Biomedical subjects

X L Chen

Publications and source records attributed to X L Chen.

At least 55 records · Page 3Linked to original sources

PubMed1999

A role for T-type Ca2+ channels in the synergistic control of aldosterone production by ANG II and K+.

Independently, plasma K+ and ANG II stimulate aldosterone secretion from adrenal glomerulosa (AG) cells, but together they synergistically control production. We studied mechanisms to mediate this synergy using bovine AG cells studied under physiological conditions (in 1.25 mM Ca2+ at 37 degrees C). Increasing K+ from 2 to 5 mM caused a potentiation of ANG II-induced aldosterone secretion and a substantial membrane depolarization ( approximately 21 mV). ANG II inhibited a K+-selective conductance in both 2 and 5 mM K+ but caused only a slight depolarization because, under both conditions, membrane potential was close to the reversal potential of the ANG II-induced current. ANG II activated calcium/calmodulin-dependent protein kinase II (CaMKII) equivalently in 2 and 5 mM K+. However, CaMKII activation caused a hyperpolarizing shift in the activation of T-type Ca2+ channels, such that substantially more current was elicited at membrane potentials established by 5 mM K+. We propose that synergy in aldosterone secretion results from K+-induced depolarization and ANG II-induced modulation of T-type channel activation, such that together they promote enhanced steady-state Ca2+ flux.

Adenosine Triphosphate↗

PubMed1998

The buffer barrier hypothesis, [Ca2+]i homogeneity, and sarcoplasmic reticulum function in swine carotid artery.

1. The goal of this study was to evaluate the buffer barrier hypothesis in an intact arterial smooth muscle. Specifically, we investigated the interrelationships between intracellular [Ca2+] ([Ca2+]i) homogeneity and sarcoplasmic reticulum function in swine carotid artery. 2. We measured focal changes in [Ca2+]i by exploiting the different characteristics of several [Ca2+]i indicators: (1) aequorin, which can detect focal increases in [Ca2+]i such as those that occur in the subplasmalemmal region ([Ca2+]pm); (2) fura-2, which is primarily a measure of mean cytoplasmic [Ca2+] ([Ca2+]c); and (3) force, which reflects increases in [Ca2+] near the contractile apparatus. We then estimated the relative degree of [Ca2+]i homogeneity with the aequorin/fura-2 ratio. Finally, we inhibited sarcoplasmic reticulum Ca2+ pumping with cyclopiazonic acid (CPA), an inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). 3. We found that, after Ca2+ depletion, the sarcoplasmic reticulum could be partially reloaded with Ca2+ by manipulations that increased the aequorin signal relatively more than the fura-2 signal. Complete reloading required large increases in the fura-2 signal. These data suggest that increases in [Ca2+]pm (as measured with aequorin) can partially reload the sarcoplasmic reticulum, but complete reloading required increases in [Ca2+]c (as measured with fura-2). Reloading could be partially inhibited by 10 microM CPA, indicating that SERCA function was important for reloading. 4. In unstimulated arteries, 10 microM CPA increased the fura-2 signal without altering the aequorin signal, thereby decreasing the aequorin/fura-2 ratio. Removal of extracellular Ca2+ without CPA also reduced the aequorin/fura-2 ratio. These data suggest that resting cells have a [Ca2+] gradient with [Ca2+]pm > [Ca2+]c; this gradient is maintained by SERCA function. 5. CPA slowed the decline in the fura-2 signal observed when histamine stimulation was removed. This result is consistent with the concept of vectorial Ca2+ efflux in which Ca2+ pumping by SERCA reduces [Ca2+]c after stimulation. 6. Ca2+ depletion by prior treatment with 100 microM histamine and CPA transiently attenuated subsequent histamine-induced aequorin and fura-2 transients. The effect on contraction was smaller: a delay in contraction of approximately 10 s. These data suggest that histamine-induced Ca2+ release has at least a small role in the initial phase of contraction; however, other contractile mechanisms appear to be able to compensate for loss of Ca2+ release with only modest changes in contraction kinetics. 7. These data suggest that there is a complex interrelationship between smooth muscle sarcoplasmic reticulum function and [Ca2+] in at least two cytoplasmic compartments. [Ca2+]pm and [Ca2+]c can differentially regulate sarcoplasmic reticulum Ca2+ filling; and sarcoplasmic reticulum function regulates [Ca2+]pm and [Ca2+]c.

