bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of cytokine inhibition of beta-adrenergic agonist stimulation of cyclic AMP in rat cardiac myocytes. Impairment of signal transduction.
Authors: Chung M K , Gulick T S , Rotondo R E , Schreiner G F , Lange L G
Published: June 2012

Studies conducted in our laboratory have demonstrated that activated immune cells produce a soluble inhibitor(s) of cardiac myocyte contractile and cyclic AMP (cAMP) responses to beta-adrenergic stimulation. To examine the mechanism of this effect, metabolic assays were conducted on cultured rat cardiac myocytes incubated in the presence and absence of supernatants harvested from rat activated splenocyte cultures. Intracellular cAMP accumulation in response to isoproterenol was inhibited by up to 74% in a dose-dependent fashion by conditioned media containing soluble cytokines from activated immune cells. By use of myocyte cultures in which contaminating nonmyocyte proliferation was inhibited by nonlethal irradiation, this phenomenon was shown to be independent of mitogenic effects. Isobutylmethylxanthine, a phosphodiesterase inhibitor, did not ablate cytokine-induced inhibition of cAMP accumulation. Parameters of beta-adrenergic receptor binding and affinity were also unaffected. cAMP suppression was maintained after cholera toxin stimulation of cAMP production via stimulatory G protein ADP-ribosylation. cAMP inhibition was not apparent when cells were stimulated with forskolin, a direct adenylate cyclase activator. Importantly, pertussis toxin treatment significantly ablated cytokine-induced cAMP inhibition. Thus, interference with agonist-occupied beta-adrenergic receptor coupling to adenylate cyclase to produce cAMP and subsequent contractile responses is induced by a factor(s) elaborated by activated immune cells. This interference occurs at the level of signal transduction across the membrane, can be overridden by pertussis toxin, and may involve changes in the coupling of the stimulatory/inhibitory G proteins to adenylate cyclase. These results demonstrate a novel mechanism of cytokine-induced myocyte dysfunction and may have important pathophysiological ramifications in immune-mediated myocardial diseases.
Effects of the spatial dispersion of acetylcholine release on the chronotropic responses to vagal stimulation in dogs.
Authors: Yang T E , Cheng J , Levy M N
Published: June 2012

We determined the effects of changing the spatial dispersion of acetylcholine release on the phase-dependent chronotropic responses to vagal stimulation in anesthetized dogs. We stimulated the vagus nerves with one brief burst of electrical pulses each cardiac cycle, and we changed the timing of the stimulus by a small, constant amount each cardiac cycle to scan the entire cycle. To vary the heterogeneity of acetylcholine release, we changed the voltage of the stimulus pulses over a range of submaximal values. To achieve the maximum homogeneity of acetylcholine release, we used supramaximal voltages, and we varied the level of acetylcholine release from each excited fiber by changing the number of pulses per burst. We used the average cardiac cycle length of the phase-response curve to assess the overall vagal effect, independent of its timing within the cardiac cycle. We found that the amplitude of the phase-response curve varied directly and the minimum-to-maximum phase difference varied inversely with the overall efficacy of vagal activity. However, for any given alteration in the overall efficacy, the specific changes in the characteristics of the phase-response curve did not depend on whether the alteration was achieved by varying the number of pulses per burst or by varying the stimulus voltage. Therefore, we conclude that although the cardiac chronotropic response is very sensitive to changes in the timing of vagal stimulation, it is not influenced appreciably by the spatial dispersion of acetylcholine release from the vagal nerve endings over a wide range of stimulation strengths.
Electrotonic influences on action potentials from isolated ventricular cells.
Authors: Tan R C , Joyner R W
Published: June 2012

This work combines a theoretical study of electrical interactions between two excitable heart cells, using a variable coupling resistance, with experimental studies on isolated rabbit ventricular cells coupled with a variable coupling resistance to a passive resistance and capacitance circuit. The theoretical results show that the response of an isolated cell to an increased frequency of stimulation is strongly altered by the presence of a coupling resistance to another cell. As the coupling resistance gradually is decreased, the stimulated cell becomes able to respond successfully to more rapid stimulation, and then, at levels of coupling resistance that allow conduction between the two cells, the coupled pair of cells exhibits arrhythmic interactions not predicted by the intrinsic properties of either cell. The experimental results show that the isolated rabbit ventricular cell is extremely sensitive to even a very small electrical load, with shortening of the action potential by 50% with electrical coupling to a model cell (of similar input resistance and capacitance to the ventricular cell) as high as 1,000 M omega, even though the action potential amplitude and current threshold are very insensitive to the electrical load.
Biphasic effects of doxorubicin on the calcium release channel from sarcoplasmic reticulum of cardiac muscle.
Authors: Ondrias K , Borgatta L , Kim D H , Ehrlich B E
Published: June 2012

