bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of acidosis on tension and [Ca2+]iin rat cerebral arteries: is there a role for membrane potential?
Authors: Peng Hong-Li , Jensen Peter E. , Nilsson Holger , Aalkjær Christian
Published: December 2017

The cellular mechanism responsible for the reduction of tension in cerebral small arteries to acidosis is not known. In this study the role of smooth muscle intracellular Ca2+concentration ([Ca2+]i) and membrane potential for the relaxation to acidosis was investigated in isolated rat cerebral small arteries. Isometric force was measured simultaneously with [Ca2+]i(fura 2) or with membrane potential (intracellular microelectrodes), and acidosis was induced by increasing[Formula: see text] or reducing[Formula: see text] of the bathing solution. Both hypercapnic and normocapnic acidosis were associated with a reduction of intracellular pH [measured with 2′,7′-bis-(carboxyethyl)-5 (and -6)-carboxyfluorescein], caused relaxation, and reduced [Ca2+]i. However, whereas hypercapnic acidosis caused hyperpolarization, normocapnic acidosis was associated with depolarization. It is concluded that a reduction of [Ca2+]iis in part responsible for the direct effect of the acidosis on the vascular smooth muscle both during normo- and hypercapnia. The mechanism responsible for the reduction of [Ca2+]idiffers between the hypercapnic and normocapnic acidosis, being partly explained by hyperpolarization during hypercapnic acidosis, whereas it is seen despite depolarization during normocapnic acidosis.
Modulation of quinidine-induced arrhythmias by temperature in perfused rabbit heart
Authors: Spear Joseph F. , Moore E. Neil
Published: December 2017

We used low temperature to slow ion channel kinetics and studied the electrophysiological effects of quinidine at different pacing rates in isolated rabbit hearts. Fifteen epicardial electrograms together with an endocardial monophasic action potential were recorded. Epicardial activation and local recovery times were measured. Arrhythmias together with the characteristics of their mode of induction and rate were analyzed by epicardial activation sequence mapping. In the presence of quinidine, arrhythmias consistent with both triggered activity and reentry were observed. At baseline, triggered activity was not inducible, even though at 25°C the recovery time was greater than that in the presence of quinidine at 36°C. Also, with quinidine, the incidence of triggered activity decreased at 30 and 25°C. Therefore prolongation of the recovery time per se does not cause triggered activity. Quinidine’s use-dependent effects on conduction and reverse use-dependent effects on recovery time were amplified by low temperatures. These findings can be understood in terms of the known temperature sensitivities of the kinetics of the membrane ion channels responsible for activation and recovery. The results demonstrate that temperature can be used as a tool to elucidate mechanisms of drug action.
Effect of tumor necrosis factor-α and interleukin-1α on heme oxygenase-1 expression in human endothelial cells
Authors: Terry Christi M. , Clikeman Jennifer A. , Hoidal John R. , Callahan Karleen S.
Published: December 2017

Heme iron exacerbates oxidant damage by catalyzing the production of free radicals. Heme oxygenase is the rate-limiting enzyme involved in heme catabolism. An inducible form of heme oxygenase, heme oxygenase-1 (HO-1), is upregulated in oxidant and inflammatory settings, and recent work suggests that HO-1 induction may serve a protective function against oxidant injury. The ability of the endogenous inflammatory mediators, interleukin (IL)-1α, tumor necrosis factor-α (TNF-α), and IL-6, to enhance HO-1 expression in cultured human endothelial cells was examined in this study. HO-1 mRNA and protein expression were upregulated by IL-1α and TNF-α exposure but not by IL-6. Induction of HO-1 mRNA by IL-1α and TNF-α occurred in a concentration- and time-dependent fashion, with maximal expression occurring by 4 h for both cytokines. Induction depended on protein synthesis and occurred at the transcriptional level. Inhibition of the AP-1 transcription factor with curcumin decreased the cytokine induction of HO-1 mRNA, suggesting the involvement of this transcription factor in cytokine signaling of HO-1. The results of this study indicate that the endogenous inflammatory cytokines IL-1α and TNF-α induce HO-1 in endothelial cells, providing further evidence that HO-1 may be an important cellular response to inflammatory stress.
Stretch-induced protection shares a common mechanism with ischemic preconditioning in rabbit heart
Authors: Gysembergh A. , Margonari H. , Loufoua J. , Ovize A. , André-Fouët X. , Minaire Y. , Ovize M.
Published: December 2017

We sought to determine whether stretch-induced preconditioning may be related to activation of adenosine receptors, ATP-sensitive K+([Formula: see text]) channels, and/or protein kinase C (PKC) in the rabbit heart. Anesthetized rabbits underwent 30 min of coronary artery occlusion followed by 3 h of reperfusion. Ischemic preconditioning was induced by one episode of 5 min of ischemia followed by 5 min of reperfusion, and stretch preconditioning was induced by a transient volume overload. The abilities of gadolinium (Gd3+), a blocker of stretch-activated channels, glibenclamide (Glib), a blocker of [Formula: see text] channels, 8-( p-sulfophenyl)-theophylline (8-SPT), a blocker of adenosine receptors, and polymyxin B (PMXB), an antagonist of PKC, to prevent the infarct size-limiting effect of stretch-induced preconditioning were evaluated. Because the infarct size-reducing effect of stretch occurred in the absence of ischemia and was prevented by previous administration of Gd3+, Glib, 8-SPT, and PMXB, we propose that activation of mechanosensitive ion channels protects the rabbit heart from subsequent sustained ischemic insult, likely through a mechanism that involves downstream activation of PKC, adenosine receptors, and/or [Formula: see text] channels.
Monkey corpus cavernosum relaxation mediated by NO and other relaxing factor derived from nerves
Authors: Okamura Tomio , Ayajiki Kazuhide , Toda Noboru
Published: December 2017