Aequorin↗

PubMed1998

Angiotensin II induces monocyte chemoattractant protein-1 gene expression in rat vascular smooth muscle cells.

Monocyte infiltration into the vessel wall, a key initial step in the process of atherosclerosis, is mediated in part by monocyte chemoattractant protein-1 (MCP-1). Hypertension, particularly in the presence of an activated renin-angiotensin system, is a major risk factor for the development of atherosclerosis. To investigate a potential molecular basis for a link between hypertension and atherosclerosis, we studied the effects of angiotensin II (Ang II) on MCP-1 gene expression in rat aortic smooth muscle cells. Rat smooth muscle cells treated with Ang II exhibited a dose-dependent increase in MCP-1 mRNA accumulation that was prevented by the AT1 receptor antagonist losartan. Ang II also activated MCP-1 gene transcription. Inhibition of NADH/NADPH oxidase, which generates superoxide and H2O2, with diphenylene iodonium or apocynin decreased Ang II-induced MCP-1 mRNA accumulation. Induction of MCP-1 gene expression by Ang II was inhibited by catalase, suggesting a second messenger role for H2O2. The tyrosine kinase inhibitor genistein and the mitogen-activated protein kinase kinase inhibitor PD098059 inhibited Ang II-induced MCP-1 gene expression, consistent with a mitogen-activated protein kinase-dependent signaling mechanism. Ang II may thus promote atherogenesis by direct activation of MCP-1 gene expression in vascular smooth muscle cells.

Angiotensin II↗

PubMed1998

Hormonal regulation of leptin mRNA expression and preadipocyte recruitment and differentiation in porcine primary cultures of S-V cells.

The hormonal regulation of leptin mRNA expression and the association between leptin expression and adipocyte differentiation were examined in primary cultures of porcine S-V cells with Northern blot and immunocytochemical analysis. Seeding for 3 days with fetal bovine serum (FBS) with varying levels of dexamethasone (Dex) increased levels of leptin mRNA in a dose-dependent manner in parallel with increases in the proportion of preadipocytes (AD-3 positive cells; AD-3, a preadipocyte marker). Six-day treatment with 10 or 850 nM insulin after FBS+Dex treatment resulted in a similar increase in leptin mRNA expression and morphological differentiation. However, significantly lower levels of leptin mRNA and smaller fat cells were observed in cultures treated with 1 nM insulin or 10 nM insulin-like growth factor-I (IGF-I). Dex-induced increases in leptin mRNA levels and AD-3 cell numbers were blocked completely by the addition of transforming growth factor-beta (TGF-beta) to FBS+Dex-treated cultures. However TGF-beta significantly increased fat cell size and leptin mRNA expression when added to ITS (insulin, 850 nM; transferrin, 5 microg/ml; and selenium, 5 microg/mL) treated cultures during the lipid-filling stage. When added with FBS+DEX for the first 3 days, growth hormone (GH) did not influence the Dex-induced increase in AD-3 cells and leptin mRNA expression, but GH reduced leptin mRNA levels when added with insulin for 6 days after FBS+Dex. These results demonstrated that regulation of leptin mRNA expression by Dex, insulin, IGF-I, TGF-beta, and GH may be associated with changes in preadipocyte number and fat cell size.

Adipocytes↗

PubMed1998

Mechanisms responsible for forskolin-induced relaxation of rat tail artery.