To define the mechanism of doxorubicin cardiotoxicity, the effects of doxorubicin and caffeine were examined on calcium release channels from cardiac sarcoplasmic reticulum. We found that calcium release from cardiac sarcoplasmic reticulum vesicles was induced by both compounds. When sarcoplasmic reticulum vesicles were incorporated into planar lipid bilayers, calcium-permeable channels were observed. Addition of caffeine (2.5-10 mM) increased channel open probability from less than 0.1% to 40%, and this effect persisted for a mean of 44 minutes. In contrast, doxorubicin (2.5-10 microM) had a biphasic effect; initially, doxorubicin activated the channel, whereas after a mean of 8 minutes, the channel became irreversibly inhibited. Although the degree of channel activation by doxorubicin was concentration dependent, the time needed to inactivate the channel was concentration independent. Pretreatment with dithiothreitol (0.2 mM) prevented doxorubicin-induced channel inactivation, and channel activity persisted for an average of 58 minutes. Dithiothreitol alone did not alter channel open probability. Our results support the hypotheses that 1) the integrity of sulfhydryl groups is important for some aspects of calcium release channel function and 2) activation and inactivation of the channel are separable processes. The biphasic effect of doxorubicin on channel function may also correspond to the clinically observed adverse effects of doxorubicin, a widely used chemotherapeutic agent that, after prolonged usage, causes a dilated cardiomyopathy.
Temporal effects of thrombolytic agents on platelet function in vivo and their modulation by prostaglandins.
Authors: Rudd M A , George D , Amarante P , Vaughan D E , Loscalzo J
Published: June 2012

To examine the temporal effects of plasmin generated in vivo on platelet function, we infused tissue-type plasminogen activator (t-PA) in rabbits over 3 hours and measured ex vivo platelet aggregation. We noted an initial increase in the aggregation response to ADP occurring 30 minutes after the start of infusion. This enhanced response was short-lived and by 180 minutes was reduced, compared with pretreatment levels. Baseline aggregation response was restored by 240 minutes. This pattern of aggregation response to t-PA infusion was also seen with thrombin as the agonist. Coinfusion of either prostaglandin I2 or prostaglandin E1 abolished the initial hyperaggregable phase induced by t-PA; the hypoaggregable phase occurred earlier (after 60 minutes) and persisted throughout the 1-hour recovery period. Similarly, streptokinase infused for 1 hour also increased platelet aggregation at early times and then reduced aggregation responses after the first hour. Plasma plasmin activity increased as expected with t-PA infusion alone, peaking at 30 minutes and returning to baseline by the first hour. Interestingly, prostaglandin E1 blunted the rise in plasma plasmin activity. This same dose of prostaglandin E1 or prostaglandin I2 used alone did not appreciably alter platelet function at any time during the experiment. Our data show that therapeutic doses of t-PA or streptokinase produce a biphasic effect on platelet aggregation response in the rabbit. Coinfusion of either of the antiplatelet agents, prostaglandin E1 or prostaglandin I2, abolishes the hyperaggregable phase and prolongs the inhibitory effects on platelet aggregation produced by t-PA. These data suggest that the effects of thrombolytic agents on platelet function are complex and can be modulated by antiplatelet prostaglandins.
Differences in transient outward currents of feline endocardial and epicardial myocytes.
Authors: Furukawa T , Myerburg R J , Furukawa N , Bassett A L , Kimura S
Published: June 2012

Whole-cell voltage-clamp experiments were performed on enzymatically dissociated single ventricular myocytes harvested from feline endocardial and epicardial surfaces. The studies were designed to test the hypothesis that the differences in the amplitude of transient outward current (Ito) contribute to the difference in action potential configuration between endocardial and epicardial myocytes. In the control state, action potentials recorded from epicardial cells demonstrated a prominent notch between phases 1 and 2, and membrane current recordings displayed a prominent Ito, whereas in endocardial cells the notch in action potentials and Ito were small. External application of 4-aminopyridine (2 mM) reduced the amplitudes of notch and Ito in epicardial cells but not in endocardial cells. After application of 4-aminopyridine (2 mM) and caffeine (5 mM), the notch and Ito were abolished completely in both endocardial and epicardial cells. The first component of Ito (Ito1) was present in all epicardial cells studied (n = 20); it was absent in 12 of the 20 endocardial cells, and a small Ito1 was present in the remaining eight endocardial cells. The mean amplitude of Ito1 was significantly greater in epicardial than in endocardial cells. At a test voltage of +80 mV, the amplitude of Ito1 was 102.0 +/- 47.7 pA/pF in epicardial cells and 3.3 +/- 3.3 pA/pF in endocardial cells (p less than 0.01). The second component of Ito (Ito2) was present in all endocardial (n = 30) and epicardial (n = 30) cells studied. The amplitude of Ito2 was significantly greater in epicardial than in endocardial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Mono-L-arginine-containing compounds dilate piglet pial arterioles via an endothelium-derived relaxing factor-like substance.
Authors: Busija D W , Leffler C W , Wagerle L C
Published: June 2012

We determined the effects of mono-L-arginine-containing compounds on pial arterioles of anesthetized piglets. A closed cranial window was implanted, and the diameter of one pial arteriole was determined by intravital microscopy. Diameter was determined during application of artificial cerebrospinal fluid containing no drugs and during application of 10(-5), 10(-4), 10(-3), and 10(-2) M L-arginine (ARG), L-arginine ethyl ester (AEE), N alpha-benzoyl-L-arginine (NBA), N alpha-benzoyl-L-arginine ester ethyl (BAEE), and L-citrulline (CIT). Initial diameters were 100-200 microns. All of these compounds dilated arterioles, but the threshold concentration needed to elicit dilation varied: 10(-5) M for NBA (n = 5), 10(-3) M for AEE (n = 9) and BAEE (n = 6), and 10(-2) M for ARG (n = 6) and CIT (n = 4). Maximal responses were 15 +/- 8% for CIT, 17 +/- 4% for ARG, 19 +/- 8% for BAEE, 28 +/- 5% for NBA, and 27 +/- 6% for AEE. Indomethacin pretreatment (5 mg/kg i.v.) did not change arteriolar responses to AEE, NBA, and BAEE. However, coadministration of methylene blue (0.5 x 10(-4) M or 0.5 x 10(-3) M) abolished dilation to 10(-3) M AEE or BAEE and attenuated dilation to 10(-5) M NBA. In addition, coadministration of hemoglobin (0.4 x 10(-4) M) abolished dilation to AEE, BAEE, or NBA. Last, intravenous (5 mg/kg) and coadministration (10(-3) M) of NG-methyl-L-arginine blocked dilation to NBA or AEE. We conclude that mono-L-arginine-containing compounds produce pial arteriolar dilation in piglets, possibly involving an endothelium-derived relaxing factor.
Intrathoracic current flow during transthoracic defibrillation in dogs. Transcardiac current fraction.
Authors: Deale O C , Lerman B B
Published: June 2012

