Alpine Skiing With total knee ArthroPlasty (ASWAP): metabolism, inflammation, and skeletal muscle fiber characteristics.

We investigated the effect of alpine skiing for 12 weeks on skeletal muscle characteristics and biomarkers of glucose homeostasis and cardiovascular risk factors. Twenty-three patients with a total knee arthroplasty (TKA) were studied 2.9 ± 0.9 years (mean ± SD) after the operation. Fourteen patients participated in the intervention group (IG) and nine in the control group (CG). Blood samples and muscle biopsies were obtained before (PRE) and 7.3 ± 0.8 days after (POST) the intervention, and blood samples again after a retention (RET) phase of 8 weeks. With skiing, glucose homeostasis improved in IG (decrease in fasting insulin, increase in muscle glycogen) but not in CG. Fiber type distribution and size, as well as capillary density and number of capillaries around the fibers (CAF), were not different between the operated and the non-operated leg in either group. The relative number of type I fibers increased with skiing in IG with no change in CG. Inflammatory biomarkers, plasma lipids, and mitochondrial proteins and activity did not change. Alpine skiing is metabolically beneficial and can be used as a training modality by elderly people with TKA.

Four New Host and Three New State Records of Dothistroma Needle Blight Caused by Dothistroma pini in the United States.

During 2010 and 2011, Dothistroma needle blight (DNB), also known as red band needle blight, was observed for the first time in Cass and Pembina counties in North Dakota (ND). In Pembina Co., DNB was observed in two sites in the Jay V. Wessels Wildlife Management Area (JWWMA). In September 2009, yellow spots on green needles were observed on some trees along the western edge of one planting. By June 2010, DNB was found on third- and fourth-year needles in both JWWMA plantings. Symptoms had developed into dark brown bands or spots on necrotic needles that contained erumpent black acervuli. In June 2011, similar DNB symptoms were observed on Pinus nigra, P. flexilis, P. ponderosa, P. cembra, and P. albicaulis in the Dale E. Herman Research Arboretum, Cass Co., ND. DNB was collected in July 2011 in Brookings Co., South Dakota (SD), from a seed source provenance planting of P. ponderosa. To identify the species causing the infections, symptomatic needles were collected in 2010 from both sites in JWWMA and then again from all four locations in 2011 on all pine species infected. Needles of P. nigra from a private residence near Fairland in Shelby County, Indiana (IN), were also included in the sample set. The rDNA-ITS was PCR-amplified either directly from conidia obtained from acervuli on the needles or from cultures obtained from isolations. Amplicons were sequenced and a BLAST search was performed in GenBank. The sequences of samples obtained from P. nigra, P. flexilis, P. cembra, and P. albicaulis in ND, P. ponderosa in SD, and P. nigra from IN showed 100% sequence homology with Dothistroma pini (Accession No. AY808302). These isolates were identical to all previously assayed isolates of D. pini from Nebraska, Minnesota, and Michigan in the United States. The P. ponderosa isolates from all three sites in ND differed from the other isolates and contained a 1-bp point mutation from a C to a T at site 72 (sequence deposited in GenBank, accession KJ933441). Mating type was determined using species-specific mating type primers for D. pini (3). All 26 samples from ND and SD were of the MAT-1 idiomorph, while the sample from IN contained the MAT-2 idiomorph. All cultures are maintained at FABI, University of Pretoria, South Africa. The two species that cause DNB, D. septosporum (G. Dorog.) M. Morelet and D. pini Hulbary, are morphologically indistinguishable and molecular characterization remains essential for correct species identification (1). Host and geographical distribution range determinations of Dothistroma spp. made without molecular methods are not valid. To date, species confirmed using DNA sequences in the United States include D. septosporum in the Pacific Northwest states of Oregon and Idaho on P. ponderosa, in Montana on P. contorta v. latifolia, and D. pini in the North Central states of Nebraska, Minnesota, and Michigan on P. nigra (1). This study documents the presence of D. pini in three additional states, including a first report of DNB in ND and SD. It also includes new records of D. pini infecting P. flexilis, P. cembra, P. albicaulis, and P. ponderosa. Results of this study have expanded the documented host range of D. pini in the United States from one (P. nigra) to five species. Globally, D. pini is now known to infect a total of 10 pine hosts (2,4,5). References: (1) I. Barnes et al. Stud. Mycol. 50:551, 2004. (2) I. Barnes et al. For. Pathol. 41:361, 2011. (3) M. Groenewald et al. Phytopathology 97:825, 2007. (4) D. Piou et al. Plant Dis. 98:841, 2014. (5) B. Piskur et al. For. Pathol. 43:518, 2013.

