bluuebunny/crossref_metadata_2025 huggingface dataset:
Ca
2+
Influx During the Cardiac Action Potential in Guinea Pig Ventricular Myocytes
Authors: Grantham C.J. , Cannell M.B.
Published: June 2012

The relative contributions of L-type Ca
2+
current (I
Ca
) and Na
+
/Ca
2+
exchange to Ca
2+
influx during the cardiac action potential (AP) are unknown. In this study, we have used an AP recorded under physiological conditions as the command voltage applied to voltage-clamped ventricular myocytes. I
Ca
(measured as nifedipine-sensitive membrane current) had a complex multiphasic time course during the AP. Peak I
Ca
was typically 4 pA/pF, after which it rapidly declined (to about 60% of peak) during the rising phase of the cell-wide Ca
2+
transient before increasing to a second, more sustained component. The initial decline in I
Ca
was sensitive to the amount of Ca
2+
released by the sarcoplasmic reticulum (SR), and conditions that reduce the amplitude of the Ca
2+
transient (such as rest or brief application of caffeine) increased net Ca
2+
influx via I
Ca
. Dissection of the Na
+
/Ca
2+
exchange current at the start of the AP suggested that Ca
2+
influx via Na
+
/Ca
2+
exchange is less than 30% of that due to I
Ca
. From these data, we suggest that I
Ca
is the primary source of Ca
2+
that triggers SR Ca
2+
release, even at the highly depolarized membrane potentials associated with the AP. However, Ca
2+
influx via Na
+
/Ca
2+
exchange is not negligible and may activate some Ca
2+
release from the SR, especially when I
Ca
is reduced. We propose that SR Ca
2+
release inhibits I
Ca
within the same beat, thereby providing a negative feedback mechanism that may serve to limit Ca
2+
influx as well as to regulate the amount of Ca
2+
stored within the SR.
Characterization of the Endothelium-Specific Murine Vascular Endothelial Growth Factor Receptor-2 (Flk-1) Promoter
Authors: Rönicke Volker , Risau Werner , Breier Georg
Published: June 2012

Flk-1, a high-affinity signaling receptor for vascular endothelial growth factor (VEGF), is strongly and specifically expressed on endothelial cells during embryonic development of the vascular system and during tumor angiogenesis. Disruption of Flk-1 gene function has recently been shown to prevent completely endothelial cell differentiation during murine embryonic development. To gain insights into the mechanisms that regulate the endothelium-specific Flk-1 expression, we have isolated the 5′-flanking region of the murine Flk-1 gene. RNase protection and primer extension analyses revealed a single transcriptional start site located 299 bp upstream from the translational start site in an initiator-like pyrimidine-rich sequence. The 5′-flanking region is rich in GC residues and lacks a typical TATA or CAAT box. A luciferase reporter construct containing a fragment from nucleotides −1900 to +299 showed strong endothelium-specific activity in transfected bovine aortic endothelial cells. Deletion analyses revealed that endothelium-specific Flk-1 expression is stimulated by the 5′-untranslated region of the first exon, which contains an activating element between nucleotides +137 and +299. In addition, two endothelium-specific negative regulatory elements were identified between nucleotides −4100 and −623. Two strong general activating elements were present in the region between nucleotides −96 and −37, which contains one potential NFκB and three potential AP-2 binding sites. This study shows that Flk-1 expression in endothelial cells is mainly regulated by an endothelium-specific activating element in the long 5′-untranslated region of the first exon and by negative regulatory elements located further upstream.
Depression of Endothelial Nitric Oxide Synthase but Increased Expression of Endothelin-1 Immunoreactivity in Rat Thoracic Aortic Endothelium Associated With Long-term, but Not Short-term, Sympathectomy
Authors: Aliev Gjumrakch , Ralevic Vera , Burnstock Geoffrey
Published: June 2012

Recent pharmacological studies have shown that perivascular nerves can influence the development and function of vascular endothelial cells (ECs). However, morphological studies have not yet been carried out to investigate whether these functional changes are associated with changes in vasoactive substances in ECs. We used postembedding electron microscopy (EM) triple gold–labeling immunocytochemistry to study the effects of short-term sympathectomy (3 days after 6-hydroxydopamine [6-OHDA] treatment) and long-term sympathectomy (guanethidine and 8 days after 6-OHDA) on the distribution of vasoactive substances in ECs of the rat thoracic aorta. The postembedding immunocytochemistry, which can detect levels of label in individual cells, showed that there was a significant decrease in endothelial NO synthase (NOS3)–labeled, serotonin (5-HT)–labeled, and substance P (SP)–labeled, but a significant increase in endothelin-1 (ET-1)–labeled, gold particles in ECs after long-term, but not after short-term (3-day), sympathectomy. In conclusion, our results show that long-term sympathectomy causes an increase in ET-1 and decrease in NOS3, 5-HT, and SP immunoreactivity in ECs of the thoracic aorta. Our data also indicate that postembedding EM triple gold–labeling immunocytochemistry is a valuable technique for quantitative studies of the content of vasoactive substances in ECs.
Coronary and Systemic Hemodynamic Effects of Sustained Inhibition of Nitric Oxide Synthesis in Conscious Dogs
Authors: Puybasset Louis , Béa Marie-Luce , Ghaleh Bijan , Giudicelli Jean-François , Berdeaux Alain
Published: June 2012

