High-resolution YAC fragmentation map of 1q21

Effects of phenoxybenzamine on the myogenic contraction in response to quick stretch were examined in the isolated dog cerebral artery. Quick stretch at a rate of 10 cm/sec by 30% of the initial muscle length (= 100%) produced a myogenic contraction in dog basilar artery. Pretreatment of the cerebral artery strips with phenoxybenzamine (10(-4) M) for 30 min inhibited the stretch-induced contraction by 50-60%. By comparison, the treatment with phenoxybenzamine abolished the cerebral artery contractions induced by 5-hydroxytryptamine and histamine. Norepinephrine-induced contraction was also suppressed to about 10% by the treatment with phenoxybenzamine. High KCl (80 mM)-induced depolarizing contraction was nearly completely inhibited by the phenoxybenzamine treatment. These findings suggest that the site(s) of mechanoreception of membrane stretch (quick stretch) leading to myogenic contraction of dog cerebral artery is not susceptible to alkylation by phenoxybenzamine. It seems likely that mechanosensor site(s) responsible for inducing myogenic contraction by quick stretch is distinct from the sites for the reception of chemical stimulations.     

Myeloid hemopoietic growth factors. Review article

The vascular system is receptive to both chemical and physical factors, and these factors elicit subsequent cellular responses such as contraction and relaxation. Quick stretch applied to cerebral and coronary arteries produces myogenic contraction by mobilization of at least two Ca2+ components, i.e., transmembrane influx and release from intracellular storage sites of Ca2+. The mechanical reception is more susceptible than pharmacological reception to chemical skinning, suggesting the importance of membrane lipids as a mechanosensor domain. Phospholipase C coupled to a cholera toxin- or pertussis toxin-insensitive GTP-binding protein, possibly a G4 class one, may play a role in the genesis of vascular contraction in response to stretch. Activation of protein kinase C may affect more strongly the maintenance phase of stretch-induced contraction through the change in Ca2+ sensitivity of the contractile elements. The contractile reaction of vascular tissue to mechanical force such as stretch is a kind of physical response and thus requires cellular signal transduction, which may be mediated through a receptive site specific for a mechanical stimulus and the pathways of Ca2+ signaling that are common to pharmacological agonists.     

Pulsatile stretch stimulates superoxide production and activates nuclear factor-kappa B in human coronary smooth muscle

There is increasing evidence that oxidative stress is of pathophysiological importance in cardiovascular disease. Mechanical forces such as pulsatility may also contribute. Using human coronary artery smooth muscle cells (HCAS), we tested the hypothesis that stretch-induced cell proliferation is associated with oxidative stress. Stretch induced DNA synthesis in HCAS, and this was prevented by the antioxidants N-acetylcysteine and pyrrolidinedithiocarbamate (PDTC). Pulsatile stretch also increased superoxide production from HCAS in a time- and stretch dependent manner. Stretch-induced superoxide production was inhibited by diphenyleneiodoniumchloride, an NADPH oxidase inhibitor, and p-chloromercuriphenylsulfonic acid, an NADH oxidase inhibitor, but not by the xanthine oxidase inhibitor oxypurinol or the cyclooxygenase inhibitor indomethacin. In electrophoretic mobility shift assays, tumor necrosis factor-alpha activated nuclear factor-kappa B (NF-kappa B) with a peak at approximately 3 hours, whereas pulsatile stretch showed sustained activation during stimulation for up to 24 hours. The sustained activation of NF-kappa B was abolished by cotreatment with N-acetylcysteine or PDTC. Furthermore, treatment of HCAS with antisense p65 and p50 oligodeoxynucleotides of NF-kappa B inhibited stretch-induced DNA synthesis. We propose that pulsatile stretch increases oxidative stress and, in turn, promotes DNA synthesis via NF-kappa B in cultured human coronary artery smooth muscle cells.     