To achieve transcardiac threshold current during transthoracic defibrillation, a considerably larger current must be delivered to the thorax to compensate for the shunting effect of the lungs, the thoracic cage, and other elements of the torso. This shunting effect is thus an important determinant of transthoracic defibrillation threshold and can be quantified by the transcardiac current fraction (FC, the ratio of transcardiac to transthoracic threshold currents). Previous estimates of FC have ranged from as low as 3% to as high as 45%. The purpose of of this study was to quantify both FC and the major intrathoracic current pathways. Transthoracic and intrathoracic voltages and currents were simultaneously measured during high-voltage transthoracic shocks in 20 dogs. With correction factors determined from another set of 12 dogs, these raw data were corrected to compensate for field distortion caused by the presence of the intrathoracic electrodes, and the adjusted data were fit to a resistive network model. The results showed that 82% of the transthoracic current was shunted by the thoracic cage, while 14% was shunted by the lungs. The remaining 4% (FC) is the portion that passed through the heart. There was good agreement between the two independent methods used to calculate FC. Analysis based on the model indicated that FC was 3.7%, whereas FC determined by direct measurement with calibrated electrodes was 4.2%. Therefore, the results of this study, in contrast to earlier estimates of FC, show that defibrillation in dogs is achieved by only 4% of the total transthoracic current.
Role of the area postrema in the modulation of the baroreflex control of heart rate by angiotensin II.
Authors: Matsukawa S , Reid I A
Published: June 2012

During angiotensin II (Ang II)-induced elevation of arterial pressure, there is an attenuation of the baroreflex control of heart rate (HR), but the site of this action of Ang II on the baroreflex is not known. To investigate the role of the area postrema, the effects of Ang II on arterial pressure and HR and on the baroreflex control of HR were compared in intact and area postrema-lesioned conscious rabbits. In intact rabbits, infusion of Ang II (2.5-100 ng/kg/min) produced dose-related increases in mean arterial pressure (MAP); the largest dose increased MAP by 32 +/- 3 mm Hg. HR decreased only at the highest dose of Ang II (21 +/- 6 beats/min). In lesioned rabbits, the increase in MAP was reduced (23 +/- 2 mm Hg, p less than 0.05) while the decrease in HR was enhanced (50 +/- 8 beats/min, p less than 0.01). The pressor and HR responses to infusion of phenylephrine (PE) (2-20 micrograms/kg/min) were not different between the two groups. In intact rabbits, the slope of the relation between HR and MAP during Ang II infusion was less than that during PE infusion; in lesioned rabbits, the slopes were not significantly different. Responses to bolus injections of Ang II and PE in intact and lesioned rabbits were similar to those obtained in the infusion study. In another series of experiments, cardiac baroreflex responses with or without background infusion of Ang II were obtained by increasing blood pressure with graded infusions of PE (2-20 micrograms/kg/min). In intact rabbits, infusion of Ang II at 10 ng/kg/min shifted the baroreflex to a higher pressure level (resetting) without changing its slope (sensitivity). Background infusion of PE caused comparable increases in blood pressure, but the subsequent baroreflex response was identical to the response without background PE. In lesioned rabbits, background infusion of Ang II did not change the slope, nor did it reset the baroreflex. The effects of Ang II on baroreflex responses during nitroprusside infusions (2-20 micrograms/kg/min) in intact and lesioned rabbits were the same as those observed during the PE infusions. These findings indicate that the attenuation of the baroreflex control of HR by Ang II results from resetting of the cardiac baroreflex and suggest that this effect is mediated via the area postrema.
Ouabain increases the calcium concentration in intracellular stores involved in stimulus-response coupling in human platelets.
Authors: Roevens P , de Chaffoy de Courcelles D
Published: June 2012

The effect of ouabain on Ca2+ homeostasis in human platelets was studied using both quin 2 and chlorotetracycline to monitor changes in cytosolic Ca2+ as well as changes in the amount of Ca2+ accumulated in intracellular storage sites. In resting platelets, ouabain induces a concentration- and time-dependent increase in cytosolic Ca2+ concentration and a marked elevation of Ca2+ in the intracellular stores. The amount of Ca2+ mobilized from these stores upon stimulation with thrombin, as well as thrombin-induced secretion of platelet 5-hydroxytryptamine, was increased after preincubation with the glycoside (3 x 10(-6) M). These data show that ouabain induces an elevation of intracellular Ca2+ levels, most likely mediated via Na(+)-Ca2+ exchange, and that this incremental amount of Ca2+ is accumulated in an intracellular store involved in stimulus-response coupling. This may explain the enhanced functional responses of platelets to agonists in the presence of ouabain and suggests a role for Na(+)-Ca2+ exchange in Ca2+ homeostasis of the human platelet.
In vivo thrombin inhibition enhances and sustains arterial recanalization with recombinant tissue-type plasminogen activator.
Authors: Jang I K , Gold H K , Leinbach R C , Fallon J T , Collen D
Published: June 2012