Sustained inhibition of NO synthesis (
N
ω
-nitro-
l
-arginine [L-NNA], 20 mg·kg
−1
·d
−1
, 7 days) was investigated at rest and during exercise in conscious dogs. At rest, L-NNA did not alter mean arterial blood pressure but markedly increased total peripheral resistance (+ 73±14%,
P
<.01). Exaggerated hypertension was observed during exercise (+132±5 mm Hg after L-NNA versus +113±5 mm Hg before L-NNA,
P
<.01). L-NNA decreased the resting coronary artery diameter by 6±1% and suppressed its exercise-induced dilation but had no effect on coronary blood flow and resistance. L-NNA decreased flow repayment volumes during reactive hyperemia, but corresponding flow debt volumes remained unchanged. The cyclooxygenase inhibitor diclofenac (10 mg/kg) had no effect on reactive hyperemia parameters before L-NNA but reduced flow repayment volumes, durations, and corresponding debt-to-repayment ratios in L-NNA–treated dogs (all
P
<.05). In vitro, indomethacin blunted the residual relaxation to bradykinin of large coronary arteries taken from L-NNA–treated, but not from control, dogs. Bradykinin-induced increase in 6-ketoprostaglandin F
1α
production was greater in coronary arteries taken from L-NNA–treated dogs (+179±41 pg/mm
2
) than from control dogs (+66±18 pg/mm
2
) (
P
<.05). These results indicate that (1) NO is of major importance in the control of systemic but not coronary resistance vessels at rest and during exercise, and (2) after L-NNA, the cyclooxygenase pathway is involved in myocardial reactive hyperemia and in the residual relaxation to bradykinin of isolated coronary arteries. Thus, in conscious dogs, the cyclooxygenase pathway might act as a protective mechanism of the coronary circulation when endothelial nitric oxide synthesis is altered.
Disruption of Hemoglobin Oxygen Transport Does Not Impact Oxygen-Dependent Physiological Processes in Developing Embryos of Zebra Fish (
Danio rerio
)
Authors: Pelster Bernd , Burggren Warren W.
Published: June 2012

Embryonic hemoglobin circulated by the developing heart in the early vertebrate embryo is widely assumed (without substantiation) to perform the same vital role of O
2
carriage that it does in fetuses and adults. In order to challenge this assumption, we measured highly O
2
-dependent physiological variables like O
2
consumption, cardiac performance, and initial swim bladder filling in the presence and absence of functional hemoglobin in the embryos and early larvae of the zebra fish,
Danio
(=
Brachydanio
)
rerio
. Functional ablation of hemoglobin by carbon monoxide or phenylhydrazine did not reduce whole-animal O
2
consumption, which was ≈85 to 90 μmol·g
−1
·h
−1
. Similarly, no differences in heart variables like ventricular pressure development or heart rate, which increased from 135 to 175 bpm between stages 36h and 96h (indicating developmental stages 36 and 96 hours after fertilization, respectively), were observed in these experiments. Initial opening of the swim bladder was not influenced in the presence of CO-occupied hemoglobin but was significantly impaired when the embryonic hemoglobin was chemically modified by incubation with phenylhydrazine. That aerobic processes continue without hemoglobin O
2
transport indicates the adequacy in the embryo of simple O
2
diffusion alone even in developmental stages with extensive convective blood circulation generated by the heart.
Protein Kinase C–Induced Changes in the Stoichiometry of ATP Binding Activate Cardiac ATP-Sensitive K
+
Channels
Authors: Light Peter E. , Sabir Aftab A. , Allen Bruce G. , Walsh Michael P. , French Robert J.
Published: June 2012

Activation of both ATP-sensitive K
+
(K
ATP
) channels and the enzyme protein kinase C (PKC) has been associated with the cardioprotective response of ischemic preconditioning. We recently showed that at low cytoplasmic ATP (≤50 μmol/L), PKC inhibits K
ATP
channel activity. This finding is surprising, as both K
ATP
channels and PKC are activated during preconditioning. However, PKC also altered ATP binding to the channel, changing the Hill coefficient from ≈2 to ≈1. This apparent change in stoichiometry would lead to a PKC-induced activation of K
ATP
channels at more physiological (millimolar) levels of ATP. The aim of the present study was to determine whether PKC activates cardiac K
ATP
channels at millimolar levels of ATP. The effects of PKC on single K
ATP
channels were studied at millimolar internal ATP levels using excised inside-out membrane patches from rabbit ventricular myocytes. Application of purified constitutively active PKC (20 nmol/L) to the intracellular surface of the patches produced an approximately threefold increase in the channel open probability. The specific PKC inhibitor peptide PKC(19-31) prevented this increase. Heat-inactivated PKC had no effect on K
ATP
channel properties. K
ATP
channel activity spontaneously returned to control levels after washout of PKC. This spontaneous reversal did not occur in the presence of 5 nmol/L okadaic acid, suggesting that the reversal of PKC's action is dependent on activity of a membrane-associated type 2A protein phosphatase (PP2A). In the presence of exogenous PP2A (7.5 nmol/L), PKC had no effect. We conclude that the PKC-induced increase in K
ATP
channel activity at millimolar ATP results from a crossing of the ATP concentration-response curves for inhibition of the phosphorylated and nonphosphorylated forms of the channel. This identifies a mechanism by which PKC activates K
ATP
channels at near physiological levels of ATP and thus could link these two components in a signaling pathway that induces ischemic preconditioning.
Time Course of Late Preconditioning Against Myocardial Stunning in Conscious Pigs
Authors: Tang Xian-Liang , Qiu Yumin , Park Seong-Wook , Sun Jian-Zhong , Kalya Anantharam , Bolli Roberto
Published: June 2012