Upregulated TRPC1 channel in vascular injury in vivo and its role in human neointimal hyperplasia.

Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.

Vascular alpha-2 adrenoceptor function is decreased in rats with congestive heart failure.

OBJECTIVE: Vascular alpha-2 adrenoceptor function of rats with congestive heart failure (CHF) was characterized in both in vivo and in vitro experiments. METHODS: CHF was induced in Sprague-Dawley rats by coronary artery ligation. Sham-operated rats served as normal controls. Postjunctional alpha-2 adrenergic responsiveness was assessed in vivo using the pithed rat model and in vitro in organ bath. Vascular alpha-2 adrenoceptor density was studied by receptor binding assay. RESULTS: Four to 6 weeks after this surgical procedure, plasma catecholamines were markedly increased in CHF rats. In vivo vascular responses to alpha-2 adrenoceptor agonists BHT933 and clonidine were significantly decreased in CHF rats (P < 0.001). Clonidine elicited dose-dependent responses in endothelium intact mesenteric arteries in both CHF and sham-operated rats. The dose-response curve in CHF was shifted to the right with a pD2 value of 5.5 +/- 0.2 compared with control rats 6.2 +/- 0.2 (P < 0.05). The response to clonidine was selectively blocked by an alpha-2 adrenergic antagonist rauwolscine in both groups. Endothelium denuded arteries showed an enhanced response to clonidine in both CHF and control rats. However, the response to clonidine is still decreased in CHF compared to sham-operated rats (P < 0.05). Alpha-2 adrenoreceptor density, as determined by [3H]yohimbine binding in membrane preparations from mesenteric arteries was decreased in CHF compared to sham-operated rats (Bmax 43 +/- 6 vs. 104 +/- 20 fmol/mg protein, P < 0.05). CONCLUSIONS: Vascular alpha-2 adrenoceptor function is decreased in rats with CHF.

Enhanced acetylcholine and P2Y-receptor stimulated vascular EDHF-dilatation in congestive heart failure.

OBJECTIVE: Congestive heart failure (CHF) is accompanied by impaired peripheral blood flow and endothelial dysfunction with decreased release of nitric oxide (NO). Strong evidence supports the existence of another vasodilatory substance, endothelium derived hyperpolarising factor (EDHF), which has not previously been studied in CHF. METHOD: CHF was induced by left coronary artery ligation resulting in a reproducible myocardial infarction in Sprague Dawley rats. Vasodilatory responses to acetylcholine and extracellular nucleotides (ATP, ADP beta S, ADP and UTP) were examined in cylindrical segments of the mesenteric artery, precontracted with noradrenaline. The combined NO- and EDHF-dilatation (after inhibition of cyclo-oxygenase pathways) was called "total dilatation", as indomethacin had only minor effects in this system. NO-dilatation was studied in segments pretreated with indomethacin and the potassium channel inhibitors charybdotoxin (10(-7.5) M) and apamin (10(-6) M), while EDHF-dilatations were studied in the presence of indomethacin (10(-5) M) and L-NOARG (10(-3.5) M). RESULTS: EDHF-dilatations in CHF were strongly up-regulated for ACh (36% vs. 73%; sham vs. CHF operated rats), ADP beta S (10% vs. 42%), ADP (0% vs. 21%) and UTP (3% vs. 35%). These dilatations were abolished by a combination of charybdotoxin and apamin, confirming that they were mediated by EDHF. The NO-dilatations on the other hand were down-regulated in CHF as compared to sham operated rats for ACh (93% vs. 76%; sham vs. CHF operated rats), ADP beta S (61% vs. 37%). ADP (60% vs. 30%), ATP (49% vs. 34%) and UTP (65% vs. 47%), while a minor decrease was seen in the total dilatation for ACh (87% vs. 75%; sham vs. CHF operated rats), ADP beta S (47% vs. 42%), ADP (59% vs. 39%), ATP (52% vs. 39%) and UTP (59% vs. 44%). CONCLUSION: In this model of non-atherosclerotic CHF there was a minor decrease in the total dilatation and a marked down-regulation of the NO-mediated dilatation, while the EDHF-dilatation was up-regulated. Increased EDHF-activity in CHF may represent a compensatory response to decreased NO-activity to preserve endothelial function and tissue perfusion.