The effects of heparin and the synthetic competitive thrombin inhibitor (2R,4R)-4-methyl-1-[N2-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfon yl)-L-arginyl]-2-piperidinecarboxylic acid monohydrate (Argatroban) on thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) was studied in groups of six or seven rabbits with arterial thrombosis. The model consisted of a whole-blood clot produced in a 1-cm isolated femoral arterial segment with superimposed endothelial damage and distal high-grade stenosis. rt-PA was injected as an intravenous bolus of 0.45 mg/kg body wt at 15-minute intervals until recanalization, or up to a maximum of four boluses. In seven rabbits given an intravenous injection of 17 mg/kg aspirin, rt-PA induced transient reflow in only one animal. In seven rabbits that received intravenous heparin (200 units/kg over 60 minutes), rt-PA administration produced reflow in five animals, which was persistent in two rabbits. Combined administration of aspirin and heparin in seven rabbits was associated with similar rt-PA-induced recanalization. rt-PA administration in six rabbits given intravenous Argatroban (100 micrograms/kg/min for 60 minutes) caused recanalization in five, with persistent patency in three. In six rabbits given aspirin and Argatroban, rt-PA caused recanalization in all, with persistent patency in five animals. Reflow occurred significantly more rapidly with Argatroban (14 +/- 7 minutes) than with heparin (35 +/- 11 minutes), reflow was obtained with fewer boluses of rt-PA in combination with Argatroban (median value of one bolus) than with heparin (median value, three boluses), and reocclusion after reflow was less frequent with Argatroban (0 of 11 versus 5 of 10 rabbits). Furthermore, the degree of thrombolysis determined by pathological analysis was significantly more extensive with Argatroban than with heparin, and patency persisted during a 3-hour observation period, despite elimination of Argatroban from the circulation. Thus, Argatroban, relative to heparin, enhances and sustains thrombolysis with rt-PA. It may offer promise as an adjunctive agent for thrombolytic therapy of arterial thrombosis.
Neuropeptide control of rat gastric mucosal blood flow. Increase by calcitonin gene-related peptide and vasoactive intestinal polypeptide, but not substance P and neurokinin A.
Authors: Holzer P , Guth P H
Published: June 2012

Submucosal blood vessels of the mammalian stomach are densely innervated by neurons containing calcitonin gene-related peptide (CGRP), substance P, neurokinin A, and vasoactive intestinal polypeptide (VIP). Because all these peptides are vasodilators in certain vascular beds, we tested the hypothesis that rat alpha-CGRP, rat VIP, substance P, and neurokinin A are candidate mediators of noncholinergic vasodilator neurons in the gastric mucosa and submucosa. The experiments were performed on urethane-anesthetized Sprague-Dawley rats. Gastric mucosal blood flow (GMBF) was measured by the hydrogen gas clearance technique, and the peptides were infused close arterially to the stomach via a catheter inserted retrogradely in the splenic artery. Basal GMBF was in the range of 35-50 ml/min/100 g. Infusion of rat alpha-CGRP (15 and 75 pmol/min) significantly increased GMBF in a dose-dependent manner, whereas mean arterial blood pressure was significantly lowered only by the higher dose of CGRP. Substance P (125 and 625 pmol/min) and neurokinin A (50 and 250 pmol/min) failed to alter GMBF, although the higher dose of each peptide led to a significant decrease in mean arterial blood pressure. Infusion of rat VIP (25 pmol/min) failed to affect GMBF and mean arterial blood pressure, whereas a fivefold higher dose of VIP (125 pmol/min) led to a significant rise of GMBF and to significant hypotension. These findings indicate that substance P and neurokinin A are unlikely to be of physiological significance for the regulation of GMBF. CGRP and VIP, however, can be considered as candidate mediators of submucosal nerve endings involved in the neural control of GMBF.(ABSTRACT TRUNCATED AT 250 WORDS)
Role of basic fibroblast growth factor in vascular lesion formation.
Authors: Lindner V , Lappi D A , Baird A , Majack R A , Reidy M A
Published: June 2012

In the present study we investigated whether basic fibroblast growth factor (bFGF) plays a role in the proliferative response of smooth muscle cells (SMCs) to denuding injury. Rat carotid smooth muscle was found to express the mRNA for bFGF, and bFGF protein was found to be present in rat aorta by immunoblot analysis. Systemically administered bFGF was a potent mitogen for vascular SMCs in arteries denuded with a balloon catheter, increasing replication from 11.5% in controls to 54.8%. Denudation with a device (filament loop), which causes only minimal damage to medial SMCs, showed a similar increase in replication (1.3% versus 43.3%) after bFGF infusion. In unmanipulated vessels, however, SMCs were unresponsive to infused bFGF. Infusion of a "mitotoxin" (bFGF conjugated to saporin) caused a greater than 50% decrease in the number of viable SMCs in the arterial wall after balloon injury. Prolonged administration of bFGF (12 micrograms/day for 2 weeks) after balloon injury caused an approximately twofold increase in intimal thickening. These results show that bFGF, which is synthesized by the arterial wall, could be a potent mitogen for SMCs in vivo and suggest that any release of endogenous bFGF may be capable of stimulating SMC proliferation, which may subsequently lead to intimal lesion formation.
Differential activation of alpha 1- and alpha 2-adrenoceptors on microvascular smooth muscle during sympathetic nerve stimulation.
Authors: Ohyanagi M , Faber J E , Nishigaki K
Published: June 2012

