Effects of unsaturated fatty acids on calcium-activated potassium current in gastric myocytes of guinea pigs.

AIM: To investigate the effects of exogenous unsaturated fatty acids on calcium-activated potassium current (I(K(Ca))) in gastric antral circular myocytes of guinea pigs. METHODS: Gastric myocytes were isolated by collagenase from the antral circular layer of guinea pig stomach. The whole-cell patch clamp technique was used to record I(K(Ca)) in the isolated single smooth muscle cells with or without different concentrations of arachidonic acid (AA), linoleic acid (LA), and oleic acid (OA). RESULTS: AA at concentrations of 2,5 and 10 micromol/L markedly increased I(K(ca)) in a dose-dependent manner. LA at concentrations of 5, 10 and 20 micromol/L also enhanced I(K(Ca)) in a dose-dependent manner. The increasing potency of AA, LA, and oleic acid (OA) on I(K(Ca)) at the same concentration (10 micromol/L) was in the order of AA>LA>OA. AA (10 micromol/L)-induced increase of I(K(ca)) was not blocked by H-7 (10 micromol/L), an inhibitor of protein kinase C (PKC), or indomethacin (10 micromol/L), an inhibitor of the cyclooxygenase pathway, and 17-octadecynoic acid (10 micromol/L), an inhibitor of the cytochrome P450 pathway, but weakened by nordihydroguaiaretic acid (10 micromol/L), an inhibitor of the lipoxygenase pathway. CONCLUSION: Unsaturated fatty acids markedly increase I(K(ca)), and the enhancing potencies are related to the number of double bonds in the fatty acid chain. The lipoxygenase pathway of unsaturated fatty acid metabolism is involved in the unsaturated fatty acid-induced increase of I(K(ca)) in gastric antral circular myocytes of guinea pigs.

Effect of actin microfilament on potassium current in guinea pig gastric myocytes.

AIM: To investigate the effect of actin microfilament on potassium current and hyposmotic membrane stretch-induced increase of potassium current in gastric antral circular myocytes of guinea pig. METHODS: Whole-cell patch clamp technique was used to record potassium current in isolated gastric myocyes. RESULTS: When the membrane potential was clamped at -60 mV, an actin microfilament disruptor, cytochanlasin-B(Cyt-B, 20 micromol/L in pipette) increased calcium-activated potassium current (I(K(Ca))) and delayed rectifier potassium current (I(K(V))) to 138.4+/-14.3% and 142.1+/-13.1% respectively at +60 mV. In the same condition, an actin microfilament stabilizer phalloidin (20 micromol/L in pipette) inhibited I(K(Ca)) and I(K(V)) to 74.2+/-7.1% and 75.4+/-9.9% respectively. At the holding potential of -60 mV, hyposmotic membrane stretch increased I(K(Ca)) and I(K(V)) by 50.6+/-9.7% and 24.9+/-3.3% at +60 mV respectively. In the presence of cytochalasin-B and phalloidin (20 micromol/L, in the pipette) condition, hyposmotic membrane stretch also increased I(K(Ca)) by 44.5+/-7.9% and 55.7+/-9.8% at +60 mV respectively. In the same condition, cytochalasin-B and phalloidin also increased I(K(V)) by 23.0+/-5.5% and 30.3+/-4.5% respectively. However, Cyt-B and phalloidin did not affect the amplitude of hyposmotic membrane stretch-induced increase of I(K(Ca)) and I(K(V)). CONCLUSION: Actin microfilaments regulate the activities of potassium channels, but they are not involved in the process of hyposmotic membrane stretch-induced increase of potassium currents in gastric antral circular myocytes of guinea pig.

Cloning, expression, and purification of HLA-A2-BSP and beta-2m in Escherichia coli.

Tetramer analysis is a novel technique in immunological research that has dramatically changed our knowledge of the immune response to pathogens, tumors and autoimmune disease. Through the formation of major histocompatibility complex (MHC)-peptide tetrameric complexes, it can provide accurate counts of antigen-specific T-cells and it allows their phenotypical and functional analysis. The tetramer is composed of the human leukocyte antigen (HLA) heavy chain, beta-2 microglobulin (beta-2m), the nominal peptide, and streptavidin. The HLA heavy chain and the beta-2m are expressed in Escherichia coli. But up to now, all laboratories have been expressing these two proteins by using isopropyl beta-d-thiogalactopyranoside IPTG. IPTG is very expensive, and it is tedious and laborious to induce expression protein. So it is difficult to scale up to express the objective protein. To address this problem, extracellular fractions of HLA-A0201 and beta-2m (absent signal peptide) genes were cloned from peripheral blood mononuclear cells (PBMCs) by RT-PCR. DNA coding for a Gly-Ser linker and a BSP (15-amino acid substrate peptide for BirA-dependent biotinylation) was added to the COOH-terminus of the extracellular fraction of HLA-A0201 by PCR, using an HLA-A0201 as the template. Then the HLA-A0201-BSP and beta-2m genes were cloned into pBV220 vector and expressed, respectively. The expressed proteins were purified and detected by ELISA and Western blot analyses. High-efficient expressions of HLA-A0201-BSP and beta-2m proteins lay a good foundation for further expression and purification in prokaryotic system and constructing MHC class I-peptide tetramer complexes to study the function of CTLs.