Innervation of human epicardial coronary veins: immunohistochemistry and vasomotility.

OBJECTIVE: The aim was to investigate the innervation and vasomotor responses to classical and putative transmitters of the coronary venous bed. METHODS: The innervation of human epicardial coronary veins was investigated using acetylcholinesterase histochemistry and immunofluorescence staining, together with antisera against the general neuronal marker protein gene product 9.5 (PGP 9.5), the catecholamine synthesising enzyme tyrosine hydroxylase, and neuropeptides [neuropeptide Y, vasoactive intestinal peptide (VIP), substance P, and calcitonin gene related peptide (CGRP)]. The vasomotor responses to noradrenaline, acetylcholine, neuropeptide Y, substance P, human alpha calcitonin gene related peptide (alpha CGRP), and VIP were tested on isolated circular human epicardial coronary vein segments. RESULTS: A network of nerve fibres was shown in the major epicardial coronary veins by means of an antiserum to PGP 9.5. The majority of the perivascular nerve fibres possessed neuropeptide Y and tyrosine hydroxylase immunoreactivity. Only a few nerve fibres displayed substance P, CGRP, and VIP immunoreactivity and acetylcholinesterase activity. Noradrenaline and acetylcholine induced powerful contractions of all the tested segments, whereas no contraction was induced by neuropeptide Y, alpha CGRP, substance P, or VIP. All segments precontracted with U46619 responded with potent relaxation to alpha CGRP, substance P, and VIP, whereas noradrenaline and acetylcholine only in low concentrations induced weak relaxation of a few of the segments. No relaxation was induced by neuropeptide Y. CONCLUSIONS: This is the first study to demonstrate comprehensively the perivascular innervation of human coronary veins and corresponding vasomotor effects, suggesting a role in regulation of the coronary venous circulation.

Congestive heart failure induces downregulation of P2X1-receptors in resistance arteries.

OBJECTIVE: Congestive heart failure (CHF) is accompanied by enhanced peripheral sympathetic nerve activity, increased vascular resistance and impaired peripheral blood flow. Besides noradrenaline and neuropeptide Y, the sympathetic nervous system also releases ATP, which has contractile effects mediated by different subtypes of P2-receptors on the vascular smooth muscle cells. The present study was designed to examine postsynaptic changes of the contractile responses to ATP and other extracellular nucleotides in CHF. METHODS: CHF was induced by left coronary artery ligation resulting in a reproducible myocardial infarction in Sprague-Dawley rats. Contractile responses were examined in cylindrical segments of aorta and the mesenteric artery after endothelium removal. To determine if an altered response was regulated on the transcriptional level, competitive reverse transcription polymerase chain reaction (RT-PCR) was used to estimate the amount of P2X1-receptor mRNA. RESULTS: ATP, which is both a P2X1- and a P2Y-receptor agonist, induced a weaker contraction in the mesenteric artery from CHF as compared to sham operated rats. A decrease in both potency and maximum contraction was shown for the selective P2X1-receptor agonist, alpha beta-MeATP, in the mesenteric artery (pEC50 = 6.04 vs. 5.76, Cmax = 57% vs. 33%, sham vs. CHF operated rats), but not in the aorta. Competitive RT-PCR also revealed decreased P2X1-receptor mRNA levels in CHF operated rats in the mesenteric artery (9106 x 10(3) vs. 714 x 10(3) molecules/microgram, sham vs. CHF operated rats), while it remained unaltered in the aorta. To study the P2Y-receptor induced contractile effects, the P2X1-receptors were first desensitised with alpha beta-MeATP (10(-5) M for 8 min). After P2X1-receptors desensitisation, UTP and UDP induced strong contractions in both the mesenteric artery and in the aorta, while ATP and ADP were much less effective. These contractions were not altered by CHF, indicating that vascular contraction mediated by P2Y-receptors are unaffected by CHF. CONCLUSION: CHF induces downregulation of P2X1-receptor stimulated contraction in the mesenteric artery depending on decreased mRNA synthesis for the receptor, while the P2Y-receptor activity remains unchanged. Downregulation of P2X1-receptors appears to be specific for peripheral resistance arteries. This may represent a compensatory response to enhanced peripheral sympathetic nerve activity and increased vascular resistance in CHF.

