bluuebunny/crossref_metadata_2025 huggingface dataset:
Rat angiotensin II (type 1A) receptor mRNA regulation and subtype expression in myocardial growth and hypertrophy.
Authors: Suzuki J , Matsubara H , Urakami M , Inada M
Published: June 2012

Two subtypes of angiotensin II (Ang II) receptors (AT1 and AT2) are distinguished by using the respective specific antagonists. In the present study, we report the regulation of cardiac AT1 type A (AT1A) receptor mRNA levels and the expression pattern of AT1 and AT2 receptors in the growth of the heart and the development and regression of cardiac hypertrophy. The ventricular AT1A mRNA level and the density of Ang II receptors at the neonatal period were significantly increased (3.5-fold and 2.5-fold, respectively) and then downregulated with maturation. The cardiac hypertrophy established in spontaneously hypertensive rats or two-kidney one-clip renovascular hypertensive rats resulted in substantial increases in ventricular AT1A mRNA levels (threefold) and Ang II receptor densities (twofold) as compared with those in respective control rats, whereas the receptor affinity was similar. The proportion of AT1 and AT2 subtypes in the specific Ang II binding in ventricular membranes prepared from normal adult rats was nearly equal. This proportion did not change significantly in the development of myocardial hypertrophy. The regression of cardiac hypertrophy by the normalization of elevated blood pressure completely reversed the increased levels of AT1A mRNA and the receptor density to the control level. Thus, AT1 and AT2 receptors are present in rat ventricular myocardium, and their expression is developmentally regulated and upregulated in response to hypertrophic change. Ang II action exerted through the increased number of Ang II receptors may contribute to the growth of the heart and thus to the maintenance of established hypertrophy as one of the hormones involved in hypertrophy development.
Effects of adenosine and its analogues on isolated intracerebral arterioles. Extraluminal and intraluminal application.
Authors: Ngai A C , Winn H R
Published: June 2012

We evaluated the responses of brain parenchymal arterioles to intraluminal and extraluminal application of adenosine and its analogues. Intracerebral arterioles (28.4- to 60.3-microns diameter) were isolated from Sprague-Dawley rats, cannulated with micropipettes, and perfused in vitro. Both extraluminal and intraluminal adenosine, 5'-(N-ethylcarboxamido)adenosine (NECA), R-N6-(phenylisopropyl)adenosine (R-PIA), and S-N6-(phenylisopropyl)adenosine (S-PIA) elicited concentration-dependent dilation of these arterioles, but intraluminal application was less potent and efficacious than extraluminal application. Inosine was not vasoactive. A common order of agonist potency (NECA > adenosine > R-PIA > or = S-PIA) was determined for both extraluminal and intraluminal application. Theophylline (10 microM) caused a rightward shift of the adenosine concentration-response curve and a 50-fold reduction in potency. Intraluminal theophylline was one sixth as effective as extraluminal theophylline in antagonizing the extraluminal adenosine response, whereas intraluminal 8-sulfophenyltheophylline, a polar theophylline derivative, was ineffective. Polyadenylic acid (PolyA, 1 microM), an adenosine polymer that does not penetrate the endothelium, induced a dilation of 44.2 +/- 5.3% when applied extraluminally but had no effect when infused intraluminally. The dilator effect of PolyA was antagonized by theophylline. We conclude that: (1) intraluminal adenosine and its analogues are effective dilators of intracerebral arterioles, (2) the dilator effects of both intraluminally and extraluminally applied adenosine are predominantly mediated by A2-type receptors, and (3) adenosine receptors mediating vasodilation are not present on the luminal surface of the endothelium.
Modification of myogenic intrinsic tone and [Ca2+]i of rat isolated arterioles by ryanodine and cyclopiazonic acid.
Authors: Watanabe J , Karibe A , Horiguchi S , Keitoku M , Satoh S , Takishima T , Shirato K
Published: June 2012

The role of the sarcoplasmic reticulum (SR) in regulating myogenic tone and [Ca2+]i was examined with ryanodine and cyclopiazonic acid (CPA) in the rat skeletal muscle arteriole (A(sk)) and mesenteric arteriole (Ams). Arterioles were cannulated at both ends to control luminal pressure in a tissue bath. Luminal diameter was measured with a video-monitored microscopic system. Fura 2-AM was loaded to measure [Ca2+]i using the fluorescence intensity ratio at excitation wavelengths of 340 to 380 nm (F340/380). The myogenic response (luminal pressure was increased from 40 to 100 mm Hg) and the intrinsic tone at 40 mm Hg were observed in A(sk) but not in Ams. Ryanodine (10(-5) M decreased the steady-state diameter of A(sk) from 138 +/- 8 to 85 +/- 9 microns (P < .05) and increased the F340/380 ratio; these effects were reversed by nifedipine or Ca(2+)-free solution. Ryanodine shifted the [Ca2+]o-contraction response curve upward. CPA (10(-5) M) also decreased the steady-state diameter of A(sk) from 131 +/- 7 to 98 +/- 11 microns (P < .05). In contrast, Ams responded to neither ryanodine nor CPA. Caffeine-induced contractions were significantly reduced by either ryanodine or CPA in both arterioles. These results indicate that SR dysfunction increased the susceptibility of the arteriolar tone to [Ca2+]o and enhanced the tone of A(sk). In conclusion, the SR function may play a critical role in regulating [Ca2+]i and the intrinsic tone of A(sk) that was myogenically active at physiological luminal pressure.
Immunohistochemical delineation of the conduction system. II: The atrioventricular node and Purkinje fibers.
Authors: Oosthoek P W , Virágh S , Lamers W H , Moorman A F
Published: June 2012