We have recently found in conscious pigs that a sequence of brief coronary occlusions induces severe myocardial stunning, but when the same sequence is repeated 24 hours later, the severity of stunning is markedly reduced (≈50%) (“late preconditioning against stunning”). As an initial step toward elucidating the mechanism and potential clinical significance of this powerful cardioprotective response, the present study was conducted to define the time course of late preconditioning against myocardial stunning. Conscious pigs underwent a sequence of ten 2-minute coronary occlusion/2-minute reperfusion cycles and then a second identical sequence at 6 hours (group I, n=7), 12 hours (group II, n=6), 24 hours (group III, n=10), 3 days (group IV, n=10), or 6 days (group V, n=11) after the first. Systolic wall thickening (WTh) in the ischemic/reperfused region remained significantly depressed for at least 3 hours after the 10th reperfusion of the first sequence, indicating myocardial stunning. When the second sequence of coronary occlusions was performed 6 hours after the first (group I), the recovery of WTh was similar to the first. In contrast, when the second sequence was repeated 12 hours after the first (group II), the recovery of WTh was improved, though not consistently, and the total deficit of WTh decreased by 41% (
P
<.05) compared with the first sequence. When the second sequence was repeated 24 hours (group III) and 3 days (group IV) after the first, the recovery of WTh was substantially enhanced, with 52% and 49% reductions in the total deficit of WTh, respectively (
P
<.01 versus the first sequence). When the second sequence was repeated 6 days later (group V), the recovery of WTh was indistinguishable from the first sequence. Thus, late preconditioning against myocardial stunning requires >6 hours to develop, lasts for at least 60 hours after its appearance (with the most effective protection present at 24 hours and 3 days), and disappears within 6 days after the preconditioning ischemia, a time course that is consistent with the synthesis and degradation of cardioprotective proteins. In view of its sustained duration, this endogenous cardioprotective mechanism is of potential clinical importance.
Inhomogeneous Disappearance of Myofilament-Related Cytoskeletal Proteins in Stunned Myocardium of Guinea Pig
Authors: Matsumura Yasushi , Saeki Eijiro , Inoue Michitoshi , Hori Masatsugu , Kamada Takenobu , Kusuoka Hideo
Published: June 2012

The decrease in Ca
2+
responsiveness of myofilaments in stunned myocardium implies that there may be structural changes in proteins composing the contractile machinery. To elucidate the lesion in stunned myocardium, isolated guinea pig hearts were subjected to global ischemia at 37°C and reperfused. SDS-PAGE revealed that the contents of desmin, α-actinin, and spectrin decreased in the myofibrillar fraction isolated from hearts reperfused after 60-minute ischemia compared with nonischemic control hearts. To examine the change of cytoskeletal proteins in stunned myocardium, immunohistochemical studies with antibodies against these proteins were performed after 15 minutes of ischemia. In stunned myocardium, the staining was largely intact, but there were some lesions where desmin was not stained and α-actinin and spectrin were only weakly identified. The percentage of normally stained areas in the myocardium (percent stained area), quantified by image processing, was significantly lower in stunned myocardium (79.6±3.6%, mean±SEM) than in nonischemic control myocardium (96.5±0.7%). Percent recovery of developed pressure significantly correlated with percent stained area (
r
=.82,
P
<.001). In hearts subjected to 15-minute ischemia but not reperfused, or in hearts reperfused with Ca
2+
-free solution after 15-minute ischemia, staining by the antibodies remained intact, suggesting that the change of the cytoskeletal proteins is mediated by Ca
2+
overload during reperfusion. In hearts treated with the protease inhibitor leupeptin (50 μmol/L) or calpain inhibitor I (100 μmol/L), both developed pressure and staining were well preserved. These results indicate that contractile dysfunction in stunned myocardium has a strong correlation with the disappearance of cytoskeletal proteins that may be mediated by a Ca
2+
-dependent intracellular protease activated during reperfusion. The disruption of cytoskeletal proteins is a possible mechanism for stunning, although it may be a secondary effect of protease activation.
Activation of β-Adrenergic Receptor Kinase During Myocardial Ischemia
Authors: Ungerer Martin , Kessebohm Kerstin , Kronsbein Kai , Lohse Martin J. , Richardt Gert
Published: June 2012

During myocardial ischemia, a local release of noradrenaline coincides with an increased density of β-adrenergic receptors. The functional activity of these receptors, however, is mainly determined by their state of phosphorylation. The β-adrenergic receptor kinase (βARK) specifically phosphorylates and thereby inactivates β-adrenergic receptors after stimulation by receptor agonists, facilitating the binding of the inhibitor protein β-arrestin to the receptors. βARK activation involves a translocation of the enzyme to the membrane. In the present study, we investigated the density and the functional activity of β-adrenergic receptors, the enzymatic activity of βARK in membranes and cytosol, the mRNA levels of βARK-1, and the expression of β-arrestin during stop-flow and low-flow ischemia in the isolated perfused rat heart. After 60 minutes of stop-flow ischemia, β-adrenergic receptor density was upregulated, but β-agonist–mediated adenylate cyclase activity was blunted. Simultaneously, βARK activity in the particulate fraction was significantly induced. The increase in βARK activity was reversible after inhibition of ischemia-evoked noradrenaline release by desipramine. Also, exposure to externally given noradrenaline increased βARK activity in the particulate fraction. Cytosolic βARK activity remained largely unchanged during stop-flow or low-flow ischemia. The steady state concentration of βARK-1 mRNA increased after 20 minutes of stop-flow ischemia and then returned to baseline values after another 20 minutes. Cardiac ischemia did not alter β-arrestin levels. During myocardial ischemia, an increase in the number of β-adrenergic receptors is paralleled by increased membrane activity of the receptor kinase βARK. This increased membrane activity may contribute to enhanced receptor phosphorylation and inactivation.
Gap Junctions in Vascular Tissues
Authors: Christ George J. , Spray David C. , El-Sabban Marwan , Moore Lisa K. , Brink Peter R.
Published: June 2012