The relative contribution of postjunctional alpha 1- and alpha 2-adrenoceptors to constriction of microvessels was examined during sympathetic nerve stimulation and sympathetic escape (difference between peak and steady-state constriction). Large arterioles (120 +/- 4 microns control diameter) and venules (174 +/- 6 microns) and small arterioles (13 +/- 4 microns) were examined in rat cremaster skeletal muscle during stimulation of the cremaster efferent innervation (decentralized lumbar sympathetic chain, 0.5-16 Hz, 2-minute train). The muscle was suspended in a tissue bath, and diameter was measured with intravital microscopy. Frequency-response curves were obtained after vehicle (prazosin or rauwolscine) was added to the bath. In large arterioles, prazosin (10(-7) M) significantly attenuated constriction by 60-80%; a fivefold higher concentration had no additional effect. In contrast, rauwolscine (1 to 5 x 10(-7) M) had no effect. Venules evidenced minimal response to nerve stimulation. In small arterioles, rauwolscine (5 x 10(-7) M) significantly attenuated constriction by 50-60%, while prazosin (10(-7) M) had no effect. These data suggest that for large arterioles, which are known to possess both receptors, alpha 1-adrenoceptors are preferentially stimulated by nerve-released norepinephrine. In contrast, sympathetic constriction of small arterioles is mediated by alpha 2-adrenoceptors. Compared with large arterioles, small arterioles exhibited greater peak and steady-state constriction at all frequencies, with maximal responses achieved over the 0.5-4 Hz range. Large arterioles exhibit graded constriction over the entire frequency range. Sympathetic escape exhibited a small, negatively correlated frequency dependence for large arterioles, tended to be greater for small arterioles, and was more evident in large arterioles during alpha 2-adrenoceptor constriction at low-frequency stimulation. This distinct neural control of large resistance vessels by alpha 1-adrenoceptors and small terminal arterioles by alpha 2-adrenoceptors may allow neurogenic regulation of these vessel segments to be differentially susceptible to modulation by other extrinsic and intrinsic vasoactive controls that preferentially interact with alpha 1- and alpha 2-adrenergic contractile mechanisms.
Mechanisms of angiotensin II- and arginine vasopressin-induced increases in protein synthesis and content in cultured rat aortic smooth muscle cells. Evidence for selective increases in smooth muscle isoactin expression.
Authors: Turla M B , Thompson M M , Corjay M H , Owens G K
Published: June 2012

Previous studies from this laboratory have demonstrated that angiotensin II (Ang II) and arginine vasopressin (AVP) are potent hypertrophic agents in cultured rat aortic smooth muscle cells. The present study identified major proteins that accumulate in Ang II-induced and AVP-induced hypertrophic cells and initiated studies of the mechanisms that contribute to their accumulation. Smooth muscle cell hypertrophy induced by Ang II and/or AVP (1 microM each) was associated with widespread increases in the content of many cellular proteins that were resolved by one- and two-dimensional gel electrophoresis. However, increases were also selective in nature, with increases in certain individual proteins, including actin (twofold to threefold), vimentin (2.5-fold to sevenfold), tropomyosin (threefold to sixfold), and myosin heavy chain, far exceeding overall increases in cellular protein content (20-40%). Increases in actin content were due largely to increased expression of smooth muscle alpha-actin (3.6- to 7.5-fold), as opposed to nonmuscle beta-actin (1.7- to 2.5-fold). Increases in smooth muscle alpha-actin were accompanied by a fivefold to eightfold increases in smooth muscle alpha-actin mRNA, indicating that these changes were not due exclusively to translational controls. Results demonstrate that contractile agonist-induced hypertrophy in cultured smooth muscle cells is due, in part, to increased expression of smooth muscle contractile proteins. Furthermore, the fact that Ang II and AVP induced selective increases in smooth muscle alpha-actin suggests that these agonists may not only regulate growth of vascular smooth muscle but may also promote expression of smooth muscle-specific contractile proteins during differentiation of vascular smooth muscle.
Different activation of the endothelial L-arginine and cyclooxygenase pathway in the human internal mammary artery and saphenous vein.
Authors: Yang Z H , von Segesser L , Bauer E , Stulz P , Turina M , Lüscher T F
Published: June 2012