The goal of the present study was to determine the physiologically relevant mechanisms for forskolin-induced relaxation of intact rat tail artery. We stimulated deendothelialized rat tail artery with phenylephrine and then relaxed the tissue with the addition of forskolin, a specific activator of adenylyl cyclase. We measured membrane potential with the use of microelectrodes, estimated intracellular Ca2+ concentration ([Ca2+]i) with the use of fura 2, and measured isometric force with a strain-gauge transducer. We found that 0.3 to 1.0 micromol/L forskolin relaxed 0.3 to 1.0 micromol/L phenylephrine-stimulated rat tail artery by decreasing the [Ca2+]i sensitivity of force as well as through repolarization. There was no evidence for forskolin-induced inhibition of Ca2+ influx beyond that associated with repolarization. There also was no evidence for forskolin-induced enhancement of Ca2+ efflux or sequestration. Inhibition of ATP-activated K+ channels with 10 micromol/L glibenclamide, Ca2+-activated K+ channels with 50 nmol/L iberiotoxin, Ca2+-activated K+ channels with 3 or 10 mmol/L tetraethylammonium ion, inwardly rectified K+ channels with 20 micromol/L Ba2+, and voltage-activated K+ channels with 0.5 mmol/L 4-aminopyridine did not significantly attenuate forskolin-induced reductions in [Ca2+]i or force. Forskolin-induced repolarization was not altered by 10 micromol/L glibenclamide or 0.5 mmol/L 4-aminopyridine. These data suggest that these K+ channels were not individually involved in forskolin-induced relaxation and that other channels and/or multiple channels are involved in forskolin-induced repolarization of intact rat tail artery. Our data also suggest that forskolin-induced relaxation of intact rat tail artery occurred primarily through repolarization and reductions in the [Ca2+]i sensitivity of force.

Animals↗

PubMed1998

[Monosynaptic transmission between mesodiencephalic neurons and motoneurons innervating extraocular inferior oblique muscle in cats].

The present study was performed to define the synaptic connection of neurons located within the dorsal mesodiencephalic junction with motoneurons innervating extraocular inferior oblique muscle (IOMN) using electrophysiologic, especially spike-triggered averaging technique in cats. In response to electric shocks applied to the medial part of Forel's field H (FFH) located in the mesodiencephalic junction, excitatory monosynaptic field potentials were observed in the IOMN pool. Moreover, while the spontaneous discharges of FFH neurons were used to trigger the activity of IOMN pool from which the induced potential changes were averaged, an excitatory monosynaptic field potential was also obtained. These results indicate that the mesodiencephalic neurons connect with and activate the IOMNs monosynaptically, and drive vertical eye movements via such a way.

Action Potentials↗

PubMed1997

Metformin relaxes rat tail artery by repolarization and resultant decreases in Ca2+ influx and intracellular [Ca2+].

BACKGROUND: Metformin treatment of type II diabetes is frequently associated with decreases in blood pressure, an effect that could result from a direct action of metformin on arterial smooth muscle. OBJECTIVE: To determine the mechanisms responsible for arterial smooth muscle relaxation induced by acute application of metformin and to evaluate the effect of insulin pretreatment on intracellular [Ca2+] ([Ca2+]i) and contraction in an intact artery. METHODS: We stimulated intact deendothelialized rat tail artery with phenylephrine, relaxed the tissue by adding increasing concentrations of metformin, and measured the membrane potential (Em), Mn2+ influx, Fura 2-estimated [Ca2+]i, and isometric force. We also evaluated the effect of insulin pretreatment on aequorin-estimated [Ca2+]i in deendothelialized swine carotid artery. RESULTS: In rat tail artery we found that a high concentration of metformin-induced repolarization associated with proportional decreases in Mn2+ influx, Fura 2-estimated [Ca2+]i, and isometric force. Incubation of swine carotid artery in 100 mU/ml insulin for 30 min or overnight (16-22 h) did not significantly alter histamine or high-K+-induced increases in [Ca2+]i or contraction. CONCLUSION: These data suggest that acute administration of high concentrations of metformin induces rat tail artery relaxation primarily by repolarization. Additionally, we found that insulin was not vasoactive in the swine carotid artery. It is possible that insulin may alter [Ca2+]i handling in other arteries, in other species, or only in cultured smooth muscle.