Enhanced Ca2+-induced contractions and attenuated alpha-adrenoceptor responses in resistance arteries from rats with congestive heart failure.

AIM: The aim of the present study was to examine the role of Ca2+-mediated contractile responses in isolated mesenteric resistance arteries from rats with congestive heart failure (CHF). METHODS: Heart failure was induced by ligation of the left coronary artery. Rats exposed to the same surgical procedure except ligation served as controls (Sham). The following experiments were conducted: (1) passive increase in radial stretch (the length-tension relationship) in Ca2+-free and in depolarizing high K+-solution; (2) the contractile responses to external application of Ca2+ and high K+-solutions in the presence of nifedipine and phentolamine; and (3) a histological evaluation of CHF and Sham vessels. RESULTS: The length-tension induced response in Ca2+-free buffer solution was significantly lower in arteries from CHF rats, starting at a very low tension (0.9+/-0.2 mN/mm for heart failure and 1.7+/-0.2 mN/mm for Sham). This difference, but at a higher degree of stretch, was also present in K+-activated vessels. The external application of Ca2+ in K+-depolarized vascular segments in the presence of phentolamine (1 microM) induced an enhanced contractile response in arteries from CHF rats compared with Sham (4.8+/-0.3 mN/mm and 3.6+/-0.6 mN/mm, respectively, P=0.059). In the absence of phentolamine the reverse response was found (4.0+/-0.4 mN/mm and 5.7+/-0.3 mN/mm for CHF vs. Sham respectively, P=0.035). Application of increasing concentrations of K+-solution induced a stronger contractile response in Sham compared with CHF arteries (Sham 4.9+/-0.4 and heart failure 4.0+/-0.3, P=0.04). Microscopic examination of vessels yielded no difference in gross morphology, media thickness or wall to lumen ratio between CHF and Sham arteries. CONCLUSION: The results indicate an attenuation of alpha-adrenoceptors and a difference of Ca2+-mediated vascular contractility in resistance arteries of congestive heart failure rats.

Enhanced endothelin-1-induced contractions in mesenteric arteries from rats with congestive heart failure: role of ET(B) receptors.

Studies of congestive heart failure (CHF) in man and in experimental CHF have demonstrated elevated circulating levels of endothelin (ET). In order to examine whether there are concomitant ET receptor alterations, the vasomotor effects of endothelin-1 (ET-1) and sarafotoxin 6c (S6c) were examined in endothelium-intact and -denuded isolated mesenteric arteries from rats with CHF. CHF was induced by ligation of the left anterior descending coronary artery. Vasomotor responses were studied using small mesenteric arteries (approx. 250 microm in diameter, determined after normalisation). The antagonists IRL2500 and FR139317 were used in order to characterise the ET-1-induced response. In mesenteric arteries with intact endothelium, ET-1-induced contractions were more potent in CHF as compared to sham (pEC(50) 9.6+/-0.2 and 9.1+/-0.1, respectively, P<0.01). In endothelium-denuded arteries, there was no difference in potency of ET-1 between CHF and sham arteries, or in maximum contraction. In the presence of IRL2500, a selective ET(B)-receptor antagonist, ET-1 was more potent in endothelium-denuded arteries of CHF rats, while this difference was not seen in sham arteries. S6c had no consistent contractile or dilatory effect in CHF and sham rats. The results indicate that the enhanced contractile effects of ET-1 noted in CHF might be due to an attenuated endothelial function and that inhibition of smooth muscle cell ET(B) receptors increase the effects of contractile ET(A) receptors in CHF rats.

