bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential sensitivity of arteriolar alpha 1- and alpha 2-adrenoceptor constriction to metabolic inhibition during rat skeletal muscle contraction.
Authors: Anderson K M , Faber J E
Published: June 2012

Our previous studies in rat skeletal muscle have determined that neural constriction of large arterioles, which regulate blood flow and peripheral resistance, is mediated by alpha 1-adrenoceptors, whereas small arterioles, which determine effective capillary density, depend on alpha 2-receptors. During physical exercise, metabolic vasodilators from contracting skeletal muscle oppose neural vasoconstriction. By mechanisms that are not understood, adrenergic constriction of small arterioles is particularly sensitive to metabolic inhibition during imbalances in oxygen supply versus demand. This sensitivity may result from the reliance of small arterioles on alpha 2-receptors and a greater sensitivity of alpha 2 constriction to metabolic dilators. We previously demonstrated selective attenuation of arteriolar alpha 2 constriction during a reduction in the oxygen supply/demand ratio subsequent to decreased skeletal muscle perfusion. In the present study, intravital microscopy of rat cremaster skeletal muscle was used to examine the effect of increased oxygen demand on adrenergic constriction of arterioles. The effect of multiple frequencies of skeletal muscle contraction (via genitofemoral nerve stimulation) on alpha 1 (norepinephrine + rauwolscine) and alpha 2 (norepinephrine + prazosin) constriction was used to evaluate neural-metabolic interactions over a wide range of metabolic conditions. Low-frequency (less than or equal to 2 Hz) skeletal muscle contraction attenuated only alpha 2 constriction; contractions greater than or equal to 4 Hz attenuated alpha 1 constriction and further reduced alpha 2 constriction. Comparison of the frequency of contraction necessary to produce inhibition of 20% of maximal dilation indicated that alpha 2 constriction was approximately 10-fold more sensitive than alpha 1 constriction to "metabolic" inhibition. High-frequency, but not low-frequency, contraction also inhibited intrinsic tone. These data suggest that release of dilator substances during moderate exercise may preferentially attenuate alpha 2 constriction to produce small arteriolar dilation and increased capillary density. In contrast, metabolic signals associated with higher frequency muscle contraction may inhibit both intrinsic tone and large arteriolar alpha 1 tone so that blood flow and oxygen delivery increase to match the elevated oxygen demand of more heavily exercising muscle.
A cellular mechanism for impaired posthypoxic relaxation in isolated cardiac myocytes. Altered myofilament relaxation kinetics at reoxygenation.
Authors: Silverman H S , Ninomiya M , Blank P S , Hano O , Miyata H , Spurgeon H A , Lakatta E G , Stern M D
Published: June 2012

Single, isolated rat ventricular myocytes were made hypoxic for 10 minutes and then reoxygenated. During hypoxia, there was a marked abbreviation of the mechanical twitch, without a decrease in its amplitude. Immediately after reoxygenation, both the time to peak shortening and the duration of relaxation were markedly prolonged, and they remained prolonged for 10-50 minutes. The alterations in contraction and relaxation were not associated with any change in the time course of either the transmembrane action potential or the cytosolic calcium transient, as recorded with the fluorescent probe indo 1. Intracellular pH, measured with a fluorescent probe (carboxyseminaphthorhodofluor), showed an acid shift during hypoxia and an alkaline rebound immediately after reoxygenation. The time courses of intracellular pH and contraction duration were not parallel during hypoxia or reoxygenation, and simulation of the alkaline pH shift by lowering PCO2 or superfusing NH4Cl (in the absence of exposure to hypoxia) did not quantitatively reproduce the prolongation of relaxation seen after reoxygenation. The prolongation of contraction after reoxygenation could be overridden by the beta-adrenergic agonist isoproterenol or the nonenzymatic phosphatase butanedione monoxime. We conclude that delayed relaxation after reoxygenation exists at the single cell level and is due to an alteration of the properties of the myofilaments. Intracellular pH is not the primary mediator of this alteration. We speculate that alteration of intracellular inorganic phosphate or covalent modification of the myofilaments might be involved.
Endothelin activation of an inwardly rectifying K+ current in atrial cells.
Authors: Kim D
Published: June 2012

Various tissues including heart express specific binding sites for endothelin. Endothelins have been reported to increase the force of contraction of cardiac muscle, presumably via specific receptors. Specific binding of endothelin to atrial tissue is particularly high. In spontaneously contracting rat atrial cells used in this study, all three isoforms of endothelin (endothelin-1, endothelin-2, and endothelin-3) decreased the rate of beating and caused an increase in inwardly rectifying K+ current in voltage-clamped whole cells. Endothelin-3 was the most potent isoform, and its effects on beating rate and K+ current were present at a concentration as low as 100 pM (Kd, approximately 1 nM). the atrial cells did not have the hyperpolarization-activated current (the pacemaker current), If. In excised inside-out patches, all three isoforms of endothelin activated a population of K+ channels with kinetic properties identical to those of acetylcholine (muscarinic)-activated K+ channels, and this was GTP dependent. Endothelin failed to decrease the beating rate or to elicit the K+ current in pertussis toxin-treated cells. These results indicate that endothelin has a potent negatively chronotropic effect by activation of the inwardly rectifying, muscarinic K+ channel and therefore could be an important regulator of heart function.
Central command increases sympathetic nerve activity during spontaneous locomotion in cats.
Authors: Hajduczok G , Hade J S , Mark A L , Williams J L , Felder R B
Published: June 2012

