured by fluorescence videomicroscopy in euvolemic and volume-expanded anesthetized Munich-Wistar rats. L-NMMA in euvolemic rats decreased vasa recta blood flow (delta QDVR, 3.97 +/- 0.80 nL/min [P < .01]; delta QAVR, 1.90 +/- 0.39 nL/min [P < .01]; n = 6) and QCC (delta QCC, 0.57 +/- 0.15 nL/min [P < .01]; n = 7) despite increases in renal perfusion pressure (RPP). Fractional excretion of sodium (FENa) remained unchanged. In volume-expanded rats, L-NMMA decreased vasa recta blood flow when RPP increased (delta QDVR, 1.42 +/- 0.79 nL/min [P = .05]; delta QAVR, 1.95 +/- 0.34 nL/min [P < .001]; n = 9) or was held constant by partial aortic occlusion (delta QDVR, 1.19 +/- 0.45 nL/min [P < .05]; delta QAVR, 1.44 +/- 0.40 nL/min [P < .01]; n = 8). QCC was unchanged by L-NMMA when RPP increased (delta QCC, 0.27 +/- 0.20 nL/min; n = 8) but decreased significantly by 0.61 +/- 0.11 nL/min (P < .01, n = 8) when increases in RPP were prevented. FENa increased when RPP increased (delta FENa, 2.47 +/- 0.51%; P < .001) and was held constant (delta FENa, 2.64 +/- 0.46%; P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)
Increased glomerular capillary pressure alters glomerular cytokine expression.
Authors: Shankland S J , Ly H , Thai K , Scholey J W
Published: June 2012

Increased glomerular capillary hydrostatic pressure (PGC) is an important hemodynamic determinant of glomerular injury, but the molecular events responsible for this association are poorly understood. PGC is normal in spontaneously hypertensive rats (SHR), but uninephrectomy leads to an increase in PGC and accelerated glomerulosclerosis. Since recent studies have implicated transforming growth factor-beta 1 (TGF-beta 1) and platelet-derived growth factor sought to determine if uninephrectomy increased mRNA levels for TGF-beta 1 and PDGF in glomeruli of SHR. Since treatment with the angiotensin-converting enzyme (ACE) inhibitor enalapril lowers PGC and prevents glomerulosclerosis in uninephrectomized SHR, we also sought to determine if ACE inhibitor lowered mRNA levels for TGF-beta 1 and PDGF in the glomeruli of uninephrectomized SHR. PGC increased from 53 +/- 1 to 64 +/- 1 mm Hg 1 week after uninephrectomy in SHR (P < .05). The increase in PGC was associated with a sixfold rise in mRNA levels for TGF-beta 1 and a twofold rise in mRNA levels for PDGF in glomeruli. mRNA levels for PDGF returned to normal 2 weeks after nephrectomy, but the increase in mRNA levels for TGF-beta 1 was sustained. An increase in TGF-beta 1 immunostaining was detectable in glomeruli 4 weeks after nephrectomy. Treatment with ACE inhibitor normalized PGC (51 +/- 1 mm Hg) and prevented the rise in glomerular mRNA levels for TGF-beta 1 and PDGF. We conclude that an acute increase in PGC leads to increased TGF-beta 1 and PDGF expression in the glomerulus, thus linking changes in PGC to cytokine gene expression.
Chloride dependence of pH modulation by beta-adrenergic agonist in rat cardiomyocytes.
Authors: Désilets M , Pucéat M , Vassort G
Published: June 2012

The effects of beta-adrenergic agonists on pHi were studied on single ventricular myocytes isolated from adult rat heart and loaded with the acetoxymethyl ester (AM) form of the pH indicator SNARF-1. In modified Krebs' solution containing 20 mmol/L HEPES and 4.4 mmol/L HCO3-, isoproterenol (1 mumol/L) caused a significant decrease of steady-state pHi from 7.20 +/- 0.02 to 7.13 +/- 0.02 (mean +/- SEM) within 2 minutes. This acidification, which was also observed in myocytes that were preloaded with the Ca2+ chelator BAPTA and superfused with nominally Ca(2+)-free solution, was blocked by propranolol as well as by the specific beta 1-antagonist CGP 20712 A but not by the beta 2-antagonist ICI 118,551. Forskolin (10 mumol/L) induced a similar reversible decrease of pHi (average decrease, 0.11 +/- 0.02 pH unit). Furthermore, adenosine (100 mumol/L) substantially attenuated the isoproterenol-induced decrease of pHi. The effect of isoproterenol was not prevented by inhibitors of the Na(+)-H+ antiport, amiloride (1 mmol/L) and 2-N,N-hexamethylene amiloride (20 mumol/L). On the other hand, blockers of Cl- transport mechanisms, DIDS (200 mumol/L) and probenecid (100 mumol/L), inhibited this acidification, Isoproterenol also failed to induce a decrease of steady-state pHi in myocytes incubated in Cl(-)-free medium. Rather, the initial rate of rise of pHi observed on removal of external Cl- ions was significantly increased in the presence of isoproterenol or dibutyryl cAMP. Because the alkalinization induced by removal of Cl- ions is mainly due to reversal of the Cl(-)-HCO3- exchanger, the augmentation of this initial rate of pHi rise directly points to a beta-adrenergic stimulation of the exchanger. Furthermore, the pHi recovery following NH4Cl exposure was accelerated by isoproterenol in the presence of probenecid, indicating that the Na(+)-HCO3- cotransport and/or the Na(+)-H+ antiport also could be activated.(ABSTRACT TRUNCATED AT 250 WORDS)