Using an antibody that reacts specifically with the myocytes of the conduction system of the bovine heart, we have studied the atrioventricular node and the spatial distribution of the Purkinje fibers in the bovine heart. This study was complemented by studying the distribution of the gap junction protein connexin43 in these areas in the bovine heart and in the human heart. The large Purkinje fibers in the bovine heart are arranged in a two-dimensional network underneath the endocardium. At discrete sites, these fibers branch to the Purkinje fibers situated between the muscle bundles of the ventricular mass. These intramural Purkinje fibers are arranged in sheets that form a complex three-dimensional network of lamellas. Contacts with the ventricular myocytes are found throughout the myocardial wall, with the exception of a subepicardial layer of 2-mm thickness, ie, 10% to 15% of the wall thickness. The spatial arrangement of the Purkinje fibers correlates well with data on electrophysiology. Connexin43 was not detected in the myocytes of the atrioventricular node, whereas in the Purkinje fibers of the atrioventricular bundle and of the bundle branches, abundant expression of connexin43 was found in both humans and cows. In the bovine Purkinje fibers, a remarkable subcellular distribution of connexin43 is found: it occupies the entire plasma membrane facing other Purkinje cells but not that facing the surrounding connective tissue. The structural differences in architecture of the ventricular conduction system in humans and cows seems not to result in substantial differences in conduction velocities. However, the Purkinje fiber network in the bovine heart may explain the efficient ventricular excitation, as reflected by the relatively short QRS complex compared with that in the human heart, where intramural Purkinje fibers are not found.
Effect of myocardial ischemia on stimulation-evoked noradrenaline release. Modulated neurotransmission in rat, guinea pig, and human cardiac tissue.
Authors: Seyfarth M , Feng Y , Hagl S , Sebening F , Richardt G , Schömig A
Published: June 2012

The effect of myocardial ischemia and its major metabolic changes, such as anoxia, acidosis, and hyperkalemia, on exocytotic noradrenaline release was investigated in rat, guinea pig, and human cardiac tissue. Noradrenaline release was evoked by electrical field stimulation, and the effect of each experimental intervention on stimulation-evoked noradrenaline release (S2) was intraindividually compared with the release induced by a control stimulation (S1). In perfused hearts, 10 minutes of global ischemia caused a reduction of noradrenaline overflow in rat hearts (mean S2/S1, 0.31), whereas the overflow was increased in guinea pig hearts (S2/S1, 1.89). This species-dependent effect may be caused by quantitatively different responses to facilitating and suppressing factors of noradrenaline release in both species. Anoxia and substrate-free perfusion increased noradrenaline overflow in guinea pig hearts (S2/S1, 2.40) but had no significant effect in rat hearts (S2/S1, 0.75). Acidosis (pH 6.0) resulted in a suppression of noradrenaline release in rat hearts (S2/S1, 0.16), whereas it had only a minor inhibiting effect in guinea pig hearts (S2/S1, 0.67). Hyperkalemia had a comparable effect in both species (S2/S1 at 15 mmol/L K+, 1.17 in rat and 1.14 in guinea pig; and S2/S1 at 20 mmol/L K+, 0.64 in rat and 0.41 in guinea pig). To obtain results regarding the modulation of noradrenaline release in human myocardium, human atrial tissue was incubated, and the effect of anoxia, acidosis, and hyperkalemia on stimulation-evoked noradrenaline release was investigated.(ABSTRACT TRUNCATED AT 250 WORDS)
Molecular structure and transcriptional function of the rat vascular AT1a angiotensin receptor gene.
Authors: Takeuchi K , Alexander R W , Nakamura Y , Tsujino T , Murphy T J
Published: June 2012

Rat vascular angiotensin receptors (AT1a receptors) are encoded by two mRNA transcripts sharing an identical receptor coding sequence but differing in their 5' and 3' untranslated sequences. We screened male Sprague-Dawley rat genomic libraries to clone the vascular AT1a receptor gene. Two sets of overlapping clones were isolated that encode over 90 kb of genomic sequence around the AT1a receptor gene. Four overlapping clones were identified from the 5' flanking portion of the gene. These contain the promoter region and two exons, 141 bp and 89 bp in size, respectively, encoding the alternatively spliced 5' untranslated mRNA sequence. Six additional clones overlap each other but do not overlap the set of clones from the 5' flanking region of the gene. These contain a single 1977-bp exon that encodes 900 bp of the 5' and 3' untranslated sequences in addition to a 1077-bp open reading frame identical to that found in vascular smooth muscle cell AT1a receptor cDNAs. Primer extension and RNase protection studies indicate that the transcription start site for this gene begins 9 bp upstream from the most 5' sequence found within the AT1a receptor cDNAs. Our mapping studies of the cloned gene, which so far includes an uncloned gap within the second intron, indicate that the transcription start site is no less than 67 kb upstream from the receptor coding exon. Promoter-reporter assays were performed by transfection of vascular smooth muscle cells with deletions of a 3.2-kb promoter region fused to a luciferase cDNA reporter plasmid. Relatively strong basal transcriptional activity is observed from the 5'-most 2 kb of the promoter and diminishes markedly with deletions within 1 kb of the early promoter region, suggesting strong promoter elements in the more upstream regions of the gene. Deletion of a 53-bp early promoter region containing the transcription start site and a putative TATA box completely abolishes the ability of upstream elements to drive transcription of the luciferase cDNA. These results indicate that we have isolated the AT1a receptor gene and its functional promoter.
Partial characterization of the rH1 sodium channel protein from rat heart using subtype-specific antibodies.
Authors: Cohen S A , Levitt L K
Published: June 2012