A controversial issue in exercise physiology is the relative contribution of central command versus afferent input from contracting muscles and baroreceptors in the regulation of sympathetic nerve activity (SNA) during exercise. Recent studies of exercising humans have suggested that central command increases cutaneous sympathetic sudomotor nerve activity but have challenged the concept that central command contributes importantly to increases in sympathetic vasoconstrictor nerve activity to skin and skeletal muscle. The purpose of this study was to examine the influence of central command on renal SNA and lumbar SNA during spontaneous locomotion in decorticate cats. Unanesthetized decorticate cats that developed locomotion spontaneously or during electrical stimulation of the subthalamic locomotor region were studied in the presence and absence of input from skeletal muscle and baroreceptor afferents. Spontaneous rhythmic locomotion in the unparalyzed state was associated with significant increases in mean arterial pressure (MAP) from 106 +/- 10 to 133 +/- 11 mm Hg (p less than 0.05) and increases in renal SNA of 301 +/- 100% (p less than 0.05). During spontaneous fictive rhythmic locomotion in paralyzed cats, there were also significant (p less than 0.05) increases in MAP (43 +/- 6%), renal SNA (183 +/- 32%), and lumbar SNA (223 +/- 83%). Baroreceptor denervation did not attenuate increases in MAP, renal SNA, and lumbar SNA during locomotion. During electrical stimulation of the subthalamic locomotor region in paralyzed cats, MAP increased by 43 +/- 17% (p less than 0.05), and renal SNA increased by 175 +/- 47% (p less than 0.05). These findings indicate that central command is capable of increasing sympathetic neural drive in unanesthetized decorticate cats. This increase in sympathetic drive occurs even in the absence of feedback from contracting muscles or from arterial and cardiopulmonary baroreceptors.
Neutrophil-mediated vasoconstriction and endothelial dysfunction in low-flow perfusion-reperfused cat coronary artery.
Authors: Ma X L , Tsao P S , Viehman G E , Lefer A M
Published: June 2012

We investigated the interaction between activated cat polymorphonuclear neutrophils (PMNs) and coronary vascular endothelial cells in vitro. It was shown that 1) 90 minutes of low-flow perfusion without reperfusion had no deleterious effects on endothelium-dependent vasodilation, whereas 90 minutes of low-flow perfusion and 20 minutes of reperfusion with a blood cell-free solution induced a 20-25% endothelial dysfunction; 2) activated PMNs produced endothelium-dependent vasoconstriction in coronary artery rings isolated from cat hearts undergoing 90 minutes of low-flow perfusion and 20 minutes of reperfusion with a blood cell-free Krebs-Henseleit solution; 3) addition of the superoxide free radical scavenger, superoxide dismutase (150 micrograms/ml), or an antibody directed against CD18 of PMN adherence glycoprotein complex (MAbR15.7, 20 micrograms/ml) attenuated PMN-induced vasoconstriction significantly, but addition of a hydroxyl radical scavenger [N-(2-mercaptopropionyl)-glycine, 150 micrograms/ml], a cyclooxygenase inhibitor, or a lipoxygenase inhibitor had no protective effect; 4) exposure of rings to a superoxide radical-generating system (i.e., xanthine and xanthine oxidase) produced significant vasoconstriction that was similar to that observed with activated PMNs and was inhibited by superoxide dismutase; and 5) activated PMNs produced a marked coronary endothelial dysfunction characterized by a decreased response to the endothelium-dependent vasodilators acetylcholine and A23187. Addition of either superoxide dismutase or MAbR15.7 protected against endothelial dysfunction. These results indicate that activated PMNs produce significant vasoconstriction and endothelial dysfunction in coronary arteries isolated from low-flow perfusion-reperfused hearts. These effects appear to be mediated primarily by superoxide radicals generated by activated PMNs that either inactivate or inhibit the synthesis and release of endothelium-derived relaxing factor. We conclude that activated PMNs are able to induce endothelial dysfunction by releasing free radicals and possibly other substances.
Differential effects of thromboxane A2 synthase inhibition, singly or combined with thromboxane A2/prostaglandin endoperoxide receptor antagonism, on occlusive thrombosis elicited by endothelial cell injury or by deep vascular damage in canine coronary arteries.
Authors: Vandeplassche G , Hemans C , Van de Water A , Xhonneux R , Wouters L , Van Ammel K , De Clerck F
Published: June 2012

In open-chest dogs, cyclic flow reductions (CFRs, 5.1-6.6/hr in controls; n = 24) caused by platelet deposition/dislodgment at sites of endothelial cell injury in critically stenosed left anterior descending coronary arteries (59% flow reduction) were attenuated to the same extent either by single thromboxane A2 (TXA2) synthase inhibition (0.31 mg/kg i.v. ridogrel; CFR, 0.16 +/- 0.16/hr; n = 6; p less than 0.05) or by a comparatively modest degree of TXA2/prostaglandin endoperoxide receptor antagonism on top of TXA2 synthase inhibition (5 mg/kg i.v. ridogrel; CFR, 0.22 +/- 0.1/hr; n = 10; p less than 0.05). By contrast, occlusive thrombosis on deep vascular damage elicited by intraluminal stimulation (150-microA anodal constant current) in nonpreconstricted canine coronary arteries (time to occlusion, 237.1 +/- 13.9 minutes; n = 7; incidence of occlusion within 300 minutes, six of seven experiments) was not affected by platelet cyclooxygenase inhibition (5 mg/kg i.v. acetylsalicylic acid; n = 7), single TXA2 synthase inhibition (1.25 mg/kg i.v. ridogrel; n = 7), or single TXA2/prostaglandin endoperoxide receptor antagonism (10 mg/kg + 10 mg/kg/hr i.v. sulotroban for 300 minutes; n = 5). However, such an occlusive thrombus formation was significantly reduced by combined TXA2 synthase/prostaglandin endoperoxide receptor inhibition (5 mg/kg i.v. ridogrel; time to occlusion greater than 300 minutes, n = 7; incidence of occlusion within 300 minutes, one of seven experiments; p less than 0.05). This study reveals 1) a differential efficacy of TXA2 synthase inhibition, singly or combined with TXA2/prostaglandin endoperoxide receptor antagonism, depending on the extent of the vessel wall lesion triggering thrombosis and the size of the thrombus required to obstruct the vascular lumen and 2) a significant synergism in preventing occlusive thrombosis of extensively damaged coronary arteries between strong TXA2 synthase inhibition and comparatively modest TXA2/prostaglandin endoperoxide receptor antagonism with ridogrel.
cGMP inhibits the activity of single calcium channels in embryonic chick heart cells.
Authors: Tohse N , Sperelakis N
Published: June 2012