Altered neuropeptide Y Y1 responses in mesenteric arteries in rats with congestive heart failure.

The aim of the present study was to elucidate if the potentiating effect of neuropeptide Y on various vasoactive agents in vitro is (1) altered in mesenteric arteries from rats with congestive heart failure and (2) mediated by the neuropeptide Y Y1 receptor. The direct vascular effects of neuropeptide Y and its modulating effects on the contractions induced by endothelin-1-, noradrenaline-, 5-hydroxytryptamine (5-HT)-, U46619-(9,11-dideoxy-11alpha, 9alpha-epoxymethano-prostaglandin F2alpha) and ATP, and acetylcholine-induced dilatations were studied in the presence and absence of the neuropeptide Y Y1 antagonist, BIBP3226 (BIBP3226¿(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl ]-D-arginine-amide¿). Neuropeptide Y, per se, had no vasoactive effect in the arteries. The potency of endothelin-1 was significantly decreased in congestive heart failure rats. Neuropeptide Y and neuropeptide Y-(13-36) potentiated the endothelin-1-induced contraction in congestive heart failure mesenteric arteries. In 20% of the congestive heart failure rats, sarafotoxin 6c induced a contraction of 31+/-4%. Neuropeptide Y also potentiated U46619- and noradrenaline-induced contractions but not 5-HT-induced contractions in congestive heart failure arteries. In sham-operated animals neuropeptide Y potentiated noradrenaline- and 5-HT-induced contractions. These potentiations were inhibited by BIBP3226. Acetylcholine induced an equipotent relaxation in both groups which was unaffected by neuropeptide Y. In conclusion, neuropeptide Y responses are altered in congestive heart failure rats. The potentiating effect differs between vasoactive substances. Neuropeptide Y Y1 and non-neuropeptide Y1 receptors are involved.

Neuropeptide Y potentiates noradrenaline-induced contraction through the neuropeptide Y Y1 receptor.

To elucidate which neuropeptide Y receptor subtype is responsible for the neuropeptide Y-induced potentiation of the noradrenaline-evoked contraction in human omental arteries we used antisense oligodeoxynucleotide (Antisense), the new selective neuropeptide Y Y1 receptor antagonist, BIBP3226 {(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl) methyl]-D-arginine-amide} and the reverse transcriptase-polymerase chain reaction (RT-PCR). Neuropeptide Y significantly potentiated the noradrenaline-induced contraction in non-incubated vessels (pEC50 6.4 +/- 0.2 vs. 5.9 +/- 0.2) and in vessels incubated with 1 microM Sense oligodeoxynucleotide (Sense) (pEC50 6.0 +/- 0.1 vs. 5.6 +/- 0.2). In vessels incubated with 1 microM Antisense the potentiating effect of neuropeptide Y was completely abolished. BIBP3226 (1 microM) inhibited the neuropeptide Y-induced potentiation in human omental arteries (pEC50 5.8 +/- 0.3 vs. 6.4 +/- 0.2). Finally, messenger RNA for the neuropeptide Y Y1 receptor was detected using RT-PCR. On the basis of our results we conclude that the neuropeptide Y-induced potentiation of the noradrenaline-induced contraction is mediated by the neuropeptide Y Y1 receptor.

A turbidimetric immunochemical method for determination of serum beta 2-microglobulin using a centrifugal analyzer.

A turbidimetric immunochemical method has been developed for quantitation of serum-beta 2-microglobulin. The serum samples are pre-treated with polyethyleneglycol to decrease the background turbidity and a centrifugal analyzer is used for the absorbance measurements. The within-assay coefficient of variation was 4.6% when 58 serum samples with a mean value of 2.0 mg/l were analyzed in duplicate. The sensitivity of the method is below 0.5 mg/l. When serum samples were analyzed by both the turbidimetric method and a radioimmunoassay a correlation coefficient of 0.989 was obtained. The turbidimetric method is simple, cheap and has a high capacity which makes it suitable for routine determinations of serum beta 2-microglobulin in a clinical laboratory.