Animals↗

PubMed1997

E-selectin gene expression in vascular smooth muscle cells. Evidence for a tissue-specific repressor protein.

E-Selectin is an inducible, endothelium-specific, cell surface adhesion molecule that mediates inflammatory responses in the vasculature. Nonendothelial cell types such as cultured human aortic smooth muscle cells (HASMCs) lack the ability to express E-selectin. We tested the hypothesis that HASMCs express a negative regulatory factor that inhibits E-selectin gene expression. E-Selectin mRNA and gene transcription were not detected in HASMCs after treatment with tumor necrosis factor-alpha (TNF-alpha) by Northern and nuclear runoff analyses, respectively. However, both E-selectin mRNA and gene transcription were dramatically induced by TNF-alpha in the same cells pretreated with the protein synthesis inhibitor cycloheximide. Lipopolysaccharide demonstrated similar effects. Furthermore, E-selectin was detected on the cell surface of HASMCs after washing out of cycloheximide. Cycloheximide pretreatment enabled immortalized human dermal microvascular endothelial cells that have lost the ability to express E-selectin to induce both E-selectin mRNA and gene transcription in response to TNF-alpha. Induction of E-selectin mRNA by lipopolysaccharide or TNF-alpha in cycloheximide-treated HASMCs was inhibited by the antioxidant pyrrolidinedithiocarbamate and the serine protease inhibitor N alpha-L-tosyl-L-phenylalanine chloromethyl ketone, suggesting that a nuclear factor-kappa B-like mechanism may play an important role in E-selectin gene expression in HASMCs. These data strongly suggest that a labile repressor protein(s) plays an important role in inhibiting E-selectin gene expression in HASMCs likely at the level of gene transcription. Except for this repressor, HASMCs and endothelial cells may share similar regulatory mechanisms for controlling E-selectin expression.

Antioxidants↗

PubMed1996

The effect of prior in vitro exposure of donor cells to trophic factors in neurotransplantation.

The ability of PC12 cells to regenerate processes is substantially enhanced in vitro if they have been previously exposed to nerve growth factor (NGF-primed), compared to cells that have not been exposed (NGF-naive). These studies were carried out to determine if the enhanced neuritogenic ability of NGF-primed cells is retained following transplantation. NGF-naive or NGF-primed PC12 cells were transplanted into the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice and allowed to survive for 2 weeks. Mice were given daily injections of cyclosporin A (CyA) to prevent anti-species graft rejection. The transplanted PC12 cells were visualized by tyrosine hydroxylase immunoreactivity. The NGF-naive transplanted cells formed dense clusters and large tumor masses in more than half the animals. Only a few of the naive PC12 cells had short processes. In contrast, many of the transplanted NGF-primed PC12 cells had processes. Furthermore, fewer of the animals transplanted with primed cells produced tumor masses in the striatum compared to animals that received NGF-naive cells. Transplantation of NGF-naive PC12 cells leads to a significant increase in the number of dopaminergic neurons in the host substantia nigra (SN) compared to MPTP-treated animals. The increase of host dopaminergic neurons was not statistically significant when NGF-primed PC12 cells were used. Following MPTP treatment and PC12 cell transplantation, injection of CyA did not affect the dopaminergic neurons in the host SN. These data suggest that exposure of cells to trophic factors, prior to transplantation, can enhance their level of differentiation and integration into the host brain.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed1996

Focal increases in [Ca2+]i may account for apparent low Ca2+ sensitivity in swine carotid artery.