Effects of cGMP on the slow (L-type) Ca2+ channels of cultured chick embryonic cardiomyocytes were investigated by a cell-attached patch-clamp method. Superfusion of the single cells with 8-bromo-cGMP, a membrane-permeable derivative of cGMP, inhibited the single-channel activity. The cyclic nucleotide decreased, in a concentration-dependent manner, the ensemble averaged currents obtained from multichannel patches. 8-Bromo-cGMP (1 mM) completely abolished the currents (n = 8), whereas 0.1 mM only slightly decreased the currents (n = 4). The influence of cGMP on the characteristics of the single Ca2+ channels was examined using 0.3 mM 8-bromo-cGMP. Unit amplitude and slope conductance of the Ca2+ channel was not changed (25 pS in control versus 24 pS in the presence of cGMP). Analysis of single-channel kinetics showed that cGMP prolonged the slow time constant for the closed-time histogram (from 6.7 to 15.4 msec); the other time constants (for the open-time and closed-time histograms) were not affected. cGMP-induced inhibition of the Ca2+ channels may be mediated by cGMP-dependent protein kinase, because 8-bromo-cGMP is a potent activator of this protein kinase and does not stimulate cAMP hydrolysis. The present results suggest that cGMP opposes the effects of cAMP on the L-type Ca2+ channels in myocardial cells.
Directional characteristics of action potential propagation in cardiac muscle. A model study.
Authors: Leon L J , Roberge F A
Published: June 2012

Propagation of an elliptic excitation wave front was studied in a two-dimensional model of a thin sheet of cardiac muscle. The sheet model of 2.5 x 10 mm consisted of a set of 100 parallel cables coupled through a regular array of identical transverse resistors. The membrane dynamics was represented by a modified Beeler-Reuter model. We defined the charging factor (CF) to represent by a single number the proportion of input current used to charge the membrane locally below threshold and showed that CF is inversely correlated with the time constant of the foot of the action potential (tau foot) during propagation on a cable. A safety factor of propagation (SF) was also defined for the upstroke of the action potential, with SF directly correlated with the maximum rate of depolarization (Vmax) and, for cablelike propagation, with propagation velocity. Propagation along the principal longitudinal axis of the elliptic wave front is cablelike but, in comparison with a flat wave front, transverse current flow provides a drag effect that somewhat reduces the propagation velocity, Vmax, SF, and CF. With a longitudinal-to-transverse velocity ratio of 3:1 or more, the wave front propagating along the principal transverse axis is essentially flat and is characterized by multiple collisions between successive pairs of input junctions on a given cable; Vmax, SF, and CF are larger than for longitudinal propagation, but CF is no longer correlated with tau foot. There are transient increases in propagation velocity and Vmax with distance from the stimulation site along both principal axes until stablized values are achieved, and a similar transient decrease in tau foot. Away from the principal axes, the action potential characteristics change progressively along the elliptic wave front.(ABSTRACT TRUNCATED AT 250 WORDS)
Human nonmuscle myosin heavy chains are encoded by two genes located on different chromosomes.
Authors: Simons M , Wang M , McBride O W , Kawamoto S , Yamakawa K , Gdula D , Adelstein R S , Weir L
Published: June 2012

We report the cloning of cDNAs encoding two different human nonmuscle myosin heavy chains designated NMMHC-A and NMMHC-B. The mRNAs encoding NMMHC-A and NMMHC-B are both 7.5 kb in size but are shown to be the products of different genes, which are localized to chromosome 22q11.2 and chromosome 17q13, respectively. In aggreement with previously reported results using avian tissues, we show that the mRNAs encoding the two myosin heavy chain isoforms are differentially expressed in rat nonmuscle and muscle tissues as well as in a number of human cell lines. The cDNA sequence encoding the 5' portion of the NMMHC-A isoform completes the previously published 3' cDNA sequence encoding a human myosin heavy chain, thus providing the cDNA sequence encoding the entire NMMHC-A amino acid sequence. Comparison of this sequence to cDNA clones encoding the amino-terminal one third of the NMMHC-B sequence (amino acids 58-718) shows them to be 89% identical at the amino acid level and 74% identical at the nucleotide level.
Ca2+ dependence of alpha-adrenergic effects on the contractile properties and Ca2+ homeostasis of cardiac myocytes.
Authors: Capogrossi M C , Kachadorian W A , Gambassi G , Spurgeon H A , Lakatta E G
Published: June 2012

