[Analysis on applications and funded projects in physiology and integrative biology of National Natural Science Foundation of China in the past decade].

Here we perform a review on applications and funded projects at Division of Physiology and Integrative Biology in Department of Life Sciences sponsored by National Natural Science Foundation of China in the past ten years. Based on the research fields of applications and funded projects and the funding cost, we analyzed the sub-disciplines of the funded applications, key support areas, research frontiers and trends in the subjects of physiology and integrative biology, which gives us an insight into the future applications to optimize the layout of research areas in Division of Physiology and Integrative Biology.

[IFTIR study on the micro-structure in the construction process of water-borne coatings].

Borne paint was studied in the article in which the solvent water was regarded as a variable factor. A series of paint samples with different percentage of water were configured before observing their storage performance and microstructures by using Fourier-transform infrared (FTIR) ATR (attenuated total reflection) spectroscopy and diffuse reflectance (DF) FTIR spectroscopy. The effects of construction process and solvent water on the coating film were examined through analyzing the changes of internal functional groups before and after coating. The results indicated that as the amount of solvent water was increased, the characteristic peak of nu(C=O) in 1 727 cm(-1) turned into acromion, the absorption peakin 871 cm(-1) was blue shifted, the intensity of the peak of hydroxyl (nu(OH)) in 3400 cm(-1) and unsaturated absorption peak of nu-CH in 3030 cm(-1) gradually weakened, the absorption peaks strengths of nu-CH3 and nu-CH2 in 2962 and 2871 cm(-1) increased, respectively. The absorption peak in 2516 cm(-1) was shifted to 2603 cm(-1) then form the acromion, the absorption peak of 1647 cm(-1) gradually changed to 1455 cm(-1), the stretching vibration absorption peak of nu(C-O) in 1107 cm(-1) was red-shifted. During the coating process of paint, the solvent water and paint molecules interacted with each other, having a significant impact on the electron cloud density distribution of the paint molecules. The results obtained in the article have an important significance for the production and construction of paint.

Cryogenic Mechanical Properties and Stability of Polymer Films for Liquid Oxygen Hoses.

To select the appropriate polymer thin films for liquid oxygen composite hoses, the liquid oxygen compatibility and the cryogenic mechanical properties of four fluoropolymer films (PCTFE, ETFE, FEP and PFA) and two non-fluoropolymer films (PET and PI) before and after immersion in liquid oxygen for an extended time were investigated. The results indicated that the four fluoropolymers were compatible with liquid oxygen before and after immersion for 60 days, and the two non-fluoropolymers were not compatible with liquid oxygen. In addition, the cryogenic mechanical properties of these polymer films underwent changes with the immersion time, and the changes in the non-fluoropolymer films were more pronounced. The cryogenic mechanical properties of the two non-fluoropolymer films were always superior to those of the four fluoropolymer films during the immersion. Further analysis indicated that the fundamental reason for these changes in the cryogenic mechanical properties was the variation in the crystalline phase structure caused by the ultra-low temperature, which was not related to the strong oxidizing properties of the liquid oxygen. Analytical results can provide useful guidance on how to select the appropriate material combination to obtain a reasonable liquid oxygen composite hose structure.

Function and biomedical implications of exosomal microRNAs delivered by parenchymal and nonparenchymal cells in hepatocellular carcinoma.

Small extracellular vesicles (exosomes) are important components of the tumor microenvironment. They are small membrane-bound vesicles derived from almost all cell types and play an important role in intercellular communication. Exosomes transmit biological molecules obtained from parent cells, such as proteins, lipids, and nucleic acids, and are involved in cancer development. MicroRNAs (miRNAs), the most abundant contents in exosomes, are selectively packaged into exosomes to carry out their biological functions. Recent studies have revealed that exosome-delivered miRNAs play crucial roles in the tumorigenesis, progression, and drug resistance of hepatocellular carcinoma (HCC). In addition, exosomes have great industrial prospects in the diagnosis, treatment, and prognosis of patients with HCC. This review summarized the composition and function of exosomal miRNAs of different cell origins in HCC and highlighted the association between exosomal miRNAs from stromal cells and immune cells in the tumor microenvironment and the progression of HCC. Finally, we described the potential applicability of exosomal miRNAs derived from mesenchymal stem cells in the treatment of HCC.

Ryanodine receptors are involved in nuclear calcium oscillation in primary pancreatic ß-cells.