Isolated monkey corpus cavernosum muscle strips contracted with prostaglandin F2αand treated with prazosin responded to transmural electrical stimulation with frequency-related relaxations that were abolished by tetrodotoxin. The nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine (l-NNA) significantly attenuated but did not abolish the response;l-arginine reversed the inhibition. The neurogenic relaxation was not influenced in the strips treated with atropine or calcitonin gene-related peptide (CGRP)-(8—37), a CGRP-receptor antagonist, and those desensitized to vasoactive intestinal polypeptide (VIP) or pituitary adenylate cyclase-activating polypeptide (PACAP). Nerve fibers containing NADPH diaphorase were histochemically demonstrated in cavernous tissues. The relaxant response resistant to the NO synthase inhibitor was abolished by high K+and tetrabutylammonium but was unaffected by glibenclamide, charybdotoxin, apamin, ouabain, SKF-525a, a cytochrome P-450 inhibitor, and oxyhemoglobin. It is concluded that neurogenic relaxations of monkey corpus cavernosum muscle is associated partly with NO released as a neurotransmitter and that other relaxing factor(s) possibly responsible for K+channel opening also participates; however, the type of K+channel involved is not determined. Acetylcholine, VIP, CGRP, PACAP, and the Na+pump do not seem to be involved in the neurogenic relaxation.
Does postsynaptic α1-adrenergic receptor supersensitivity contribute to autonomic dysreflexia?
Authors: Landrum Lisa M. , Thompson Gerald M. , Blair Robert W.
Published: December 2017

Quadriplegics often experience periods of severe hypertension known as autonomic dysreflexia. Clinically, these events have been well documented, but the mechanisms for mediating autonomic dysreflexia remain unclear. We used a chronic rat model to investigate the potential development of supersensitivity at postsynaptic α1-adrenergic receptors as a contributing factor to the exaggerated sympathetic response characteristic of autonomic dysreflexia. Adult male Wistar and Sprague-Dawley rats were anesthetized and given spinal transection at T5. After 30 days, rats were reanesthetized and arterial and venous catheters implanted. Twenty-four hours later, colorectal distension (CRD) was used to evoke autonomic dysreflexia in conscious, spinalized rats. To gauge changes in α1-receptor sensitivity, we assessed mean arterial pressure (MAP) in response to intravenous phenylephrine (PE) infusions. No consistent differences were observed between intact and spinalized rats. Therefore, supersensitivity of α1-receptors cannot completely account for the hypertensive bouts associated with autonomic dysreflexia. In addition, while attempting to develop an appropriate model for autonomic dysreflexia, we discovered that spinalized Wistar rats exhibited MAP responses characteristic of autonomic dysreflexia, whereas lesioned Sprague-Dawley rats did not, when subjected to CRD. Thus Wistar rats provide a better animal model for autonomic dysreflexia.
Cardiac myocyte calcium transport in phospholamban knockout mouse: relaxation and endogenous CaMKII effects
Authors: Li Li , Chu Guoxiang , Kranias Evangelia G. , Bers Donald M.
Published: December 2017

Increases in heart rate are accompanied by acceleration of relaxation. This effect is apparent at the single myocyte level and depends on sarcoplasmic reticulum (SR) Ca transport and Ca/calmodulin dependent protein kinase [CaMKII; see R. A. Bassani, A. Mattiazzi, and D. M. Bers. Am. J. Physiol. 268 ( Heart Circ. Physiol. 37): H703–H712, 1995]. Because phosphorylation of phospholamban (PLB) by CaMKII can stimulate SR Ca transport, it is a plausible candidate mechanism. We examined this issue using ventricular myocytes isolated from wild-type (WT) mice and those in which the PLB gene was ablated by gene targeting (PLB-KO). During steady-state (SS) stimulation, twitch relaxation and intracellular Ca concentration ([Ca]i) decline were significantly faster than after a rest in both WT and PLB-KO myocytes. Furthermore, the CaMKII inhibitor KN-93 (1 μM) abolished the stimulation-dependent acceleration of twitch [Ca]idecline in PLB-KO. This indicates that neither PLB nor its phosphorylation are required for the CaMKII-dependent acceleration of the SS twitch [Ca]idecline and relaxation. Other quantitative aspects of Ca transport in WT and PLB-KO myocytes were also examined. As expected, the time constant (τ) of [Ca]idecline during the SS twitch is much faster in PLB-KO than in WT myocytes (112 ± 6 vs. 188 ± 14 ms, P < 0.0001). There was also an increase in SS SR Ca load, based on the change of [Ca]iduring rapid caffeine-induced contractures (CafC) with Na/Ca exchange blocked (565 ± 74 nM for WT, 1118 ± 133 nM for PLB-KO, P < 0.01). Accounting for cytosolic Ca buffering, this implies a 37% increase in SR Ca content. The τ for [Ca]idecline of the CafC with Na present indicated slower extrusion by Na/Ca exchange in the PLB-KO mouse (2.2 ± 0.2 s in WT vs. 3.2 ± 0.2 s in PLB-KO, P < 0.01), although exchanger protein expression was unchanged. Integrated Ca flux analysis in WT and PLB-KO myocytes, respectively, shows that 90 and 96% of Ca during twitch relaxation is removed by the SR Ca-ATPase, 9 and 3.4% by Na/Ca exchange, and 0.5 and 0.1% by slow mechanisms (mitochondria Ca uniporter and sarcolemmal Ca-ATPase). We conclude that the PLB-KO myocytes retain a CaMKII-dependent acceleration of SS twitch [Ca]idecline. The PLB-KO (vs. WT) myocytes also have higher SR Ca pump activity, higher SR Ca load, and reduced Na/Ca exchange activity.
The trabecula culture system: a novel technique to study contractile parameters over a multiday time period
Authors: Janssen P. M. L. , Lehnart S. E. , Prestle J. , Lynker J. C. , Salfeld P. , Just H. , Hasenfuss G.
Published: December 2017