alpha-Adrenergic stimulation is known to enhance myocardial contractility. Adult rat left ventricular myocytes bathed in 1 mM [Ca2+] (Ca0) and electrically stimulated at 0.2 Hz responded to alpha-adrenergic stimulation with 50 microM phenylephrine and 1 microM propranolol with an increase in twitch amplitude to 177.1 +/- 25.6% of control (mean +/- SEM). In contrast, when cell Ca2+ loading was increased by bathing cells in 5 mM Ca0, alpha-adrenergic stimulation decreased twitch amplitude to 68.6 +/- 8.2% of control. Time-averaged cytosolic [Ca2+] of cells in 1.0 mM Ca0 is enhanced via an increase in the frequency of electrical stimulation. When myocytes were stimulated at 2 Hz in 1 mM Ca0, alpha-adrenergic stimulation did not increase twitch amplitude (103.8 +/- 12.4% of control). In myocytes loaded with the Ca2+ probe into-1, alpha-adrenergic effects during stimulation at 0.2 Hz (an increase in twitch amplitude in 1 mM Ca0 and a decrease in twitch amplitude in 5 mM Ca0) were associated with similar changes in the indo-1 transient. In 5 mM Ca0, spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR) occurred in the diastolic interval between twitches (2.9 +/- 1.4 spontaneous SR Ca2+ oscillations/min; n = 7); alpha-adrenergic stimulation abolished these oscillations in six of seven cells. Thus, an increase in the frequency of spontaneous diastolic SR Ca2+ release (i.e., Ca2+ overload) is not the mechanism for the negative inotropic effect of alpha-adrenergic stimulation in 5 mM Ca0. In experiments with unstimulated myocytes, we determined whether the effect of alpha-adrenergic stimulation on cell Ca2+ homeostasis and oscillatory SR Ca2+ release observed in 5 mM Ca0 occurs only during electrical stimulation, when voltage-dependent currents are operative, or also at rest. Unstimulated rat ventricular myocytes in 5 mM Cao exhibit oscillatory SR Ca2+ release; alpha-adrenergic stimulation decreased the frequency of these oscillations to 53.9 +/- 8.9% of control, and this effect was blocked by 1 microM prazosin. In unstimulated indo-1-loaded myocytes alpha-adrenergic stimulation decreased the resting indo-1 fluorescence ratio in 5 mM Ca0, whereas it had no effect in 1 mM Ca0. Additional experiments were aimed at defining a role for Ca(2+)-activated, phospholipid-dependent protein kinase C (PKC) for the negative inotropic effect of alpha-adrenergic stimulation in 5 mM Ca0. Short-term preexposure to 0.1 microM 4 beta-phrobol 12-myristate 13-acetate (PMA) has been shown to maximally activate PKC.(ABSTRACT TRUNCATED AT 400 WORDS)
Inhibition of coronary artery superoxide dismutase attenuates endothelium-dependent and -independent nitrovasodilator relaxation.
Authors: Omar H A , Cherry P D , Mortelliti M P , Burke-Wolin T , Wolin M S
Published: June 2012

Isolated bovine coronary arteries were treated with 10 mM diethyldithiocarbamate (DETCA) for 30 minutes to deplete the cytosolic ZnCu form of superoxide dismutase (SOD). This treatment completely inhibited the endothelium- and cGMP-dependent relaxation to acetylcholine (mediated via the endothelium-derived relaxing factor, which is thought to be nitric oxide) without significantly inhibiting endothelium-dependent relaxation to arachidonic acid (mediated by prostaglandins). DETCA treatment of endothelial cells cultured from the coronary arteries inhibited bradykinin-elicited release of endothelium-derived relaxing factor, which was detected by bioassay on an isolated rabbit aorta in the presence of extracellular SOD. DETCA also inhibited cGMP-associated relaxations to nitric oxide and to vasodilators thought to function via the generation of this mediator (nitroglycerin and nitroprusside), but cAMP-associated relaxations to isoproterenol and papaverine were not altered. The inhibitory effects of DETCA against the relaxation to nitroprusside and nitroglycerin were attenuated by severe hypoxia. DETCA treatment of isolated coronary arterial smooth muscle or cultured endothelial cells produced an increase of chemiluminescence elicited in the presence of lucigenin, a detector of superoxide anion generation. The addition of SOD markedly attenuated the effects of DETCA treatment on arterial relaxation and chemiluminescence. Therefore, control of cellular superoxide anion levels by endogenous SOD appears needed for the release of endothelium-derived relaxing factor and relaxation of vascular smooth muscle to nitrovasodilators mediated via cGMP in the bovine coronary artery, but SOD is not critical for other endothelium-dependent or cAMP-associated relaxant mechanisms.
Endothelial, not hemodynamic, differences are responsible for preferential leukocyte rolling in rat mesenteric venules.
Authors: Ley K , Gaehtgens P
Published: June 2012