Ryanodine receptors (RyRs) are mainly located on the endoplasmic reticulum (ER) and play an important role in regulating glucose-induced cytosolic Ca(2+) oscillation in pancreatic ß-cells. However, subcellular locations and functions of RyRs on other cell organelles such as nuclear envelope are not well understood. In order to investigate the role of RyRs in nuclear Ca(2+) oscillation we designed and conducted experiments in intact primary pancreatic ß-cells. Immunocytochemistry was used to examine the expression of RYRs on the nuclear envelope. Confocal microscopy was used to evaluate the function of RYRs on the nuclear envelope. We found that RyRs are expressed on the nuclear envelope in single primary pancreatic ß-cells and isolated nuclei. Laser scanning confocal microscopy studies indicated that application of glucose to the cells co-incubated with Ca(2+) indicator Fluo-4 AM and cell-permeable nuclear indicator Hoechst 33342 resulted in nuclear Ca(2+) oscillation. The pattern of glucose-induced Ca(2+) oscillation in the nucleus and cytosol was similar. The reduction of Ca(2+) oscillation amplitude by ryanodine was much greater in the nucleus though both the cytosol and the nucleus Ca(2+) amplitude decreased by ryanodine. Our results suggest that functional ryanodine receptors not only exist in endoplasmic reticulum but are also expressed in nuclear envelope of pancreatic ß-cells.

[Investigation of FTIR spectra analysis of gallstones from Xuzhou region by solvent extraction].

The present paper is a preliminary exploration of the possible way the gallstones are formed. Five categories of gallstones from clinical surgery in Xuzhou region were extracted by a series of solvents. Fourier transform infrared spectroscopy (FTIR) was used to characterize the structure of morphological changes between gallstone and residue by extracting. The gallstone samples are from clinical surgeries in Xuzhou region where gallstone disease is quite common. Samples were extracted with a series of solvents, and then FTIR and other instrumental analysis were applied to characterize the composition, structure and morphological changes of the samples both before and after the extraction. The results show that the gallstone samples can be classified as 5 types: cholesterol-type, cholesterol-based hybrid type with salt, bilirubin and protein as its insoluble substances, brown pigment type and black pigment type gallstones. The results also indicate that protein plays a key role in gallstone nucleation process by providing a sediment matrix for the formation of gallstones. Both cholesterol and carbonated hydroxyapatite are found in the insoluble substances of the samples. It is possible that cholesterol was accompanied by carbonated hydroxyapatite and there are interactions between them, and these interactions contribute to the crystallization process and speed up the formation of gallstones. All the results above may provide useful references for the clinical diagnosis, treatment and prevention of gallstones.

FKBP12.6-knockout mice display hyperinsulinemia and resistance to high-fat diet-induced hyperglycemia.

FK506 binding protein 12.6 kDa (FKBP12.6), a protein that regulates ryanodine Ca(2+) release channels, may act as an important regulator of insulin secretion. In this study, the role of FKBP12.6 in the control of insulin secretion and blood glucose is clarified using FKBP12.6(-/-) mice. FKBP12.6(-/-) mice showed significant fed hyperinsulinemia but exhibited normoglycemia, fasting normoinsulinemia, and normal body weight compared with wild-type (WT) littermate control mice. Deletion of FKBP12.6 resulted in enhanced glucose-stimulated insulin secretion (GSIS) both in vivo and in vitro, a result that is due to enhanced glucose-induced islet Ca(2+) elevation. After a high-fat dietary challenge (HF diet) for 3 mo, FKBP12.6(-/-) mice displayed higher body weight, hyperinsulinemia, and lower fed blood glucose concentrations compared with WT mice. FKBP12.6(-/-) mice displayed hyperinsulinemia, and resistance to HF diet-induced hyperglycemia, suggesting that FKBP12.6 plays an important role in insulin secretion and blood glucose control, and raising the possibility that it may be a potential therapeutic target for the treatment of type 2 diabetes.

[Investigation of FTIR spectra analysis on carbon dioxide absorption with improved amine solution].