The endothelium releases substances controlling vascular tone and platelet function. We investigated mediators of endothelium-dependent responses in human internal mammary arteries and saphenous veins. The inhibitor of nitric oxide formation, NG-monomethyl L-arginine, enhanced the sensitivity to norepinephrine (fivefold) and evoked more pronounced endothelium-dependent contractions in internal mammary arteries (19 +/- 6% of 100 mM KCl) than in saphenous veins (2 +/- 1%; p less than 0.005). In internal mammary arteries, NG-monomethyl L-arginine, but not indomethacin, markedly reduced endothelium-dependent relaxations to acetylcholine (from 95 +/- 2% to 39 +/- 7%; p less than 0.005) and prevented those to histamine (78 +/- 6% to 4 +/- 3%; p less than 0.005). In saphenous veins, endothelium-dependent relaxations to acetylcholine were weak (24 +/- 11%), while nitric oxide caused comparable relaxations (85 +/- 3%) as in internal mammary arteries (80 +/- 5%; NS). NG-Monomethyl L-arginine prevented the relaxations to acetylcholine and unmasked endothelium-dependent contractions (30 +/- 10%). Indomethacin and the thromboxane synthetase inhibitor CGS-13080 augmented relaxations of saphenous veins to acetylcholine from 24 +/- 11% to 46 +/- 9% (p less than 0.05). Histamine-evoked contractions were converted to endothelium-dependent relaxations by indomethacin and the thromboxane A2/endoperoxide receptor antagonist SQ-30741 (38 +/- 3% and 40 +/- 6%; p less than 0.05) but not CGS-13080. Thus, 1) nitric oxide mediates endothelium-dependent relaxations in human arteries and veins; 2) internal mammary arteries release more nitric oxide than do saphenous veins, and 3) in saphenous veins, the effects of nitric oxide are reduced by endothelium-derived contracting factors originating from the cyclooxygenase pathway.
Transmural myocardial deformation in the ischemic canine left ventricle.
Authors: Villarreal F J , Lew W Y , Waldman L K , Covell J W
Published: June 2012

The myocardium is a complex three-dimensional structure consisting of myocytes interconnected by a dense collagen weave that courses in different directions. Regional ischemia can be expected to produce complex changes in ventricular deformation. In the present study, we examined the effects of ischemia on two- and three-dimensional finite strains during acute transmural myocardial ischemia in 13 open-chest anesthetized dogs. In contrast to systolic deformation observed during the control period in which circumferential shortening exceeded longitudinal shortening, our results indicate that after 5 minutes of acute ischemia, end-systolic in-plane lengthening across the left ventricular wall occurs in approximately equal amounts in the circumferential and longitudinal directions. Along with these changes in extensional strains, there were significant negative transverse shearing deformations during ischemia. Myocardial ischemia also resulted in a loss of the normal end-systolic transmural gradients of shortening and thickening. Three-dimensional end-diastolic strains indicate that the left ventricular wall undergoes a significant passive reconfiguration that varies transmurally with lengthening in the epicardial tangent plane and wall thinning increasing from the epicardium toward the endocardium. The large systolic changes in shearing deformations with ischemia could potentially influence collateral blood flow and certainly indicate that uniaxial measurements of deformation in the ischemic myocardium, which do not account for shearing deformation, are incomplete and must be interpreted with caution. Moreover, normal transmural systolic gradients in deformation, which would be anticipated on geometric grounds, are lost during ischemia, implying that the material properties of ischemic tissue or the loading conditions imposed on the ischemic region by partially impaired adjacent myocardium vary transmurally.
Effects of antibodies to myosin light chain kinase on contractility and myosin phosphorylation in chemically permeabilized smooth muscle.
Authors: De Lanerolle P , Strauss J D , Felsen R , Doerman G E , Paul R J
Published: June 2012

We have used an immunological approach to investigate the role of myosin light chain phosphorylation (MLC-Pi) in the control of contractility in smooth muscle. Our aim was to specifically inhibit myosin light chain kinase (MLCK) in the presence of physiologically activating levels of Ca2+ so that other putative Ca2(+)-dependent regulatory systems could be unmasked. Fab fragments were prepared by papain digestion of immunoglobulin G (IgG) molecules obtained from goats immunized with turkey gizzard MLCK. Anti-MLCK Fab was then purified by chromatography on an MLCK-Sepharose 4B column. These affinity-purified Fab fragments inhibit the activity of MLCK purified from turkey gizzard smooth muscle and interact monospecifically with MLCK in various mammalian smooth muscles as demonstrated by a Western blot analysis. The effect of these Fab fragments on the contractile properties was tested in guinea pig taenia coli made permeable (skinned) using Triton X-100. Skinned fibers, approximately 100 microns in diameter and 4 mm long, were mounted for isometric measurements and immersed in calcium-EGTA buffers. Fibers preincubated with anti-MLCK Fab in relaxing solution (Ca2+ less than 1 nM) for 75 minutes developed about 25% of the isometric force of a parallel control contraction when transferred to contracting solution (Ca2+ = 0.5 microM). When added to contracting solution at the peak of a contracture, anti-MLCK Fab elicited a relaxation that was complete in about 120 minutes despite the presence of Ca2+. No significant effect on isometric force was observed when fibers were incubated with another affinity-purified mouse Fab raised against the Fc region of human IgG (control Fab).(ABSTRACT TRUNCATED AT 250 WORDS)
Evidence that neuropeptide Y and norepinephrine mediate electrical field-stimulated vasoconstriction of rabbit middle cerebral artery.
Authors: Van Riper D A , Bevan J A
Published: June 2012