At the onset of the inflammatory process, leukocytes roll along venular but not arteriolar walls before they firmly attach and emigrate. To test whether differences in hydrodynamic flow conditions are responsible for the preferential occurrence of leukocyte rolling in venules, we varied wall shear rate, gamma w, between 30 and 2,000 sec-1 by selective micro-occlusion of side branches in venules and arterioles (diameter, 20-37 microns) of the exposed mesentery of anesthetized rats. In venules, 39% (range, 6-77%) of all passing leukocytes were found interacting with the endothelium (rolling), whereas this fraction was only 0.6% in arterioles. The fraction of rolling leukocytes in venules decreased from 49 +/- 13% at gamma w less than 100 sec-1 (N = 12) to 24 +/- 13% at gamma w greater than 400 sec-1 (N = 12). Mean leukocyte rolling velocity in venules increased with gamma w, but the most frequent rolling velocity class was 20-40 microns/sec at all shear rates. In arterioles, even prolonged (up to 90 minutes) conditions of reduced flow (gamma w less than 150 sec-1) did not induce leukocyte rolling. Radial distribution of freely flowing leukocytes not different in arterioles and venules. The data indicate that hemodynamic factors are not responsible for the difference of leukocyte adhesion between arterioles and venules. The venular endothelium appears to be specialized to support leukocyte adhesion during inflammation. This finding correlates with reports on preferential expression of various endothelial-leukocyte adhesion molecules on venular endothelial cells.
Relation between ventricular and myocyte remodeling with the development and regression of supraventricular tachycardia-induced cardiomyopathy.
Authors: Spinale F G , Zellner J L , Tomita M , Crawford F A , Zile M R
Published: June 2012

Chronic supraventricular tachycardia (SVT) causes left ventricular (LV) dilatation and dysfunction. Termination of SVT appears to reduce LV size and improve function. However, changes in myocyte structure and morphology that accompany the development and regression of SVT-induced cardiomyopathy have not been studied. Accordingly, we measured LV function using echocardiography and catheterization in three groups of six pigs each: 3 weeks of atrial pacing (SVT; 240 beats/min), 4-week recovery from SVT (PST), and sham-operated controls. At each of these three end points, isolated myocyte dimensions and nuclear number were measured using fluorescence, and the volume percent of myocytes and myofibrils was computed from tissue sections using stereological techniques. SVT resulted in reduced LV fractional shortening (15 +/- 3% versus 31 +/- 2%, p less than 0.05), increased end-diastolic dimension (5.6 +/- 0.8 versus 3.8 +/- 0.2 cm, p less than 0.05), and no change in mass (2.6 +/- 0.1 versus 2.6 +/- 0.2 g/kg, p = NS) compared with controls. Myocyte length significantly increased with SVT (171 +/- 9 versus 109 +/- 11 microns, p less than 0.05), without significant changes in cell width (28 +/- 2 versus 26 +/- 2 microns). Nuclear number did not change with SVT; however, nuclear area/myocyte area significantly increased compared with controls (9.5 +/- 0.8 versus 8.7 +/- 0.8 x 10(-2), p less than 0.05). The volume percent of myocytes within the ventricular wall and the volume percent of myofibrils within myocytes decreased with SVT compared with controls (72 +/- 3% versus 80 +/- 3% and 45 +/- 5% versus 63 +/- 4%, respectively, p less than 0.05), with no change in total myocyte volume (54.2 +/- 2.7 versus 54.3 +/- 1.8 microns3 x 10(12)). In the PST group, LV fractional shortening returned to control values; however, there was persistent dilatation (end-diastolic dimension: 4.2 +/- 0.1 cm, p less than 0.05), and LV hypertrophy developed (3.3 +/- 0.3 g/kg, p less than 0.05). Increased myocyte length (158 +/- 5 microns, p less than 0.05) and width (33 +/- 2 microns, p less than 0.05) were observed in the PST group. The volume percent of myocytes and myofibrils returned to control values, with total myocyte volume significantly increased in the PST group compared with the control and SVT groups (74.5 +/- 2.6 microns3 x 10(12), p less than 0.05). In addition, the number of nuclei per myocyte in the PST group significantly increased from control values (5.1 +/- 0.1 versus 4.0 +/- 0.1, p less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)
Remodeling of coronary vessels during aging in purebred beagles.
Authors: Tomanek R J , Aydelotte M R , Torry R J
Published: June 2012

We compared six young (1 year) and six senescent (11 years) purebred beagles to determine the effects of aging on the coronary microvasculature. The hearts were perfusion-fixed in vitro, and myocardial specimens were subjected to microscopic image analysis. Absolute left ventricular mass increased by 55% with age, while cardiocyte cross-sectional area increased by 10% and 30% in the midmyocardium and endomyocardium, respectively. Although capillary numerical density was lower in the senescent dogs (16% in midmyocardium and 19% in endomyocardium), volume density was similar in the two groups because capillary diameter increased significantly with age in both regions. Capillary length density was reduced by 27% with age in the endomyocardium of the old beagles. Wall/lumen ratios of arterioles of three size classes (less than or equal to 15, 16-25, and 26-50 microns) were found to be nearly identical for the two age groups. Mean arteriolar diameter increased within the smallest size class in both ventricular regions. In contrast, a twofold or greater increase in wall thickness with age occurred in the left anterior descending coronary artery and its first branch, which was mainly due to expansion of the medial interstitium. The connective tissue fraction of the myocardium was significantly higher in the senescent than in the young in the epimyocardium and midmyocardium but not in the endomyocardium. These data provide evidence for three conclusions regarding aging of coronary vessels in beagles. First, a decline in capillary length density is limited to the endomyocardium.(ABSTRACT TRUNCATED AT 250 WORDS)
Myocyte mitotic division in the aging mammalian rat heart.
Authors: Anversa P , Fitzpatrick D , Argani S , Capasso J M
Published: June 2012