Carbon dioxide is a major sort of greenhouse gas as well as important carbon resource. With the developments of industries, emission of carbon dioxide has increased sharply. Hence, controls of carbon dioxide emission and resource transformation have become the hotspot of current study. As a new kind of carbon resource, the fields of CO2 research and application are very extensive. Among those methods, the amine absorption has good qualities of faster absorption rate, higher efficiency and so on, so it has been widely studied. But organic amine have such shortcomings: high consumption of heat energy, strong corrosive and easy oxidated, now pursuer mainly focused on the organic amine modified. The results showed that, when the time the amount of antioxidant 1010 is 0.152, the absorption capacity is 2503.53 mL. the volume of analysis is 982.00 mL, and the absorption rate changes more slowly, by FTIR, Samples of its renewable-OH associating is not apparent that the antioxidant content in 1010, oxidation products of the MEA is acid or less oxidation and antioxidant 1010 product in early to respond fully to form stable non-radical compounds. Therefore, the best dosage of antioxidant 1010 is 0.15%. When the time that the amount of Na2SO3 is 0.15%, the absorption capacity is 2922.88 mL. Analysis of the volume is 723.00 mL, by FTIR, which reveals the oxidation products of the MEA is amide -C=O which in alkaline solution can be transiting into primary amine, and be easy absorbing CO2. Comparing the antioxygenic proerty of antioxidant 1010 with Na2SO3, from the absorption rate, the amount of absorption , Na2SO3's antioxidant properties is superior than antioxidant 1010; by infrared spectral analysis, 1010/20% MEA solution's oxidation products is the acid, Na2SO3/20% MEA solutions, the oxidation product is amide, amide solution is advantaged for absorbing CO2, So Na2SO3's antioxidant properties is superior than antioxidant 1010.

Extracellular ATP-induced nuclear Ca2+ transient is mediated by inositol 1,4,5-trisphosphate receptors in mouse pancreatic beta-cells.

Extracellular ATP (eATP) induces an intracellular Ca(2+) transient by activating phospholipase C (PLC)-associated P2X4 purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent Ca(2+) release from intracellular stores in mouse pancreatic beta-cells. Using laser scanning confocal microscopy, Ca(2+) indicator fluo-4 AM, and the cell permeable nuclear indicator Hoechst 33342, we examined the properties of eATP-induced Ca(2+) release in pancreatic beta-cell nuclei. eATP induced a higher nuclear Ca(2+) transient in pancreatic beta-cell nuclei than in the cytosol. After pretreatment with thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pumps, the amplitude of eATP-induced Ca(2+) transients in the nucleus was still much higher than those in the cytosol. This effect of eATP was not altered by inhibition of either the plasma membrane Ca(2+)-ATPase (PMCA) or the plasma membrane Na(+)/Ca(2+) exchanger (NCX) by LaCl(3) or by replacement of Na(+) with N-Methyl-Glucosamine. eATP-induced nuclear Ca(2+) transients were abolished by a cell-permeable IP3R inhibitor, 2-aminoethoxydiphenyl borate (2-APB), but were not blocked by the ryanodine receptor (RyR) antagonist ryanodine. Immunofluorescence studies showed that IP3Rs are expressed on the nuclear envelope of pancreatic beta-cells. These results indicate that eATP triggers nuclear Ca(2+) transients by mobilizing a nuclear Ca(2+) store via nuclear IP3Rs.

[Periodic and fractal precipitation in ATP-Co(2+)-decoxycholate gel system].

In the simulation experiments in vitro of the formation of gallstone, adenosine-triphosphate(ATP)-Co(2+)-deoxycholic acid(DC) gel system was chosen to study the periodic precipitation progress. The effect of ATP on the Co(2+)-DC gel system was also determined, and the structure of the periodic precipitation formed was characterized by FTIR. The results show that the patterns formed in the systems with ATP are different, ATP affected the rate and structure of precipitation through its variable participation in the metal coordination complexes as judged by the phosphate P=O bands and the deoxycholate COO- symmetric and asymmetric vibration bands as measured by FTIR Theses spectroscopic differences were correlated with color and pattern differences in the precipitates. ATP has a more remarkable function than AMP to the modes of patterns, meanwhile the system patterns transform from fractal to periodic precipitation. There is a complex interaction among ATP, sodium deoxycholic and Co2+ with a transparent crystal produced. The crystal is deoxycholic acid and the periodic precipitation is composed of ATP and DC covalent to Co2+. These results indicate that stone formation and remodeling is a dynamic, nonlinear progress. Much of the precipitate, as judged by local differences in composition, is not in equilibrium with the general gel environment. The authors conclude that the formation of gallstone features complex and nonlinear chemical character, in which nucleotides as living material play a very important role.