[Construction of HLA-A2-peptide tetramer and application in HBV/HCV infection].

OBJECTIVE: To construct HBV and HCV-specific HLA-A2-peptide tetramers, and to direct clinical therapy. METHODS: Recombinant class I HLA-A2 heavy chains and beta-2 M were produced in Escherichia coli cells transformed with pBV220 vectors. Only the extracellular domain of class I heavy chain was expressed, following modification by replacement of the C-terminal domain with a substrate sequence for BirA biotinylation. HLA-A2-BSP was folded in the presence of beta-2 microglobulin and a specific peptide to form a peptide-MHC complex. The MHC complexes were biotinylated using purified BirA enzyme. Biotinylated MHC-peptide complexes were purified. Tetramers were generated by mixing biotinylated protein complex with streptavidin-PE at a molar ratio of 4:1. Then analysis of stained PBMCs was performed using FACScan and CellQuest software. RESULTS: The expression levels of pBV220-HLA-A2-BSP and beta-2M were 46% and 48% of total bacterial proteins estimated from SDS - PAGE, respectively. And they were mainly located in the insoluble fraction of the cell as inclusion bodies and the proportion were about 85% and 90%, respectively. The purity of pBV220-HLA-A2-BSP and beta-2M was above 95% analyzed by SDS-PAGE, and the concentration of pBV220-HLA-A2-BSP and beta-2M was about 1.5 g/L and 1.2 g/L, respectively. Using the constructed HLA-A2-peptide tetramer to detect the HBV/HCV-specific CTL, the HBV-specific CD8(+) frequencies were 1.84% and 0.02% - 0.68% of the total CD8(+) T cells in acute and chronic HBV hepatitis, respectively. As an additional control, an HLA-A2/HCV tetramer was tested in the acute and chronic HBV hepatitis. The frequencies never exceeded 0.02% of the total CD8(+) T cell number. Similar low levels of background staining were also detected in the HLA-A2(+) or A2(-) healthy control. The HCV-specific CD8(+) frequency was 0.02 - 0.72% of the total CD8(+) T cells in chronic HCV hepatitis. The same frequencies of control were detected. CONCLUSION: High-efficient expressions of HLA-A0201-BSP and beta-2m proteins lay a good foundation for further expression and purification in prokaryotic system and constructing MHC class I-peptide tetramer complexes to study the function of CTLs. Especially, using these two HBV and HCV-specific tetramer can detect the frequencies of the HBV/HCV-specific CD8(+) T cells directly in vitro.

Inhibitory effect of C-type natriuretic peptide on spontaneous contraction in gastric antral circular smooth muscle of rat.

AIM: To investigate the effect of natriuretic peptides on gastric motility and its possible mechanism. METHODS: Spontaneous contraction of gastric antral circular muscle of rats was recorded by four channel physiograph. The concentration of cyclic guanosine monophosphate (cGMP) was measured by radioimmunoassay. The distribution of natriuretic peptide receptors (NPR) was analyzed by autoradiograph. RESULTS: NPR existed in different regions of rat stomach and its density was the largest in gastric antrum. Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) all inhibited the spontaneous contraction of gastric antral circular smooth muscle. Among them, the inhibitory effect of CNP on the spontaneous contraction was the most potent and exhibited a dose-dependent manner. CNP-induced inhibition was diminished by LY83583 (a kind of inhibitor of guanylate cyclase) and potentiated by zaprinist (a kind of inhibitor of cGMP sensitive phosphoesterase). CNP markedly enhanced the concentration of cGMP in antral smooth muscle. The inhibitory effect of CNP on spontaneous contraction was also inhibited by tetraethylammonium (a nonselective potassium channel blocker, TEA). CONCLUSION: The distribution density of NPR is the most in gastric antrum. CNP significantly inhibits gastric motility in rat gastric antrum. The inhibitory effect occurs via a cGMP dependent pathway and potassium channel is also involved in the relaxation induced by CNP in gastric antrial circular smooth muscle of rat.

Role of calcium mobilization in sodium nitroprusside-induced increase of calcium-activated potassium currents in gastric antral circular myocytes of guinea pig.