Three subtype-specific antisera were generated against peptides corresponding to portions of the amino terminus, interdomain 1-2, and carboxy terminus of the rH1 sodium channel primary sequence to confirm the expression of this protein in the adult rat heart and to determine selected biochemical properties of this protein that might contribute to its subtype-specific characteristics. All three antisera identify a 240-kD band on Western blots of partially purified cardiac membrane proteins and by immunoprecipitation of iodinated partially purified membrane proteins. Unlike other characterized mammalian sodium channels, no beta subunit is detected in association with the rH1 alpha subunit. The rH1 alpha subunit is a complex sialoglycoprotein as evidenced by its interaction with wheat germ agglutinin-Sepharose and by reduction in its apparent molecular weight after treatment with neuraminidase; deglycosylation with N-glycanase confirms that the rH1 protein contains significantly less carbohydrate than other sodium channel proteins characterized to date (5% versus 25% to 30%). Consistent with electrophysiological studies indicating a role of phosphorylation in channel regulation, the rH1 alpha subunit can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase A. The possible functional significance of these findings is discussed.
Regulation of Ca2+ channels by cAMP and cGMP in vascular smooth muscle cells.
Authors: Ishikawa T , Hume J R , Keef K D
Published: June 2012

Whole-cell Ca2+ channel currents in rabbit portal vein cells were recorded using the amphotericin B-perforated patch-clamp technique at 35 degrees C. This technique allowed recording of stable inward currents in the absence of run-down for more than 30 minutes. Depolarizing voltage steps from a holding potential of -70 mV elicited voltage-dependent inward currents. The voltage dependence of inward currents measured in either 2.5 mmol/L Ba(2+)- or 2.5 mmol/L Ca(2+)-containing solution were very similar. However, maximum Ba2+ current (obtained at around +10 mV) was approximately 1.5-fold larger than maximum Ca2+ current. Changing the holding potential from -70 to -40 mV decreased inward currents but did not shift the voltage dependence significantly. Inward currents were also completely blocked by the dihydropyridine Ca2+ channel blocker, nicardipine (10 mumol/L), suggesting the presence of predominantly L-type Ca2+ channels in rabbit portal vein cells. Isoproterenol caused small increases in the amplitude of Ba2+ currents in a concentration-dependent manner (10 nmol/L to 1 mumol/L), which were reversed with propranolol. Forskolin (1 mumol/L) or 8-bromo-cAMP (0.1 mmol/L) also caused small increases in the amplitude of Ba2+ currents, suggesting that the stimulatory actions of isoproterenol are importantly linked to the production of cAMP. Higher concentrations of of isoproterenol (10 mumol/L) or forskolin (10 mumol/L) caused a transient increase in Ba2+ currents followed by f decrease in current amplitude. Higher doses of 8-bromo-cAMP (1 mmol/L) and low doses of 8-bromo-cGMP (0.1 mmol/L) inhibited Ba2+ currents, increased the rate of current inactivation, and produced a negative voltage shift in steady-state availability. These results indicate that low concentrations of intracellular cAMP produce modest increases in Ca2+ channel activity, whereas cGMP and higher concentrations of cAMP result in inhibition of Ca2+ channel activity in vascular smooth muscle cells. The observed similarities of cGMP and high concentrations of cAMP on Ba2+ current amplitude, kinetics, and steady-state inactivation suggest mediation by a common mechanism, possibly involving activation of cGMP-dependent protein kinase.
Determinants of loaded shortening velocity in single cardiac myocytes permeabilized with alpha-hemolysin.
Authors: Sweitzer N K , Moss R L
Published: June 2012

Force-velocity relations were obtained from single cardiac myocytes isolated by enzymatic digestion of rat myocardium and permeabilized with the pore-forming staphylococcal toxin alpha-hemolysin. Single cardiac myocytes were attached to a force transducer and piezoelectric translator and viewed with an inverted microscope to allow periodic monitoring of sarcomere length during experiments. Permeabilized cells were activated by immersion in a bath of known [Ca2+]. We report that the Ca2+ sensitivity of cells obtained by enzymatic digestion and permeabilized using alpha-hemolysin is similar to that reported previously for mechanically disrupted ventricular myocardium; however, the tension-pCa relation is less steep in the new preparation. During isotonic measurements, force was clamped to various loads using a rapid-response servo system. All recordings of shortening under load were distinctly curvilinear, and analysis of data involved fitting each shortening recording with a single exponential curve and calculating the value of the slope at the initial time of the load clamp. In addition, the presence of significant resting force at initial sarcomere lengths in these cells required that the possibility of alteration of velocity due to the presence of resting force be addressed. The maximum shortening velocity in fully Ca(2+)-activated single ventricular myocytes studied by this method was 2.83 muscle lengths per second on average. The basis for curvilinear shortening is postulated to be multifactorial in cardiac muscle, involving a combination of shortening inactivation and one or more passive elasticities that resist stretch or compression depending on sarcomere length. Shortening velocity shows a dependence on myosin isoform content when cells from a single heart are compared; however, this relation does not hold when cells from different hearts are compared. The behavior of single alpha-hemolysin-permeabilized myocyte shortening under loaded conditions at lower levels of Ca2+ is also described. During submaximal Ca2+ activation, initial shortening velocities are faster than those observed in maximally activated cells. This may be due to contributions of high passive force to increase shortening velocity under conditions of low active force generation, when passive force in the cell is a greater proportion of the total force and there are fewer bound crossbridges.
Intraluminal flow-initiated hyperpolarization and depolarization shift the membrane potential of arterial smooth muscle toward an intermediate level.
Authors: Bevan J A , Wellman G C
Published: June 2012