Improvement of SSO-mediated gene repair efficiency by nonspecific oligonucleotides.

Targeted gene repair mediated by single-stranded DNA oligonucleotides (SSOs) is a promising method to correct the mutant gene precisely in prokaryotic and eukaryotic systems. We used a HeLa cell line, which was stably integrated with mutant enhanced green fluorescence protein gene (mEGFP) in the genome, to test the efficiency of SSO-mediated gene repair. We found that the mEGFP gene was successfully repaired by specific SSOs, but the efficiency was only approximately 0.1%. Then we synthesized a series of nonspecific oligonucleotides, which were single-stranded DNA with different lengths and no significant similarity with the SSOs. We found the efficiency of SSO-mediated gene repair was increased by 6-fold in nonspecific oligonucleotides-treated cells. And this improvement in repair frequency correlated with the doses of the nonspecific oligonucleotides, instead of the lengths. Our evidence suggested that this increased repair efficiency was achieved by the transient alterations of the cellular proteome. We also found the obvious strand bias that antisense SSOs were much more effective than sense SSOs in the repair experiments with nonspecific oligonucleotides. These results provide a fresh clue into the mechanism of SSO-mediated targeted gene repair in mammalian cells.

Cooperativeness of the higher chromatin structure of the beta-globin locus revealed by the deletion mutations of DNase I hypersensitive site 3 of the LCR.

High-level transcription of the globin genes requires the enhancement by a distant element, the locus control region (LCR). Such long-range regulation in vivo involves spatial interaction between transcriptional elements, with intervening chromatin looping out. It has been proposed that the clustering of the HS sites of the LCR, the active globin genes, as well as the remote 5' hypersensitive sites (HSs) (HS-60/-62 in mouse, HS-110 in human) and 3'HS1 forms a specific spatial chromatin structure, termed active chromatin hub (ACH). Here we report the effects of the HS3 deletions of the LCR on the spatial chromatin structure of the beta-globin locus as revealed by the chromatin conformation capture (3C) technology. The small HS3 core deletion (0.23 kb), but not the large HS3 deletion (2.3 kb), disrupted the spatial interactions among all the HS sites of the LCR, the beta-globin gene and 3'HS1. We have previously demonstrated that the large HS3 deletion barely impairs the structure of the LCR holocomplex, while the structure is significantly disrupted by the HS3 core deletion. Taken together, these results suggest that the formation of the ACH is dependent on a largely intact LCR structure. We propose that the ACH indeed is an extension of the LCR holocomplex.

Site-specific base changes in the coding or promoter region of the human beta- and gamma-globin genes by single-stranded oligonucleotides.

SSOs (single-stranded oligonucleotides) can mediate site-specific alteration of base-pairs in episomal and chromosomal target genes in mammalian cells. The TNE (targeted nucleotide exchange) can result in either repair or mutation of a gene sequence and is mediated through endogenous DNA repair pathway(s). Thus the approach provides a technique for the treatment of monogenic disorders associated with specific point mutations such as SCD (sickle cell disease). We studied the potential application of SSOs for SCD by introducing either an A to T substitution at the sixth codon of the human beta-globin gene (sickle locus) or a C to G mutation at -202 of the Ggamma-globin gene promoter region. The latter TNE is an alternative strategy to ameliorate the clinical manifestations of sickle cell anaemia by re-activating fetal haemoglobin gene expression in adult erythrocytes. A sensitive and valid PCR assay system was developed, which allows detection of point mutations as low as 0.01% at these sites. Using this system, TNE between 0.01 and 0.1% at the sickle locus or gamma-globin gene promoter region was detected after transfection with SSOs in cultured human cell lines. TNE in the Ggamma-globin promoter region exhibited varying degrees of strand bias that was dependent on SSO design and the cell's DNA mismatch repair activity. The results suggest that the endogenous DNA repair machinery may permit SSO correction of the sickle defect by modification of the beta- and/or gamma-globin genes.

[FTIR investigation of organic phase containing rare earth ion in naphthenic acid-phosphonate ester-heptane system].