Estimates of [Ca2+]i sensitivity in intact smooth muscle are frequently obtained by measuring [Ca2+]i with indicators such as aequorin or Fura-2. We investigated whether focal increases in [Ca2+]i could impair such measures of [Ca2+]i sensitivity. Stimulation of swine carotid artery with 10 microM histamine increased aequorin estimated [Ca2+]i, Fura-2 estimated [Ca2+]i and Ca2+ sensitivity without significantly altering the aequorin/Fura-2 ratio (an estimate of [Ca2+]i homogeneity). Subsequent inhibition of Na+/Ca2+ exchange by replacement of Na+ in the PSS with choline+ significantly increased aequorin-estimated [Ca2+]i but only minimally increased Fura-2 estimated [Ca2+]i, myosin light chain (MLC) phosphorylation and force. This resulted in a large increase in the aequorin/Fura-2 ratio, suggesting an increase in [Ca2+] inhomogeneity. Addition of 100 microM histamine to tissues in the choline+ buffer initially increased both aequorin and Fura-2 estimated [Ca2+]i, but after 10 min exposure both of the [Ca2+]i estimates declined to pre-histamine levels. Histamine addition significantly increased MLC phosphorylation and force, indicating increased Ca2+ sensitivity, but the aequorin/Fura-2 ratio remained elevated and unchanged from pre-histamine values. These data show that under certain conditions, aequorin and Fura-2 can yield widely differing estimates of [Ca2+]i and thus can cause misleading assessments of Ca2+ sensitization mechanisms. These discrepancies may arise from inhomogeneous or focal increases in [Ca2+]i which can be evaluated with the aequorin/Fura-2 ratio.

Aequorin↗

PubMed1996

Nitroglycerin relaxes rat tail artery primarily by lowering Ca2+ sensitivity and partially by repolarization.

At least five mechanisms are hypothesized to account for guanosine 3',5'-cyclic monophosphate (cGMP)-induced relaxation of arterial smooth muscle: 1) repolarization, 2) inhibition of Ca2+ release, 3) inactivation of L-type Ca2+ channels, 4) enhancement of Ca2+ efflux/sequestration, and 5) decreasing the intracellular Ca2+ concentration ([Ca2+]i) sensitivity of force. The goal of this study was to investigate the physiological relevance of these five mechanisms in the intact rat tail artery. We stimulated deendothelialized rat tail artery with phenylephrine or high extracellular K+ concentration ([K+]o) and then relaxed the tissue by adding nitroglycerin to increase guanosine 3',5'-cyclic monophosphate concentration. We measured membrane potential (Em) with microelectrodes, [Ca2+]i with fura 2, and isometric force with a strain-gauge transducer. We found that decreases in the [Ca2+]i sensitivity of force accounted for most of the nitroglycerin-induced relaxation of tissues prestimulated with maximal (1 microM) phenylephrine or 30 mM [K+]o. In submaximally (0.1-0.3 microM) phenylephrine-prestimulated tissues, nitroglycerin-induced relaxation was caused primarily by a decrease in the [Ca2+]i sensitivity of force and partially by repolarization and the resultant decrease in [Ca2+]i. Nitroglycerin also partially attenuated transient increases in [Ca2+]i and force induced by 100 microM phenylephrine in the absence of extracellular Ca2+, indicating that nitroglycerin also inhibited intracellular Ca2+ release. Nitroglycerin-induced relaxation was not associated with inactivation of Ca2+ channels or enhancement of Ca2+ efflux/sequestration. These data suggest that nitroglycerin relaxes precontracted rat tail artery primarily by decreasing the [Ca2+]i sensitivity of force.

Animals↗

PubMed1996

[The effect of early chest physiotherapy on blood gas and circulatory function in old patients after thoracotomy].

In this study, 15 old patients undergoing thoracotomy were selected for observing the influence of effective chest physiotherapy started early in postoperation on blood gas and circulatory function. Following ultrasonic aerosol inholation for 20 minutes, turning the body, and chest percussion in head-up were started at 6th hour after operation. The result showed that PaO2, SaO2 were significantly increased and PaCO2 were decreased at 30 minutes after treatment comparied with it before treatment. In other side, although the hemodynamics could maintain stabilization, cardiac arrhythmias were occured during treatment in three cases, whose ECG were abnormal in preopration. It concluded that effectively chest physiotherapy, started early in postoperation, could prevent postoperative hypoxemia in old thoracotomy patients, But, ECG monitoring is necessary during treatment in patients whose ECG was abnormal before operation.