We examined the effect of intraluminal flow of physiological saline on the membrane potential of vascular smooth muscle cells in isolated rabbit cerebral arteries. Intraluminal flow (20 microL/min) caused a depolarization of 4.8 +/- 0.7 mV in muscle cells with a resting membrane potential of -62.5 +/- 1.2 mV (n = 19). However, when cells were depolarized to -48.7 +/- 1.8 mV using histamine and serotonin, the response to intraluminal flow was the opposite, a hyperpolarization of 5.6 +/- 1.0 mV (n = 9). These opposing effects of flow on membrane potential appear to balance at -57.8 +/- 1.1 mV (n = 31). Our results suggest that intraluminal flow may affect the level of basal tone present in arteries in vivo through modulating the membrane potential of vascular smooth muscle cells by concurrently activated depolarization and hyperpolarization.
Brefeldin A defines distinct pathways for atrial natriuretic factor secretion in neonatal rat atrial and ventricular myocytes.
Authors: De Young M B , Keller J C , Graham R M , Wildey G M
Published: June 2012

The intracellular pathways for basal atrial natriuretic factor (ANF) secretion from the heart and their correlation with ANF processing to the active form were characterized in cultured neonatal rat atrial and ventricular myocytes. Brefeldin A, a fungal antimetabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was used to inhibit nascent protein trafficking. Thus, release of newly synthesized hormone was blocked, but release of stored hormone was unaffected. Whereas brefeldin A inhibited basal ventricular ANF release to 10% of the control value, basal ANF release from atrial cells was enhanced. Furthermore, basal atrial ANF secretion was inhibited by agents preventing myocyte depolarization, Ca2+ influx, release of Ca2+ from intracellular stores, or activation of protein kinase C, whereas ventricular ANF secretion was unaffected by these agents. Brefeldin A did not alter maturational processing of pro-ANF to ANF-(99-126) in either atrial or ventricular cultures. These findings indicate that (1) basal secretion of ANF from ventricular cells relies largely on newly synthesized hormone and is probably constitutive, (2) basal secretion of ANF from atrial cells is independent of transport of newly synthesized protein and occurs via a regulated pathway controlled at least in part by signaling changes associated with myocyte beating, and (3) processing of pro-ANF occurs either with constitutive or regulated secretion of hormone, which may indicate multiple cellular locations for the processing enzyme.
Subcellular mechanism for Ca(2+)-dependent enhancement of delayed rectifier K+ current in isolated membrane patches of guinea pig ventricular myocytes.
Authors: Nitta J , Furukawa T , Marumo F , Sawanobori T , Hiraoka M
Published: June 2012

Intracellular Ca2+ augments delayed rectifier K+ current (IK) in cardiac myocytes, which may play a major modulatory role in repolarization of action potentials. We investigated subcellular mechanisms for Ca(2+)-induced enhancement of IK in large-pipette inside-out membrane patches excised from isolated guinea pig ventricular myocytes. When [Ca2+]i was raised from 10(-8) to 10(-6) mol/L, the amplitude of IK measured at +80 mV was increased from 12.0 +/- 2.2 to 19.5 +/- 3.3 pA (P < .01). The enhancement of IK by Ca2+ was dose dependent, with an EC50 of 3.8 x 10(-8) mol/L. A calmodulin antagonist, W7 (50 mumol/L), calmidazolium (100 mumol/L), or HT-74 (20 mumol/L), added to the intracellular solution abolished enhancement of IK by Ca2+, whereas the inactive form of the W7 analogue, W5, had no effect on IK. In the presence of a protein kinase inhibitor with a relatively high specificity for protein kinase C (H7), for protein kinase A (H8 or peptide-type inhibitor PKI), or for calmodulin kinase II (KN-62) or a nonspecific inhibitor of serine/threonine protein kinases (staurosporine), increases in [Ca2+]i still enhanced IK. Ca(2+)-induced enhancement of IK was also observed when Mg2+ and ATP were omitted from the intracellular solution to delete exogenous phosphate donors and when adenylylimidodiphosphate was added to preclude trapped cytoplasmic substrates. Thus, cardiac IK was enhanced by increases in [Ca2+]i at a physiological range via a calmodulin-dependent pathway, which did not involve a phosphorylation process.
Peripheral adaptations in trained aged rats with femoral artery stenosis.
Authors: Yang H T , Ogilvie R W , Terjung R L
Published: June 2012

The development and functional significance of exercise-induced peripheral adaptations were evaluated in aged animals with peripheral arterial insufficiency. Fisher 344 male rats (21 months old) were subjected to bilateral stenosis of the femoral arteries sufficient to limit active hyperemia but not to impair resting blood flow. Beginning the third day after stenosis, animals were (1) exercised by walking (n = 12) on a treadmill at 20 m/min at 15% inclination, twice a day, 5 days per week, or (2) limited to cage activity (n = 10). Exercise tolerance improved from approximately 5 to approximately 35 minutes (P < .001) over the 8 weeks of the training program but increased only marginally to approximately 8 minutes for the sedentary group. An isolated hind limb preparation perfused at equivalent blood flows (approximately 1 ml.min-1 x g-1 with an arterial blood oxygen content of approximately 20 vol%) was used to assess the functional and metabolic impact of muscle-specific adaptations during sequential contraction periods at 4, 8, 15, 30, 45, 60, 75, and 90 tetani per minute. An initially similar force development of approximately 10 N/g was better maintained (P < .001) by the trained group. The peak oxygen consumption attained by the trained group of 5.68 +/- 0.34 mumol.min-1 x g-1 was greater (P < .01) than that of the sedentary group (4.34 +/- 0.29 mumol.min-1 x g-1). This was due to a greater oxygen extraction, since oxygen delivery was the same (approximately 10 mumol.min-1 x g-1) to muscles of both groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of ischemia and reperfusion on cardiac ryanodine receptors--sarcoplasmic reticulum Ca2+ channels.
Authors: Zucchi R , Ronca-Testoni S , Yu G , Galbani P , Ronca G , Mariani M
Published: June 2012