AIM: To investigate the role of calcium mobilization in the calcium-activated potassium currents [IK(Ca)] increased by sodium nitroprusside (SNP), a nitric oxide (NO) donor, in gastric antral circular myocytes of the guinea pig. METHODS: A perforated patch-clamp technique was used, and the myocytes were isolated by collagenase. RESULTS: SNP 100 mol/L significantly increased IK(Ca), and enhanced the spontaneous transient outward currents (STOC). SNP-induced increase of IK(Ca) was not blocked by extracellular calcium-free solution (containing egtazic acid 10 micromol/L and nicardipine 5 micromol/L, an L-type calcium channel blocker. And SNP 100 micromol/L suppressed the L-type calcium currents (ICa). SNP-induced increase of STOC was inhibited by heparin 3 g/L, a potent inhibitor of inositol triphosphate receptor (InsP3R). However, ryanodine 10 micromol/L, an inhibitor of calcium-induced calcium release (CICR), did not inhibit the effect of SNP-induced increase of STOC. Methylene blue (1 micromol/L), an inhibitor of soluble guanylate cyclase, also inhibited such an effect. CONCLUSION: The increase of IK(Ca) caused by SNP may be mediated by cGMP via IP3-sensitive calcium pools, however, extracellular Ca2+ may not be involved in the process.

Role of actin microfilament in hyposmotic membrane stretch-induced increase in muscarinic current of guinea-pig gastric myocytes.

To investigate the relationship between cytoskeleton and hyposmotic membrane stretch-induced increase in muscarinic current, the role of actin microfilament in hyposmotic membrane stretch-induced increase in muscarinic current was studied with the whole-cell patch clamp technique in guinea-pig gastric myocytes. In this study, the muscarinic current was induced by carbachol (50 micromol/L) or GTPgammaS (0.5 mmol/L). The results showed that hyposmotic superfusate (202 mOsmol/L) increased carbachol-induced current (I(CCh)) by 145+/-27% and increased GTPgammaS-induced current by 183+/-30%; but in the presence of cytochalasin-B (Cyt-B, 20 micromol/L), an actin cytoskeleton disruptor, hyposmotic membrane stretch increased I(CCh) by 70+/-6%. However, hyposmotic membrane stretch induced increase in I(CCh) was potentiated to 545+/-81% by phalloidin (20 micromol/L), an actin microfilament stabilizer. The results demonstrated that hyposmotic membrane stretch increased the muscarinic currents induced by carbachol or GTPgammaS and that the actin microfilament is involved in the process in guinea-pig gastric myocytes.

Role of unsaturated fatty acids in the enhancement of muscarinic current by hyposmotic membrane stretch in guinea pig smooth muscle cells.

To investigate the function of exogenous unsaturated fatty acids in hyposmotic membrane stretch enhancement of muscarinic current (ICCh) in antral circular smooth muscle cells of guinea pig, we recorded the membrane current with the conventional whole cell patch-clamp technique. I(CCh) elicited by 50 micromol/L carbachol (CCh) at the holding potential of 20 mV under isosmotic condition was taken as control. Hyposmotic membrane stretch increased I(CCh) to 226.0+/-21.0%. When the cells were pretreated with 5 micromol/L arachidonic acid (AA), linoleic acid (LA) or oleic acid (OA), I(CCh)was inhibited to 3.8+/-0.6%, 35.2+/-0.8% and 66.6+/-0.6% respectively. Hyposmotic membrane stretch increased I(CCh) to 106.0+/-2.5%, 173.2+/-6.8% and 222.1+/-11.0% of the control respectively. Five micromol/L AA inhibited hyposmotic membrane stretch-enhanced I(CCh) by 51.2+/-3.8%, while the control I(CCh) under isosmotic condition was inhibited by 96.2+/-1.6%. The results suggest that unsaturated fatty acids inhibited I(CCh) and the inhibitory effect is more significant when the unsaturation degree is increased. However, the unsaturated fatty acids are not involved in the increase of I(CCh) induced by hyposmotic membrane stretch.

Intracellular calcium was involved in muscarinic currents increased by hypoosmotic membrane stretch in gastric myocytes of guinea pig.

AIM: To investigate the role of intracellular calcium in muscarinic currents increased by hypoosmotic membrane stretch in gastric antral circular myocytes of the guinea pig. METHODS: The whole cell patch-clamp technique was used, and the myocytes were isolated by collagenase. Cells were swelled by hypoosmotic solution (200 Osmmol/kg). RESULTS: The hypoosmotic membrane stretch markedly increased carbachol-induced muscarinic currents (ICCh). The ICCh and the increase of ICCh were completely blocked by quinidine 3 micromol/L, a specific muscarinic current blocker. In external calcium-free solution hypoosmotic membrane stretch could not increase ICCh, but in the presence of nicadipine 5 micromol/L, a L-type calcium channel blocker or gadolinium chloride 100 nmol/L, a stretch-activated cation channel blocker, the ICCh was still increased by hypoosmotic membrane stretch. When both nicardipine and gadolinium chloride were added into external solution, ICCh were not increased by hypoosmotic membrane stretch any more. Ryanodine, a calcium-induced calcium release (CICR) agonist completely blocked hypoosmotic membrane stretch-induced increase of ICCh. CONCLUSION: Hypoosmotic membrane stretch increased ICCh and the increment was related to influx of external calcium and CICR.