To determine whether myocyte mitotic division occurs in the adult mammalian heart and whether this cellular process is affected by aging, we measured the percentage of myocyte nuclei showing metaphase chromosomes in myocytes isolated from the left and right ventricles of rats at 8-12, 19-24, and 28-32 months after birth. Metaphase chromosomes were found at all ages in both ventricles. However, from 8-12 to 28-32 months, the fraction of nuclei exhibiting metaphase chromosomes increased 6.3-fold and 2.3-fold in the left and right ventricles, respectively. Thus, myocyte cellular hyperplasia is present in the adult and aging myocardium as a compensatory mechanism to regenerate tissue mass and recover function, which are lost with the progression of life and senescence.
Identification and functional significance of troponin I isoforms in neonatal rat heart myofibrils.
Authors: Martin A F , Ball K , Gao L Z , Kumar P , Solaro R J
Published: June 2012

We investigated the mechanism(s) responsible for differences in the effects of acidic pH on Ca2+ activation of the activity of adult and neonatal rat heart myofilaments. Studies on preparations of myofilaments reconstituted with adult troponin-tropomyosin (Tn-Tm) and either adult or neonatal thick filaments indicated that the difference in effect of acidic pH is related to differences in Tn-Tm and not other myofilament proteins. Immunoblotting analysis showed that development of the rat heart myofibrils is associated with isoform switching from slow skeletal TnI to cardiac TnI and from a slow mobility isoform of TnT (TnT1) to a faster Mr isoform (TnT2. Expression of slow skeletal TnI was associated with a relative insensitivity of myofilament Ca2+ activation to deactivation by acidic pH. Moreover, the effect of acidic pH on Ca2+ activation of ATPase activity of soleus myofibrils, which contain cardiac TnC and slow skeletal TnI, was essentially the same as the effect of acidic pH on rat cardiac myofibrils in the early neonatal period. Neonatal myofilaments also contained a relative abundance of a set of polypeptides copurifying with the thin filaments. We have identified these proteins as histones. The relative amount of histones among a variety of preparations from different species was not correlated with the pH sensitivity of myofibrillar Ca2+ activation. Shifts in TnT isoforms among these species were also not correlated with an altered response to acidic pH. Our data provide evidence in support of the hypothesis that the relative insensitivity of neonatal myofilament activity to acidic pH is due to the presence of slow skeletal TnI in the thin-filament regulatory complex.
Contractile deactivation and uncoupling of crossbridges. Effects of 2,3-butanedione monoxime on mammalian myocardium.
Authors: Gwathmey J K , Hajjar R J , Solaro R J
Published: June 2012

We investigated the effects of 1 and 3 mM 2,3-butanedione monoxime (BDM, diacetyl monoxime) on excitation and contraction of cardiac muscle in several types of preparations at various levels of organization. We selected a concentration of BDM that was not expected to affect sarcolemmal calcium flux and action potential duration in cardiac tissue. Two indicators were used to record intracellular calcium. Aequorin, a bioluminescent calcium indicator, was used in studies with ferret papillary muscle preparations, and fura-2, a fluorescent calcium indicator, was used in studies with guinea pig cardiac myocytes. In both cases, addition of BDM resulted in a reduction of peak intracellular calcium released from the sarcoplasmic reticulum and a reduction of peak twitch force. The duration of the action potential of isolated myocytes was slightly abbreviated in the presence of BDM. In studies on the calcium current in the myocytes, addition of BDM was associated with reduced calcium current at any potential. Peak calcium current was reduced by 7.9 +/- 1% in the presence of BDM. In tetanized ferret papillary muscles, BDM reduced maximal calcium-activated force by 30 +/- 5% and increased the calcium ion concentration required for half-maximal force by 0.1 +/- 0.01 microM. The Hill coefficient was reduced from 5.00 +/- 0.11 to 3.40 +/- 0.20. Maximal shortening velocity of ferret papillary muscles was increased in the presence of BDM from 1.55 +/- 0.24 to 2.04 +/- 0.33 mm/sec. Ca2+ binding to troponin C in skinned fiber preparations from guinea pig, bovine, and canine hearts was unaffected by addition of up to 10 mM BDM. Our results indicate that BDM affects both calcium availability and responsiveness of the myofilaments to Ca2+. Uncoupling of contractile activation from excitation may also result from altered crossbridge kinetics.
Evidence for isoproterenol-induced phosphorylation of phosphatase inhibitor-1 in the intact heart.
Authors: Neumann J , Gupta R C , Schmitz W , Scholz H , Nairn A C , Watanabe A M
Published: June 2012

The positive inotropic effect of the beta-adrenoceptor agonist isoproterenol is accompanied by inhibition of phosphatase type 1 activity in myocardium. Indirect assays suggest that this effect is due to activation of protein phosphatase inhibitor-1, which inhibits phosphatase activity only when phosphorylated. To test this hypothesis directly, electrically stimulated (3 Hz) guinea pig ventricular preparations were perfused according to the Langendorff method with physiological buffers with or without 5 mCi 32P/heart, and then various concentrations of isoproterenol were applied. Contractility was recorded. Hearts were freeze-clamped and cAMP and inhibitor-1 activities were measured. In 32P-labeled hearts a protein at about 26 kd on autoradiograms of 12% sodium dodecyl sulfate gels was detected. Isoproterenol (1 microM) increased rate of tension development to 238% of the predrug value, cAMP concentrations 1.5-fold, and inhibitor-1 activity threefold. Concomitantly, there was an increase in a 32P-labeled band at about 26 kd from 380 to 540 pmol 32P/mg protein. This protein at about 26 kd, after transfer to nitrocellulose, was recognized by an antiserum prepared against rabbit skeletal muscle inhibitor-1. More radioactive protein of about 26 kd could be immunoprecipitated by the antiserum from isoproterenol-treated than from untreated hearts. It is concluded that a protein, probably identical to phosphatase inhibitor-1, is phosphorylated in vivo in the heart in the presence of isoproterenol. Phosphorylation of inhibitor-1 with consequent modification of type 1 phosphatase activity may contribute to the effects of isoproterenol in the heart.
Control of the Na-Ca exchanger in isolated heart cells. I. Induction of Na-Na exchange in sodium-loaded cells by intracellular calcium.
Authors: Haworth R A , Goknur A B , Hunter D R
Published: June 2012