We investigated the effect of ischemia and reperfusion on the cardiac ryanodine receptor, which corresponds to the sarcoplasmic reticulum Ca2+ channel. Isolated working rat hearts were subjected to 10 to 30 minutes of global ischemia, followed or not by reperfusion. Ischemia produced significant reduction in the density of high-affinity 3H-ryanodine binding sites, determined either in whole-heart homogenate (Bmax, 220 +/- 22, 203 +/- 12, and 228 +/- 14 fmol/mg protein after 10, 20, and 30 minutes of ischemia versus 298 +/- 18 fmol/mg protein in the control condition; P < .01) or in a fraction enriched in sarcoplasmic reticulum (Bmax, 1.08 +/- 0.15 pmol/mg protein after 20 minutes of ischemia versus 1.69 +/- 0.08 pmol/mg protein in the control condition; P < .01). The Kd (1.5 +/- 0.1 nmol/L) and the Ca2+ dependence of high-affinity 3H-ryanodine binding were not affected by ischemia. The density of low-affinity 3H-ryanodine binding sites was also reduced after 20 minutes of ischemia (14.0 +/- 2.3 versus 34.0 +/- 8.2 pmol/mg protein in the sarcoplasmic reticulum fraction, P < .05), without significant changes in Kd (4.7 +/- 1.2 versus 2.4 +/- 1.0 mumol/L). All these changes persisted after 20 minutes of reperfusion. Analysis of tissue fractions showed that 55% of the ryanodine binding sites were retained in the pellet of a low-speed centrifugation ("nuclear pellet") and that the effects of ischemia concerned only the receptors released in the supernatant ("postnuclear supernatant"). In parallel experiments, we evaluated the effect of ryanodine on oxalate-supported Ca2+ uptake, which represents sarcoplasmic reticulum Ca2+ uptake. As expected, we found that high concentrations of ryanodine stimulated Ca2+ uptake, owing to channel blockade. The response to 900 mumol/L ryanodine was slightly reduced in crude homogenate and significantly reduced in postnuclear supernatant obtained from ischemic hearts. In conclusion, the number of ryanodine receptors is reduced after ischemia; this effect concerns a subpopulation of the receptors, persists after reperfusion, and might contribute to modify sarcoplasmic reticulum function.
Cocaine-induced microvascular spasm in Yucatan miniature swine. In vivo and in vitro evidence of spasm.
Authors: Núñez B D , Miao L , Wang Y , Núñez M M , Klein M A , Sellke F W , Ross J N , Susulic V , Paik G Y , Carrozza J P
Published: June 2012

The purpose of the present study was to determine the maximal coronary flow reserve (CFR) before and after the administration of successive cocaine doses (0.1, 0.5, 3, and 7 mg/kg IV) for 2 minutes at 10-minute intervals in eight miniature swine. CFR was assessed by the administration of adenosine (0.03, 0.3, and 3 mg IC). Hemodynamic and flow measurements were performed 3 minutes after each dose. Coronary flow (CF) was measured with a Doppler-tipped wire in the proximal left anterior descending coronary artery (LAD). Also, microvessels were dissected, and vessel diameters were measured by a videoelectronic dimension analyzer. In vivo, LAD CF increased fourfold, CFR increased twofold, and coronary vascular resistance (CVR) decreased fourfold after the administration of adenosine. In contrast, LAD CF decreased threefold, CFR decreased onefold, and CVR increased sixfold 3 minutes after the administration of cocaine. Adenosine (3 mg) was repeated 4 minutes after the administration of cocaine, and LAD CF increased 1.4-fold, CVR increased 2.5-fold, and CFR decreased onefold. Thus, adenosine partially reversed the potent cocaine constrictor effect. In vitro, 10(-9) mol/L cocaine decreased the diameter of the coronary microvessels from 129 +/- 12 to 127 +/- 12 microns, and 10(-4) mol/L cocaine decreased coronary microvessel diameter to 114 +/- 15 microns (P < .05). In conclusion, cocaine in vivo decreases CFR, and consistent with the in vivo effect, cocaine in vitro produced constriction of vessels < 200 microns. These results indicate that cocaine can produce profound microvascular spasm. This may contribute to the ischemia/infarction reported in patients who abuse cocaine and who are subsequently found to have normal epicardial coronary arteries.
Molecular determinants of reperfusion-induced leukocyte adhesion and vascular protein leakage.
Authors: Kurose I , Anderson D C , Miyasaka M , Tamatani T , Paulson J C , Todd R F , Rusche J R , Granger D N
Published: June 2012