Aged↗

PubMed1995

Focal [Ca2+]i increases detected by aequorin but not by fura-2 in histamine- and caffeine-stimulated swine carotid artery.

1. We hypothesized that the homogeneity of intracellular [Ca2+] ([Ca2+]i) varies and is regulated in arterial smooth muscle. 2. We evaluated this hypothesis by exploiting the different characteristics of several [Ca2+]i indicators: (1) aequorin, which theoretically can measure focal increases in [Ca2+]i, (2) fura-2, which is predominantly a measure of mean cytoplasmic [Ca2+], and (3) myosin light chain phosphorylation and force, which reflect increases in [Ca2+] near the contractile apparatus. 3. From the differences in the observed aequorin and fura-2 signals, we developed an index of the relative degree of [Ca2+]i homogeneity as the ratio of the aequorin signal and fura-2 signal. 4. Stimulation with intermediate concentrations of histamine (1 and 10 microM) or high [K+]o (25 and 40 mM) increased [Ca2+]i and contractile stress. Relative [Ca2+]i homogeneity, estimated from the aequorin/fura-2 ratio, remained similar to levels observed in unstimulated tissues. 5. Higher concentrations of histamine (100 microM) also increased [Ca2+]i and stress, but the aequorin/fura 2 ratio declined , indicating increased [Ca2+]i homogeneity. Similarly, the aequorin/fura-2 ratio decreased when extracellular Ca2+ was removed. 6. Stimulation with histamine in low extracellular [Ca2+] transiently increased [Ca2+]i and the aequorin/fura-2 ratio. Similarly, in tissues treated with low extracellular [Ca2+], restoration of extracellular Ca2+ transiently increased both [Ca2+]i and the aequorin/fura-2 ratio. Although both of these experiments demonstrated a transient decrease in [Ca2+]i homogeneity, only histamine stimulation led to increased myosin light chain phosphorylation and force. These results indicate that the focal increases in [Ca2+]i observed with histamine stimulation and Ca2+ restoration occurred in different cellular regions. 7. Addition of caffeine (20 mM) increased [Ca2+]i and [cAMP], but this was not accompanied by sustained increased myosin light chain phosphorylation or contraction. Phosphorylation of myosin light chain kinase did not appear to underlie the lack of increase in myosin light chain phosphorylation. Rather, caffeine induced a sustained increase in the aequorin/fura-2 ratio, suggesting that caffeine inhibits smooth muscle contraction by localizing increases in [Ca2+]i to a region distant from the contractile apparatus. 8. These data suggest that there can be transient and sustained focal increases in [Ca2+]i. Aequorin detected increased [Ca2+]i in small regions of the cytoplasm during release from and refilling of the intracellular Ca2+ store and with caffeine stimulation. Dual use of aequorin and fura-2 permits determination of relative [Ca2+]i homogeneity in smooth muscle.

Aequorin↗

PubMed1995

Phenylephrine contracts rat tail artery by one electromechanical and three pharmacomechanical mechanisms.