In the intact heart, various triggers induce alterations in gene expression that impact on contractile function. Because changes in gene expression reflect altered protein expression patterns after 12–48 h, we developed a system in which intact twitching cardiac trabeculae can be studied for multiday periods. Right ventricular trabeculae from pentobarbital sodium-anesthetized rabbits were mounted in a sterile, closed muscle chamber. Over the first 48 h, developed force (Fdev) did not significantly change: 102.3 and 98.9% of the initial Fdev was observed after 24 and 48 h, respectively ( n = 8). Also, neither diastolic force, time from peak to 50% relaxation (RT50), nor protein synthesis measured by a [3H]leucine incorporation assay changed significantly over time. Contractile response after >48 h to an increase in extracellular calcium concentration (1.8 to 2.5 mM; Fdevincreased 43.5%, n = 2) or to 1 μM isoproterenol (Fdevincreased 138.6% and RT50decreased 34.9%, n = 2) was similar to those observed in freshly dissected preparations. In conclusion, this system can investigate contractile function of multicellular preparations under well-defined physiological conditions after events that alter gene and consequent protein expression.
Insulin improves contractile function during moderate ischemia in canine left ventricle
Authors: Tune Johnathan D. , Mallet Robert T. , Downey H. Fred
Published: December 2017

This study determined the effects of insulin on myocardial contractile function and glucose metabolism during moderate coronary hypoperfusion. Coronary perfusion pressure (CPP) was lowered from 100 to 60, 50, and 40 mmHg in the left anterior descending coronary artery of anesthetized, open-chest dogs. Regional glucose uptake (GU), lactate uptake, myocardial O2consumption, and percent segment shortening (%SS) were measured without ( n = 12) or with intravenous (4 U/min, n = 12) or intracoronary insulin (4 U/min, n = 6). Glucose metabolites were also measured in freeze-clamped biopsies of control heart ( n = 6) and hearts treated with intravenous insulin ( n = 6) at the completion of the protocol (40 mmHg CPP). GU increased with intravenous and intracoronary insulin ( P < 0.01). In all groups, GU was unaffected by reduced CPP, although lactate uptake decreased significantly ( P < 0.01). Myocardial O2consumption fell ( P < 0.05) as CPP was lowered in all groups and was not altered significantly by intravenous or intracoronary insulin treatment. Without insulin, %SS decreased 72% ( P < 0.05) at 40 mmHg CPP, but in hearts treated with intravenous and intracoronary insulin, %SS was not reduced ( P > 0.05). Myocardial glycogen, alanine, lactate, and pyruvate contents were not significantly different in untreated hearts and hearts treated with intravenous insulin. Thus, in moderately ischemic canine myocardium, insulin markedly improved regional contractile function and did not appreciably increase the products of anaerobic glucose metabolism.
Arterial baroreflex during pregnancy and renal sympathetic nerve activity during parturition in rabbits
Authors: O’Hagan Kathleen P. , Casey Susan M.
Published: December 2017

The arterial baroreflex control of renal sympathetic nerve activity (RSNA) was evaluated in nine term pregnant (P) and 12 nonpregnant (NP) conscious New Zealand White rabbits. In an additional four P rabbits, the RSNA response to spontaneous parturition was measured. The blood pressure (BP)-RSNA relationship was generated by sequential inflations of aortic and vena caval perivascular occluders. Rest BP (P: 61 ± 2 vs. NP: 73 ± 2 mmHg) and the centering point of the baroreflex (P: 57 ± 2 vs. NP: 70 ± 2 mmHg) were lower ( P < 0.05) in term pregnancy. Baroreflex range (P: 246 ± 14% vs. NP 263 ± 24% of rest RSNA) was not affected by pregnancy. However, maximal reflex gain was moderately depressed (−44%) in P rabbits (P: −15 ± 1 vs. NP: −27 ± 4% of rest RSNA/mmHg; P < 0.05) due to a significant reduction in the slope coefficient. Delivery of a fetus was associated with strong renal sympathoexcitation. Peak RSNA averaged 80 ± 37% of smoke-elicited RSNA or 1,221 ± 288% of rest RSNA (mean ± SD). These results suggest that, in contrast to rat pregnancy, depressed arterial baroreflex control of RSNA in rabbit pregnancy is due primarily to a reduction in maximal gain rather than a reduction in the maximal sympathetic response to hypotension.
Intraretinal oxygen distribution in urethan-induced retinopathy in rats
Authors: Yu Dao-Yi , Alder Valerie A. , Cringle Stephen J. , Su Er-Ning , Burns Margaret
Published: December 2017

This study was performed to explore the interaction between chronic neural degeneration and the subsequent vascular remodeling. Weekly urethan administration in rats from birth produces a retinopathy model characterized by photoreceptor degeneration, retinal vascular regression, and retinal pigment epithelium (RPE) neovascularization. We investigated the hypothesis that altered oxygen distribution across the retina and choroid could be involved in the vascular changes seen in this retinopathy. We compared measurements of vitreal, intraretinal, and choroidal oxygen tension ([Formula: see text]) distribution in anesthetized and ventilated control and urethan-treated rats at 8 and 16 wk of age with the use of oxygen-sensitive microelectrodes. Striking differences were observed in both choroidal and intraretinal [Formula: see text]distribution in urethan-treated rats compared with controls. At both ages, intrachoroidal [Formula: see text] was much lower in the urethan-treated rats. In addition, the intraretinal[Formula: see text] distribution across the inner retinal layers was more uniform than normal. A small elevation in[Formula: see text] was present at 8 wk in the inner nuclear layer where subsequent vascular regression occurred, and a small reduction in [Formula: see text] was present at the RPE, which recovered to normal values by 16 wk in regions where RPE vessel networks were first evident. Although the retinal arteries were considerably thinner at both ages in the urethan-treated rats, the vitreal [Formula: see text] profiles and superficial retinal [Formula: see text] remained normal. The unexpected and large reduction in the oxygen delivery from the choroid found in the urethan-treated rats may account for the lack of major hyperoxia within the pathological retina and the lower oxygen tension in the RPE before the vascular proliferation in this region. We propose that tissue [Formula: see text] is an important determinant of the vascular remodeling, which is seen in this model of neural degeneration and that the [Formula: see text]distribution changes described in this study help provide a new view of the pathogenesis of this model.
Genistein elicits biphasic effects on L-type Ca2+current in feline atrial myocytes
Authors: Wang Yong G. , Lipsius Stephen L.
Published: December 2017