Naphthenic acid (NA) was mixed with PC88A in heptane, and 10 moL x L(-1) KOH aqueous solution was used to saponify the solution subsequently. The transparent and clear appearance of the obtained solution indicated the formation of w/o micell and microemusion. In the present study, micell with high saponification percentage (80%) was used to extract neodymium. The organic phase structure was characterized using FTIR spectroscopy in the extraction process. IR spectra indicate that the characteristic peaks of nu(COOH), nu(s)(COO-) and nu(P=O) shift to lower wavenumbers as the loading of Nd in organic phase increases. The results demonstrated that Nd(III) are coordinated to P=O and COO- groups simultaneously, and the supramolecules with self-assembly structure was formed containing PC88A and NA, which were binding with Nd(III) in the extracted organic phase.

[Study on the structure of periodic and chaotic patterns of AMP-DC-Cu-gel systems].

This is an in vitro model to mimic the conditions present during gallstone formation. Adenosine mono-phosphate (AMP), an important bio-molecule, was chosen. Its effect on the formation of periodic/chaotic patterns in the deoxycholate-CuCl2-gel and deoxycholate-CuCl2-glucose-gel systems were studied. FTIR spectroscopy was used to identify the structure of the precipitates. The results indicate that the patterns formed in the systems with AMP are different. And FTIR spectra show that AMP may also be involved in the coordination sphere of Cu2+ ion. The results are more complicated in the deoxycholate-CuCl2 system containing glucose, suggesting that the nonlinear scientific concept should be considered in understanding gallstone formation.

ERp44 C160S/C212S mutants regulate IP3R1 channel activity.

Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release (IICR) via IP(3)R(1), but the mechanism remains largely unexplored. Using extracellular ATP to induce intracellular calcium transient as an IICR model, Ca(2+) image, pull down assay, and Western blotting experiments were carried out in the present study. We found that extracellular ATP induced calcium transient via IP(3)Rs (IICR) and the IICR were markedly decreased in ERp44 overexpressed Hela cells. The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331-377) mutants of ERp44 on IICR were significantly decreased compared with ERp44. However, the binding capacity of ERp44 to L3V domain of IP(3)R(1) (1L3V) was enhanced by ERp44 C160S/C212S mutation. Taken together, these results suggest that the mutants of ERp44, C160/C212, can more tightly bind to IP(3)R(1) but exhibit a weak inhibition of IP(3)R(1) channel activity in Hela cells.

Atrial natriuretic peptide regulates Ca channel in early developmental cardiomyocytes.

BACKGROUND: Cardiomyocytes derived from murine embryonic stem (ES) cells possess various membrane currents and signaling cascades link to that of embryonic hearts. The role of atrial natriuretic peptide (ANP) in regulation of membrane potentials and Ca(2+) currents has not been investigated in developmental cardiomyocytes. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the role of ANP in regulating L-type Ca(2+) channel current (I(CaL)) in different developmental stages of cardiomyocytes derived from ES cells. ANP decreased the frequency of action potentials (APs) in early developmental stage (EDS) cardiomyocytes, embryonic bodies (EB) as well as whole embryo hearts. ANP exerted an inhibitory effect on basal I(CaL) in about 70% EDS cardiomyocytes tested but only in about 30% late developmental stage (LDS) cells. However, after stimulation of I(CaL) by isoproterenol (ISO) in LDS cells, ANP inhibited the response in about 70% cells. The depression of I(CaL) induced by ANP was not affected by either Nomega, Nitro-L-Arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, or KT5823, a cGMP-dependent protein kinase (PKG) selective inhibitor, in either EDS and LDS cells; whereas depression of I(CaL) by ANP was entirely abolished by erythro-9-(2-Hydroxy-3-nonyl) adenine (EHNA), a selective inhibitor of type 2 phosphodiesterase(PDE2) in most cells tested. CONCLUSION/SIGNIFICANCES: Taken together, these results indicate that ANP induced depression of action potentials and I(CaL) is due to activation of particulate guanylyl cyclase (GC), cGMP production and cGMP-activation of PDE2 mediated depression of adenosine 3', 5'-cyclic monophophate (cAMP)-cAMP-dependent protein kinase (PKA) in early cardiomyogenesis.

Ca(2+) release induced by cADP-ribose is mediated by FKBP12.6 proteins in mouse bladder smooth muscle.