Mechanisms of stretch-induced changes in [Ca2+]i in rat atrial myocytes: role of increased troponin C affinity and stretch-activated ion channels

To study the effects of stretch on the function of rat left atrium, we recorded contraction force, calcium transients, and intracellular action potentials (APs) during stretch manipulations. The stretch of the atrium was controlled by intra-atrial pressure. The Frank-Starling behavior of the atrium was manifested as a biphasic increase of the contraction force after increasing the stretch level. The development of the contraction force after step increase of the stretch (intra-atrial pressure from 1 to 3 mm Hg) was accompanied by the increase in the amplitude of the calcium transients (P<0.05, n=4) and decrease in the time constant of the Ca2+ transient decay. The APs of the individual myocytes were also affected by stretch; the duration of the AP was decreased at positive voltages (AP duration at 15% repolarization level, P<0.001; n=13) and increased at negative voltages (AP duration at 90% repolarization level, P<0. 01; n=13). To study the mechanisms causing these changes we developed a mathematical model describing [Ca2+]i and electrical behavior of single rat atrial myocytes. Stretch was simulated in the model by increasing the troponin (TnC) sensitivity and/or applying a stretch-activated (SA) calcium influx. We mimicked the Ca2+ influx by introducing a nonselective cationic conductance, the SA channels, into the membrane. Neither of the 2 plausible mechanosensors (TnC or SA channels) alone could produce similar changes in the Ca2+ transients or APs as seen in the experiments. The model simulated the effects of stretch seen in experiments best when both the TnC affinity and the SA conductance activation were applied simultaneously. The SA channel activation led to gradual augmentation of Ca2+ transients, which modulated the APs through increased Na+/Ca2+-exchanger inward current. The role of TnC affinity change was to modulate the Ca2+ transients, stabilize the diastolic [Ca2+]i, and presumably to produce the immediate increase of the contraction force after stretch seen in experiments. Furthermore, we found that the same mechanism that caused the normal physiological responses to stretch could also generate arrhythmogenic afterpotentials at high stretch levels in the model.     

Biophysical signals underlying myogenic responses in rat interlobular artery

-To assess cellular mechanisms mediating myogenic responses of interlobular artery (ILA), experiments were performed with the use of isolated perfused hydronephrotic kidneys. ILAs were divided into 3 groups according to their basal diameters: proximal (>60 microm), intermediate (40 to 60 microm), and distal (<40 microm) ILAs. Myogenic responses were obtained by stepwise increase in perfusion pressure. Greater myogenic responsiveness was observed in ILAs with smaller diameters. Diltiazem (10 micromol/L) inhibited myogenic responses of all segments of ILAs. Furthermore, gadolinium (10 micromol/L), a mechanosensitive cation channel blocker, abolished myogenic responses of distal but not proximal ILA. In contrast, 2-nitro-4-carboxyphenyl-N, N-diphenyl-carbamate (200 micromol/L), an inhibitor of phospholipase C, prevented myogenic responses of proximal but not distal ILA. Finally, basal proximal ILA diameters were increased by treatment with 50 nmol/L of staurosporine (P<0.05), and subsequent addition of thapsigargin (1 micromol/L) blocked myogenic contraction of proximal ILAs. Myogenic responses of intermediate ILAs exhibited characteristics between those of distal and proximal ILAs. Our data indicate that underlying mechanisms for myogenic responses differ in distinct segments of ILAs. The present results suggest that mechanosensitive cation channels are involved in myogenic constriction of distal ILAs. Finally, our findings provide evidence that the stimulation of phospholipase C mediates myogenic contraction of proximal ILAs.     

The effects of 1,25-dihydroxycholecalciferol and phytase on the natural phytate phosphorus utilization by laying hens

Biochemical changes that are associated with the growth phase of stretch-induced skeletal muscle hypertrophy are better understood than events that maintain the increased muscle mass. One purpose of this study was to determine whether changes that occur during the period of rapid muscle hypertrophy persist during periods when muscle growth plateaus or the rate of enlargement slows. Serum response factor (SRF), myogenin, MyoD, and actin mRNA expression patterns were examined. SRF protein interactions with serum response element-1 (SRE1) of the chicken skeletal alpha-actin gene were also characterized. Anterior latissimus dorsi (ALD) wet weight (132% and 122%) and total RNA concentration (29% and 19%) increased after 2 and 3 weeks of stretch overload, respectively. Myogenin mRNA per microgram RNA increased after 3 (775%), 6 (1073%), 14 (227%), and 21 days (133%) of stretch overload. At 6 days, myogenin mRNA levels were increased in the distal, middle and proximal regions of the ALD. Serum response factor (SRF) mRNA per microgram total RNA was not increased after 2 or 3 weeks of stretch overload. MyoD and skeletal alpha-actin mRNAs per microgram total RNA were also unchanged after 2 and 3 weeks of stretch. Gel mobility shift assays demonstrated that SRF bound to SRE1 from 14-day-stretched ALD nuclear extracts had an increased mobility compared to control, and this difference in mobility was maintained in nuclear extracts from ALD muscle whose mass was declining. These results indicate that the expression of myogenin mRNA and total RNA remains elevated during either slow or maintenance periods of stretch-induced increases in ALD mass, when SRF mRNA has returned to control levels. Additionally, stretch-induced alterations in SRF binding to SRE1, from the skeletal alpha-actin promoter, occur regardless of the rate of stretch-induced growth.     