Integration and coordination of responses among vascular wall cells are critical to the local modulation of vasomotor tone and to the maintenance of circulatory homeostasis. This article reviews the vast literature concerning the principles that govern the initiation and propagation of vasoactive stimuli among vascular smooth muscle cells, which are nominally the final effectors of vasomotor tone. In light of the abundance of new information concerning the distribution and function of gap junctions between vascular wall cells throughout the vascular tree, particular attention is paid to this integral aspect of vascular physiology. Evidence is provided for the important contribution of intercellular communication to vascular function at all levels of the circulation, from the largest elastic artery to the terminal arterioles. The thesis of this review is that the presence of gap junctions, in concert with the autonomic nervous system, pacemaker cells, myogenic mechanisms, and/or electrotonic current spread (both hyperpolarizing and depolarizing waves through gap junctions), confers a plasticity, adaptability, and flexibility to vasculature that may well account for the observed diversity in regulation and function of vascular tissues throughout the vascular tree. It is hoped that the summary information provided here will serve as a launching pad for a new discourse on the mechanistic basis of the integrative regulation and function of vasculature, which painstakingly accounts for the undoubtedly complex and manifold role of gap junctions in vascular physiology/dysfunction.
Conditional Transgene Expression in the Heart
Authors: Yu Zhihui , Redfern Charles S. , Fishman Glenn I.
Published: June 2012

Conditional transgene expression is a potentially useful approach to investigate complex biological systems in vivo. We recently demonstrated that tetracycline-responsive promoters could be employed to achieve regulated, cardiac-specific expression of target genes in transgenic mice. To more fully define the quantitative and spatial parameters associated with tetracycline-regulated gene expression in the heart, we crossed transgenic mice harboring either a firefly luciferase or a nuclear-localized bacterial
lacZ
target gene with strains expressing a tetracycline-controlled transactivator (tTA) under the regulatory control of 2.9 kb of 5′ flanking sequence from the rat α-myosin heavy chain gene. Luciferase activity was induced nearly 300-fold in the hearts of binary-transgenic mice compared with mice carrying only the luciferase reporter gene. No significant transactivation was observed in any other tissues examined. Binary transgenics harboring the
lacZ
reporter gene showed substantial β-galactosidase activity throughout the heart, but the response of individual cardiac myocytes was heterogeneous. For both reporter genes, tetracycline treatment fully repressed tTA-dependent transactivation. These data provide important insights into the nature of studies that can be successfully addressed using the tetracycline-regulated gene expression system in the heart.
Thrombin Activates the Sarcolemmal Na
+
-H
+
Exchanger
Authors: Yasutake Masahiro , Haworth Robert S. , King Anna , Avkiran Metin
Published: June 2012

Thrombin can activate the plasma membrane Na
+
-H
+
exchanger in a variety of noncardiac cells. We have studied (1) the effect of thrombin on the activity of the sarcolemmal Na
+
-H
+
exchanger in freshly isolated quiescent ventricular myocytes from the adult rat heart and (2) the signaling mechanism(s) underlying any effect. Reverse-transcription polymerase chain reaction analysis revealed thrombin receptor mRNA expression in a myocyte-enriched cell preparation. As an index of Na
+
-H
+
exchanger activity, acid efflux rates (
J
H
s) were determined in single myocytes (n=4 to 11 per group) loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 after two consecutive intracellular acid pulses (induced by transient exposure to 20 mmol/L NH
4
Cl) in bicarbonate-free medium. At a pH
i
of 6.9,
J
H
did not change significantly during the second pulse relative to the first in control cells. However, when the second pulse occurred in the presence of 0.2, 1, or 5 U/mL thrombin,
J
H
increased by 30%, 62% (
P
<.05), and 87% (
P
<.05), respectively. A hexameric thrombin receptor–activating peptide (SFLLRN) mimicked the effect of thrombin and increased
J
H
by 73% (
P
<.05) at 25 μmol/L. In contrast, an inactive control peptide (FLLRN) was without effect at 25 μmol/L. In cells pretreated with 100 nmol/L GF109203X or 5 μmol/L chelerythrine (protein kinase C inhibitors), neither 5 U/mL thrombin nor 25 μmol/L SFLLRN produced a significant increase in
J
H
. In the presence of 10 μmol/L HOE-694 (a Na
+
-H
+
exchanger inhibitor), pH
i
did not recover after an acid load, even during exposure to 5 U/mL thrombin or 25 μmol/L SFLLRN, confirming that the Na
+
-H
+
exchanger was the primary acid efflux mechanism under the conditions used. Neither 5 U/mL thrombin nor 25 μmol/L SFLLRN affected resting pH
i
and Ca
2+
or background acid loading. We conclude that (1) adult rat ventricular myocytes express a functional thrombin receptor, whose stimulation results in increased activity of the sarcolemmal Na
+
-H
+
exchanger, and (2) this effect appears to occur through a protein kinase C–mediated mechanism.
Insulin-like Growth Factor II Induces DNA Synthesis in Fetal Ventricular Myocytes In Vitro
Authors: Liu Qingquan , Yan Huajun , Dawes Nicola J. , Mottino Giuliano A. , Frank Joy S. , Zhu Hong
Published: June 2012

