Biomedical subjects

X Han

Publications and source records attributed to X Han.

At least 127 records · Page 7Linked to original sources

PubMed1999

[Studies on ACE gene insertion/deletion polymorphism, serum ACE activity, and diabetic retinopathy in type II diabetic patients].

To clarify the relationship among angiotensin 1-converting enzyme(ACE) gene insertion/deletion(I/D) polymorphism, serum ACE activity, and diabetic retinopathy in Type II diabetic patients, we examined 36 healthy controls, 40 Type II diabetic patients without diabetic retinopathy, and 68 Type II diabetic patients with diabetic retinopathy. All of patients suffered from Type II diabetes beyond 5 years and matched in age and body mass index. An insertion/deletion polymorphism of ACE gene was identified by polymerase chain reaction(PCR). Serum ACE activity was determined using spectrophotometry. The distribution of DD, ID, and II genotypes of the ACE gene did not differ among the three groups (16.7%, 33.3%, and 50.0% in the healthy controls; 22.5%, 35.0%, and 42.5% in Type II diabetic patients patients without diabetic retinopathy; and 20.6%, 30.9%, and 48.5% in Type II diabetic patients with diabetic retinopathy; respectively). The frequency of ACE I/D genotypes was not significantly different between non-proliferative retinopathy and proliferative retinopathy of Type II diabetic patients with diabetic retinopathy (P > 0.05). Serum ACE activity of three groups was similar (P > 0.05). These results do not support the hypothesis that the DD genotype of the ACE gene would be a clinically useful genetic marker for predicting the development of diabetic retinopathy in Type II diabetic patients. There is no association between ACE gene I/D polymorphism and the prognosis of diabetic retinopathy in Type II diabetic patients. ACE may not involve in forming diabetic retinopathy in Type II diabetic patients.

Adult↗

PubMed1999

[Relationship between serum angiotensin I-converting enzyme activity and diabetic nephropathy in patients with type II diabetes].

To clarify the relationship between serum angiotensin I-converting enzyme (ACE) activity and Type II diabetic patients with or without diabetic nephropathy. We examined 36 healthy controls, 58 Type II diabetic patients without diabetic nephropathy, and 50 Type II diabetic with diabetic nephropathy. The diabetic duration of all of Type II diabetic patients was more than 5 years. All patients matched well in age and BMI. An insertion/deletion polymorphism of ACE gene was identified by polymerase chain reaction(PCR). Serum ACE activity was determined using spectrophotometry. Although the distribution of DD, ID, and II genotypes of the ACE gene did not differ among the three groups, serum ACE activity was significantly higher in Type II diabetic patients with diabetic nephropathy than in that without diabetic nephropathy(P < 0.05), especially in the groups with D allele of ACE gene. Serum ACE activity had no difference between healthy controls and Type II diabetic patients without diabetic nephropathy. There is no association between serum ACE activity and Type II diabetic patients without diabetic nephropathy in any genotype of ACE gene. The increasing serum ACE activity plays a role in the initiation of diabetic nephropathy or may serve as a risk marker for later development of overt diabetic nephropathy in Type II diabetic patients.

Adult↗

PubMed1999

[Relationship between nitric oxide and oxygen free radicals in different duration of diabetes in rat kidney].

To study the relationship between nitric oxide(NO) and oxygen free radicals in rat kidney of diabetes mellitus(DM), the authors examined the changes of renal tissue NO level and nitric oxide synthase(NOS) activity, lipid peroxidation(LPO) level and superoxide dismutase(SOD), peroxidase(POD) and catalase (CAT) activities in streptozotocin(STZ)-induced diabetic rats and normal controls(NC) at the 2nd, 8th- and 16th-week. The results showed that renal-tissue NO level and NOS activity were increased at the 2nd week, but decreased at the 8th- and 16th-week; LPO levels were significantly elevated in all DM groups than those in NC groups. SOD, POD and CAT activities decreased significantly in the 8th and 16th week DM groups compared with those in NC groups. The results suggest that NO level in kidney of STZ-induced diabetic rats dynamically change with the disease course of development. This change may be related with the change of anti-oxidation in kidney which high glucose induces.

Animals↗

PubMed1999

[Effect of feishuling on production of tumor necrosis factor alpha from hamster alveolar macrophages].