A perforated patch recording method was used to determine the effects of genistein (Gen), a protein tyrosine kinase (PTK) inhibitor, on basal L-type Ca2+current ( ICa,L) in feline atrial myocytes. Gen (50 μM) elicited biphasic changes in ICa,L: an initial inhibition (−55 ± 4%; phase 1) followed by a secondary stimulation (34 ± 9%; phase 2) of ICa,L. Withdrawal of Gen elicited a further potentiation of ICa,L(152 ± 19%; phase 3) above control ( n = 46). In general, phase 1 inhibition and phase 3 potentiation varied directly with Gen concentration, and phase 2stimulation exhibited biphasic concentration-dependent changes compared with control. When cells were dialyzed using a ruptured patch recording method, Gen elicited only inhibition of ICa,L; phases 2 and 3 were abolished. Vanadate (1 mM), an inhibitor of protein tyrosine phosphatase, abolished both Gen-induced inhibition and stimulation of ICa,L. Daidzein (50 μM), a weakly active analog of Gen, exerted no significant effects on ICa,L, and withdrawal of daidzein failed to potentiate ICa,L. In a few cells, Gen elicited a prominent vanadate-sensitive stimulation of ICa,Lin the absence of any significant inhibition of ICa,L. Gen-induced changes in ICa,Lwere unaffected by either 100 μM 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM) or 1 μM ryanodine, agents that alter intracellular Ca2+; 4 μM H-89 or 50 μM Rp diastereomer of adenosine 3′,5′-monophosphothioate (RP-cAMPS), inhibitors of protein kinase A (PKA); 0.1 μM calphostin C or 2 μM chelerythrine, inhibitors of protein kinase C (PKC); or 100 μM NG-monomethyl-l-arginine (l-NMMA), an inhibitor of nitric oxide (NO) synthase. We conclude that in feline atrial myocytes, Gen acts via membrane-bound PTKs to inhibit ICa,Land via cytosolic PTKs to stimulate ICa,L. Gen-induced changes in ICa,Lare not related to changes in intracellular Ca2+or to secondary interactions with either PKA, PKC, or NO signaling pathways. These results indicate that in atrial myocytes ICa,Lis regulated by two independent and competing PTK signaling mechanisms.
Mechanisms of cardiac hypertrophy in canine volume overload
Authors: Matsuo Takeshi , Carabello Blase A. , Nagatomo Yoshitatsu , Koide Masaaki , Hamawaki Masayoshi , Zile Michael R. , McDermott Paul J.
Published: December 2017

This study tested whether the modest hypertrophy that develops in dogs in response to mitral regurgitation is due to a relatively small change in the rate of protein synthesis or, alternatively, is due to a decreased rate of protein degradation. After 3 mo of severe experimental mitral regurgitation, the left ventricular (LV) mass-to-body weight ratio increased by 23% compared with baseline values. This increase in LV mass occurred with a small, but not statistically significant, increase in the fractional rate of myosin heavy chain (MHC) synthesis (Ks), as measured using continuous infusion with [3H]leucine in dogs at 2 wk, 4 wk, and 3 mo after creation of severe mitral regurgitation. Translational efficiency was unaffected by mitral regurgitation as measured by the distribution of MHC mRNA in polysome gradients. Furthermore, there was no detectable increase in translational capacity as measured by either total RNA content or the rate of ribosome formation. These data indicate that translational mechanisms that accelerate the rate of cardiac protein synthesis are not responsive to the stimulus of mitral regurgitation. Most of the growth after mitral regurgitation was accounted for by a decrease in the fractional rate of protein degradation, calculated by subtracting fractional rates of protein accumulation at each time point from the corresponding Ksvalues. We conclude that 1) volume overload produced by severe mitral regurgitation does not trigger substantial increases in the rate of protein synthesis and 2) the modest increase in LV mass results primarily from a decrease in the rate of protein degradation.
Tyrosine phosphorylation of paxillin/pp125FAKand microvascular endothelial barrier function
Authors: Yuan Yuan , Meng F. Y. , Huang Q. , Hawker James , Mac Wu H.
Published: December 2017

The transendothelial movement of solutes is a dynamic process controlled by a complex interaction between the cytoskeleton and adhesion proteins. The aim of this study was to examine whether protein tyrosine phosphorylation is involved in the regulation of endothelial barrier function. The apparent permeability coefficient of albumin ( Pa) was measured in isolated and perfused coronary venules. Tyrosine phosphatase inhibitors, including phenylarsine oxide and sodium orthovanadate, dose and time dependently increased basal Pa. Western blot analysis of cultured coronary venular endothelial cells revealed that inhibition of tyrosine phosphatase induced an increase in phosphotyrosine content in a number of proteins, including bands at 65–70 and 120–130 kDa, which were identified as paxillin and focal adhesion kinase (pp125FAK), respectively. The time course and dose responsiveness of protein tyrosine phosphorylation were tightly correlated with those of increases in Pa. Furthermore, stimulation of endothelial cells with histamine or phorbol myristate acetate (PMA) enhanced tyrosine phosphorylation of paxillin and pp125FAK, which was blocked by the tyrosine kinase inhibitor damnacanthal. Correspondingly, the increases in venular permeability elicited by histamine and PMA were abolished in damnacanthal-treated venules. Taken together, the data suggest a possible involvement of protein tyrosine phosphorylation in the control of endothelial barrier function. Paxillin and its associated focal adhesion proteins may play a specific role in agonist-induced hyperpermeability responses in the endothelium of exchange vessels.
Arterial response during cold pressor test in borderline hypertension
Authors: Lafleche A. B. , Pannier B. M. , Laloux B. , Safar M. E.
Published: December 2017