Possible mechanism of the potent vasoconstrictor actions of ryanodine on femoral arteries from spontaneously hypertensive rats

1. The Ca2+ buffering function of sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) was examined. Differences in the effects of ryanodine that removes the function of SR, on tension and cellular Ca2+ level were assessed in endothelium-denuded strips of femoral arteries from 13-week-old SHR and normotensive Wistar-Kyoto rats (WKY). 2. The addition of ryanodine to the resting strips caused a concentration-dependent contraction in SHR. This contraction was extremely small in WKY. In the presence of 10(-5) M ryanodine, caffeine (20 mM) failed to cause a further contraction in SHR, but it caused a small contraction in WKY. After washout of the strips with a Krebs solution, the resting tone was greatly elevated in SHR when compared with WKY. 3. The elevated resting tone in SHR strips was abolished by 10(-7) M nifedipine. The ryanodine-induced contraction was also abolished by 10(-7) M nifedipine. Nifedipine itself caused a relaxation from the resting tone of SHR strips, suggesting the maintenance of myogenic tone. 4. In strips preloaded with fura-PE3, the addition of 10(-5) M ryanodine caused a large and moderate elevation of cytosolic Ca2+ level ([Ca2+]i) in SHR and WKY, respectively. After washout, the resting [Ca2+]i was greatly elevated in SHR. The ryanodine-induced elevation of [Ca2+]i was decreased by 5 x 10(-6) M verapamil in SHR. Verapamil itself caused a decrease in resting [Ca2+]i which was significantly greater in SHR than in WKY, and caused a relaxation only in SHR. 5. The resting Ca2+ influx in arteries measured by a 5 min incubation with 45Ca was significantly increased in SHR when compared with WKY. The resting Ca2+ influx was not increased by 10(-5) M ryanodine in both SHR and WKY. The net cellular Ca2+ uptake in arteries measured by a 30 min incubation with 45Ca was decreased by 10(-5) M ryanodine in both strains. 6. The resting Ca2+ influx was decreased by 10(-7) M nifedipine in the SHR artery, but it was unchanged in the WKY artery. 7. These results suggest that (1) the Ca2+ influx via L-type voltage-dependent Ca2+ channels was increased in the resting state of the SHR femoral artery, (2) the greater part of the increased Ca2+ influx was buffered by Ca2+ uptake into the SR and some Ca2+ reached the myofilaments resulting in the maintenance of the myogenic tone, and (3) therefore the functional removal of SR by ryanodine caused a potent contraction in this artery.     

The physiology of spasticity

The contractile effects of ATP and related purine compounds on the isolated rabbit detrusor were investigated. It was found that ATP produced an initial rapid, phasic contraction followed by a slowly developing and maintained increase in tension. ADP caused a contraction closely mimicking the tonic response to ATP. The ADP induced contraction and the tonic response to ATP could both be abolished by indomethacin. beta, gamma-methylene ATP (APPCP), which is not degraded to ADP, elicited a rapid, phasic response, which could be abolished by nifedipine. AMP, dibutyryl-cAMP, and adenosine in low concentrations had no contractile effects; high concentrations of adenosine and 2-chloroadenosine, which is resistant to adenosine deaminase, decreased tone and spontaneous activity. The amplitude of the ATP induced contraction was positively correlated to the Ca2+-concentration in the extracellular medium; removal of Ca2+ abolished the ATP contraction before the responses to high K+ and carbachol disappeared. Responses to electrical field stimulation, mediated by non-cholinergic, non-adrenergic mechanisms were abolished by nifedipine and significantly reduced by indomethacin. It is concluded that in isolated rabbit detrusor, a direct contractile response can be elicited only by tripolyphosphates (ATP and APPCP), and that the diphosphate moiety ADP stimulates synthesis of prostaglandins The similarity between the effects of stimulation of non-cholinergic, non-adrenergic neurones and the phasic response to ATP supports the view that in rabbit detrusor ATP may be involved in excitation.     