There are at least four mechanisms hypothesized to account for excitation-contraction coupling in arterial smooth muscle. 1) Contractile agonists and changes in extracellular [K+] ([K+]o) induce contraction by depolarization, which increases Ca2+ influx; this is the only mechanism involving a change in membrane potential (Em). 2) Contractile agonists release Ca2+ from the intracellular Ca2+ store. 3) Contractile agonists increase Ca2+ influx without changing Em either by activating voltage-dependent L-type Ca2+ channels or by opening other Ca(2+)-permeable channels. 4) Contractile agonists increase intracellular Ca2+ ([Ca2+]i) sensitivity of force; this is the only mechanism that does not involve changes in [Ca2+]i. Each of these mechanisms has been demonstrated in intact, skinned, or dissociated smooth muscle preparations. However, these four mechanisms have not been compared in the same preparation. The goal of this study was to determine which of these four contractile mechanisms are physiologically relevant in the intact rat tail artery. We stimulated deendothelialized rat tail artery with phenylephrine and high [K+]o. We then measured Em with microelectrodes, [Ca2+]i with fura 2, and isometric force with a strain gauge transducer. We find that all four mechanisms contributed to phenylephrine-induced rat tail artery contraction. The majority of phenylephrine-induced contraction was caused by depolarization and by increases in the [Ca2+]i sensitivity of force. Low concentrations of phenylephrine also increased [Ca2+] independent of changes in Em, potentially by increases in Ca2+ influx. Release of Ca2+ from intracellular stores was only observed with high concentrations of phenylephrine. Smooth muscle appears to invoke multiple mechanisms for excitation-contraction coupling.

Animals↗

PubMed1995

pHi, [Ca2+]i, and myosin phosphorylation in histamine- and NH4(+)-induced swine carotid artery contraction.

We examined the interaction among changes in pHi, [Ca2+]i, myosin light-chain phosphorylation, and contraction in arterial smooth muscle stimulated by histamine, NH4+, Tris+, and/or changes in extracellular pH (pHo). We loaded swine carotid medial tissues with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein to measure pHi or aequorin to measure [Ca2+]i. Incubation of tissues in NH4+ increased pHi, [Ca2+]i, myosin phosphorylation, and force. Washout of NH4+ decreased pHi and transiently further increased in [Ca2+]i and force. Incubation of tissues in a similar concentration of Tris+ or increasing pHo also increased pHi; however, there were only modest changes in [Ca2+]i and force. Increasing extracellular pH coincidentally with washout of NH4+ prevented the decrease in pHi but did not affect the NH4+ washout-induced contraction. These data suggest that NH4+ altered [Ca2+]i and contraction by mechanisms other than its effects on pHi. The type of pH buffer did not affect the [Ca2+]i, myosin phosphorylation, or stress response to histamine stimulation. The time course of changes in pHi was much slower than the time course of histamine-induced changes in [Ca2+]i, myosin phosphorylation, and stress. Addition of 10 mmol/L NH4+ concurrently with histamine aborted the histamine-induced decrease in pHi and significantly slowed the histamine-induced increase in [Ca2+]i, myosin phosphorylation, and stress. There was little effect on histamine-induced increases in [Ca2+]i, myosin phosphorylation, or contraction when three other protocols aborted the histamine-induced decrease in pHi. These data show that incubation in NH4+ can alter [Ca2+]i and contraction in both unstimulated and histamine-stimulated smooth muscle. However, these effects were not caused by NH4(+)-dependent changes in pHi.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia↗

PubMed1995

Influences of phenylephrine on delayed afterdepolarization and triggered activity in sheep Purkinje fibers.

AIM: To compare the effects of phenylephrine (Phe) on DAD and TA in the absence or presence of propranolol (Pro). METHODS: Using intracellular microelectrode methods to record transmembrane potentials in sheep Purkinje fibers. RESULTS: Perfused Phe 1 mumol.L(-1) for 60 min, in the absence of Pro, Phe increased the amplitude of DAD from 7.5 +/- 1.2 to 9.0 +/- 2.0 mV (n = 8, P < 0.05), nonsignificantly changed the duration of DAD and induced TA in 3/11 preparations; but in the presence of Pro 0.5 mumol.L(-1) blocking beta-adrenoceptors, the amplitude of DAD increased from 7.2 +/- 1.8 to 8.3 +/- 2.1 mV at the first 20 min (n = 8, P < 0.01), and then decreased to 6.3 +/- 1.6 mV, the duration of DAD prolonged from 192 +/- 17 to 280 +/- 27 ms (n = 8, P < 0.01 vs control), meanwhile Phe could suppress the TA induced by acetylstrophanthidin and this effect was blocked by prazosin. CONCLUSION: When Pro is efficiently used in abolishing arrhythmias evoked by catecholmines, apart from directly blocking beta-adrenoceptors, relatively aggravating excitation of alpha-adrenoceptors to suppress DAD and TA is one of the important reasons.