We observed previously that sympathetic activation produced by lower body negative pressure increases pulse-pressure amplification with little change in mean pressure. Whether the cold pressor test (CPT) might produce a similar hemodynamic pattern has been ignored. Ten subjects with borderline hypertension and ten age- and sex-matched normotensive controls were compared to investigate carotid-brachial pulse-pressure amplification (aplanation tonometry) and changes in brachial and carotid distensibility (echotracking technique) before and during CPT. The maneuver markedly increased blood pressure without a change in heart rate. Pulse-pressure amplification tended to disappear as a consequence of a higher increase in carotid than in brachial pulse pressure, due to an earlier return of wave reflections at the carotid site. CPT caused a significant decrease in carotid and brachial distensibility. Both results were more pronounced in controls than in borderline hypertensives. Thus CPT reduces pulse-pressure amplification. In humans, this change may greatly influence the calculation of arterial distensibility, although this point is usually minimized in animal experiments. Furthermore, in young subjects, sympathetic stimulus may induce different arterial responses, depending on the mechanism involved: reflex or global nonspecific stimulus.
Alterations in basal protein kinase C activity modulate renal afferent arteriolar myogenic reactivity
Authors: Kirton C. Adam , Loutzenhiser Rodger
Published: December 2017

Myogenic vasoconstriction of the renal afferent arteriole contributes to the autoregulation of renal blood flow, glomerular filtration rate, and glomerular capillary pressure (PGC). The reactivity of the afferent arteriole to pressure and the efficiency of PGCcontrol are subject to physiological and pathophysiological alterations, but the determinants of the myogenic response of this vessel are largely unknown. We used the in vitro perfused hydronephrotic rat kidney to investigate the role of protein kinase C (PKC) in the control of this response. Inhibition of PKC by 1 μM chelerythrine attenuated myogenic reactivity but did not affect the afferent arteriole vasoconstrictor response to KCl (35 mM)-induced depolarization. Low concentrations of phorbol ester (10 nM phorbol 12-myristate 13-acetate) and low levels of ANG II or endothelin-1 (3 pM) potentiated myogenic vasoconstriction without affecting basal afferent arteriolar diameters. These actions were blocked by 1 μM chelerythrine, suggesting a PKC-dependent mechanism. Finally, although PKC inhibition attenuated basal myogenic responses, full reactivity to pressure was restored by 1 mM 4-aminopyridine, a pharmacological inhibitor of delayed rectifier K channels, which are known to be modulated by PKC. The ability of 4-aminopyridine to circumvent the effects of PKC inhibition militates against a direct role of PKC in myogenic signaling. We interpret these observations as indicating that basal PKC activity is an important determinant of myogenic reactivity in the renal afferent arteriole. However, PKC activation does not appear to play an obligate role in myogenic signaling in this vessel. We suggest that basal PKC activity directly modulates voltage-gated K channel activity, thereby indirectly affecting myogenic reactivity.
Ox-LDL induces apoptosis in human coronary artery endothelial cells: role of PKC, PTK, bcl-2, and Fas
Authors: Li Dayuan , Yang Baichun , Mehta Jawahar L.
Published: December 2017

Oxidized low-density lipoprotein (ox-LDL) plays a critical role in the development of atherosclerosis. Recent studies show that ox-LDL may induce apoptosis of cultured rabbit smooth muscle cells and human macrophages. This study was designed to determine the modulation by ox-LDL of apoptosis in cultured human coronary arterial endothelial cells (HCAEC) during hypoxia-reoxygenation and to determine underlying mechanisms. When HCAEC were ∼85% confluent, the cells were exposed to hypoxia (24 h)-reoxygenation (3 h), native LDL, or ox-LDL. Fragmented DNA end-labeling, DNA laddering, and light and electron microscopy were used to determine changes characteristic of apoptosis. Ox-LDL (20 μg/ml) increased apoptosis during hypoxia-reoxygenation compared with hypoxia-reoxygenation alone ( P < 0.05). Low concentrations of ox-LDL (5 μg/ml) and native LDL (20 μg/ml) under identical conditions had no effect on the degree of apoptosis. Ox-LDL markedly decreased endogenous superoxide dismutase activity and increased lipid peroxidation in HCAEC. The presence of ox-LDL, but not native LDL, in cultured HCAEC resulted in the activation of protein kinase C (PKC) and protein tyrosine kinase (PTK). The specific PKC and PTK inhibitors significantly reduced ox-LDL-mediated apoptosis of HCAEC ( P < 0.05). Hypoxia-reoxygenation significantly increased Fas expression and decreased bcl-2 expression in HCAEC lysate as determined by Western analysis. Ox-LDL further increased Fas expression and decreased bcl-2 expression. These data indicate that ox-LDL enhances hypoxia-reoxygenation-mediated apoptosis in HCAEC. Ox-LDL-mediated apoptosis of HCAEC appears to involve activation of PKC and PTK. In addition, ox-LDL modulates Fas and bcl-2 protein expression in HCAEC. This study also suggests that ox-LDL is more important than native LDL in hypoxia-reoxygenation-induced apoptosis.
Measurement of synaptic vesicle exocytosis in aortic baroreceptor neurons
Authors: Hay Meredith , Hasser Eileen M.
Published: December 2017

