Pharmacological Characterization of Bradykinin Receptors Coupled to Phosphoinositide Turnover in SV40-Immortalized Human Trabecular Meshwork Cells

This study sought to pharmacologically characterize bradykinin receptors on SV40-immortalized human trabecular meshwork (HTM3) cells. Phosphoinositide (PI) turnover studies were conducted using [³H]myo-inositol-labeled HTM3 cells and anion exchange chromatography to quantify [³H]inositol phosphates generated in response to bradykinin (BK) and various BK analogs. The blockade of these responses was studied using two potent and receptor-subtype selective antagonists. BK and T-kinin (Ile-Ser-BK; TK) induced a 4.2–4.4 fold stimulation of PI turnover above base levels at 1–10 μM. Several other peptides unrelated to BK, including angiotensin II, endothelin, cholecystokinin, bombesin and peptide YY tested at 1–10 μMwere essentially inactive. The molar potencies (EC₅₀) of BK, TK and close analogs were: BK=4.5±0.5 nM(n=6), Lys-BK=6.5±0.7 nM(n=3), TK=38.8±6.6 nM(n=8), Met-Lys-BK=41.5±13.4 nM(n=4), Des-Arg⁹-BK=2093±626 nM(n=4). All the latter BK-related peptides>were full agonists. The actions of BK and TK were potently and competitively antagonized by Hoe-140 (molar potency=0.6–1 nM;pA₂n=8.97–9.21,n=3–4) and byD-Arg⁰[Hyp³,-Thi^5,8,-DPhe⁷]-BK (molar potency=251 nM;-log potency, pK_b=6.6), two selective B₂-type BK antagonists. In conclusion, rank order of potency of BK agonists and the blockade of BK- and TK-induced PI turnover by the selective antagonists are consistent with the classification of the BK receptors on HTM3 cells as the B₂-receptor subtype.     

CULTURING RAT NEONATAL MYOCYTES CAUSES CHANGES IN THE PHOSPHATIDYLINOSITOL TURNOVER PATHWAY

1. Cultured neonatal myocytes are commonly used as a model system for the study of cardiac phosphatidylinositol (PI) turnover. 2. In neonatal myocytes stimulation with noradrenaline causes the release of the Ca2(+)-releasing compound inositol-1,4,5-trisphosphate and the generation of the Ca2(+)-regulatory compound inositol-1,3,4,5-tetrakisphosphate. 3. Addition of noradrenaline to intact, neonatal rat hearts stimulates the release of inositol-1,4,5-trisphosphate, but not inositol-1,3,4,5-tetrakisphosphate. 4. These findings show that the isolation and culture of the neonatal myocyte causes changes in the PI turnover pathway so that it becomes similar to that described in other cell types and different from that in intact myocardial tissue. 5. The neonatal myocyte is not a useful model for the study of cardiac PI turnover.     

牛磺酸防护高能冲击波致肾损伤的实验研究

目的 探讨牛磺酸对高能冲击波(HESW)致家兔肾损伤的防护作用及机制.方法20只雄性新西兰成年家兔随机分2组,每组10只,均予12.75 kV冲击波冲击左肾下极1500次.实验组在冲击的同时静脉滴注牛磺酸150 mg/kg,对照组给予等容积的生理盐水,在冲击前及冲击后3 d测定血丙二醛(MDA)、超氧化物歧化酶(SOD)的变化,检测肾脏组织学变化及热休克蛋白70(HSP70)的表达.结果 冲击前实验组和对照组血MDA分别为(2.2±0.4)、(2.1±0.4)mmol/L,SOD分别为(56.4±9.9)、(55.8±10.1)μU/L,差异均无统计学意义(P＞0.05).冲击后实验组血MDA、SOD分别为(2.7±0.7)mmol/L、(55.3±5.7)μU/L,与冲击前相比,差异无统计学意义(P＞0.05);对照组血MDA、SOD分别为(5.7±0.7)mmol/L、(33.3±3.5)μU/L,与冲击前相比,差异有统计学意义(P＜0.05).冲击后实验组和对照组肾小管评分分别为7.2±0.8和31.8±1.9,HSP70分别为25.2±4.1和6.4±0.9,差异均有统计学意义(P＜0.05).结论 牛磺酸对HESW致兔肾损伤有良好的防护作用,防护机制可能与其抗氧化作用和诱导HSP70合成增多有关.     