Increase in cardiac P2X1-and P2Y2-receptor mRNA levels in congestive heart failure.

We wanted to study the expression of P2-receptors at the mRNA-level in the heart and if it is affected by congestive heart failure (CHF). To quantify the P2 receptor mRNA-expression we used a competitive RT-PCR protocol which is based on an internal RNA standard. The P2 receptor mRNA-expression was quantified in hearts from CHF rats and compared to sham-operated rats. Furthermore, the presence of receptor mRNA was studied in the myocardium from patients with heart failure. In the sham operated rats the G-protein coupled P2Y-receptors were expressed at a higher level than the ligand gated ion-channel receptor (P2X1). Among the P2Y-receptors the P2Y6-receptor was most abundantly expressed (P2Y6 > P2Y1 > P2Y2 = P2Y4 > P2X1). A prominent change was seen for the P2X1- and P2Y2-receptor mRNA levels which were increased 2.7-fold and 4.7-fold respectively in the myocardium from the left ventricle of CHF-rats. In contrast, the P2Y1-, P2Y4- and P2Y6-receptor mRNA levels were not significantly altered in CHF rats. In human myocard the P2X1-, P2Y1-, P2Y2-, P2Y6- and P2Y11-receptors were detected by RT-PCR in both right and left atria and ventricles, while the P2Y4-receptor band was weak or absent. In conclusion, most of the studied P2-receptors were expressed in both rat and human hearts. Furthermore, the P2X1- and P2Y2-receptor mRNA were upregulated in CHF, suggesting a pathophysiological role for these receptors in the development of heart failure.

Renal and cardiovascular role of the neuropeptide Y Y1 receptor in ischaemic heart failure rats.

The cardiovascular role of the neuropeptide Y Y1 receptors in-vivo and in-vitro in ischaemic heart failure was evaluated by using the novel neuropeptide Y Y1 selective antagonist BIBP 3226 (R-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-D-arginine-amid e). In pithed rats, incremental doses of BIBP 3226 inhibited the exogenous neuropeptide Y induced pressor response in a dose-related fashion and a bolus injection of BIBP 3226 (0.5 mg kg(-1)) significantly shifted the pressor response curve of exogenous neuropeptide Y to the right. The potentiation effect to exogenous neuropeptide Y on the pressor response to preganglionic sympathetic nerve stimulation in ischaemic heart failure rats as well as on the contractile response to noradrenaline in renal arteries in sham-operated animals were also inhibited by the neuropeptide Y Y1 antagonist. In conscious ischaemic heart failure rats, incremental doses of BIBP 3226 (0.125-1 mg kg(-1)) significantly reduced basal blood pressure and heart rate. Compared with sham-operated rats, neuropeptide Y by itself induced no contraction and no potentiation on noradrenaline elicited contraction in renal artery of the ischaemic heart failure rat. Furthermore, under in-vivo conditions, BIBP 3226 did not influence basal renal function or the response to exogenous neuropeptide Y on urinary volume, urinary sodium and urinary potassium. Our results demonstrate that although there is a downregulation of the Y1 receptors by ischaemic heart failure, Y1 receptors are still mainly involved in cardiovascular actions of exogenous neuropeptide Y and play a role in maintaining basal blood pressure and heart rate in ischaemic heart failure. However, our data do not imply any significant role of Y1 receptors on basal renal function in the ischaemic heart failure rat model.

Estrogen attenuates vascular expression of inflammation associated genes and adhesion of monocytes to endothelial cells.