The purpose of this study was to evaluate the use of the fluorescent membrane label FM1–43 as a measure of synaptic terminal exocytosis during stimulation of labeled aortic baroreceptor and unlabeled nodose ganglia neurons. Activation of the nerve terminals with electrical stimulation or depolarization with 90 mM KCl in the presence of 2.0 μM FM1–43 resulted in bright, punctate staining of synaptic boutons. Additional depolarization in the absence of dye resulted in destaining with a time course that was consistent and repeatable in multiple boutons within a given terminal. Destaining was dependent on calcium influx and was blocked by bath application of 100 μM CdCl2. Whole cell patch-clamp studies have reported that depolarization-induced calcium influx in aortic baroreceptor cell bodies is predominantly caused by the activation of ω-conotoxin GVIA (ω-CgTx)-sensitive N-type calcium channels. In addition, these N-type channels have been shown to be inhibited by activation of metabotropic glutamate receptors. In the present study, exocytosis in aortic baroreceptor terminals was not affected by bath application of 5 μM nifedipine and only partially inhibited by bath application of 2.0 μM ω-CgTx. However, depolarization-induced exocytosis was significantly inhibited by bath application of 200 μM L-AP4, a type III metabotropic glutamate receptor agonist. Results from this study suggest that 1) FM1–43 can be used to measure synaptic vesicle exocytosis in baroreceptor neurons; 2) the N-type calcium channel may not be involved in the initial phase of vesicle exocytosis; and 3) activation of L-AP4-sensitive metabotropic glutamate receptors inhibits 90 mM KCl-induced vesicle release.
Estrogens modulate bovine vascular endothelial cell permeability and HSP 25 expression concomitantly
Authors: Delarue F. , Daunes S. , Elhage R. , Garcia A. , Bayard F. , Faye J.-C.
Published: December 2017

The atheroprotective properties of estrogens are supported by clinical data from postmenopausal women who use estrogen replacement therapy. However, the mechanisms mediating activity remain unknown, and it has been suggested that estrogens may help to modulate endothelial permeability to atherogenic lipoproteins. In these studies we used bovine vascular endothelial cells as an in vitro model to show that estrogens were able to regulate low-density lipoprotein transport and permeability of the endothelial monolayer. Macromolecular transport was observed to be a second-order polynomial function of estrogen concentration. Moreover, this regulation was correlated with expression of heat shock protein (HSP) 25, which is known to influence fluid phase pinocytosis and cytoskeleton remodeling, thus suggesting a role for HSP 25 in the estrogenic control of transcellular permeability of the endothelium monolayer.
Effects of coronary artery disease on expression and microvascular response to VEGF
Authors: Métais Caroline , Li Jianyi , Li Jian , Simons Michael , Sellke Frank W.
Published: December 2017

The effects of coronary artery disease (CAD) on human coronary microvascular responses to vascular endothelial growth factor (VEGF) and the alterations of the myocardial expressions of VEGF and its flk-1 and flt-1 receptors were examined in 48 patients. Microvascular studies were performed in vitro with video microscopy. The expressions of VEGF and its receptors were examined using Northern analysis of total mRNA, and the expressions of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) were examined by RT-PCR. VEGF and hepatocyte growth factor (HGF) caused potent relaxations of microvessels. These responses were reduced in the presence of NG-nitro-l-arginine and the tyrosine kinase inhibitor genistein or in microvessels from patients with CAD. Relaxations to substance P and sodium nitroprusside were similar in both groups. The substance P response was abolished in the presence of NG-nitro-l-arginine. The expression of VEGF and its receptors and the expression of cNOS and iNOS were not altered in patients with CAD. In conclusion, VEGF and HGF elicit the release of nitric oxide through activation of tyrosine kinase receptors. CAD is associated with reduced vascular responses to both VEGF and HGF; this is not likely due to a reduced expression of VEGF or flt-1 or flk-1 receptors and not due to a generalized endothelium dysfunction despite the presence of mild hypercholesterolemia in these patients with CAD. These findings may have important implications regarding the efficacy of endogenous and exogenous VEGF in patients with risk factor for CAD.
Role of K+channels in arteriolar vasodilation mediated by integrin interaction with RGD-containing peptide
Authors: Platts Steven H. , Mogford Jon E. , Davis Michael J. , Meininger Gerald A.
Published: December 2017

Integrins are transmembrane adhesion receptors found on most cells, including vascular smooth muscle cells. Several integrins bind to the conserved amino acid sequence Arg-Gly-Asp (RGD), and synthetic RGD-containing peptides can cause endothelium-independent arteriolar vasodilation by interacting with the αvβ3-integrin expressed by vascular smooth muscle. We hypothesized that RGD peptide-induced vasodilation involves K+channels. Rat cremaster arterioles were treated with cRGD (GPenGRGDSPCA) in the presence or absence of the nonselective K+channel inhibitor tetraethylammonium (TEA, 20 mM). TEA caused arterioles to constrict by 19 ± 5% and inhibited cRGD-induced vasodilation ( n = 7, P < 0.05). Vessels preconstricted with phenylephrine (5 × 10−7M) showed no significant inhibition of the dilatory response to cRGD, indicating that inhibition by TEA was not related to increased vasomotor tone. Further evidence for the involvement of K+channels was obtained by addition of 100 mM KCl ( n = 5), which inhibited vasodilation caused by cRGD. Inhibition of large and small conductance, Ca2+-activated K+channels with iberiotoxin (100 nM) or apamin (25 nM), respectively, had no effect on cRGD-induced vasodilation. Partial inhibition of vasodilation was observed with inhibitors of voltage-gated (4-aminopyridine, 1 mM), ATP-sensitive (glibenclamide, 1 μM), and inward rectifying (barium, 50 μM) K+channels. These data support the hypothesis that integrin-signaling pathways leading to arteriolar vasodilation may involve modulation of K+channel function.
Properties of a Cl−current activated by cell swelling in rabbit portal vein vascular smooth muscle cells
Authors: Greenwood I. A. , Large W. A.
Published: December 2017