The adherence and emigration of leukocytes have been implicated as a rate-limiting step in the microvascular dysfunction associated with reperfusion of ischemic tissues. The objective of the present study was to define the relation between leukocyte-endothelial cell adhesion and albumin leakage in rat mesenteric venules exposed to ischemia and reperfusion (I/R). Leukocyte adherence and emigration as well as albumin extravasation were monitored in single post-capillary venules using intravital fluorescence microscopy. Ischemia (0, 10, 15, or 20 minutes) was induced by complete occlusion of the superior mesenteric artery, and all parameters were monitored for 30 minutes after reperfusion. The magnitude of the leukocyte adherence and emigration and albumin leakage elicited by I/R was positively correlated with the duration of ischemia. The albumin leakage response was also highly correlated with the number of adherent and emigrated leukocytes. Monoclonal antibodies against the adhesion glycoproteins CD18, CD11b, intercellular adhesion molecule-1 (ICAM-1) (at 10 and 30 minutes), and L-selectin (at 10 minutes), but not P- or E-selectin, reduced I/R-induced leukocyte adherence and emigration as well as albumin leakage. Platelet-leukocyte aggregates were formed in postischemic venules; the number of aggregates was reduced by antibodies against P-selectin, CD11b, CD18, and ICAM-1, but not E- or L-selectin. These results indicate that reperfusion-induced albumin leakage is tightly coupled to the adherence and emigration of leukocytes in postcapillary venules. This adhesion-dependent injury response is primarily mediated by CD11b/CD18 on activated neutrophils and ICAM-1 on venular endothelium and appears to require L-selectin-dependent leukocyte rolling.
Atrial natriuretic factor and isosorbide dinitrate modulate the gating of ATP-sensitive K+ channels in cultured vascular smooth muscle cells.
Authors: Kubo M , Nakaya Y , Matsuoka S , Saito K , Kuroda Y
Published: June 2012

The effects of atrial natriuretic factor (ANF) and isosorbide dinitrate (ISDN), activators of particulate and soluble guanylate cyclase, respectively, on K+ currents were investigated in patch-clamp recordings of smooth muscle cells cultured from rat thoracic aorta. In the cell-attached patch configuration, ANF enhanced Ca(2+)-activated K+ (KCa) channel activities as reported previously. When KCa channels were blocked with 1 mmol/L tetraethylammonium or 10(-7) mol/L charybdotoxin, ANF and ISDN applied to the bathing solution activated ATP-sensitive K+ (KATP) channels without altering channel conductance. Pretreatment with methylene blue, a guanylate cyclase inhibitor, abolished the effects of ISDN on KATP channels, whereas 8-bromo-cGMP activated these channels, suggesting that the effects of ISDN on KATP channels were mediated by cGMP. Our results suggest that vasorelaxant agents that increase intracellular cGMP concentrations modulate the gating of two major potassium channels, ATP sensitive and Ca2+ activated, that might play an important role in controlling vascular tone by changing the membrane potential.
Lysophosphatidylcholine in oxidized low-density lipoprotein increases endothelial susceptibility to polymorphonuclear leukocyte-induced endothelial dysfunction in porcine coronary arteries. Role of protein kinase C.
Authors: Sugiyama S , Kugiyama K , Ohgushi M , Fujimoto K , Yasue H
Published: June 2012

We have shown that transferred lysophosphatidylcholine (lysoPC) from oxidized low-density lipoprotein (Ox-LDL) to endothelial surface membrane activates protein kinase C (PKC) in endothelial cells, suggesting that Ox-LDL could alter endothelial functions through PKC activation. The purposes of the present study were to examine whether the endothelial susceptibility to polymorphonuclear leukocytes (PMNs) may be altered in Ox-LDL-treated coronary arteries, which have properties closely resembling those observed in atherosclerotic arteries, and to determine the mechanism(s) by which Ox-LDL may affect the endothelial susceptibility to PMNs. Isolated porcine coronary arteries were cannulated and perfused with oxygenated culture medium with or without LDLs or lipids at a constant flow (37 degrees C, pH 7.4). The treatment of porcine coronary arteries with Ox-LDL increased endothelial adhesiveness to PMNs and augmented PMN-induced impairment of endothelium-dependent arterial relaxation (EDR). Furthermore, Ox-LDL stimulated the expression of intercellular adhesion molecule-1 (ICAM-1) in the porcine coronary arterial endothelium. These effects of Ox-LDL were not mediated by the scavenger-receptor-mediated process but were attributed to lysoPC in Ox-LDL. Blocking of the PMN adherence to endothelium by using anti-CD18 monoclonal antibody abolished the PMN-induced impairment of EDR. Coincubation with staurosporine or calphostin C, inhibitors of PKC, during treatment of the arteries with Ox-LDL or lysoPC attenuated the augmentative effects of Ox-LDL and lysoPC on endothelial ICAM-1 expression, endothelial adhesiveness to PMNs, and PMN-induced EDR impairment. Treatment of the arteries with phorbol 12-myristate 13-acetate, a potent stimulator of PKC, induced ICAM-1 expression and enhanced the endothelial adhesiveness to PMNs and PMN-induced EDR impairment, mimicking the effects of Ox-LDL. These results suggest that lysoPC in Ox-LDL induces endothelial ICAM-1 expression, which facilitates PMN adherence to endothelium and the subsequent augmentation of PMN-induced EDR impairment. PKC activation in endothelial cells by lysoPC in Ox-LDL may at least in part be involved in these effects of Ox-LDL. LysoPC in Ox-LDL increases endothelial susceptibility to PMN-induced endothelial dysfunction.
Brain "ouabain" mediates the sympathoexcitatory and hypertensive effects of high sodium intake in Dahl salt-sensitive rats.
Authors: Huang B S , Leenen F H
Published: June 2012