High K+-induced contraction requires depolarization-induced Ca2+ release from internal stores in rat gut smooth muscle

Aim:

Depolarization-induced contraction of smooth muscle is thought to be mediated by Ca²⁺ influx through voltage-gated L-type Ca²⁺ channels. We describe a novel contraction mechanism that is independent of Ca²⁺ entry.

Methods:

Pharmacological experiments were carried out on isolated rat gut longitudinal smooth muscle preparations, measuring isometric contraction strength upon high K⁺-induced depolarization.

Results:

Treatment with verapamil, which presumably leads to a conformational change in the channel, completely abolished K⁺-induced contraction, while residual contraction still occurred when Ca²⁺ entry was blocked with Cd²⁺. These results were further confirmed by measuring intracellular Ca²⁺ transients using Fura-2. Co-application of Cd²⁺ and the ryanodine receptor blocker DHBP further reduced contraction, albeit incompletely. Additional blockage of either phospholipase C (U 73122) or inositol 1,4,5-trisphophate (IP₃) receptors (2-APB) abolished most contractions, while sole application of these blockers and Cd²⁺ (without parallel ryanodine receptor manipulation) also resulted in incomplete contraction block.

Conclusion:

We conclude that there are parallel mechanisms of depolarization-induced smooth muscle contraction via (a) Ca²⁺ entry and (b) Ca²⁺ entry-independent, depolarization-induced Ca²⁺-release through ryanodine receptors and IP₃, with the latter being dependent on phospholipase C activation.     

Laparoscopic treatment of a calcium fluorophosphate stone within a seminal vesicle cyst

Stones in the seminal vesicles are extremely rare. We present a 62-year-old patient with a stone within a seminal vesicle cyst, who was cured by laparoscopic treatment. The operative time was 80 rnin, and the estimated blood loss was 90 mL. Scanning electron microscope examination of the stone showed a compact crystal image externally and sparse spherical crystal structure in kernel. Composition of the stone was calcium fluorophosphate on X-ray diffractometer. The follow-up time was 15 months with no recurrence of cyst or stone. To our knowledge, this case is the first to describe laparoscopic removal of a stone within a seminal vesicle cyst, and the first to describe calcium fluorophosphate as the composition of seminal vesicle stones. （Asian JAndro12008 Mar; 10： 337-340）     

Low‐temperature particulate calcium phosphates for bone regeneration

Background: Low‐temperature synthesized calcium phosphates are produced by mixing calcium phosphate powders in an aqueous solution resulting in a precipitated phase. These compounds can be formulated in several forms (e.g. injectable cements and implantable blocks), and are commonly used as bone substitutes and drug delivery systems for the treatment of bone defects. As bone substitutes, calcium phosphates in general offer the advantages of being biocompatible and osteoconductive. Aims: The present work employed a machine‐based process to derive a reproducible preparation method for low‐temperature calcium phosphate particulate (LTCP). The in vivo outcomes of LTCP were compared with those of three commercially available bone substitutes by histomorphometric measurements of bone formation and material degradation in a rat femur implantation model. Materials & Methods: Specifically, LTCP, anorganic bovine bone (AB), bioactive glass (BG), and demineralized bone matrix (DBM) were implanted in defects created in the distal aspect of rat femora. Reparative bone and particulate volumes of these biomaterials were evaluated post‐operatively using micro‐computed tomography and histological analyses at 3, 6, 12, and 16 weeks. Results & Discussion: Results showed that, despite invoking bone formation, AB, BG, and DBM were found un‐resorbed in situ at 16 weeks. Conversely, LTCP showed an early increase in bone formation as well as clear evidence of complete degradation and reparative bone remodelling, resulting in the total reconstitution of the marrow cavity and marrow tissue. Conclusion: LTCP promoted increased early bone formation, associated with an improved degradation rate, compared with the other three bone‐substitute biomaterials tested. To cite this article:
Araújo MVF, Mendes VC, Chattopadhyay P, Davies JE. Low‐temperature particulate calcium phosphates for bone regeneration.
Clin. Oral Impl. Res. 21 , 2010; 632–641.     