In rabbit portal vein smooth muscle cells, application of a hypotonic external solution caused cell swelling and evoked an outwardly rectifying Cl− current. The hypotonicity-activated current was markedly reduced by the anti-estrogen tamoxifen (10 μM) and was inhibited by DIDS in a voltage-dependent manner [the concentration required to inhibit the current by 50% (IC50) at −50 and +100 mV was 21 and 5 μM DIDS, respectively]. Indanyloxyacetic acid 94 (IAA-94) and niflumic acid also inhibited the hypotonicity-activated current, with 50% inhibition produced at concentrations of ∼200 and 100 μM, respectively. In isotonic conditions, application of tamoxifen and DIDS to cells decreased the holding current due to the inhibition of a resting conductance that was outwardly rectifying and reversed at the Cl− equilibrium potential. These data show that rabbit portal vein myocytes have a resting Cl− conductance that is enhanced by cell swelling; its possible physiological role is discussed.
Endothelin-receptor blockade does not alter closure of the ductus arteriosus
Authors: Fineman Jeffrey R. , Takahashi Yasushi , Roman Christine , Clyman Ronald I.
Published: December 2017

Endothelin-1 (ET-1) is synthesized within the wall of the ductus arteriosus (DA) and is a potent constrictor of the DA in vitro. However, the role of endogenous ET-1 in closure of the DA at birth remains unclear. Therefore, we studied the effects of a selective ETA-receptor antagonist (PD-156707), or its vehicle, on DA closure in 13 late-gestation fetal lambs during the first 5 h after birth. We also studied the effects of ETA-receptor blockade on DA constriction induced by oxygen, indomethacin (a cyclooxygenase inhibitor), and LY-83583 (a soluble guanylate cyclase inhibitor) in vitro ( n = 9 ductus arteriosus rings). In vehicle-treated lambs in vivo, the DA constricted during the 5-h study period after birth: DA resistance increased (from 0.007 ± 0.01 to 3.406 ± 4.15 mmHg ⋅ ml−1 ⋅ min ⋅ kg−1; P < 0.05); the pressure gradient across the DA increased (from 1.4 ± 2.1 to 25.2 ± 9.4 mmHg; P < 0.05); and DA blood flow decreased (from 193.5 ± 48.0 to 19.3 ± 14.3 ml ⋅ kg−1 ⋅ min−1; P < 0.05). In vitro, the DA was constricted by exposure to 30% oxygen (23 ± 14% net active tension; P < 0.05), indomethacin (5 × 10−6 M, 22 ± 5% net active tension; P < 0.05), LY-83583 (10−5 M, 24 ± 10% net active tension; P < 0.05), and ET-1 (10−7 M, 19 ± 4% net active tension; P < 0.05). Although PD-156707 blocked both the in vivo and in vitro effects of exogenous ET-1, it had no effect on postnatal ductus constriction nor on in vitro ductus contractile responses to oxygen, indomethacin, or LY-83583. This study suggests that endogenous ET-1 does not play an important role in closure of the DA at birth.
Interaction between carotid baroregulation and the pulsating heart: a mathematical model
Authors: Ursino Mauro
Published: December 2017

A mathematical model of short-term arterial pressure control by the carotid baroreceptors in pulsatile conditions is presented. The model includes an elastance variable description of the left and right heart, the systemic (splanchnic and extrasplanchnic) and pulmonary circulations, the afferent carotid baroreceptor pathway, the sympathetic and vagal efferent activities, and the action of several effector mechanisms. The latter mechanisms work, in response to sympathetic and vagal action, by modifying systemic peripheral resistances, systemic venous unstressed volumes, heart period, and end-systolic elastances. The model is used to simulate the interaction among the carotid baroreflex, the pulsating heart, and the effector responses in different experiments. In all cases, there has been satisfactory agreement between model and experimental results. Experimental data on heart rate control can be explained fairly well by assuming that the sympathetic-parasympathetic systems interact linearly on the heart period. The carotid baroreflex can significantly modulate the cardiac function curve. However, this effect is masked in vivo by changes in arterial and atrial pressures. During heart pacing, cardiac output increases with frequency at moderate levels of heart rate and then fails to increase further because of a reduction in stroke volume. Shifting from nonpulsatile to pulsatile perfusion of the carotid sinuses decreases the overall baroreflex gain and significantly modifies operation of the carotid baroreflex. Finally, a sensitivity analysis suggests that venous unstressed volume control plays the major role in the early hemodynamic response to acute hemorrhage, whereas systemic resistance and heart rate controls are a little less important.
Atrial natriuretic peptide clearance receptor participates in modulating endothelial permeability
Authors: Hempel Albrecht , Noll Thomas , Bach Christoph , Piper Hans Michael , Willenbrock Roland , Höhnel Klaus , Haller Hermann , Luft Friedrich C.
Published: December 2017