To assess whether brain ouabain-like activity (OLA) mediates the hypertensive effects of high sodium intake in Dahl salt-sensitive (Dahl S) rats, the effects of blockade of brain OLA on mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) were evaluated in conscious Dahl salt-resistant (Dahl R) and Dahl S rats on a regular (120 mumol/g) or high sodium (1370 mumol/g) diet from 4 to 7 weeks of age. Dahl S rats given high sodium showed higher basal MAP and augmented responses of MAP and RSNA to air stress and to intracerebroventricular injection of the alpha 2-adrenergic receptor agonist guanabenz as compared with Dahl R rats or Dahl S rats given regular sodium. In contrast, the sympathoexcitatory and pressor responses to intracerebroventricular injection of ouabain (0.3 and 1.0 microgram) were markedly attenuated in Dahl S rats given high sodium. Intracerebroventricular preinjection of 0.3 microgram ouabain significantly enhanced blood pressure and RSNA responses to air stress and intracerebroventricular guanabenz in Dahl S rats given regular sodium to the levels observed in Dahl S rats given high sodium. Intracerebroventricular digoxin-specific antibody Fab (DAF) fragments (132 micrograms/8 microL for 5 minutes) did not change basal MAP and RSNA during the first 4 hours after administration in Dahl S rats on a high sodium diet for 3 weeks. However, 18 hours after the injection of DAF fragments, basal MAP and RSNA were significantly decreased, reaching values for Dahl S rats on a regular sodium diet. The magnitude of increases or decreases in MAP and RSNA to air stress or intracerebroventricular guanabenz were significantly attenuated by the DAF fragments in Dahl S rats on a high sodium but not regular sodium diet. Concomitant intracerebroventricular infusion of DAF fragments (200 micrograms per day) prevented the development of hypertension after a high sodium diet in Dahl S rats and prevented an augmentation in pressor and sympathoexcitatory responses to air stress. After discontinuing the infusion of DAF fragments, resting MAP gradually increased to the high levels found in Dahl S rats given high sodium treated with gamma-globulins. These results support the concept that high sodium intake may cause hypertension in Dahl S rats by increasing endogenous brain OLA, thereby enhancing sympathetic outflow and basal blood pressure as well as sympathoexcitatory and pressor responses to stress.
Imipramine blocks rapidly activating and delays slowly activating K+ current activation in guinea pig ventricular myocytes.
Authors: Valenzuela C , Sánchez-Chapula J , Delpón E , Elizalde A , Pérez O , Tamargo J
Published: June 2012

Imipramine is a tricyclic antidepressant drug that also exhibits antiarrhythmic effects and whose clinical spectrum of activity is similar to that of quinidine. It has been previously demonstrated that imipramine inhibits the aggregate time-dependent outward K+ current (IK). IK is composed of at least two components: a slowly activating La(3+)-resistant delayed rectifying current (IK,s) and a rapidly activating La(3+)-sensitive current (IK,r). To assess the effects of imipramine on IK,r and IK,s, single guinea pig ventricular myocytes were studied using the nystatin-perforated patch-clamp technique in the absence and in the presence of La3+. Imipramine inhibited IK,r and IK,s in a concentration-dependent manner. The effects of imipramine on the aggregate time-dependent outward current were more marked than those on IK,s alone. Thus, 1 mumol/L imipramine decreased the tail currents elicited on return to -30 mV after long depolarizing pulses (5 seconds, from -40 to +50 mV) in the absence and in the presence of La3+ by 27 +/- 4% and 15 +/- 3% (n = 6), respectively. Moreover, the inhibition induced by imipramine was greater after short (0.5-second) pulses than after 5-second depolarizing pulses, both in the absence and in the presence of La3+ (53 +/- 3% and 30 +/- 5%, respectively; n = 6; P < .05). Imipramine did not significantly modify either the activation midpoint or the slope factor of the aggregate IK and IK,s activation curves. The reduction of IK,s by imipramine was voltage dependent and was more marked at negative membrane potentials. In the presence of 1 mumol/L imipramine, the ratio of tail current to time-dependent current remained constant at 0.37 +/- 0.03, regardless of the test pulse duration at +50 mV. Thus, the envelope-of-tails test was satisfied in the presence of 1 mumol/L imipramine, which indicates that imipramine, at this concentration, blocks IK,r. Imipramine (1, 5, and 10 mumol/L) had no effect on the kinetics of the later phase of IK activation but delayed the beginning of the activation of IK,s by 62 +/- 22, 74 +/- 23, and 155 +/- 53 milliseconds in the presence of 1, 5, and 10 mumol/L imipramine, respectively. These results suggest that imipramine preferentially blocks rapidly activating K+ channels. In addition, experiments performed in the presence of 30 mumol/L La3+ suggest that the drug preferentially binds, but maybe not exclusively, to a closed state of the slowly activating K+ channel.
Cardiovascular development. Prospects for a genetic approach.
Authors: Fishman M C , Stainier D Y
Published: June 2012

Genetics is a powerful tool, especially when used in combination with embryology, in the seeking of genes necessary for assembly of the cardiovasculature. The first questions must address the types of cellular decisions that are made during development. As for simpler systems in C elegans and D melanogaster, the lineage and cell-fate decisions of the cardiovascular progenitors need to be assessed. In addition it is likely that new paradigms will emerge for multicellular assembly. The study of cardiovascular mutations will define individual genetic steps that define organotypic decisions. A genetic approach is a natural extension of embryology, physiology, and anatomy, fields of great sophistication with regard to the cardiovasculature, because, like them, it focuses on integrative biology and on the intact organism. The zebrafish is particularly well suited to a combination genetic-embryologic study of the fashioning of the cardiovasculature.
Restricted distribution of connexin40, a gap junctional protein, in mammalian heart.
Authors: Gros D , Jarry-Guichard T , Ten Velde I , de Maziere A , van Kempen M J , Davoust J , Briand J P , Moorman A F , Jongsma H J
Published: June 2012