OBJECTIVE: To observe the effect of the traditional Chinese medicine Feishuling on the production of tumor necrosis factor alpha (TNF-alpha) from hamster alveolar macrophages in vitro and investigate the mechanism of its preventive and therapeutic effect on chronic obstructive pulmonary diseases(COPD). METHOD: Alveolar macrophages were harvested through bronchoalveolar lavage and cultured, Feishuling were added to the culture medium with lipopolysaccharide(LPS) simultaneously or 6 h after LPS. TNF-alpha production in the culture supernatants was tested by ELISA assay and the expression of TNF-alpha mRNA in the cells was measured through in situ hybridization. RESULT: After administration of Feishuling, the content of TNF-alpha in the supernatants and the expression of TNF-alpha mRNA in the cells were decreased more significantly than those in LPS stimulation group(P < 0.01), and there was a dose-effect relationship, but no significant differences were observed as compared with the simple culture medium group(P > 0.05). CONCLUSION: The effect of reducing the production of TNF-alpha from alveolar macrophages and inhibiting the damage mediated by TNF-alpha might be a part of the mechanism of Feishuling to prevent and treat COPD.

Animals↗

PubMed1999

[Molecular clone and sequence analysis of cDNA fragments of hog cholera virus strain C].

The 5' nonencoding region, p23 and p14 encoding region and E1 gene of hog cholera virus (HCV) strain C were amplified from total RNA extracted from HCV strain C infected rabbit spleen by reverse transcription and nested or half--nested PCR. The PCR products were cloned into pGEM-T vector. Nucleotide sequencing was performed using an ABI PRISM sequencing device; based on the incorporation of fluoresect labelled dideoxynuclotide teminators. The obtained sequences on 5' noncoding region and part of p23 and p14 encoding region were compared with HCV strains Shimen and C sequenced in Moormann's lab. The result showed that the homology between HCV strains C sequenced in this report and in Moormann's lab was 99.19%, and the homology between HCV strains Shimen, the standard virulent HCV strain in China, and C sequenced in this report was 94.69%. It was also discovered that the base C at 244 of the genome of HCV strains Shimen and C sequenced in this report was absent at the genome of C strain sequenced in Moormann's lab et al.

5' Untranslated Regions↗

PubMed1999

[The modulation of collagen synthesis and expressions of type I and III collagen mRNAs in aorta smooth muscle cell by PDGF and heparin].

OBJECTIVE: The effect of platelet derived growth factor (PDGF) and heparin on the modulation of human aorta smooth muscle cell(hASMC) proliferation, collagen synthesis, expressions of type I and III collagen mRNAs as well as transforming growth factor-beta (TGF-beta) mRNA were investigated. METHODS: 3H-TdR and 3H-proline incorporation and Northern blot analysis were done. 3H-TdR and 3H-proline incorporation was statistically analyzed with t test among different experimental groups. RESULTS: In comparison to the control group, PDGF possessed the ability in promoting markedly the DNA synthesis (3H-TdR incorporation: PDGF group vs control, P < 0.01), synthesis and secretion of collagen protein (3H-proline incorporation of SMC and medium: PDGF group vs control, P < 0.01), expressions of type I and type III collagen mRNAs, and transforming growth factor mRNA in hASMC. Whereas heparin significantly decreased the DNA synthesis (heparin group vs control, P < 0.01), synthesis and secretion of collagen protein (heparin group vs control, P < 0.01) of hASMC in vitro. Heparin also inhibited the promoting effect of PDGF on DNA synthesis, the synthesis and secretion of collagen protein, and reduced the up-regulation of expressions of type I and type III collagen mRNAs as well as TGF-beta mRNA that stimulated by PDGF. CONCLUSIONS: PDGF could promote the collagen synthesis through upregulating type I and type III collagen mRNA expressions while heparin just inhibiting SMC DNA and collagen synthesis effected in the way of anti-atherosclerosis.

Anticoagulants↗

PubMed1999

[The morphometric analysis of lung on protective effect of Chinese herbs 814 on elastase-induced emphysema in hamsters].

OBJECTIVE: To determine the protective effect of Chinese herbal medicine 814 on elastase-induced emphysema in hamsters. METHODS: Animals were injected intratrachealy with elastase for emphysematous models, prophylactic-therapeutic groups were administrate with 814 through esophagus two weeks before the instillation of elastase untill animals were killed at three different time at first, second, and third month. Pulmonary artery pressure, blood gas analysis, heart index and the ratio of dried weight to wet weight of lungs (WW/DW) were examined. The lung paraffin sections were measured mean linear intercept (MLI), mean alveolar number (MAN), ratio of parenchyma area to total area (PA/TA) by the microscope-computer morphometric analysis system. RESULTS: WW/DW in the prophylactic-therapeutic groups was recovered at the same level with the controls, whereas the emphysematous were significantly increased (P < 0.05); and compared with the emphysematous groups, the prophylactictherapeutic groups significantly decreased in MLI, increased in MAN and PA/TA (P < 0.001 or P < 0.05). CONCLUSIONS: Administration of 814 could partly inhibit the development of emphysema induced by elestase in hamsters.