We investigated the contractile response of isolated rabbit middle cerebral artery (MCA) segments to electrical field stimulation (EFS). The dynamics of the EFS contraction were compared with a similar-sized branch of rabbit ear artery. In comparison with the ear artery, the EFS contractions of the MCA displayed a longer latency and a higher stimulus frequency threshold. Greater stimulation train lengths were required to attain equilibrium, and the time course of EFS response--including force development, plateau, and return to rest tension--was significantly slower than in the ear artery. Morphological and pharmacological studies of the MCA showed that it receives sympathetic adrenergic innervation: whole-mount preparations displayed catecholamine histofluorescence; electron micrographs of MCA sections revealed a population of varicosities containing chromaffin positive large and small vesicles; and EFS contractions were blocked by tetrodotoxin (30 nM) and guanethidine (5 microM) and by chronic surgical sympathectomy. Exposure to prazosin (10 microM) or phenoxybenzamine (1 microM) blocked norepinephrine contractions but did not significantly influence the EFS contraction. Procedures and drugs that antagonized the responses to neuropeptide Y, serotonin, or histamine were also ineffective in blocking the EFS contraction. The involvement of ATP could not be assessed, since the purinergic P2 agonist alpha,beta-methylene ATP was ineffective in blocking ATP-mediated contractions. The EFS contraction, however, could be blocked by a combination of neuropeptide Y desensitization and phenoxybenzamine (30 nM) or prazosin (0.1 microM). These results suggest that norepinephrine and neuropeptide Y are released from sympathetic nerves and mediate EFS contraction by occupation of postjunctional alpha-adrenoceptor and neuropeptide Y receptors. Since the blockade of only one of these components does not diminish the response to EFS, the adrenergic neuroeffector system in this artery may involve complex prejunctional regulatory mechanisms.
Effect of aging and hypertension on myosin biochemistry and gene expression in the rat heart.
Authors: Buttrick P , Malhotra A , Factor S , Greenen D , Leinwand L , Scheuer J
Published: June 2012

The mechanisms by which the aged heart adapts to a superimposed pressure load such as hypertension have not been described. We therefore investigated biochemical and molecular genetic adaptations in the 24-month-old rat heart subjected to renovascular hypertension. Compared with 4-month-old rats, aging was associated with a 68% increase in left ventricular mass without any change in heart weight-to-body weight ratio, a 33% reduction in calcium-activated myosin ATPase activity, and a shift from a V1 to a V3 predominant myosin heavy chain (MHC) isoform distribution. A 46% reduction in alpha-MHC mRNA and a reciprocal increase in beta-MHC mRNA was seen. When hypertension was superimposed, there was a further 75% increase in ventricular mass, a 63% increase in heart weight-to-body weight ratio, and a 19% reduction in myosin ATPase. Myosin isozyme distribution was further shifted to V3, and the ratio of alpha-MHC to beta-MHC mRNA was reduced. In addition, with hypertension a significant (greater than 50%) reduction in the mRNA level of the cardiac sarcoplasmic reticular calcium-activated ATPase was seen. These data demonstrate that the aged myocardium is able to respond to a superimposed pressure load with a molecular genetic and protein synthetic pattern of hypertrophy analogous to that seen in younger animals.
Contribution of cAMP-phosphodiesterase inhibition and sensitization of the contractile proteins for calcium to the inotropic effect of pimobendan in the failing human myocardium.
Authors: Böhm M , Morano I , Pieske B , Rüegg J C , Wankerl M , Zimmermann R , Erdmann E
Published: June 2012

Previous studies have shown reduced effects of cAMP-dependent positive inotropic agents in the failing human myocardium; thus other cAMP-independent mechanisms of action may be useful to increase force of contraction in this condition. The purpose of this investigation was to determine whether a positive inotropic effect of the cAMP-phosphodiesterase (PDE) inhibitor pimobendan is observed in the failing human myocardium and to study whether other factors, such as an increase in the Ca2+ sensitivity of myofilaments, play a functional role in the increase in force of contraction. Pimobendan produced a positive inotropic effect in isolated preparations from nonfailing donor hearts; however, in moderately (New York Heart Association class II-III, NYHA II-III) and severely (NYHA IV) failing myocardium, this effect was reduced. In addition, in NYHA IV specimens pimobendan inhibited the crude cAMP-PDE (crude PDE) and the isoenzymes I-III (PDE I-III) in a concentration-dependent way. As judged from the IC50 values found in this tissue for the inhibition of PDE III and of crude PDE, the potency of the compound was 18.1 times greater on PDE III. Consistent with a cAMP-PDE-dependent mechanism of action, the positive inotropic effect was potentiated by isoproterenol and inhibited by adenosine in failing myocardium. In failing myocardium, pimobendan also increased the sensitivity of skinned cardiac fibers to Ca2+ and shifted the Ca(2+)-tension relation to the left. This sensitizing effect began at 0.01 mumol/l in NYHA II-III and NYHA IV and rose to about 200% at 300 mumol/l in both groups. In contrast, the demethylated metabolite UD-CG 212 Cl failed to produce positive inotropic effects in failing myocardium alone, but in the presence of isoproterenol, it exerted an increase in force of contraction. The potency of UD-CG 212 Cl for PDE III inhibition in NYHA IV was greater than that of pimobendan. The metabolite pronouncedly decreased the sensitivity of skinned cardiac fibers to Ca2+ at 30-300 mumol/l in NYHA II-III and NYHA IV. It is concluded that in the failing human heart pimobendan inhibited PDE III and sensitized contractile proteins for Ca2+. Both effects appear to be involved in the positive inotropic effect of the compound, because its metabolite, UD-CG 212 Cl, had no effect on force of contraction and on the Ca2+ sensitivity of skinned cardiac fibers but inhibited PDE III even more potently than pimobendan.(ABSTRACT TRUNCATED AT 400 WORDS)
Sodium-induced cardiac hypertrophy. Cardiac sympathetic activity versus volume load.
Authors: Fields N G , Yuan B X , Leenen F H
Published: June 2012