Stretch-induced alkalinization of feline papillary muscle: an autocrine-paracrine system

Myocardial stretch is a well-known stimulus that leads to hypertrophy. Little is known, however, about the intracellular pathways involved in the transmission of myocardial stretch to the cytoplasm and nucleus. Studies in neonatal cardiomyocytes demonstrated stretch-induced release of angiotensin II (Ang II). Because intracellular alkalinization is a signal to cell growth and Ang II stimulates the Na+/H+ exchanger (NHE), we studied the relationship between myocardial stretch and intracellular pH (pHi). Experiments were performed in cat papillary muscles fixed by the ventricular end to a force transducer. Muscles were paced at 0.2 Hz and superfused with HEPES-buffered solution. pHi was measured by epifluorescence with the acetoxymethyl ester form of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM). Each muscle was progressively stretched to reach maximal developed force (Lmax) and maintained in a length that was approximately 92% Lmax (Li). During the "stretch protocol," muscles were quickly stretched to Lmax for 10 minutes and then released to Li; pHi significantly increased during stretch and came back to the previous value when the muscle was released to Li. The increase in pHi was eliminated by (1) specific inhibition of the NHE (EIPA, 5 micromol/L), (2) AT1-receptor blockade (losartan, 10 micromol/L), (3) inhibition of protein kinase C (PKC) (chelerythrine, 5 micromol/L), (4) blockade of endothelin (ET) receptors with a nonselective (PD 142,893, 50 nmol/L) or a selective ETA antagonist (BQ-123, 300 nmol/L). The increase in pHi by exogenous Ang II (500 nmol/L) was also reduced by both ET-receptor antagonists. Our results indicate that after myocardial stretch, pHi increases because of stimulation of NHE activity. This involves an autocrine-paracrine mechanism in which protein kinase C, Ang II, and ET play crucial roles.     

Early prognostic biochemical indicators of fulminant hepatic failure

This article reviews related studies from the authors' laboratory, which focus on the regulation of vascular Na+,K+-ATPase in hypertension. Earlier studies, including the authors', suggested that Na-pump activity in cardiovascular tissues is subject to regulation during hypertension; most of these studies report a stimulation of the vascular enzyme during established stages of hypertension. To test hypothesis that in vascular smooth muscle, strain resulting from elevated pressure may be a signal initiating a cascade of events leading to increased expression of Na+,K+-ATPase, the authors used cell culture and the Flexercell Strain Unit to apply cyclical stretch to rat aortic smooth muscle cells (ASMC) for several days. These studies demonstrated that mechanical strain induces the upregulation of both the alpha-1 and alpha-2 subunits of Na+,K+-ATPase. Mechanisms underlying these changes appear to involve a transient increase in intracellular sodium entering the cell through stretch-activated channels. Calcium entering the cell via L-type channels did not affect stretch-induced upregulation of the alpha isoforms. In addition, protein kinase C inhibition resulted in inhibition of the Na-pump during stretch, but not under nonstretch conditions. The authors conclude that the stretch component of vascular pressure upregulates the Na+,K+-ATPase catalytic subunits. Intracellular sodium may be a signal for this regulation. In addition, phosphorylation by PKC may be important in stretch-induced short-term regulation of the vascular Na-pump.     