Animals↗

PubMed1999

[New solution of simultaneous equations in spectrophotometry for the determination of aspirin and acetaminophen in xiaoer tuishao pian].

The contents of aspirin and acetaminophen in xiaoer tuishao pian were determined by using the new solution of simultaneous equations without any preliminary separation. The average recoveries and variation coefficients of the analysis are 99.95% and 1.74% for aspirin and 100.1% and 0.93% for acetaminophen, respectively. The method was simple, rapid and accurate.

Acetaminophen↗

PubMed1998

Reconstitution of membrane fusion between pancreatic islet secretory granules and plasma membranes: catalysis by a protein constituent recognized by monoclonal antibodies directed against glyceraldehyde-3-phosphate dehydrogenase.

An isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isolated and purified from rabbit brain cytosol has previously been demonstrated to catalyze membrane fusion (Glaser and Gross, Biochemistry 33 (1994) 5805-5812; Glaser and Gross, Biochemistry 34 (1995) 12193-12203). Herein, we provide evidence suggesting that this GAPDH isoform can reconstitute in vitro protein-catalyzed fusion between naturally occurring subcellular membrane fractions involved in insulin exocytosis. Utilizing purified rat pancreatic beta-cell plasma membranes and secretory granules, we show that a brain cytosolic factor catalyzed the rapid and efficient fusion of these two purified membrane fractions which could be inhibited by a monoclonal antibody directed against the brain isoform of GAPDH. Moreover, the brain cytosolic factor also catalyzed the fusion of reconstituted vesicles prepared from lipid extracts of islet plasma membranes and secretory granules. Although the brain cytosolic factor rapidly catalyzed membrane fusion between islet plasma membranes and secretory granules, it did not catalyze fusion between one secretory granule population with another. To identify the potential importance of brain cytosolic factor catalyzed membrane fusion in islet cells, we examined extracts of hamster insulinoma tumor cells (HIT cells) for fusion-catalyzing activity. A protein constituent was present in HIT cell cytosol which was immunologically similar to the rabbit brain GAPDH isoform. Although native HIT cell cytosol did not catalyze membrane fusion, removal of an endogenous protein inhibitor unmasked the presence of the protein which catalyzed membrane fusion activity and such fusion was ablated by a monoclonal antibody directed against the brain isoform of GAPDH. Collectively, these results suggest the possibility that an isoform of brain GAPDH, also evident in HIT cells, can catalyze fusion between the two naturally occurring subcellular membrane compartments involved in insulin secretion and suggest a novel paradigm potentially coupling glycolytic flux with insulin release.

Animals↗

PubMed1998

Characterization of a slowly inactivating outward current in adult mouse ventricular myocytes.

We recently have reported that suppression of the slowly inactivating component of the outward current, Islow, in ventricular myocytes of transgenic mice (long QT mice) overexpressing the N-terminal fragment and S1 segment of Kv1.1 resulted in a significant prolongation of action potential duration and the QT interval. Here we describe the detailed biophysical properties and physiological role of Islow by applying the whole-cell patch-clamp technique at both room temperature and 37 degreesC. This current activates rapidly with time constants ranging from 3.8+/-0.8 ms at -20 mV to 2.1+/-0.5 ms at 50 mV at room temperature. The half-activation voltage and slope factor are -12.5+/-2.6 mV and 7. 7+/-1.0 mV, respectively. The inactivation of this current is slow compared with the fast inactivating component Ito, with time constants of approximately 100 ms at 37 degreesC. The steady-state inactivation of Islow is not temperature-dependent, with half-inactivation voltages and slope factors of -35.1+/-1.3 and -5. 4+/-0.4 mV at 37 degreesC, and -37.6+/-1.8 and -5.8+/-0.6 mV at room temperature. Double exponentials were required to describe the time-dependent recovery of Islow from steady-state inactivation, with time constants of 233+/-34 and 3730+/-702 ms at 37 degreesC, and 830+/-240 and 8680+/-2410 ms at room temperature. Islow is highly sensitive to 4-aminopyridine but is insensitive to tetraethylammonium, alpha-dendrotoxin, and E-4031. Stimulation with action-potential waveforms under voltage-clamp mode revealed that this current plays an important role in the early and middle phases of repolarization of the cardiac action potential. We conclude that the biophysical properties and pharmacological profiles of Islow are similar to those of Kv1.5-encoded currents.