Adrenergic alpha-Agonists↗

PubMed1994

Assay of pulmonary microvascular endothelial angiotensin-converting enzyme in vivo: comparison of three probes.

We monitored the activity of pulmonary microvascular endothelial-bound angiotensin-converting enzyme (ACE) in vivo by means of multiple indicator-dilution-type techniques, utilizing three different probes: the hydrolysis of two substrates, [3H]-benzoyl-Phe-Ala-Pro (BPAP) and [14C]benzoyl-Ala-Gly-Pro (BAGP), and the binding of the inhibitor [3H]RAC-X-65 (RAC), all measured during a single transpulmonary passage in anesthetized rabbits, placed on total heart bypass, so that both systemic and pulmonary circulations were fully supported by means of a two-channel extracorporeal pump. Experiments were performed at pulmonary blood flows (Qb) of 250, 400, 560, and 800 ml/min in control or indomethacin-pretreated rabbits. ACE activity was also compared to that of pulmonary microvascular endothelial-bound 5'-nucleotidase, by measuring the dephosphorylation of its natural substrate 5'-[14C]AMP. We calculated substrate utilization, mean lung transit time (t), and volume of distribution (i.e., central blood volume) of all substrates, as well as inhibitor binding. We also calculated Amax/Km and Bmax products of enzyme mass and kinetic constants for substrates and inhibitor, respectively. As Qb increased, Amax/Km values for all three substrates and Bmax increased linearly, indicating microvascular recruitment. In experiments in which either BPAP and 5'-AMP metabolism or BAGP metabolism and RAC binding were studied concomitantly, a linear relationship was observed between Qb-induced changes in Amax/Km values of BPAP vs 5'-AMP as well as in Amax/Km of BAGP vs Bmax of RAC. Similarly, increasing Qb increased central blood volume and decreased t. Indomethacin had no effect on most of the hemodynamic or enzyme parameters measured. We conclude that in vivo assays of ACE proceed as predicted by Michaelis-Menten kinetics and offer insights into pulmonary endothelial pathophysiology.

Animals↗

PubMed1994

Radiation-induced early pulmonary endothelial ectoenzyme dysfunction in vivo: effect of indomethacin.

We investigated the early effects of radiation on pulmonary endothelial function in vivo 7-8 hr after exposure of rabbits to a single dose of 30 Gy to the chest. Utilizing multiple indicator-dilution techniques, we measured rates and kinetics of hydrolysis of the synthetic substrates [3H]benzoyl-Phe-Ala-Pro (BPAP) and [14C]benzoyl-Ala-Gly-Pro (BAGP) by endothelial-bound angiotensin-converting enzyme (ACE) and of 5'[14C]-AMP by endothelial-bound 5'-nucleotidase (NCT) and binding of the synthetic ACE inhibitor [3H]RAC-X-65 during a single transpulmonary passage in anesthetized, artificially ventilated, open-chest rabbits in which both systemic and pulmonary circulations were fully supported by an extracorporeal pump. We have shown that these techniques and the use of the aforementioned probes provide reliable information on pulmonary endothelial function in vivo. Radiation to the chest produced endothelial ectoenzyme dysfunction, as reflected in altered available perfused capillary surface area and altered enzyme kinetics of all probes (decreases in substrate hydrolysis, inhibitor binding, first- and second-order kinetic constants) over a wide range of pulmonary blood flow values (reflecting approximately 60-200% of normal cardiac output). Indomethacin prevented most of these alterations in partially as well as fully recruited lungs. We conclude that impairment of endothelial ectoenzyme activity is an early event in the pathogenesis of radiation-induced lung damage, which occurs independently of hemodynamic influences and may involve synthesis of arachidonic acid metabolites.

Animals↗