Expression of fibroblast growth factor family during postnatal skeletal muscle hypertrophy

The potential role of the fibroblast growth factor (FGF) family during stretch-induced postnatal skeletal muscle hypertrophy was analyzed by using an avian wing-weighting model. After 2 or 11 days of weighted stretch, anterior latissimus dorsi (ALD) muscles were, on average, 34 (P < 0.01) and 85% (P < 0.01) larger, respectively, than unweighted ALD control muscles. By using quantitative RT-PCR, FGF-1 mRNA expression was found to be significantly decreased in ALD muscles stretched for 2 or 11 days. In contrast, FGF-4 and FGF-10 mRNA expression was significantly increased 2 days after initiation of stretch. FGF-2, FGF-10, fibroblast growth factor receptor 1, and FREK mRNA expression was significantly increased at 11 days poststretch. Increases in FGF-2 and FGF-4 protein could be detected throughout the myofiber periphery after 11 days of stretch. On a cellular level, FGF-2 and FGF-4 proteins were differentially localized. This differential expression pattern and protein localization of the FGF family in response to stretch-induced hypertrophy suggest distinct roles for individual FGFs during the postnatal hypertrophy process.     

The effect of alterations to action potential duration on beta-adrenoceptor-mediated aftercontractions in human and guinea-pig ventricular myocytes

Aftercontractions induced by beta-adrenoceptor stimulation in human and guinea-pig cardiomyocytes may be related to changes in action potential duration (APD). We investigated the effects of altering APD during the occurrence of isoproterenol-induced aftercontractions, using the KATP channel openers cromakalim and lemakalim or the action potential voltage clamp technique, in guinea-pig and human ventricular cardiomyocytes. Contractile responses were measured at 32 degrees C using a video-based edge-detection system. In guinea-pig myocytes, action potentials, Indo-1 fluorescence and contraction were measured at 22 degrees C. Isoproterenol (< or = 12 nM) had variable effects on APD but induced aftercontractions, the majority (14/19 cells) of which occurred during the action potential. Short action potentials were produced using K+ channel openers. These compounds reduced or completely abolished the isoproterenol-induced aftercontractions. Increasing isoproterenol in the presence of K+ channel opener restored the main contraction to a level similar to or above those with isoproterenol alone, but without the reappearance of aftercontractions. When cells were stimulated to contract under action potential voltage clamp, isoproterenol-induced aftercontractions were abolished by voltage clamping with action potentials of short duration. It was possible to induce aftercontractions in some cells without application of isoproterenol if voltage clamp-imposed action potentials of very long duration were used. These aftercontractions were also abolished by shortening action potential duration. We conclude that K+ channel openers or the imposition of action potentials of short duration can dissociate positively inotropic beta-adrenoceptor stimulation from aftercontraction formation and that action potentials of long duration can be pro-arrhythmic.     

Role of epidermal growth factor and its receptor in mechanical stress-induced differentiation of human periodontal ligament cells in vitro

The periodontal ligament (PDL) contains precursor cells for osteoblasts and cementoblasts. It has been shown that epidermal growth factor (EGF) inhibits dexamethasone-induced differentiation and up-regulates EGF-receptor (EGF-R) expression, whereas EGF-R is down-regulated in the course of differentiation. Thus it was suggested that EGF and its receptors act as a negative regulator of osteoblastic differentiation in PDL cells. In order to investigate further this hypothesis, human PDL cells were now used to elucidate the role of EGF and EGF-R in their proliferation and differentiation under mechanical stress-loaded conditions in vitro, as the PDL regularly receives mechanical stress from occlusal forces. As a model of mechanical stress, a cyclic stretch of 9 or 18% elongation was applied to the cells with a Flexercell cell-strain unit system. Alkaline phosphatase activity and osteocalcin mRNA expression were significantly induced by loading cyclic stretch for more than 4 days, whereas stretch slightly inhibited cell proliferation. Visualization of the actin stress fibres of the cells by rhodamine phalloidin revealed that approx. 10% of the total number of cells had become aligned perpendicularly to the direction of the stretch. The effects of stretch on alkaline phosphatase activity and cell proliferation were totally abolished by the presence of 10 ng/ml EGF. Western blotting of EGF-R protein demonstrated that stretch-induced differentiation accompanied the decreased expression of EGF-R protein in the cells. However, the amount of tyrosine-phosphorylated EGF-R upon EGF stimulation was restored to the control level in stretched cells. These results suggest that the EGF/EGF-R system acts as a negative regulator of differentiation of PDL cells regardless of the type of differentiation stimuli. Also, interaction between mechanical stress and the EGF/EGF-R system may participate in the osteoblastic differentiation of PDL cells and thereby regulate the source of cementoblasts and osteoblasts.     