4-Aminopyridine↗

PubMed1998

Up-regulation of human secreted frizzled homolog in apoptosis and its down-regulation in breast tumors.

In the screening of apoptosis-related genes, an elevated 4.5-kb transcript representing the full-length cDNA of human secreted frizzled-related protein (hsFRP) was cloned. To investigate its possible role in the regulation of cell proliferation, gene expression of hsFRP was examined in human immortalized breast epithelial cell line HBL-100 during growth arrest and apoptosis. Serum deprivation caused G arrest and induction of hsFRP. When serum was re-introduced into the cell culture, the expression of hsFRP declined. Adriamycin treatment induced accumulation of hsFRP mRNA and decrease of beta-catenin. This indicates that the regulation of hsFRP may be involved in the cell-cycle/apoptosis mechanism and possibly in the wnt signaling pathway. hsFRP transcripts were undetectable in cells derived from malignant breast carcinomas, but detectable in 3 immortalized non-malignant breast epithelial cell lines, indicating the involvement of hsFRP in the breast malignant transformation. When tumor and adjacent normal tissues from the same patients were examined, lower expression was found in 5/5 of breast tumors, 2/4 of ovary tumors and 3/5 of kidney tumors. These data suggest the possible involvement of hsFRP in regulation of cell proliferation and breast tumorigenesis.

Apoptosis↗

PubMed1998

Isolation of a protein Z-dependent plasma protease inhibitor.

Human protein Z (PZ) is a 62,000-Mr, vitamin K-dependent plasma protein whose structure is similar to coagulation factors VII, IX, X, protein C, and protein S, but whose function is not known. The procoagulant activity of factor Xa in a one-stage plasma coagulation assay is reduced when factor Xa is first incubated with PZ. This apparent inhibitory effect is time dependent, requires the presence of calcium ions and procoagulant phospholipids (rabbit brain cephalin), and appears predominantly related to the incubation period of PZ with cephalin. In serum the initial rate of inhibition of factor Xa with calcium ions and cephalin also is enhanced in the presence PZ. A PZ-dependent protease inhibitor (ZPI) has been isolated from plasma. ZPI is a 72,000-Mr single-chain protein with an N-terminal amino acid sequence of LAPSPQSPEXXA (X = indeterminate) and an estimated concentration in citrate-treated plasma of 1.0-1.6 microg/ml. In systems using purified components, the factor Xa inhibition produced by ZPI is rapid (>95% within 1 min by coagulation assay) and requires the presence of PZ, calcium ions, and cephalin. The inhibitory process appears to involve the formation of a factor Xa-PZ-ZPI complex at the phospholipid surface.

Amino Acid Sequence↗

PubMed1998

FTIR study of the thermal denaturation of alpha-actinin in its lipid-free and dioleoylphosphatidylglycerol-bound states and the central and N-terminal domains of alpha-actinin in D2O.

Fourier transform infrared (FTIR) spectroscopy has been carried out to investigate the thermal denaturation of alpha-actinin and its complexes with dioleoylphosphatidylglycerol (DOPG) vesicles. The amide I regions in the deconvolved spectra of alpha-actinin in the lipid-free and DOPG-bound states are both consistent with predominantly alpha-helical secondary structure below the denaturation temperatures. Studies of the temperature dependence of the spectra revealed that for alpha-actinin alone the secondary structure was unaltered up to 40 degrees C. But, in the presence of DOPG vesicles, the thermal stability of the secondary structure of alpha-actinin increased to 55 degrees C. The thermal denaturation mechanisms of the lipid-free and DOPG-bound states of alpha-actinin also vary. The secondary structure of the lipid-free alpha-actinin changed to be predominantly unordered upon heating to 65 degrees C and above. Whereas, the original alpha-helical structure in the DOPG-bound alpha-actinin retained even at 70 degrees C, the highest temperature we examined. Analysis of the reduction in amide II intensities, which is due to peptide H-D exchange upon heating alpha-actinin in D2O, showed that partially unfolded states with increased solvent accessibility but substantial secondary structures could be observed from 35 to 40 degrees C only if DOPG vesicles were present. A so-called "protamine precipitation" method has been developed to purify the N-terminal domain of alpha-actinin by use of the fact that the central domain of alpha-actinin is negatively charged but the N-terminal domain is positively charged. Thermal denaturation of the central and N-terminal domains of alpha-actinin were then investigated with FTIR. The secondary structure of the N-terminal domain of alpha-actinin was found to be thermally sensitive below 35 degrees C, which is characterized as the increase of the alpha-helical structure at the expense of the random coil upon heating the N-terminal domain from 4 to 35 degrees C. The membrane-binding ability of the N-terminal domain of alpha-actinin was proposed in terms of the analysis of the local electrostatic properties of alpha-actinin and the assignment of the amide II bands in the FTIR spctra of alpha-actinin.