Insulin-like growth factor II (IGF2) belongs to a family of growth factors that includes insulin and insulin-like growth factor I (IGF1). Although the accumulating evidence indicates that IGF1 is involved in regulating proliferation of ventricular myocytes, the role of IGF2 is less clear. To gain more insight into the functions of IGF2, rat ventricular expression of IGF2 mRNA at four developmental stages was examined by Northern analysis. An abundant IGF2 mRNA of ≈3.8 kb was detected in fetal ventricles. It was dramatically decreased in neonatal ventricles and became undetectable in juvenile and adult ventricles. Similar expression patterns of the mRNA encoding IGF1 receptor and IGF2 receptor were observed. Since the results of Northern analysis strongly suggest the importance of IGF2 in regulating proliferation of fetal rat ventricular myocytes, the effects of an exogenous IGF2 on DNA synthesis in cultured rat ventricular myocytes were determined. DNA synthesis, which was monitored by measuring 5-bromo-2′-deoxyuridine (BrdU) and [
3
H]thymidine incorporation, was increased by twofold to threefold in IGF2-stimulated fetal ventricular myocytes, whereas no change in BrdU or [
3
H]thymidine incorporation was observed in neonatal ventricular myocytes. Instead, IGF2 seemed to induce hypertrophy in neonatal ventricular myocytes. An antisense oligonucleotide against rat IGF2 mRNA was able to significantly reduce BrdU incorporation, and this effect was quantitatively reversed by the addition of exogenous IGF2. Reversion by exogenous IGF2 was abolished by a monoclonal antibody against IGF1 receptor. In conclusion, our results suggest that IGF2 directly regulates proliferation of fetal rat ventricular myocytes in a paracrine/autocrine fashion.
Ceramide Is Not a Signal for Tumor Necrosis Factor–Induced Gene Expression but Does Cause Programmed Cell Death in Human Vascular Endothelial Cells
Authors: Slowik Mark R. , De Luca Linda G. , Min Wang , Pober Jordan S.
Published: June 2012

Tumor necrosis factor (TNF) activates transcription of endothelial leukocyte adhesion molecule-1 (CD62E) in endothelial cells (ECs) through the binding to the gene promoter of the p50/p65 heterodimeric form of nuclear factor-κB (NF-κB) and of the N-terminal phosphorylated form of the ATF2/c-Jun transcription factor, which is phosphorylated by Jun N-terminal kinase (JNK). However, the intracellular signaling pathways that activate endothelial NF-κB and JNK in TNF-induced responses are unknown. In this study we have examined the role of a recently described TNF signaling pathway involving sphingomyelin activation to generate ceramide, a potential intracellular mediator. We find that concentrations of TNF that strongly activate NF-κB and JNK within 15 minutes do not produce either a measurable decline in sphingomyelin or a measurable generation of ceramide in cultured human umbilical vein ECs at any time examined. Stimulation of ECs with purified sphingomyelinase (SMase) enzyme causes a rapid 60% to 80% decrease in cellular sphingomyelin content and a large increase in ceramide. However, SMase treatment only minimally activates NF-κB, achieving levels that are insufficient to initiate gene transcription. Extracellular SMase does not have access to intracellular sphingomyelin, but treatment of ECs with membrane-permeant ceramide analogues still completely fails to activate NF-κB and only activates JNK at late times. Neither SMase nor ceramide analogues induce gene transcription or surface expression of endothelial leukocyte adhesion molecules that are readily induced by TNF. Strikingly, low concentrations of membrane-permeant ceramide cause programmed cell death in ECs, a finding not observed at any concentrations of TNF tested. We conclude that ceramide is not an important second messenger for TNF signaling of gene transcription in ECs but may be a second messenger for cell death in response to as-yet-unidentified signals.
cAMP-Mediated Vascular Protection in an Orthotopic Rat Lung Transplant Model
Authors: Naka Yoshifumi , Roy Dilip K. , Liao Hui , Chowdhury Nepal C. , Michler Robert E. , Oz Mehmet C. , Pinsky David J.
Published: June 2012

Prostaglandin E
1
(PGE
1
) is often added to the donor pulmonary flush solution to enhance clinical lung preservation for transplantation. Although PGE
1
is thought to act as a pulmonary vasodilator during the harvest period, the precise mechanism(s) of action whereby PGE
1
enhances lung preservation is unknown. Because cAMP levels decline in endothelial and vascular smooth muscle cells exposed to hypoxia, we hypothesized that a PGE
1
-mediated increase in cAMP levels within the preserved lungs might improve pulmonary vascular homeostasis following lung transplantation. Rat lungs demonstrated a time-dependent decline in cAMP levels during hypothermic storage, with cAMP levels significantly increased by PGE
1
supplementation (≈2-fold by 6 hours,
P
<.0005). To test whether augmenting cAMP levels may enhance lung preservation, experiments were performed using an orthotopic rat left lung transplant model. Compared with controls, supplementing the preservation solution with the membrane-permeable cAMP analogue dibutyryl-cAMP resulted in dose-dependent preservation enhancement, marked by reduced pulmonary vascular resistance (6.0-fold,
P
<.01), improved arterial oxygenation (3.0-fold,
P
<.01), reduced graft neutrophil infiltration (1.5-fold,
P
<.05), and improved recipient survival (7.0-fold,
P
<.005). Similar preservation enhancement was observed with another cAMP analogue (8-bromo-cAMP) or the phosphodiesterase inhibitor indolidan. Stimulating the cAMP second messenger system by PGE
1
supplementation resulted in marked hemodynamic benefits and improved recipient survival, in parallel with reduced graft neutrophil infiltration, vascular permeability, and platelet deposition. These beneficial effects of PGE
1
were abrogated by simultaneous administration of the cAMP-dependent protein kinase antagonist Rp-cAMPS. Although an arterial vasodilator (minoxidil) resulted in significant pulmonary vasodilation during harvest, it lacked other nonvasodilating effects of PGE
1
and resulted in poor preservation. These data show that harvest vasodilation by itself is insufficient to enhance lung preservation and that PGE
1
enhances lung preservation by stimulating the cAMP-dependent protein kinase and promoting nonvasodilatory mechanisms of pulmonary protection.
Vascular Smooth Muscle α
v
β
3
Integrin Mediates Arteriolar Vasodilation in Response to RGD Peptides
Authors: Mogford Jon E. , Davis George E. , Platts Steven H. , Meininger Gerald A.
Published: June 2012