Tyrosine kinase inhibitors suppress prostaglandin F2alpha-induced phosphoinositide hydrolysis, Ca2+ elevation and contraction in iris sphincter smooth muscle

We investigated the effects of the protein tyrosine kinase inhibitors, genistein, tyrphostin 47, and herbimycin on prostaglandin F2alpha- and carbachol-induced inositol-1,4,5-trisphosphate (IP3) production, [Ca2+]i mobilization and contraction in cat iris sphincter smooth muscle. Prostaglandin F2alpha and carbachol induced contraction in a concentration-dependent manner with EC50 values of 0.92 x 10(-9) and 1.75 x 10(-8) M, respectively. The protein tyrosine kinase inhibitors blocked the stimulatory effects of prostaglandin F2alpha, but not those evoked by carbachol, on IP3 accumulation, [Ca2+]i mobilization and contraction, suggesting involvement of protein tyrosine kinase activity in the physiological actions of the prostaglandin. Daidzein and tyrphostin A, inactive negative control compounds for genistein and tyrphostin 47, respectively, were without effect. Latanoprost, a prostaglandin F2alpha analog used as an antiglaucoma drug, induced contraction and this effect was blocked by genistein. Genistein (10 microM) markedly reduced (by 67%) prostaglandin F2alpha-stimulated increase in [Ca2+]i but had little effect on that of carbachol in cat iris sphincter smooth muscle cells. Vanadate, a potent inhibitor of protein tyrosine phosphatase, induced a slow gradual muscle contraction in a concentration-dependent manner with an EC50 of 82 microM and increased IP3 generation in a concentration-dependent manner with an EC50 of 90 microM. The effects of vanadate were abolished by genistein (10 microM). Wortmannin, a myosin light chain kinase inhibitor, reduced prostaglandin F2alpha- and carbachol-induced contraction, suggesting that the involvement of protein tyrosine kinase activity may lie upstream of the increases in [Ca2+]i evoked by prostaglandin F2alpha. Further studies aimed at elucidating the role of protein tyrosine kinase activity in the coupling mechanism between prostaglandin F2alpha receptor activation and increases in intracellular Ca2+ mobilization and identifying the tyrosine-phosphorylated substrates will provide important information about the role of protein tyrosine kinase in the mechanism of smooth muscle contraction, as well as about the mechanism of the intraocular pressure lowering effect of the prostaglandin in glaucoma patients.     

The scimitar syndrome: clinical spectrum and surgical treatment

The stretch-induced myogenic response (MR) of large-capacitance pulmonary arteries were studied in normal and pulmonary hypertensive fetuses as well as normal newborn and adult sheep. Pulmonary hypertension in the fetus was induced by ligation of the ductus arteriosus for 12 d. The MR was obtained by stretching the vessel segments in vitro from their resting diameter (no load) to the diameter at which the muscle fibers were at optimal length (Lo), and the response was measured as a percentage of force obtained after supramaximal electrical stimulation (Po). In five control and four pulmonary hypertensive fetuses, the MR was also obtained after a stretch of 140% of Lo. The pulmonary hypertensive fetal arteries had a lower stress (1.3 +/- 0.4 versus 4.0 +/- 0.5 mN/mm2; p < 0.001) and shortening capacity compared with the fetal control (5.1 +/- 1.6 versus 9.9 +/- 0.8% of Lo; p < 0.01). The MR was observed in 21% of the control and 30% of the experimental fetuses, and it was of greater magnitude in the latter (14.8 +/- 1.9 of Po versus 34.3 +/- 2.5%, respectively; p < 0.01). When stretched to 140% of Lo, the MR was also greater in the experimental (514 +/- 148% of Po) than the control fetuses (142 +/- 68; p < 0.05). Postnatally, the MR was present in 67% of the newborn and 15% of the adult pulmonary artery segments, and the response was greatest in the newborn (23.1 +/- 4.2% of Po) compared with the adult (2.3 +/- 0.8; p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulsatile stretch stimulates vascular endothelial growth factor (VEGF) secretion by cultured rat cardiac myocytes