Actinin↗

PubMed1998

The upf3 protein is a component of the surveillance complex that monitors both translation and mRNA turnover and affects viral propagation.

The nonsense-mediated mRNA decay pathway functions to degrade aberrant mRNAs that contain premature translation termination codons. In Saccharomyces cerevisiae, the Upf1, Upf2, and Upf3 proteins have been identified as trans-acting factors involved in this pathway. Recent results have demonstrated that the Upf proteins may also be involved in maintaining the fidelity of several aspects of the translation process. Certain mutations in the UPF1 gene have been shown to affect the efficiency of translation termination at nonsense codons and/or the process of programmed -1 ribosomal frameshifting used by viruses to control their gene expression. Alteration of programmed frameshift efficiencies can affect virus assembly leading to reduced viral titers or elimination of the virus. Here we present evidence that the Upf3 protein also functions to regulate programmed -1 frameshift efficiency. A upf3-Delta strain demonstrates increased sensitivity to the antibiotic paromomycin and increased programmed -1 ribosomal frameshift efficiency resulting in loss of the M1 virus. Based on these observations, we hypothesize that the Upf proteins are part of a surveillance complex that functions to monitor translational fidelity and mRNA turnover.

Alleles↗

PubMed1998

Nonesterified fatty acids induce transmembrane monovalent cation flux: host-guest interactions as determinants of fatty acid-induced ion transport.

Nonesterified fatty acids are key intermediates in cellular metabolism whose intracellular concentration is regulated by multiple anabolic, catabolic, and oxidative enzymatic cascades. Herein, we demonstrate that fatty acids induce transmembrane monovalent cation flux with an apparent rate constant kapp = 10(-)4 - 10(-)3 s-1. Fatty acid-induced cation efflux exploits the ionic association of the cation with the carboxylate anion of the fatty acid and the subsequent transmembrane flip-flop of the fatty acid-cation complex. Rates of fatty acid-induced transmembrane cation flux were dependent upon complex host-guest interactions between the fatty acid-cation complex and the phospholipid constituents which comprise the membrane bilayer including (1) the degree of unsaturation of the fatty acid guest and the regiospecificity and stereospecificity of its olefinic linkages; (2) the phospholipid subclass and individual molecular species which constitute the host membrane phospholipids; (3) impedance matching of host and guest hydrophobic characteristics; and (4) the cholesterol content of the membrane bilayer. Arrhenius analysis demonstrated that fatty acid-induced K+ efflux was facilitated largely by changes in the entropy of activation of ion translocation and not the energy of activation. Moreover, Arrhenius analysis demonstrated that the energy of activation of ion translocation was phospholipid subclass specific. For example, arachidonic acid-induced cation efflux in membranes comprised of 16:0-18:1 plasmenylcholine possessed an Ea = 5.3 +/- 0.4 kcal/mol, while that for 16:0-18:1 phosphatidylcholine was 7.2 +/- 0.5 kcal/mol. Electrophysiologic measurements of planar lipid membranes containing 10 mol % arachidonic acid as a substitutional impurity confirmed the ability of physiologically relevant amounts of fatty acid to induce ion translocation with a specific conductance of 2.6 +/- 0.3 microS/cm2. Collectively, these results demonstrate that fatty acids facilitate transmembrane cation flux by an ion carrier type mechanism and suggest that fatty acid-mediated ion transport contributes to the leakage current present in many cell types and thus potentially modulates cellular responsivity during signal transduction where the intracellular content of fatty acids changes dramatically.

Arachidonic Acid↗

PubMed1998

Characteristics of nitric oxide-mediated cholinergic modulation of calcium current in rabbit sino-atrial node.