OBJECTIVE: Investigate effects of estrogen at gene expression and functional levels in vascular wall cells treated with bacterial lipopolysaccharide (LPS). MATERIALS AND METHODS: Aortic segments from ovariectomized mice were treated with LPS for 24 h in the absence or presence of 17beta-estradiol (E2). Gene activity was determined by Affymetrix microarray analysis and real-time RT-PCR. Adhesion of [3H]-thymidine labelled human THP-1 monocytes to mouse bEnd.3 endothelial cells was determined by measuring radioactivity of DNA from co-culture homogenates. RESULTS: Analysis of global gene expression profiles revealed that 10 nM E2 attenuates LPS-induced (10 ng/ml) expression of genes coding for well-known acute-phase proteins, such as alpha-trypsin inhibitor heavy chain 4, serum amyloid A3 and lipocalin 2. The E2-induced down-regulation of these three genes observed by microarray was confirmed by realtime RT-PCR. Treatment with 500 ng/ml LPS increased adhesion of monocytes to endothelial cells more than two fold. Importantly, LPS-induced monocyte adhesion was fully prevented by 50 nM E2. CONCLUSION: Estrogen reduces expression of acute-phase protein genes and inhibits LPS-induced moncocyte adhesion to endothelial cells, suggesting that estrogen might have a vasculoprotective effect via this mechanism.

Relationships between plasma levels of catecholamines and neuropeptides and the survival time in patients with congestive heart failure.

OBJECTIVES: To evaluate the importance of various variables reflecting neuroendocrine activation in relation to prognosis in patients with congestive heart failure (CHF). DESIGN: Plasma levels of noradrenaline, adrenaline, neuropeptide Y, substance P, calcitonin gene-related peptide, vasopressin and atrial natriuretic peptide were measured in patients presenting with CHF and related to survival time. Survival time was calculated from the time-point of this investigation until the month when the patient died or until December 1992. The follow-up period ranged up to 60 months. Seven of the 31 patients were still alive at the end of this period. SETTING: Lund University Hospital, Sweden. SUBJECTS: Thirty-one patients with CHF due to ischaemic heart disease. Six patients had a degree of heart failure corresponding to NYHA I-II and 25 corresponding to NYHA III-IV. Ten of these 25 patients were on angiotensin converting enzyme (ACE) inhibition in addition to therapy with digoxin and diuretics. The catecholamine and neuropeptide levels were compared to those of a control group of 31 healthy subjects aged 20-80 years. INTERVENTION: There were not any specific intervention in addition to the treatment for heart failure as outlined above. MAIN OUTCOME MEASURES: Survival time until death from heart disease or until the end of the follow up period in December 1992. RESULTS: There were inverse relationships between survival time on one hand and plasma values of noradrenaline (r = -0.49; P < 0.01) and atrial natriuretic peptide (r = -0.49; P < 0.01) on the other hand. There was a significant correlation between the plasma values of noradrenaline and atrial natriuretic peptide (r = 0.50; P < 0.01). CONCLUSION: Increased plasma levels of noradrenaline and atrial natriuretic peptide are of major importance as prognostic markers in patients with CHF.

Increased store-operated Ca2+ entry into contractile vascular smooth muscle following organ culture.

Ca2+ inflow via store-operated Ca2+ channels was investigated in rings of rat tail and basilar arteries kept in serum-free organ culture, which is known to preserve the contractility of the vascular smooth muscle. After culture for 3-4 days, Ca2+ release from intracellular stores in response to caffeine (20 mM) was augmented 2- to 4-fold. Following depletion of intracellular Ca2+ stores by caffeine and thapsigargin (10 microM), addition of Ca2+ (2.5 mM) caused an increase in the intracellular Ca2+ concentration which was 2-3 times greater in cultured than in freshly dissected rings, and was not affected by verapamil (10 microM). In contrast, L-type Ca2+ channel currents were decreased by 20% after culture. While freshly dissected rings developed no or very little force in response to the addition of Ca2+ after store depletion, cultured rings developed 42% (tail artery) and 60% (basilar artery) of the force of high-K+-induced contractions. These contractions in cultured vessels were insensitive to verapamil but could be completely relaxed by SKF-96365 (30 microM). Store depletion by caffeine increased the Mn2+ quench rate 3- to 4-fold in freshly dissected as well as cultured tail artery, while there was no increase in freshly dissected basilar artery, but a 3-fold increase in cultured basilar artery. Uptake of Ca2+ into intracellular stores was twice as rapid in cultured as in freshly dissected tail artery. This study shows that organ culture of vascular smooth muscle tissue causes changes in Ca2+ handling, resembling the pattern seen in dedifferentiating smooth muscle cells in culture, although contractile properties are maintained.