Evidence has accumulated that vascular endothelial growth factor (VEGF) is expressed in the heart, and its expression is markedly increased in response to hypoxia. Recently, it was shown that pulsatile myocardial stretch in vivo markedly enhanced VEGF mRNA level in the heart. To investigate whether pulsatile mechanical stretch really stimulates VEGF expression by cardiac myocytes, using an in vitro preparation, we examined the secretion of VEGF into the culture media from cardiac myocytes subjected to pulsatile stretch. We found that pulsatile mechanical stretch induced rapid secretion of VEGF by cultured rat cardiac myocytes and mRNA expression of VEGF and VEGF receptors in the cardiac myocytes. We also found that the stretch-induced secretion of VEGF was at least in part mediated by TGF-beta. These data provide the direct evidence that mechanical overload itself can induce VEGF secretion by cardiac myocytes, which may play a role in ameliorating the relative myocardial hypoxia.     

Understanding the interactions between physician assistants and their supervising physicians in the provision of patient care

Both contractile and relaxant responses to tetrapentylammonium ions (TPA+) were studied in rat isolated mesenteric artery. TPA+ (5-10 micromol/l) caused a sustained increase of muscle tension. The contractile effect of TPA+ (10 micromol/l) was dependent upon the presence of extracellular Ca2+ but independent of the presence of endothelium. TPA+ (10-50 micromol/l) induced biphasic contraction, and the amplitude of peak and sustained tension decreased with increasing TPA+ concentration. TPA+ (100-300 micromol/l) only produced monophasic contraction. TPA+ (50 micromol/l) abolished the transient contraction induced by caffeine (10 mmol/l) or phenylephrine (1 micromol/l) in the absence of extracellular Ca2+. Nifedipine and verapamil concentration-dependently reduced the TPA+-induced contraction with respective IC50 values of 1.34 +/- 0. 24 and 9.46 +/- 1.36 nmol/l, these values were similar to 1.35 +/- 0. 21 and 16.07 +/- 1.71 nmol/l, respectively, for the inhibitory effects of nifedipine and verapamil on the high K+ (60 mmol/l)-induced contraction. TPA+ (>10 micromol/l) concentration-dependently reduced the phenylephrine (1 micromol/l)-, U46619 (30 nmol/l)-, endothelin I (10 nmol/l)- and high K+ (60 mmol/l)-induced sustained tension with respective IC50 values of 53. 7 +/- 9.5, 31.9 +/- 5.3, 30.9 +/- 3.4 and 20.9 +/- 2.8 micromol/l. The present results indicate that TPA+ at low concentrations could contract the arterial smooth muscle probably through promoting Ca2+ influx. At higher concentrations (>20 micromol/l), TPA+ relaxes arterial smooth muscle probably through inhibition of both nifedipine-sensitive Ca+ channels and internal Ca2+ release. TPA+, unlike other quaternary ammonium ions, could therefore act at multiple sites in arterial smooth muscle.     

Role of endothelin receptors, calcium and nitric oxide in the potentiation by endothelin-1 of the sympathetic contraction of rabbit ear artery during cooling

1. To examine further the potentiation by endothelin-1 on the vascular response to sympathetic stimulation, we studied the isometric response of isolated segments, 2 mm long, from the rabbit central ear artery to electrical field stimulation (1-8 Hz), under different conditions, at 37 degrees C and during cooling (30 degrees C). 2. Electrical stimulation produced frequency-dependent contraction, which was reduced (about 63% for 8 Hz) during cooling. At 30 degrees C, but not at 37 degrees C, endothelin-1 (1, 3 and 10 nM) potentiated the contraction to electrical stimulation in a dose-dependent way (from 43 +/- 7% to 190 +/- 25% for 8 Hz). 3. This potentiation by endothelin-1 was reduced by the antagonist for endothelin ETA receptors BQ-123 (10 microM) but not by the antagonist for endothelin ETB receptors BQ-788 (10 microM). The agonist for endothelin ETB receptors IRL-1620 (0.1 microM) did not modify the contraction to electrical stimulation. 4. The blocker of L-type Ca2+ channels verapamil (10 microM l-1) reduced (about 72% for 8 Hz) and the unspecific blocker of Ca(2+)-channels NiCl2 (1 mM) practically abolished (about 98%), the potentiating effects of endothelin-1 found at 30 degrees C. 5. Inhibition of nitric oxide synthesis with NG-nitro-L-arginine (L-NOARG, 0.1 mM) increased the contraction to electrical stimulation at 30 degrees C more than at 37 degrees C (for 8 Hz, this increment was 297 +/- 118% at 30 degrees C, and 66 +/- 15% at 37 degrees C). Endothelium removal increased the contraction to electrical stimulation at 30 degrees C (about 91% for 8 Hz) but not at 37 degrees C. Both L-NOARG and endothelium removal abolished the potentiating effects of endothelin-1 on the response to electrical stimulation found at 30 degrees C. 6. These results in the rabbit ear artery suggest that during cooling, endothelin-1 potentiates the contraction to sympathetic stimulation, which could be mediated at least in part by increasing Ca2+ entry after activation of endothelin ETA receptors. This potentiating effect of endothelin-1 may require the presence of an inhibitory tone due to endothelial nitric oxide.     

