Influence of Anoxia and Dinitrophenol on Ca2+ Efflux and Phosphorylase a Activity in Rabbit Colon Smooth Muscle

Abstract: It was observed in earlier studies that when the phosphorylase a activity of rabbit colon smooth muscle was increased by anoxia or 2,4-dinitrophenol (DNP), the calcium content of the mitochondrial fraction decreased. Despite this, under basal conditions there was no significant increase in the Ca²⁺ content in the fraction consisting of microsomes and cytoplasm. In the present study it was therefore investigated whether mitochondrial Ca²⁺ released by anoxia and DNP is translocated from the smooth muscle cells into the extracellular fluid. The Ca²⁺ efflux from rabbit colon into a Ca²⁺-free Krebs-Ringer bicarbonate buffer was measured by using a Ca²⁺-selective electrode. Both anoxia and DNP (6.6 × 10^-5M) increased the Ca²⁺ efflux from the smooth muscle cells. The local anaesthetic D-mepivacaine, at a concentration of 1×10 ³M, reduced the increase in Ca²⁺ efflux and simultaneously enhanced the anoxic or DNP-induced rise in phosphorylase a activity. The replacement of external Na⁺ by choline was found to reduce the basal Ca²⁺ efflux and to moderately increase the phosphorylase a activity. On addition of DNP to the Na⁺-free and Ca²⁺-free buffer there was no significant change in the Ca²⁺ efflux, but the increase in phosphorylase a activity was greater than in the physiological buffer containing 137 mM Na⁺. These observations support the suggestion that anoxia and DNP, by releasing Ca²⁺ from the mitochondria, increase the phosphorylase a activity of smooth muscle.     

Biochemical and Morphological Characterization of Subcellular Fractions Isolated from Rabbit Colon Muscle

Abstract From a homogenate of rabbit colon muscle subcellular fractions were isolated by differential centrifugation. The crude microsomal fraction could be separated into subfractions, a fraction of vesicular microsomes at 35% sucrose, a fraction containing sarcolemma, mitochondrial fragments and microsomal vesicles at 35–45% sucrose and a small protein fraction at 45–55% sucrose. Their biochemical properties and their morphological characterization were investigated. The cholesterol and the phospholipid content was equally distributed between the microsomal fractions 35% and 35–45% while the RNA was localized to the mitochondria and the microsomal fraction 35%. The enzyme cytochrome c oxidase was found to be concentrated in the mitochondria while a high contamination was found in the microsomal fractions 35–45%. The NADH-oxidase activity was highest in the 35% fraction and the 5′-nucleotidase activity in the 40,000 × g supernatant. The microsomal subfractions contained the enzymes ATPase, adenylate cyclase and phosphodiesterase. In the 35% fraction Ca stimulated the hydrolysis of ATP. The binding of [³H]-ouabain and the incorporation of [³H]-leucine was most pronounced in the 35% fraction. In a K⁺ -free Krebs Ringer medium the binding of the glucoside was stimulated in all the fractions. From these results we concluded that the fraction 35% sucrose may be mainly derived from the endoplasmic reticulum and the plasma membrane while the 35–45% originates from the plasma membrane, mitochondria and to a lesser extent the endoplasmic reticulum.     

Cyclic AMP and Ca-binding in Microsomal Fractions Isolated from Rabbit Colon Smooth Muscle