Dilatory responses to acetylcholine, calcitonin gene-related peptide and substance P in the congestive heart failure rat.

It was examined to what extent congestive heart failure (CHF) in rats, induced by ligation of the left coronary artery, affects the vascular responses to the vasodilatory substances acetylcholine (ACh), calcitonin gene-related peptide (CGRP), and substance P (SP). After induction of CHF status, the basilar, mesenteric and renal arteries and the iliac vein were studied in vitro. Dilatory responses were determined in relation to pre-contraction by the thromboxane mimetic U46619. Sham-operated animals (Sham) served as controls. U46619 induced stronger contraction in CHF basilar and renal arteries compared with the corresponding segments in Sham. ACh induced concentration-dependent dilations in all vessels examined with no difference of maximum relaxation or potency between CHF and Sham. SP induced weak dilations in all arteries examined while the response was markedly attenuated in CHF iliac veins compared with Sham (Emax% 12.2 +/- 3.4 vs. 32.3 +/- 4.8, P = 0.01). The CGRP induced dilation in the CHF basilar artery was weaker (Emax% 18.6 +/- 6.5 vs. 66.9 +/- 5.0, P < 0.001) and less potent (pEC50: 8.2 +/- 0.2 vs. 9.0 +/- 0.2, P = 0.01) compared with Sham. Further, CGRP was less potent in the renal artery of CHF rats compared with Sham (pEC50: 8.1 +/- 0.2 vs. 9.5 +/- 0.3, P < 0.01). In the CHF iliac vein, CGRP was more potent compared with Sham (pEC50: 9.7 +/- 0.4 vs. 8.3 +/- 0.4, P < 0.05). It can be concluded CHF is accompanied by alterations in the vascular response to the dilatory substances studied. The changes differ between vascular beds and between the different substances.

Modulation of vascular contractile responses to alpha 1- and alpha 2-adrenergic and neuropeptide Y receptor stimulation in rats with ischaemic heart failure.

In order to evaluate adaptational changes in vascular function in congestive heart failure (CHF), we studied the contractile responses of isolated arterial and venous blood vessels from rats suffering from CHF induced by coronary artery ligature, resulting in a myocardial infarction. The contractile responses of the basilar, femoral and renal arteries and of the iliac vein were examined in relation to adrenergic and neuropeptide Y (NPY) receptor function by the action of the alpha 1 agonist phenylephrine, the alpha 2 agonist clonidine and NPY. The contractile force was measured (in mN) and in % of K(+)-induced contraction as well as pD2 to each agonist. When stimulated by a 60 mM K(+)-buffer solution, the femoral and renal arteries from CHF rats responded with a stronger contraction (Emax; 9.4 +/- 0.6 and 9.8 +/- 0.6 mN) than the corresponding Sham vessels (Emax; 6.2 +/- 0.7 and 5.6 +/- 0.4 mN respectively, P < 0.001). On the contrary, the iliac vein of CHF responded less to K+ than the Sham iliac vein (Emax 2.5 +/- 0.2 and 3.7 +/- 0.5 mN, P < 0.01). The CHF iliac vein responded with a weaker contraction when stimulated with phenylephrine (Emax 1.9 +/- 0.4 mN) and showed a lower sensitivity (pD2 5.6 +/- 0.1) than the corresponding sham vessel (Emax 5.7 +/- 2.3 mN and pD2 6.3 +/- 0.5, P < 0.05). The CHF renal artery was less sensitive to clonidine (pD2 6.4 +/- 0.6) than the Sham renal artery (pD2 7.2 +/- 0.1, P < 0.05). The results indicate differences between CHF and Sham vessel segments according to both contractile capacity induced by K(+)-depolarization and to agonist induced contractile capacity and sensitivity. The differences are not of general nature but vary according to the vascular bed examined.