The atrial natriuretic peptide (ANP)-C receptor is generally believed to clear ANP; however, the ANP-C receptor may serve to reduce cAMP by inhibiting adenylate cyclase. ANP decreases endothelial permeability in coronary endothelial cell monolayers. We tested the hypothesis that part of this effect might be mediated by the ANP-C receptor. We used an endothelial cell monolayer from rat coronary endothelium and measured albumin flux. We applied either ANP or a ring-deleted ANP (C-ANP), which only stimulates the ANP-C receptor. ANP and C-ANP both decreased permeability from 100 pM to 100 nM by 60 and 30%, respectively. ANP increased endothelial cGMP contents 5.5-fold, whereas C-ANP had no effect. ANP reduced endothelial cAMP contents by 75%, which was only partly blocked by pertussis toxin. C-ANP also reduced cAMP; however, this effect was completely blocked by pertussis toxin. Protein kinase G inhibition blocked the ANP-mediated decrease in permeability by 50%. In contrast, pretreatment with pertussis toxin, in the face of protein kinase G inhibition, blocked the effect completely. C-ANP decreased permeability by half the amount of ANP. This C-ANP effect was completely blocked by pertussis toxin but not by protein kinase G inhibition. Isoproterenol (10 μM) increased permeability by almost 50%, which was completely blocked by ANP but only partially blocked by C-ANP. The C-ANP effect was blocked completely by pertussis toxin. Isoproterenol increased cAMP threefold, which was abolished by ANP. C-ANP reduced the isoproterenol-induced increase in cAMP by 50%. Isoproterenol had no effect on cGMP. We conclude that agonist binding to the ANP-C receptor inhibits cAMP production via a Giprotein-coupled signaling system. This inhibition may contribute to the decreased endothelial permeability evoked by ANP in this system.
Endothelial vasodilator production by uterine and systemic arteries. III. Ovarian and estrogen effects on NO synthase
Authors: Vagnoni Karen E. , Shaw Cynthia E. , Phernetton Terrance M. , Meglin Beth M. , Bird Ian M. , Magness Ronald R.
Published: December 2017

During the follicular phase of the ovarian cycle, when the local estrogen-to-progesterone ratio is elevated, uterine blood flow is elevated. This vasodilatory response is reproduced by exogenous 17β-estradiol (E2β) administration via a nitric oxide (NO)-mediated mechanism. We hypothesized that endogenous ovarian estrogen and exogenous E2β treatment elevate expression of endothelial cell-derived NO synthase (eNOS) in uterine, but not in systemic, arteries. Uterine, mammary, and systemic (renal and/or omental) arteries were collected from 1) ewes synchronized to the follicular ( day −1 to day 0) or luteal ( day 10) phases of the ovarian cycle ( n = 4 per phase), 2) ovariectomized ewes 120 min after systemic vehicle or E2β (5 μg/kg iv) treatment, and 3) ovariectomized ewes on days 0, 3, 6, 8, and 10 of E2β (5 μg/kg iv, followed by 6 μg/kg per day) treatment. Expression of eNOS was localized primarily to the endothelium rather than vascular smooth muscle (VSM) in all arteries examined by immunohistochemistry and Western analysis; inducible NOS was not detected in either endothelium or VSM. Expression of eNOS protein was greater ( P < 0.05) in uterine, but not in systemic, artery endothelium-isolated protein collected from follicular versus luteal phase ewes. Acute systemic E2β treatment of ovariectomized ewes increased ( P < 0.05) eNOS protein levels in uterine artery endothelium. Prolonged E2β administration progressively increased uterine, but not systemic, artery endothelial eNOS protein expression. Therefore, the increased local estrogen-to-progesterone ratio during the follicular phase locally elevates eNOS expression, which possibly elevates uterine blood flow. These responses can be partly reproduced with E2β administration.
Phosphatidylinositol 3-kinase γ mediates shear stress-dependent activation of JNK in endothelial cells
Authors: Go Young-Mi , Park Heonyong , Maland Matthew C. , Darley-Usmar Victor M. , Stoyanov Borislav , Wetzker Reinhard , Jo Hanjoong
Published: December 2017

Shear stress differentially activates extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) by mechanisms involving Gαi2and Gβ/γ proteins, respectively, in bovine aortic endothelial cells (BAEC). The early events in this signaling mechanism by which G proteins regulate ERK and JNK in response to shear stress have not been defined. Here we show that BAEC endogenously express a G protein-dependent form of phosphatidylinositol 3-kinase, PI3Kγ, and its activity is stimulated by shear stress. PI3Kγ activity was measured in vitro using BAEC that were transiently transfected with an epitope-tagged PI3Kγ (vsv-PI3Kγ). Exposure of BAEC to shear stress rapidly and transiently stimulated the activity of vsv-PI3Kγ (maximum by 15 s, with a return to basal after 1-min exposure to 5 dyn/cm2shear stress). Activity of vsv-PI3Kγ was stimulated by shear stress intensities as low as 0.5 dyn/cm2. Treatment of BAEC with an inhibitor of PI3K, wortmannin, inhibited shear-dependent activation of JNK but had no effect on that of ERK. Furthermore, expression of a kinase-inactive mutant (PI3KγK799R) in BAEC inhibited the shear-dependent activation of JNK but not ERK. Taken together, these results suggest that PI3Kγ selectively regulates the shear-sensitive JNK pathway. This differential and novel signaling pathway may be responsible for coordinating various mechanosensitive events in endothelial cells.
Essential roles for G1cyclin-dependent kinase activity in development of cardiomyocyte hypertrophy
Authors: Tamamori Mimi , Ito Hiroshi , Hiroe Michiaki , Terada Yoshio , Marumo Fumiaki , Ikeda Masa-Aki
Published: December 2017

Although cardiomyocytes undergo terminal differentiation soon after birth, irreversibly withdrawing from the cell cycle, growth stimulation induces cell hypertrophy. Such growth stimulation is also responsible for the upregulation of G1cyclins and cyclin-dependent kinase (CDK) activity in proliferating cells. We sought to determine whether G1CDK activity is involved in the hypertrophy of rat neonatal cardiomyocytes in culture. We show that serum stimulation promoted the G1CDK activity without induction of DNA synthesis in cardiomyocytes. Furthermore, overexpression of CDK inhibitors p16INK4aand p21CIP1/WAF1by use of the adenovirus vector effectively prevented cell enlargement and depressed serum-induced protein synthesis and expression of skeletal α-actin and atrial natriuretic factor, genetic markers of cardiac hypertrophy. These results suggest that the G1CDK activity promoted by serum stimulation is required for the induction of cardiomyocyte hypertrophy and provide novel evidence for understanding the regulation of cardiac hypertrophy by cell cycle regulators.