Arteriolar vasodilation and the resultant increase in blood flow are characteristic vascular responses to tissue injury. The dilatory mediators signaling these responses are incompletely understood. We show that integrin-binding peptides containing the Arg-Gly-Asp (RGD) tripeptide sequence cause immediate and, in some instances, sustained vasodilation when applied to isolated rat cremaster arterioles. The vasodilation is dependent on interaction of the soluble RGD sequence with the α
v
β
3
integrin expressed by smooth muscle cells in the arteriolar wall. Possible in vivo sources of soluble RGD sequences are fragments of extracellular matrix proteins that are generated after tissue injury. Indeed, protease-generated fragments of denatured collagen type I (a major source of RGD sequences) also cause cremaster arteriolar vasodilation through the α
v
β
3
integrin. Thus, extracellular matrix protein fragments containing the RGD sequence may act as vascular wound recognition signals to regulate blood flow to injured tissue.
Sustained Increase in Aortic Endothelial Nitric Oxide Synthase Expression In Vivo in a Model of Chronic High Blood Flow
Authors: Nadaud Sophie , Philippe Monique , Arnal Jean-Francois , Michel Jean-Baptiste , Soubrier Florent
Published: June 2012

Physiological adaptation of normal blood vessels to acute or chronic changes in blood flow is endothelium dependent. In vitro studies have shown that, among other genes, NO synthase (NOS) 3 mRNA and protein expression is enhanced by acute elevation of shear stress in endothelial cells. We have investigated the effect of chronic high blood flow on NOS3 mRNA and protein expression in rat aorta. NOS3 mRNA levels were measured by quantitative polymerase chain reaction (PCR) in the aortas of 12 rats with arteriovenous fistulas and 9 sham-operated control rats. The PCR assay indicated that NOS3 mRNA levels were significantly enhanced (twofold) during high blood flow. Western blots showed that immunoreactive NOS3 levels were also increased to a similar extent. Furthermore, the Ca
2+
-dependent NOS activity, measured by the
l
-arginine to
l
-citrulline conversion assay, and the cGMP content were also significantly increased in the proximal aortic wall submitted to the arteriovenous shunt. These results indicate that NOS3 mRNA and protein expression is enhanced in vivo during chronic high blood flow.
Mildly Oxidized LDL Evokes a Sustained Ca
2+
-Dependent Retraction of Vascular Smooth Muscle Cells
Authors: Augé Nathalie , Fitoussi Guylène , Bascands Jean-Loup , Pieraggi Marie-Thérèse , Junquero Didier , Valet Philippe , Girolami Jean-Pierre , Salvayre Robert , Nègre-Salvayre Anne
Published: June 2012

Oxidized low density lipoprotein (LDL) is thought to play a major role in atherogenesis. Atherosclerotic arteries exhibit structural changes associated with profound alterations in vascular tone that are potentially involved in arterial spasm and ischemic heart disease. We report here the role of oxidized LDL in the retraction of vascular smooth muscle cells. Mildly oxidized LDL elicited a broad and sustained peak in cytosolic calcium concentration ([Ca
2+
]
i
) in cultured arterial smooth muscle cells. Concomitant with the [Ca
2+
]
i
rise, oxidized LDL evoked a sustained and intense retraction of smooth muscle cells, as shown by the changes in cross-sectional area of single cells. Cell retraction was dependent on time, the concentration of oxidized LDL, and the level of LDL oxidation (native LDL induced neither a significant [Ca
2+
]
i
rise nor cell retraction). Oxidized LDL but not native LDL also elicited a delayed (12±2 hours) and sustained (14±2 hours) increase in isometric tension in deendothelialized arterial rings only, thus suggesting a protective role of intact endothelium. When triggered by nontoxic doses of oxidized LDL, retraction of cultured cells and the contractile response of aortic rings was reversible, whereas with higher (toxic) doses (≥200 μg apoB/mL), cell retraction was irreversible and led progressively to detachment and cell death. Cell retraction can be prevented in three ways: (1) by inhibiting LDL oxidation with supplements of antioxidants (indirect inhibition); (2) by blocking the pathogenic intracellular signaling elicited by oxidized LDL (direct inhibition), eg, by inhibiting calcium influx with EGTA or the calcium channel blocker nisoldipine or by blocking intracellular signaling (at a still-unknown step) by the lipophilic antioxidant α-tocopherol; and (3) by directly inhibiting myosin light chain kinase by 1-(5-chloronaphthalene-1-sulfonyl)-
1H
-hexahydro-1,4-diazepine. In conclusion, oxidized LDL evoked a sustained and intense calcium-dependent retraction of cultured smooth muscle cell, which can be prevented by inhibiting LDL oxidation or by blocking the intracellular signaling induced by oxidized LDL.
Macromolecular Composition of Stress Fiber–Plasma Membrane Attachment Sites in Endothelial Cells In Situ
Authors: Kano Yumiko , Katoh Kazuo , Masuda Michitaka , Fujiwara Keigi
Published: June 2012

Stress fibers (SFs) are present along the apical (apical SF) and the basal (basal SF) portions of cultured cells. We have recently shown that apical SFs are anchored to the apical plasma membrane (PM) in a manner similar to how basal SFs are attached to focal adhesion sites. We propose calling such apical SF–membrane attachment sites “apical plaques.” To study the macromolecular composition of the apical plaque and the focal adhesion in endothelial cells (ECs) in situ, we examined by confocal laser scanning and fluorescence microscopy guinea pig aortae stained with various antibodies against focal adhesion–associated proteins. Basal SFs oriented parallel to the blood flow direction were mainly located in the upstream half of the cell. Thin apical SFs were also observed. Spotty staining patterns were observed in the basal and the apical portions of cells stained with anti-vinculin, anti-talin, anti-paxillin, or anti-fibronectin receptor, indicating the presence of focal adhesions and apical plaques in ECs in situ. Although fibronectin receptors were present in the apical plaque, fibronectin was not detected on the apical cell surface. Our data suggest that the molecules responsible for the SF-PM association are the same between in vitro and in situ cells. Our results appear to support a hypothesis that the SF system is involved in sensing and/or signal transduction of fluid mechanical forces.
Role of Endothelium-Related Mechanisms in the Pathophysiology of Renal Ischemia/Reperfusion in Normal Rabbits
Authors: Caramelo C. , Espinosa G. , Manzarbeitia F. , Cernadas M.R. , Tejerizo G. Pe´rez , Tan D. , Mosquera J.R. , Digiuni E. , Monto´n M. , Milla´s I. , Hernando L. , Casado S. , Lo´pez-Farre´ A.
Published: June 2012