Abstract From a homogenate of rabbit colon muscle two ATP dependent Ca-accumulating microsomal fractions were isolated by differential centrifugation on a sucrose density gradient at 35 % and 35-45 % sucrose. Adenylate cyclase and phosphodiesterase activities were found in the fractions. The Ca-accumulation and the ATPase activity of these fractions were stimulated by cyclic AMP (10-⁵ M) at an ATP concentration of 0.35 mM ATP. In the presence of higher concentrations of ATP (5 mM) cyclic AMP had no effect on the Ca-binding. The higher concentration of ATP markedly increased the cyclic AMP formation in relation to the activity found at the lower concentration of ATP. Isoprenaline (2 × 10-⁶ M) stimulated the Ca-accumulation in the 35-45 % fraction and increased the hydrolysis of ATP. These effects were absent in the fraction isolated at 35 % sucrose. In the former fraction isoprenaline also stimulated the adenylate cyclase activity at 0.35 mM but not at 5 mM ATP. Both the effect of isoprenaline on the Ca-binding and the adenylate cyclase activity were inhibited by the adrenergic β-receptor blocking agent sotalol. In the 35-45 % fraction papaverine (1 × 10-³ M) stimulated the Ca-accumulation and inhibited the phosphodiesterase activity. It is suggested that cyclic AMP and agents which influence the cyclic AMP metabolism in the microsomes may have a regulatory role on the Ca-binding of the microsomes.     

动脉粥样硬化兔血管平滑肌细胞大电导钙激活钾通道NO直接激活研究

目的:研究NO是否可以直接激活VSMC BKCa以及AS时血管平滑肌细胞BKCa对NO的反应性是否发生改变,为治疗AS提供新思路.方法:3月龄新西兰兔20只,雌雄各半,随机分为实验组(AS组)和对照组(正常组),每组10只.实验组兔喂高脂饲料8周以建立AS模型,对照组兔喂普通饲料8周.以ISMN为外源性NO供体,应用单通道膜片钳技术检测NO对兔VSMC BKCa的直接激活作用及两组兔对NO的反应性差别.结果:在inside-out patches(膜电位 40 mV)下,ISMN显著增加BKCa通道开放事件数,缩短Tc,增加Po,且具有浓度依赖性.10-6 mol·L-1的ISMN(膜电位 40 mV)可分别使AS组和对照组BKCa的Po增加4.917±1.475倍和9.616±3.227倍(P＜0.01).结论:NO对VSMCBKCa有直接激活作用,但在AS时VSMC BKCa对NO的激活敏感性是降低的.     

Effect of Glyceryltrinitrate and 8–Br–cGMP on Tension and Phosphorylase a Activity in Vascular Smooth Muscle

Abstract: The aim of the present study was to examine the effect of glyceryltrinitrate (GTN) and 8–Br–cGMP on tension and cytosolic calcium concentration in pre–contracted bovine mesenteric arteries (BMA). The activity of glycogen phosphorylase a was used as a measure of the cytosolic calcium concentration. The activity of this enzyme is regulated by the cytosolic calcium concentration and/or cAMP. Since the cAMP level was not found to be affected by GTN–treatment, the use of phosphorylase a activity to monitor changes in the cytosolic calcium concentration can be justified. The vessels were contracted with phenylephrine (10 μM) or 100 mM K⁺–depolarization, which caused an increase in phosphorylase a activity. Addition of 1 μM GTN to the phenylephrine–contracted vessels resulted in a 3–4–fold rise in intracellular cGMP level, which was accompanied by a large decrease in tension and phosphorylase a activity. The K⁺–depolarized vessels, on the other hand, were largely resistant to the relaxant action of GTN, and there was only a slight reduction of the phosphorylase a activity. In phenylephrine–contracted vessels, made tolerant to GTN by incubation at elevated pH in the presence of GTN (0.44 mM), no changes in tension and phosphorylase a activity were seen after stimulation with a test dose of GTN (1 μM). The cGMP response was also markedly blunted in the tolerant vessels. Relaxation of phenylephrine–contracted BMA induced by 8–Br–cGMP (0.5 mM) was also accompanied by a reduction in phosphorylase a activity. The close association between the relaxation of BMA and the reduction in phosphorylase a activity suggests that GTN and 8–Br–cGMP exert their effects mainly by reducing the cytosolic free calcium concentration although a contributing effect on the contractile apparatus can not be excluded     

Determination of Free Extracellular Levels of Methotrexate by Microdialysis in Muscle and Solid Tumor of the Rabbit