1. We have previously shown that nitric oxide (NO) production is essential for cholinergic inhibition of the beta-adrenergic stimulated L-type calcium current (ICa-L) in rabbit pacemaker (sino-atrial node (SAN)) cells. The present experiments demonstrate the presence of constitutive nitric oxide synthase (cNOS) in SAN cells, and characterize the NO-mediated cholinergic response. 2. Immunohistochemical staining, using an antibody prepared against endothelial cNOS, demonstrated that this enzyme was present in single myocytes obtained from the SAN. 3. The activation of cNOS is known to be Ca2+ and calmodulin dependent. Strongly buffering intracellular Ca2+ with the membrane-permeable chelator BAPTA-AM (10 microM) significantly reduced (and in some cases abolished) the attenuation of ICa-L by the muscarinic agonist carbamylcholine (CCh). In contrast, the CCh-induced activation of an outward K+ current, IK,ACh, was unaffected by buffering of [Ca2+]i. The calmodulin inhibitor 48/80 (20 microM) also abolished the attenuation of ICa-L by CCh, with no change in the activation of IK,ACh. 4. Neither thapsigargin nor ryanodine (5-10 microM), agents which deplete intracellular Ca2+ stores, significantly changed the attenuation of ICa-L by CCh. 5. Pertussis toxin (PTX) completely abolished both the inhibitory action of CCh on ICa-L and the activation of IK,ACh. This establishes that a PTX-sensitive GTP-binding protein links the muscarinic receptor to NO synthase activation in SAN cells. 6. Our hypothesis is that NO leads to activation of a cyclic GMP (cGMP)-activated phosphodiesterase (PDE II) as a mechanism for enhanced cyclic AMP breakdown and ICa-L attenuation. This was supported by showing that a specific inhibitor of PDE II, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), blocks the effect of CCh on ICa-L, but not on IK,ACh. Using reverse transcriptase-polymerase chain reaction techniques, we have established that PDE II is the dominant cyclic nucleotide phosphodiesterase isoform in SAN cells.

1-Methyl-3-isobutylxanthine↗

PubMed1998

The surveillance complex interacts with the translation release factors to enhance termination and degrade aberrant mRNAs.

The nonsense-mediated mRNA decay pathway is an example of an evolutionarily conserved surveillance pathway that rids the cell of transcripts that contain nonsense mutations. The product of the UPF1 gene is a necessary component of the putative surveillance complex that recognizes and degrades aberrant mRNAs. Recent results indicate that the Upf1p also enhances translation termination at a nonsense codon. The results presented here demonstrate that the yeast and human forms of the Upf1p interact with both eukaryotic translation termination factors eRF1 and eRF3. Consistent with Upf1p interacting with the eRFs, the Upf1p is found in the prion-like aggregates that contain eRF1 and eRF3 observed in yeast [PSI+] strains. These results suggest that interaction of the Upf1p with the peptidyl release factors may be a key event in the assembly of the putative surveillance complex that enhances translation termination, monitors whether termination has occurred prematurely, and promotes degradation of aberrant transcripts.

Fungal Proteins↗

PubMed1998

Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase.

Cardiac myocytes have been shown to express constitutively endothelial nitric oxide synthase (eNOS) (nitric oxide synthase 3), the activation of which has been implicated in the regulation of myocyte L-type voltage-sensitive calcium channel current (ICa-L) and myocyte contractile responsiveness to parasympathetic nervous system signaling, although this implication remains controversial. Therefore, we examined the effect of the muscarinic cholinergic agonist carbachol (CCh) on ICa-L and contractile amplitude in isoproterenol (ISO)-prestimulated ventricular myocytes isolated from adult mice, designated eNOSnull mice, with targeted disruption of the eNOS gene. Although both eNOSnull and wild-type (WT) ventricular myocytes exhibited similar increases in ICa-L in response to ISO, there was no measurable suppression of ICa-L by CCh in cells from eNOSnull mice, in contrast to cells from WT mice. These results were reflected in the absence of an effect of CCh on the positive inotropic effect of ISO in eNOSnull myocytes. Also, unlike myocytes from WT animals, eNOSnull myocytes failed to exhibit an increase in cGMP content in response to CCh. Nevertheless, the pharmacologic nitric oxide donors 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein increased cGMP generation and suppressed ISO-augmented ICa-L in eNOSnull cells, suggesting that the signal transduction pathway(s) downstream of eNOS remained intact. Of importance, activation of the acetylcholine-activated K+ channel by CCh was unaffected in atrial and ventricular eNOSnull myocytes. These results confirm the obligatory role of eNOS in coupling muscarinic receptor activation to cGMP-dependent control of ICa-L in cardiac myocytes.

Animals↗