The present study addressed the effect of interventions aimed to increase NO in the setting of acute renal ischemia/reperfusion (I/R) in uninephrectomized rabbits. In the 60-minute post-I/R period,
l
-arginine+superoxide (O
2
•−
) dismutase (SOD) synergistically improved the renal functional (69.4% versus 10.4% of the pre-I/R glomerular filtration rate with or without
l
-arginine+SOD, respectively;
P
<.01) and histological parameters (82.9% decrease of medullary congestion in
l
-arginine+SOD,
P
<.01 versus vehicle) and blocked the I/R-dependent neutrophil accumulation (89.3% reduction). In spite of these results over the short term, a second set of experiments disclosed that the protection by
l
-arginine+SOD was no longer present at 24 and 48 hours (plasma creatinine in vehicle-treated versus
l
-arginine+SOD–treated animals [mg/100 mL]: 24 hours after I/R, 9.4±1.9 versus 8.07±0.65; 48 hours after I/R, 11.6±3.6 versus 9.7±0.9;
P
=NS in all the cases). Additional experiments were conducted using a milder 30-minute ischemic model, which showed no significant functional or histological protection by using
l
-arginine+SOD. In conclusion, our experiments disclosed the following: (1) the critical importance of the interaction between NO and O
2
•−
in the acute protective effect of
l
-arginine (this effect not only improved renal function and histology but also reduced neutrophil accumulation) and (2) the discordance existing between the immediate protection afforded by
l
-arginine+SOD and the lack of protection observed at 24 and 48 hours. This finding suggests that a punctual intervention on the NO system at the time of I/R is not sufficient to reduce renal damage over the long term.
Selectively Enhanced Cellular Signaling by G
i
Proteins in Essential Hypertension
Authors: Pietruck Frank , Moritz Albrecht , Montemurro Michael , Sell Alexandra , Busch Stefan , Rosskopf Dieter , Virchow Sebastian , Esche Helmut , Brockmeyer Norbert , Jakobs Karl H. , Siffert Winfried
Published: June 2012

Recent studies have shown an enhanced signaling capacity of receptors coupled to pertussis toxin (PTX)–sensitive guanine nucleotide–binding proteins (G proteins) in immortalized B lymphoblasts from patients with essential hypertension. In the present study, we analyzed (1) whether such alterations would also be expressed in nontransformed cells of these individuals and (2) whether other G protein–mediated signaling pathways were also altered. Therefore, we established primary cultures of skin fibroblasts from previously characterized normotensive and hypertensive individuals (NT and HT cells, respectively). [Ca
2+
]
i
rises induced by
lyso
-phosphatidic acid (LPA), thrombin, and sphingosine-1-phosphate as well as the formation of inositol 1,4,5-trisphosphate and [
3
H]thymidine incorporation evoked by LPA were PTX sensitive and enhanced twofold in HT fibroblasts. In contrast, cellular responses induced by bradykinin, endothelin-1, and angiotensin II (all PTX insensitive) were similar in NT and HT cells. Formation of cAMP induced by stimulation of G
s
with isoproterenol was identical in NT and HT cells. Western blot analysis yielded no evidence for an overexpression of Gα
i2
, Gα
i3
, Gβ
2
, and Gβ
4
. Furthermore, sequencing of cDNAs encoding for the ubiquitously expressed PTX-sensitive G protein subunits Gα
i2
, Gα
i3
, Gβ
1
, and Gβ
2
from NT and HT cell lines yielded no evidence for mutations in these genes. Although the molecular mechanisms remain to be defined, these data support the concept of a selective enhancement of signal transduction via PTX-sensitive G proteins in essential hypertension.
Effects of FK-506 on Contraction and Ca
2+
Transients in Rat Cardiac Myocytes
Authors: McCall Eileen , Li Li , Satoh Hiroshi , Shannon Thomas R. , Blatter Lothar A. , Bers Donald M.
Published: June 2012

FK-506 binding protein (FKBP) has been reported to be closely associated with the ryanodine receptor in skeletal and cardiac muscle and to modulate sarcoplasmic reticulum (SR) Ca
2+
release channel gating in isolated channels. FK-506 can inhibit the activity of FKBP, thereby reversing its effects on SR Ca
2+
release. We investigated the function of FKBP during normal contractions and Ca
2+
transients in intact rat ventricular myocytes loaded with fluorescent Ca
2+
indicators. FK-506 significantly increased steady state twitch Ca
2+
transients and contraction amplitudes even under conditions in which the SR Ca
2+
load and Ca
2+
current were unaltered, suggesting that FK-506 increases the fraction of SR Ca
2+
released during excitation-contraction (E-C) coupling. Action potentials were somewhat prolonged, consistent with the larger Ca
2+
transients causing greater inward Na
+
-Ca
2+
exchange current. FK-506 did not affect SR Ca
2+
uptake but modestly decreased Ca
2+
extrusion via Na
+
-Ca
2+
exchange in intact cells (although no effect on Na
+
-Ca
2+
exchange was seen in sarcolemmal vesicles). In most cells, FK-506 caused an increase in SR Ca
2+
content during steady state stimulation, as assessed by caffeine-induced contractures. This was probably due to the inhibition of Ca
2+
efflux via Na
+
-Ca
2+
exchange. FK-506 also accelerated the rest decay of SR Ca
2+
content and increased the frequency of resting Ca
2+
sparks about fourfold. The increase in frequency of these basic Ca
2+
release events was not associated with changes in the amplitude or duration of the Ca
2+
sparks. We conclude that FK-506 increases the fraction of SR Ca
2+
released during normal twitches and enhances the rate of SR Ca
2+
release during rest. FK-506 also inhibits Na
+
-Ca
2+
exchange, although this effect may be indirect. These effects are consistent with an important SR-stabilizing effect of FKBP in intact rat ventricular myocytes.
Further Studies of the Effects on Myocardial Energy Utilization Elicited by Nitroglycerin
Authors: DARBY T. D. , ALDINGER E. E.
Published: June 2012