Purpose. To determine of the pharmacokinetic profile of methotrexate (MTX) in blood and extracellular fluid (ECF) of VX2 tumor and muscle in rabbits. Methods. Microdialysis probes were inserted into VX2 tumor and in muscle tissue. Following intravenous administration of MTX (30 mg/ kg), serial collection of arterial blood samples and dialysates of muscle and tumor ECF for 4 h was carried out. Quantitation of MTX and determination of free plasma concentrations was performed by fluorescence polarization immunoassay and ultrafiltration, respectively. Correlations were established between the unbound plasma and ECF MTX concentrations. Results. Total and free plasma concentrations exhibited a parallel three exponential decay in both healthy and tumorigenic animals. Total clearance (8.9 vs 6.5 ml^–1.min^–1.kg^–1) and volume of distribution (4.0 vs 2.9 1.kg^–1), however, tended to decrease in the tumor-bearing group. The ECF/plasma AUC ratio equaled 14.2 ± 8.8% in muscle and 23.9 ± 15.9% in tumor. The concentration-time profile of muscle ECF MTX was parallel and highly correlated (r = 0.97) to that determined in plasma. In contrast, free MTX plasma levels were not correlated with tumor ECF concentrations (r = 0.564). Conclusions. In addition to the well-known pharmacological variability in the concentration-effect relationship, the important inter-individual variability in tumor exposure to MTX may partly explain that studies in patients with solid tumors have often failed to demonstrate firm correlations between MTX blood pharmacokinetics and the chemotherapeutic response.     

Effects of Cholinergic Drugs and Imidazole on Ca Release and Cyclic AMP Formation in Microsomal Fractions from Rabbit Colon

Abstract Following the addition of carbachol or acetylcholine to microsomal fractions isolated from rabbit colon which were preloaded with Ca, the ions were rapidly released. In the 35-45% fraction Ca was completely released within 10 min., but in the 35 % fraction only 30 % was released. Carbachol reduced the adenylate cyclase activity of the 35-45 % fraction. Both these effects were blocked by atropine. Exogenous cyclic AMP completely inhibited the Ca-releasing action of carbachol in the 35 % fraction and markedly reduced it in the 35-45 % fraction. Imidazole released Ca from the 35-45 % fraction and stimulated its phosphodiesterase activity. It is suggested that the microsomal fractions are parts of a Ca-sequestering system in smooth muscle which are able to bind Ca and which on the addition of some contracting drugs release the ions and thereby activate the contractile system. The release of Ca may partly at least be due to a reduction of the adenylate cyclase activity, although other mechanisms must also be considered.     

Mechanisms of the responses to Non-Cholinergic,Non-Adrenergic Nerve Stimulation and to ATP in Isolated Rabbit Urinary Bladder: Evidence for ADP Evoked Prostaglandin Release

Abstract: 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. β, γ-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.     

Synthesis and Smooth Muscle Calcium Channel Antagonist Effects of Dialkyl 1,4-Dihydro-2,6-dimethyl-4-aryl-3,5-pyridinedicarboxylates Containing a Nitrooxy or Nitrophenyl Moiety in the 3-Alkyl Ester Substituent

A group of racemic 3-[2-nitrooxyethyl(1,3-dinitrooxy-2-propyl or 4-nitrophenylethyl)] 5-isopropyl 1,4-dihydro-2,6-dimethyl-4-[2-trifluoromethylphenyl (2-nitrophenyl or 3-nitrophenyl)]-3,5-pyridinedicarboxylates 13–15 were prepared using the Hantzsch reaction that involved the condensation of 2-nitrooxyethyl 9a , 1,3-dinitrooxy-2-propyl 9b or 4-nitrophenylethyl 9c acetoacetate with isopropyl 3-aminocrotonate 11 and 2-trifluoromethyl 12a , 2-nitro 12b or 3-nitro 12c benzaldehyde. In vitro calcium channel antagonist activities were determined using a guinea pig ileum longitudinal smooth muscle assay. Compounds 13–15 exhibited superior, or equipotent, calcium channel antagonist activity (10^?8 to 10^?10 M range) relative to the reference drug nifedipine (IC₅₀ = 1.43 × 10^?8 M). The R¹ C-3 ester substituent was a determinant of calcium channel antagonist activity where the potency order was CH₂CH₂ONO₂ > CH₂CH₂-C₆H₄-4-NO₂ ≥ CH(CH₂ONO₂)₂. In contrast, the C-4 R²-aryl substituent (2-CF₃-C₆H₄-, 2-O₂N-C₆H₄- or 3-O₂N-C₆H₄-) was not a major determinant of activity. Compounds 13a–15a , which possess a 3-(2-nitrooxyethyl) ester substituent exhibit superior calcium channel antagonist smooth muscle relaxant activity (IC₅₀ = 10^?10 M range) relative to nifedipine, could serve as potential probes to investigate the in vivo release of nitric oxide (NO) which induces vascular muscle relaxation.     