Connexin40 (Cx40) is a member of the connexin family of gap junction proteins. Its mRNA, abundant in lung, is also present in mammalian heart, although in lower amount. Rabbit antipeptide antibodies directed to the COOH terminus (residues 335 to 356) of rat Cx40 were characterized to investigate the distribution of Cx40 in rat and guinea pig cardiac tissues. The affinity-purified antibodies detect specifically a major protein (M(r), 40,000) in immunoblots of total extracts from rat lung and rat and guinea pig heart. In sections of guinea pig atrial tissue treated for immunofluorescence, a strong labeling associated with myocytes was seen with a distribution consistent with that of intercalated disks. The results of immunoelectron microscopy carried out with guinea pig atrial tissue showed that epitopes recognized by these antibodies were exclusively associated with gap junctions. These results, added to those of control experiments, demonstrate that antibodies 335-356 are specific for Cx40. Double-labeling experiments carried out with lung sections using anti-factor VIII and anti-Cx40 antibodies suggest that Cx40 is expressed in blood vessel endothelial cells. In guinea pig and rat heart sections, investigated using both immunofluorescence and immunoperoxidase techniques, a signal was also found to be associated with vascular walls. In guinea pig heart, only atrial myocytes are Cx40-positive. No labeling was detected in ventricular myocytes, including those of the His bundle and the bundle branches, which otherwise do express connexin43 (Cx43). In rat heart Cx40-expressing myocytes are localized in branches, and the Purkinje fibers. Cx43 is not detected either in the His bundle or in the proximal parts of the bundle branches, and consequently, Cx40 is the first connexin demonstrated in this region of the rat conduction system. Cx40 was not detected in the working ventricular myocytes. Double-labeling experiments carried out with hen anti-Cx43 antibodies and rabbit anti-Cx40 antibodies demonstrated that, in tissues expressing both Cx43 and Cx40, these two connexins were localized in the same immunoreactive sites. A few sites, however, appear to contain only one or the other of these two connexins.
Distinct receptors and signaling pathways in alpha-thrombin- and thrombin receptor peptide-induced vascular contractions.
Authors: Tesfamariam B
Published: June 2012

The vasoactive mechanisms of the serine protease alpha-thrombin were examined in isolated coronary arteries from dogs. In resting coronary arteries with endothelium, alpha-thrombin caused concentration-dependent contractions that were characterized by an initial transient relaxation followed by slowly developing sustained contractions. The vascular actions of alpha-thrombin were mimicked by the thrombin receptor-activating peptide (TRAP) SFLLRNP, a synthetic peptide based on the cleaved terminus of the thrombin receptor domain. Treatment of the arteries with N omega-nitro-L-arginine or removal of endothelium abolished the transient relaxations and enhanced the contractions, indicating that the transient relaxations were mediated by the concurrent release of endothelium-derived nitric oxide. alpha-Thrombin that had been catalytically inactivated with the irreversible inhibitor by use of D-Phe-Pro-Arg-chloromethyl ketone did not cause contractions, indicating the requirement of proteolytic cleavage by alpha-thrombin to induce contractions. In contrast to TRAP, alpha-thrombin-induced contractions were blocked by hirudin (a specific thrombin inhibitor), nifedipine and diltiazem (Ca2+ channel blockers), or staurosporine and calphostin C (protein kinase C inhibitors). Unlike alpha-thrombin, which undergoes homologous desensitization, TRAP failed to cause desensitization to subsequent stimulation by alpha-thrombin or TRAP. These observations support the hypothesis that vasoactive actions of alpha-thrombin are mediated by a mechanism that involves cleavage at the active site to expose a new NH2 terminus that activates the thrombin receptor. Further, the dissociation between alpha-thrombin and the synthetic receptor peptide in signal transduction and dissimilar desensitizing properties suggest the existence of distinct thrombin receptor subtypes and/or signaling events in vascular smooth muscle.
Extracellular ATP induces immediate-early gene expression but not cellular hypertrophy in neonatal cardiac myocytes.
Authors: Zheng J S , Boluyt M O , O'Neill L , Crow M T , Lakatta E G
Published: June 2012

It is well-documented that norepinephrine (NE) induces the expression of immediate-early genes (IEGs), such as c-fos, c-jun, and jun-B, in cultured neonatal heart cells and leads to cell growth without cell division (ie, hypertrophy). Although purinergic receptors activated by ATP are present on cardiac myocytes and ATP is coreleased with NE from sympathetic nerve endings within the heart, the potential role of the purinergic system in the cascade of events that leads to cardiac hypertrophy is unknown. We report in the present study that stimulation of purinergic receptors by micromolar concentrations of extracellular ATP increased the levels of c-fos and jun-B mRNA as well as FOS and JUN-B proteins in neonatal cardiac myocytes. The magnitude of response to micromolar ATP was comparable to that elicited by NE. The increase in IEG expression induced by ATP was preceded by a rapid transient increase in cytosolic Ca2+. Pretreatment of myocytes with the intracellular Ca2+ chelator BAPTA-AM prevented the ATP-stimulated increase in cytosolic Ca2+ and attenuated the ATP-stimulated increase in c-fos expression. In contrast, NE did not increase cytosolic Ca2+ in quiescent myocytes, and pretreatment with BAPTA-AM did not inhibit the NE-stimulated increase in c-fos gene expression. Furthermore, although NE markedly increased [14C]phenylalanine incorporation into protein and myocyte hypertrophy measured by cell size, ATP did not. These results demonstrate that stimulation of purinergic receptors by ATP activates IEGs via a Ca(2+)-dependent pathway in cardiac myocytes that differs from the NE stimulated activation of these genes.(ABSTRACT TRUNCATED AT 250 WORDS)