We examined the role and molecular mechanism of cADPR action on Ca(2+) spark properties in mouse bladder smooth muscle. Dialysis of cADPR with patch pipettes increased frequency and amplitude of spontaneous transient out currents (STOCs) to 6.1+/-0.87 currents/min from 1.2+/-0.36 currents/min (control) and to 179.8+/-48.7pA from 36.4+/-22.6pA (control), respectively, in wildtype (WT) cells, and the effects of cADPR on STOCs were significantly blocked by JVT-591, a RYR2 stabilizer. In contrast, no significant changes were observed in FKBP12.6 null cells. Further studies indicated that Ca(2+) spark properties were altered by cADPR in WT but not FKBP12.6 null cells, namely, Ca(2+) spark frequency was increased by about 3.4-fold, peak Ca(2+) (F/F0) increased to 1.72+/-0.57 from 1.56+/-0.13, size increased to 2.86+/-0.26 microM from 1.92+/-0.14 microM, rise time and half-time decay were prolonged 1.6-fold and 2.3-fold, respectively, in WT cells. Furthermore, in the presence of thapsigargin cADPR still altered Ca(2+) spark properties, and cADPR increased F/F0 without affecting Ca(2+) spark decay time in voltage clamping cells. Dissociation studies demonstrated that application of cADPR resulted in significant removal of FKBP12.6 proteins from sarcoplasmic reticulum (SR) microsomes, and that treatment of the RyR2 immunoprecipitation complexes with cADPR or FK506 disrupted the interaction between RyR2 and FKBP12.6. Finally, cADPR altered SR Ca(2+) load in WT myocytes but not in FKBP12.6-null myocytes. Taken together, these results suggest that Ca(2+) release induced by cADPR is mediated by FKBP12.6 proteins in mouse bladder smooth muscle.

The higher structure of chromatin in the LCR of the beta-globin locus changes during development.

The beta-globin locus control region (LCR) is able to enhance the expression of all globin genes throughout the course of development. However, the chromatin structure of the LCR at the different developmental stages is not well defined. We report DNase I and micrococcal nuclease hypersensitivity, chromatin immunoprecipitation analyses for histones H2A, H2B, H3, and H4, and 3C (chromatin conformation capture) assays of the normal and mutant beta-globin loci, which demonstrate that nucleosomes at the DNase I hypersensitive sites of the LCR could be either depleted or retained depending on the stages of development. Furthermore, MNase sensitivity and 3C assays suggest that the LCR chromatin is more open in embryonic erythroblasts than in definitive erythroblasts at the primary- and secondary-structure levels; however, the LCR chromatin is packaged more tightly in embryonic erythroblasts than in definitive erythroblasts at the tertiary chromatin level. Our study provides the first evidence that the occupancy of nucleosomes at a DNase I hypersensitive site is a developmental stage-related event and that embryonic and adult cells possess distinct chromatin structures of the LCR.

Nitric oxide mediates stretch-induced Ca2+ release via activation of phosphatidylinositol 3-kinase-Akt pathway in smooth muscle.

BACKGROUND: Hollow smooth muscle organs such as the bladder undergo significant changes in wall tension associated with filling and distension, with attendant changes in muscle tone. Our previous study indicated that stretch induces Ca(2+) release occurs in the form of Ca(2+) sparks and Ca(2+) waves in urinary bladder myocytes. While, the mechanism underlying stretch-induced Ca2+ release in smooth muscle is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We examined the transduction mechanism linking cell stretch to Ca(2+) release. The probability and frequency of Ca(2+) sparks induced by stretch were closely related to the extent of cell extension and the time that the stretch was maintained. Experiments in tissues and single myocytes indicated that mechanical stretch significantly increases the production of nitric oxide (NO) and the amplitude and duration of muscle contraction. Stretch-induced Ca(2+) sparks and contractility increases were abrogated by the NO inhibitor L-NAME and were also absent in eNOS knockout mice. Furthermore, exposure of eNOS null mice to exogenously generated NO induced Ca(2+) sparks. The soluble guanylyl cyclase inhibitor ODQ did not inhibit SICR, but this process was effectively blocked by the PI3 kinase inhibitors LY494002 and wortmannin; the phosphorylation of Akt and eNOS were up-regulated by 204+/-28.6% and 258+/-36.8% by stretch, respectively. Moreover, stretch significantly increased the eNOS protein expression level. CONCLUSIONS/SIGNIFICANCE: Taking together, these results suggest that stretch-induced Ca2+ release is NO dependent, resulting from the activation of PI3K/Akt pathway in smooth muscle.