Isolated adult rat heart cells in suspension were loaded with sodium by incubation with ouabain in the absence of calcium for 30 minutes. Addition of low levels of calcium induced accelerated rates of sodium influx and efflux, as measured with 22Na. The magnitude of calcium-induced 22Na efflux was 50-fold greater than the net rate of calcium uptake and required extracellular sodium, but not extracellular calcium, once some calcium was taken up. Calcium did not induce 86Rb efflux. The accelerated rate of 22Na efflux was prevented by verapamil, but verapamil was ineffective when added after calcium. Addition of EGTA after calcium reversed the effect of calcium, but only after incubation. Dichlorobenzamil, unlike verapamil, both prevented and reversed the induction of sodium fluxes by calcium. We conclude 1) that intracellular calcium induces Na-Na exchange through the Na-Ca exchanger in sodium-loaded cells exposed to calcium; and 2) that Na-Na exchange can be activated by calcium that enters the cell through calcium channels. We propose that this Na-Na exchange reflects the intrinsic activity of the Na-Ca exchanger.
Effects of ischemia and hypercarbic acidosis on myocyte calcium transients, contraction, and pHi in perfused rabbit hearts.
Authors: Mohabir R , Lee H C , Kurz R W , Clusin W T
Published: June 2012

The time courses of changes in pHi and cytosolic calcium were compared in isolated perfused rabbit hearts with the use of the calcium-sensitive fluorescent indicator indo-1 and the pH indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Cell-permeant forms of these indicators were loaded into myocytes by arterial infusion or by direct infusion into the extravascular space. Indo-1 fluorescence was recorded from the epicardial surface of the left ventricle at an excitation wavelength of 360 nm and emission wavelengths of 400 and 550 nm. BCECF fluorescence was recorded at an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Calibration procedures were developed for each indicator that allowed [Ca2+]i and pHi to be quantified during ischemia. Global ischemia decreased contractility and caused a rapid increase in both the systolic and end-diastolic levels of the calcium transients. Ninety seconds of ischemia increased peak systolic [Ca2+]i from 609 +/- 29 to 1,341 +/- 159 nM, while end-diastolic [Ca2+]i increased from 315 +/- 25 to 553 +/- 52 nM. The observed increase in diastolic [Ca2+]i, was shown not to arise from indo-1-loaded endothelial cells. The initial increase in [Ca2+]i was followed by a gradual decline and then a secondary rise occurring between 5 and 15 minutes of ischemia. In contrast, ischemia caused a monotonic decrease in pHi from a baseline of 7.03 +/- 0.06 to 6.83 +/- 0.02 after 2 minutes, 6.32 +/- 0.1 after 10 minutes, and 6.11 +/- 0.04 after 15 minutes. Perfusion of hearts with acidified (hypercarbic) saline increased the systolic and diastolic levels of the calcium transients, but only when pHi fell below a threshold value, which was more acidic than values achieved during the first 2 minutes of ischemia (6.83 +/- 0.03). Lesser degrees of acidification caused a decrease in contractility but did not affect the calcium transients. Effects of pHi on the calcium transients were not due to altered calcium sensitivity of indo-1. These results suggest that cytosolic acidification may contribute to the increase in [Ca2+]i during the first 15 minutes of global ischemia, but the [Ca2+]i increase during the first 2 minutes is mediated by other factors.
Effect of Nucleosides on Acute Left Ventricular Failure in the Isolated Dog Heart
Authors: BUCKLEY N. M. , TSUBOI K. K. , ZEIG N. J.
Published: June 2012

The effect of nucleosides on acute left ventricular failure was studied in 15 isolated dog hearts. The unilateral failure was produced by exposure of the isolated left ventricle to elevated aortic pressure. Parameters of left ventricular function used to evaluate the control, failure and nucleoside periods included ventricular stroke work and output, contractility and distensibility. Adenosine and cytidine were found to be negative inotropic substances. Guanosine, inosine, thymidine and uridine were found to he positive inotropic substances, restoring the control level of ventricular function.
Endothelium-dependent relaxation and hyperpolarization in aorta from control and renal hypertensive rats.
Authors: Van de Voorde J , Vanheel B , Leusen I
Published: June 2012