Vascular Smooth Muscle Relaxation by Nitro Compounds: Reduced Relaxation and cGMP Elevation in Tolerant Vessels and Reversal of Tolerance by Dithiothreitol

Abstract: Smooth muscle preparations obtained from bovine mesenteric artery were incubated with high concentrations of nitroglycerin (NG) or nitroprusside (NP) at elevated pH. This treatment was found to make the vessels tolerant towards both the relaxant and cGMP elevating action of a challenging dose of nitrocompounds when tested on the histamine contracted vessels. Also, some cross-tolerance between NP and NG could be found since pretreatment of the vessels with NP caused a reduction in both the relaxant and the cGMP elevating action of NG. The NG induced tolerance could be partly reversed by treatment with the disulfide reducing agent dithiothreitol (DTT). These results are suggested to strenghten the evidence for cGMP as a mediator of vascular smooth muscle relaxation induced by nitrocompounds. The ability of DTT to partly reverse this tolerance indicates the existence of critical tissue sulfhydryl groups with which the nitrocompounds interact. There also seemed to exist certain differences in the mechanism of action between NG and NP since the tolerance induced against NG was much more pronounced than was the case for NP.     

Regulation by FK506 and rapamycin of Ca2+ release from the sarcoplasmic reticulum in vascular smooth muscle: the role of FK506 binding proteins and mTOR

Background and purpose:

The sarcoplasmic reticulum (SR), regulates the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyto) in vascular smooth muscle. Release from the SR is controlled by two intracellular receptor/channel complexes, the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (IP₃R). These receptors may be regulated by the accessory FK506-binding protein (FKBP) either directly, by binding to the channel, or indirectly via FKBP modulation of two targets, the phosphatase, calcineurin or the kinase, mammalian target of rapamycin (mTOR).

Experimental approach:

Single portal vein myocytes were voltage-clamped in whole cell configuration and [Ca²⁺]_cyto measured using fluo-3. IP₃Rs were activated by photolysis of caged IP₃ and RyRs activated by hydrostatic application of caffeine.

Key results:

FK506 which displaces FKBP from each receptor (to inhibit calcineurin) increased the [Ca²⁺]_cyto rise evoked by activation of either RyR or IP₃R. Rapamycin which displaces FKBP (to inhibit mTOR) also increased the amplitude of the caffeine-evoked, but reduced the IP₃-evoked [Ca²⁺]_cyto rise. None of the phosphatase inhibitors, cypermethrin, okadaic acid or calcineurin inhibitory peptide, altered either caffeine- or IP₃-evoked [Ca²⁺]_cyto release; calcineurin did not contribute to FK506-mediated potentiation of RyR- or IP₃R-mediated Ca²⁺ release. The mTOR inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, like rapamycin, decreased IP₃-evoked Ca²⁺ release.

Conclusions and implications:

Ca²⁺ release in portal vein myocytes, via RyR, was modulated directly by FKBP binding to the channel; neither calcineurin nor mTOR contributed to this regulation. However, IP₃R-mediated Ca²⁺ release, while also modulated directly by FKBP may be additionally regulated by mTOR. Rapamycin inhibition of IP₃-mediated Ca²⁺ release may be explained by mTOR inhibition.