To investigate the possible contributions of cardiac volume overload and cardiac sympathetic hyperactivity in the effects of sodium on cardiac mass, we evaluated the effects of treatment with saline (1%) and deoxycorticosterone acetate + saline (DOCA/saline) for 10 days and 3 and 6 weeks on ventricular anatomy and intracardiac pressures. Sympathetic activity in the heart and other tissues was assessed at 10 days and 3 weeks by catecholamine turnover rates and tyrosine hydroxylase activity. Both saline and DOCA/saline produced concentric left ventricular (LV) hypertrophy. Right ventricular weight showed only small increases. Saline treatment did not affect LV end-systolic pressure, whereas DOCA/saline caused a moderate increase (to 159 mm Hg). Right atrial pressure was not affected by either treatment, whereas LV end-diastolic pressure increased but only after the development of LV hypertrophy. Both saline and DOCA/saline decreased LV norepinephrine concentration; only DOCA/saline decreased norepinephrine content per LV. However, neither treatment altered the norepinephrine turnover rate constant, the absolute turnover rate, or the tyrosine hydroxylase activity. The results demonstrate that increased saline intake or DOCA/saline produces concentric LV hypertrophy without any increase in blood pressure in the case of saline and with increases in LV filling pressure following rather than preceding the appearance of LV hypertrophy. The lack of an increase in LV norepinephrine turnover and tyrosine hydroxylase activity suggests that the hypertrophy is not mediated through increased cardiac neuronal sympathetic activity.
Involvement of a GTP-binding protein in stimulating action of angiotensin II on calcium channels in vascular smooth muscle cells.
Authors: Ohya Y , Sperelakis N
Published: June 2012

The possible involvement of a GTP-binding protein in the regulation of Ca2+ channels by angiotensin II (Ang II) in vascular muscle cells was investigated by the whole-cell voltage-clamp method. Single cells were freshly isolated from guinea pig portal vein. The pipette solution contained high Cs+ to inhibit K+ currents and thereby isolate the Ca2+ channel current. Ba2+ (2 mM) was in the bath solution as a charge carrier for the Ca2+ channel. Application of Ang II (0.1-100 nM) produced an increase in peak amplitude of the Ba2+ current, with a shift of the current-voltage curve in the negative direction. These effects were inhibited by pretreatment with an antagonist of the Ang II receptor, [Sar1,Ile8]-Ang II. Presence of 0.1 mM GTP in the pipette solution stabilized the Ang II action, but 0.3-1.0 mM GDP-beta-S and 1.0 mM GTP-gamma-S inhibited it. GTP-gamma-S alone produced a slowly progressing increase in the basal (unstimulated) current amplitude. Preincubation of muscle tissues with pertussis toxin (1 micrograms/ml, for up to 6 hours at 36 degrees C) or intracellular application of preactivated pertussis toxin (1 micrograms/ml) plus NAD (1 mM) did not inhibit the Ang II action. Cholera toxin (10 micrograms/ml) also had no effect on the Ang II action. These results suggest that the Ang II stimulation of Ca2+ channels in smooth muscle of guinea pig portal vein may be mediated by a G protein that is insensitive to both pertussis toxin and cholera toxin.
Adenine nucleotide release from isolated perfused guinea pig hearts and extracellular formation of adenosine.
Authors: Borst M M , Schrader J
Published: June 2012

The quantification of adenine nucleotides released from the heart is hampered by their rapid dephosphorylation to adenosine in the extracellular space catalyzed by highly active ectonucleotidases. To determine the total release of adenine nucleotides from isolated Langendorff-perfused guinea pig hearts, ecto 5'-nucleotidase was effectively blocked by infusion of alpha, beta-methylene-ADP (AOPCP, 50 microM). Adenine nucleotides were measured in the coronary venous effluent by the luciferin-luciferase method after enzymatic rephosphorylation to ATP. In hearts perfused at a constant flow rate (10 ml/min) with normoxic buffer (95% O2, 5% CO2) the release +/- SEM of adenine nucleotides and adenosine was 0.06 +/- 0.01 (n = 11) and 0.04 +/- 0.01 (n = 13) nmol/min. In the presence of AOPCP, the release of adenine nucleotides increased to 0.43 +/- 0.04 nmol/min (n = 9; p less than 0.05), whereas adenosine remained unchanged. Hypoxic perfusion (10% O2, 85% N2, 5% CO2) caused a threefold increase in adenine nucleotide release but a 40-fold increase in adenosine. In contrast, global ischemia (30 seconds) caused adenine nucleotide and adenosine release to rise to similar values of 1.06 +/- 0.10 and 0.80 +/- 0.14 nmol/min (n = 9). Stimulation of hearts with isoproterenol (4 nM) likewise increased the release of adenine nucleotides (0.50 +/- 0.04 nmol/min) and adenosine (0.87 +/- 0.21 nmol/min) (n = 6). To determine the cellular source of adenine nucleotides released from the heart, the coronary endothelial adenine nucleotide pool was selectively prelabeled by [3H]adenosine. Global ischemia increased the specific radioactivity of released adenine nucleotides by 57%. The findings indicate that 1) adenine nucleotides and adenosine are released at the same order of magnitude from the well-oxygenated heart; 2) beta-adrenergic stimulation and ischemia stimulate the release of adenine nucleotides and adenosine, both purines reaching vasoactive concentrations in the effluent perfusate; 3) during hypoxic perfusion only the release of adenosine is greatly enhanced; and 4) the coronary endothelium preferentially contributes to the ischemia-induced adenine nucleotide release.