Endothelium-dependent relaxations are depressed in hypertension. In this study we investigated the possible involvement of endothelium-dependent smooth muscle hyperpolarization in this phenomenon. In isolated aortic segments from control rats, acetylcholine (10(-8)-10(-5) M) elicits relaxations after precontraction with norepinephrine (10(-7) M), and acetylcholine or carbachol (10(-5) M) induce smooth muscle hyperpolarization (10.6 +/- 0.9 mV). Both effects disappear after removal of the endothelium and are depressed by tetraethylammonium (3 x 10(-3) M), a rather nonspecific blocker of K+ channels, but not by glibenclamide (10(-5) M), a potent blocker of the ATP-regulated K+ channels, which has a marked effect on the relaxation induced by BRL 38227. The relaxation effect of acetylcholine is impaired in norepinephrine-contracted preparations from hypertensive rats but is not further depressed by tetraethylammonium. In aorta from hypertensive rats, hyperpolarization induced by carbachol was significantly reduced to a mean of only 21.8% of the values obtained in preparations from normotensive rats. From the relaxation-hyperpolarization relation obtained with BRL 38227 (opening K+ channels), it is derived that the endothelium-dependent hyperpolarization (approximately 10 mV) contributes for at least 20-30% of the maximal relaxation effect of acetylcholine on rat aorta. It is concluded that the diminished endothelium-dependent hyperpolarization may contribute to the depression of the endothelium-dependent relaxation in hypertension.
Gentamicin effects on renal ischemia/reperfusion injury.
Authors: Zager R A
Published: June 2012

This study assessed gentamicin's effects on ischemia/reperfusion renal injury to better understand when and how it worsens postischemic acute renal failure. Rats were subjected to 25 minutes of renal pedicle occlusion with and without preischemic (15-minute) or postischemic (15-minute or 8-hour) gentamicin treatment (100 mg/kg, by itself a subtoxic dose). Gentamicin's impact on hypoxia/reoxygenation injury to isolated rat proximal tubular segments was also assessed. Preischemic and postischemic gentamicin worsened the severity of acute renal failure to the same degree, suggesting that pretreatment induces its effect in the reperfusion period. Gentamicin paradoxically lessened hypoxic damage to proximal tubular segments (assessed by lactate dehydrogenase release), again implying no adverse impact on oxygen deprivation-induced tubular injury. From 0-4 hours of reperfusion, gentamicin approximately halved ATP/ADP ratios (due to increased ADP), indicating a drug-induced defect in cellular energetics. This abnormality temporally correlated with evolving morphological damage. Although antioxidants (deferoxamine and sodium benzoate) have been reported to protect against pure aminoglycoside nephrotoxicity, they did not mitigate gentamicin's adverse impact on postischemic acute renal failure. Gentamicin did not influence ischemia/immediate reperfusion deacylation/reacylation (assessed by renal free fatty acid content) despite its known antiphospholipase activity. Although in the normal kidney gentamicin preferentially accumulated in cortex, in the postischemic kidney, both cortex and outer medullary stripe developed striking (approximately threefold to fivefold) and comparable gentamicin increments. In conclusion, gentamicin appears to exacerbate postischemic acute renal failure by adversely influencing the reperfusion, not the ischemic injury, process. This may occur because increased gentamicin accumulation negatively impacts on reperfusion cellular energetics.
Reduced production of cGMP underlies the loss of endothelium-dependent relaxations in the canine basilar artery after subarachnoid hemorrhage.
Authors: Kim P , Schini V B , Sundt T M , Vanhoutte P M
Published: June 2012

Endothelium-dependent relaxations are inhibited during chronic vasospasm after subarachnoid hemorrhage in the canine basilar artery, although the luminal release of endothelium-derived relaxing factor (EDRF) is maintained. The present study investigated the mechanisms underlying the impaired vascular reactivity and in particular whether the loss of responsiveness of the smooth muscle to EDRF is due to an impaired production of cGMP. Bradykinin and nitric oxide evoked concentration-dependent relaxations in isolated canine basilar arteries with and without endothelium, respectively, which were reduced in the subarachnoid hemorrhage group. Relaxations evoked by M&B22,948 (an inhibitor of cGMP phosphodiesterases) were smaller, but those evoked by the lipophilic cGMP analogue 8-bromo-cGMP were potentiated slightly in the subarachnoid hemorrhage group. The resting levels of cGMP in rings with endothelium (reflecting the effect of spontaneous release of EDRF) and those evoked by bradykinin in rings with endothelium and by nitric oxide in rings without endothelium were diminished in the subarachnoid hemorrhage group. These data indicate that the altered endothelium-mediated relaxations of the smooth muscle after subarachnoid hemorrhage is due, at least in part, to an impaired activation of soluble guanylate cyclase leading to a reduced production of cGMP in the smooth muscle.
Protein p67. A calcium-binding protein localized at the sarcolemma of secretory atrial myocytes.
Authors: Iida H , Nishitani H , Shibata Y
Published: June 2012

Bovine heart 67-kd protein (p67) was coisolated with calpactin I complex by cycles of Ca(2+)-dependent precipitation followed by solubilization with EGTA-containing buffer. Using affinity-purified anti-p67 antibody and anti-p36 (36-kd subunit of calpactin I) antibody, we examined the localization of the two proteins in secretory atrial myocytes and other endocrine tissues of adult rats. Immunofluorescence microscopy showed that p67 was expressed both in the atrial myocytes in situ and in cultured atrial myocytes in which we failed to detect p36 and that p67 appeared to be closely associated with the cell surface. We also found that p67 was colocalized with p36 in the thyroid follicle epithelium and zona reticularis of the adrenal gland. On the other hand, neither p67 nor p36 was detectable in pancreas islet cells. Immunoelectron microscopy revealed that p67 was localized at the sarcolemma in the atrial myocytes in situ. The p67, which was shown to be a globular molecule with a diameter of 18-25 nm by a low-angle rotary shadowing method, bound radioactive Ca2+ on a nitrocellulose membrane. The results suggest that Ca(2+)-binding proteins expressed in endocrine cells seem to vary from tissue to tissue and that p67 may function in Ca(2+)-mediated events at the plasma membrane of secretory atrial myocytes and some types of endocrine cells expressing this protein.