Since sympathetic stimulation could play an important part in the short term attacks of angina pectoris, the effects of nitroglycerin on the myocardial response to levarterenol were investigated. Levarterenol infusions directly increased the isometric systolic tension (IST) developed by the cardiac muscle. Nitroglycerin had no apparent direct effect on this increase in IST either in the normal heart or in the heart following partial coronary ligation. In experiments in which IST was measured with a lever system adjusted to the end-diastolic length and tension occurring just prior to the contraction, nitroglycerin was found to have an indirect depressant effect on the levarterenol response. This decrease in contractility was shown to be secondary to a decrease in hemodynamic work load elicited by nitroglycerin. It is suggested that the chief effect of nitroglycerin in the relief of angina pectoris is due to this agent's ability to decrease a work load imposed on the myocardium by sympathetic stimulation.
The Electrocardiogram of the Normal and Hypertensive Rat
Authors: SAMBHI MOHINDER P. , WHITE FRED N.
Published: June 2012

Electrocardiograms of normal rats and those rendered hypertensive by potassium deficiency have been compared. The unipolar chest leads were found to be useful in the recognition of spurious patterns among normal animals as well as in evaluating the severity of the hypertension. Backward orientation of the spatial QRS vector in the horizontal phane was the single most consistent finding in the hypertensive animals.
The Influence of Polyvinyl Chloride (P.V.C.) Tubing on the Isolated Perfused Rat's Heart
Authors: MEYLER F. L. , WILLEBRANDS A. F. , DURRER D.
Published: June 2012

Perfusion of the isolated rat's heart through certain types of polyvinyl chloride tubing results in a deterioration of cardiac performance. Since plastics are commonly used in heart-lung machines and transfusion-sets, this observation may be of some importance.
Importance of Myocardial Reserve in Determining Changes in Heart Rate during Levarterenol Infusion
Authors: SOLOFF LOUIS A. , CORTEZ FELIX , WINTERS WILLIAM L.
Published: June 2012

Levarterenol-induced hypertension may slow, increase or produce no change in the cardiac rate of persons with cardiovascular disease. The degree of cardiac disability at least partly determines the type of response.
Action of Long-Chain Polymers on Kidney Juxtaglomerular Cells and Connective Tissue Mast Cells
Authors: CORBASCIO ALDO N.
Published: June 2012

The histochemical nature and the biologic characteristics of the specific cytoplasmic granulations of the juxtaglomerular (J-G) cells of the kidney have been reinvestigated. In the rat, it appears that these cells are mesenchymal in origin and capable of reacting as part of the reticuloendothelial system when exposed to the action of long-chain polymers like dextran or ovomucoid.

The substance contained in the granules is probably constituted by a mixture of polysaccharide sulfate esters and lipoids, accounting for their staining properties with metachromatic dyes. Hyaluronic acid is not responsible for the latter reaction.

The J-G granules respond in a manner strikingly similar to that of mast-cell granules after treatment with dextran or ovomucoid. The attempt to quantitate the amount of histamine liberated by degranulation of J-G cells has been less successful than with degranulated connective tissue mast cells, owing to the low J-G cell/parenchyma ratio in the kidney.

The presence of mast-cell-like elements at the vascular pole of the kidney glomerulus is stressed in view of their possible role in the functional activity of the nephron, owing to the fact that degranulation of these cells is capable of releasing anticoagulant and vasoactive substances like heparin, histamine, and serotonin proximal to 1 of the most important circulatory districts of the body.

The finding of elements with analogous characteristics in the subintimal layer of arterioles in other organs raises the question as to whether J-G cells play a less specialized role in the kidney than hitherto supposed.
Butter, Corn Oil and Fibrinolysis in Rats
Authors: TILLMAN ROOSEVELT L. , O'NEAL ROBERT M. , THOMAS WILBUR A. , HIXON BARBARA BARTELS
Published: June 2012

We have recently reported a dietary method for the production of thrombosis and infarcts in rats and have suggested that part of the mechanism of thrombosis in these animals was interference by the diet with some hematologic factor, possibly fibrinolysis. In the study reported herein, we have developed a method for studying fibrinolysis in rats in vitro, utilizing an electronic device called the Thrombelastograph. Results of this study correlate well with the previously demonstirated thrombogenic properties of 2 of the diets. Rats on a "butter-thiouracil-cholesterol-bile salt" diet (known to produce thrombi and infarcts) have prolonged clotlysis times, as compared with rats on a similar diet (and not thus far associated with thrombi and infarcts) in which corn oil is substituted for butter. Although the in vitro test is performed under highly artificial conditions, they are carefully standardized and the fact that the results correlate well with previously demonstrated thrombogenic properties of the diet would suggest that it has practical value for the study of thrombogenic effects of various diets in experimental animals. It is possible that it will prove to be even more useful in the study of fibrinolysis in man.