gp130 is involved in stretch-induced MAP kinase activation in cardiac myocytes

We have recently reported that mitogen activated protein kinase (MAP kinase) is activated by the stretch of the cultured cardiac myocytes in the angiotensin II deficient state in the angiotensinogen-deficient mice (Atg-/-), suggesting that factors other than the cardiac renin-angiotensin system are involved in the stretch-induced MAP kinase activation. We examined the contribution of cytokines using RX435, an anti-gp130 antibody. Leukemia inhibitory factor, which is one of the cytokines and has the common receptor subunit gp130, activated MAP kinase and the response was completely blocked by pretreatment of the Atg-/- cardiac myocytes with RX435. RX435 pretreatment greatly reduced stretch-induced activation of MAP kinase in Atg-/- cardiac myocytes. Interestingly, the same results were obtained in the cardiac myocytes of control mice. These results suggest that cytokine-gp130 may play a role in the stretch-induced MAP kinase activation independently of Ang II in cardiac myocytes.     

Functional properties of spinal interneurons activated by muscular free nerve endings and their potential contributions to the clasp-knife reflex

1. The goal of this study was to characterize the functional properties of spinal interneurons that are excited by muscular free nerve endings and to assess their contributions to the clasp-knife reflex. 2. The patterns of activity of 82 spinal interneurons that were excited by squeezing the Achilles tendon or manipulation of the muscle surfaces, preferential stimuli for muscular free nerve endings, were extracellularly recorded in lamina V-VII of the L5-S1 spinal cord in decerebrated and spinalized cats. 3. Interneurons were uniformly excited by increases in muscular length and force. Responses to muscle stretch exhibited gradual decay during maintained stretch, afterdischarge after stretch release, and adaptation to repeated stretch. Responses to isometric contraction induced by electrical stimulation of motor axons was also prolonged after contraction, but did not decay during maintained contraction. For similar increases in force, stretch evoked greater excitation than contraction, indicating that both stretch and contraction contributed to interneuronal activity. Overall, the time course and magnitude of the interneuronal responses to stretch and contraction paralleled the time course and magnitude of the clasp-knife reflex. 4. Interneurons were powerfully excited by muscular free nerve endings, which mediate the clasp-knife reflex, and by cutaneous receptors. Only occasionally were they excited by primary spindle or Golgi tendon organ afferents, which suggests that activation of muscular free nerve endings mediated the interneuronal responses to stretch and contraction. 5. Simultaneous recordings of interneuronal activity and the clasp-knife reflex revealed a broad correlation between interneuronal activity and clasp-knife inhibition. 6. Because the patterns of activity of free nerve ending-responsive interneurons during stretch and contraction were similar to the clasp-knife reflex, were closely correlated with clasp-knife inhibition during simultaneous interneuronal and reflex recordings, and were powerfully excited by muscular free nerve endings, it is likely that the interneurons described above contributed to the clasp-knife reflex. 7. In contrast, a small number (n = 16) of interneurons were recorded that were only weakly excited by muscular free nerve endings but strongly excited by group I afferents, exhibited less spontaneous and evoked activity, and had significantly different responses to stretch and contraction. These interneurons are less likely to have contributed to the clasp-knife reflex.