Solution Ca/P ratio affects calcium phosphate crystal phases

Summary Previous studies on brushite formation and dissolution concluded that brushite was stable only in acidic solutions with pH<6.5. Francis in 1965 predicted that, at pH range 3.5–6.5, brushite would be the preferred phase at low solution Ca/P ratio and hydroxyapatite at high Ca/P ratio. This work presents room-temperature experimental evidence that solution Ca/P ratio affects calcium phosphate crystal phases and that brushite can be the preferred phase in certain supersaturated aqueous solutions containing Ca⁺⁺ and Pi, with or without Mg⁺⁺, even at pH 7.0. Solution mixtures were prepared containing initial [CaCl₂]=0.1−10.0 mM, [Na₂HPO₄]=0.1−100.0 mM, [MgCl₂]=0 or 3 mM. By addition of NaCl, the ionic strengths in terms of osmolarity were also varied from 100 to 800 mosM. Precipitates were isolated from solutions on the 7th, 14th and 21st days and identified by x-ray diffraction. Results indicated that in the presence of 3 mM Mg⁺⁺ and relatively high initial apparent activity products (Ca⁺⁺)(Pi), brushite, whitlockite and hydroxyapatite were obtained as transformation products of initially formed amorphous calcium phosphate when solution Ca/P ratios had low, medium and high values, respectively. In the absence of Mg⁺⁺, whitlockite was not found. However, with or without Mg⁺⁺, when (Ca⁺⁺)(Pi) was relatively low, only brushite was formed by direct crystallization. We concluded that although solutions were mixed at 23°C, pH 7.0, the results were useful in explaining the in vivo calcium phosphate crystal phases observed in renal and dental calculi.     

磷酸钙骨水泥人工骨的制备及修复兔桡骨大段骨缺损的实验研究

目的制备水泥型羟基磷灰石(CPC)人工骨,并研究CPC人工骨修复兔桡骨大段骨缺损的成骨效果.方法高温法制备出磷酸四钙,然后在模拟体内环境下将其与无水磷酸氢钙发生水化固化反应,合成水泥型羟基磷灰石人工骨,将自制的CPC人工骨植入新西兰兔桡骨大段骨缺损模型中,术后4、8、12、16周取材,采用HE染色和Masson三色染色分析评价骨缺损修复效果.结果 CPC植入后4周有新生软骨形成及未成熟的骨组织,可见大量的软骨细胞、成骨细胞、胶原组织及较多的小血管.8～16周新骨继续生成,人工骨逐渐改建为成熟的板层骨、骨小梁和髓腔结构.结论 CPC人工骨制备简单,有良好的生物相容性和成骨效果,可能是一种较理想的骨移植替代材料.     

Impaired β-adrenergic control of immune function in patients with chronic heart failure: reversal by β1-blocker treatment

BACKGROUND: In patients with chronic heart failure (CHF) and heightened sympathetic activity alterations in immune function have been described. OBJECTIVES: To find out whether, in CHF patients, beta-blocker treatment might beneficially affect immune function. METHODS: We studied activation of circulating lymphocytes (assessed as concanavalin A (CON A)-induced inositol phosphate (IP) formation and proliferation ([3H]-thymidine incorporation) from 8 CHF patients on standard medication (Group A, mean age 54 +/- 6 yrs, NYHA class II - IV, mean 3.1 +/- 0.3) and in 9 CHF patients on standard medication and additional treatment with the beta1-blocker metoprolol (Group B, mean age: 56 +/- 3 yrs, NYHA class II - IV, mean 2.9 +/- 0.2); 8 age-matched healthy volunteers (mean age 49 +/- 3 yrs) served as controls. RESULTS: Compared to controls, in group A isoprenaline-induced lymphocyte cyclic AMP-increase was reduced, CON A-evoked IP formation significantly enhanced and isoprenaline-induced inhibition of CON A-evoked IP formation and proliferation almost abolished. In group B, however, all these parameters were not significantly different from controls. CONCLUSION: In CHF patients lymphocyte cyclic AMP response to beta-adrenoceptor stimulation is blunted and the inhibitory effect of cyclic AMP on lymphocyte activation is almost abolished; this could result in a non-regulated increased production and release of proinflammatory cytokines that might contribute to the progression of the disease. Chronic treatment of CHF patients with the beta1-blocker metoprolol (at least partly) restores lymphocyte cyclic AMP responses to beta-adrenoceptor stimulation and the inhibitory effects of cyclic AMP on lymphocyte activation; the resulting "normalization" of the immune function might contribute to the beneficial effects of beta1-blockers in treatment of CHF.