Biomedical subjects

D Kim

Publications and source records attributed to D Kim.

At least 199 records · Page 11Linked to original sources

PubMed2000

Apoptosis in dilated cardiomyopathy.

OBJECTIVE: Cardiomyopathy, a popular diagnosis that always obscures more than it reveals, nevertheless has several characteristic histological features. These prominently include widespread focal myocardial fibrosis and associated hypertrophy of surviving cardiac myocyte. In fact, focal noninflammatory degeneration (not necrosis) has been demonstrated as a feature of many forms of cardiac hypertrophy. We hypothesized that this loss of myocardial cells in dilated cardiomyopathy (DCMP) may result from cell death by apoptosis. METHODS: Endomyocardial biopsy specimens from the right ventricles of six patients who suffered from DCMP were studied, and myocardial specimens from two persons who died in motor vehicle accidents were used as negative controls. For identification of apoptosis, immunohistochemistry with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling was performed. In addition, apoptosis was confirmed morphologically by confocal laser scanning microscopy with propidium iodide. RESULTS: Apoptosis, that was represented by an apoptotic index ranging from 19.8 to 25.4%, could be extensively seen in myocytes and also rarely in non-myocytes of interstitium and vascular endothelium. Morphologically, there were a lot of nuclei with clumps of condensed chromatin, suggestive of apoptosis. CONCLUSION: The present study demonstrated that myocyte loss in DCMP might be mainly due to the apoptosis of myocytes and interstitial cells, rather than inflammation or cell necrosis.

Adult↗

PubMed2000

MR imaging findings of Rathke's cleft cysts: significance of intracystic nodules.

BACKGROUND AND PURPOSE: Rathke's cleft cysts often may be difficult to differentiate from other intrasellar or suprasellar masses on radiologic studies. The purpose of this study was to describe the significance of intracystic nodules, a diagnostic characteristic found in Rathke's cleft cysts, on MR images. METHODS: A retrospective review of MR studies was conducted for 13 patients who, after pathologic analysis, were diagnosed as having Rathke's cleft cyst. These patients underwent unenhanced and contrast-enhanced T1- and T2-weighted axial and coronal spin-echo sequential imaging. The signal intensity and incidence of the intracystic nodules on T1- and T2-weighted images were analyzed. The signal intensity of the nodule was compared with that of white matter and surrounding cyst fluid. The signal intensity of cyst fluid was compared with the intraoperative appearance of the cyst fluid. Biochemical and pathologic analyses of the intracystic nodules were conducted in two cases. RESULTS: An intracystic nodule having high signal intensity on T1-weighted images and low signal intensity on T2-weighted images was observed in 10 (77%) of the cases. At surgery, intracystic nodules were yellow, waxy, solid masses. Pathologic analysis showed this nodule to be a mucin clump. Biochemical analysis of the intracystic nodules showed cholesterol and proteins as the main constituents. In the Rathke's cleft cyst with intracystic nodules, cyst fluid revealed low signal intensity to isointensity relative to the intensity of the nodules on T1-weighted images, and isointensity to high signal intensity on T2-weighted images. Intracystic nodules were clearly visible on T2-weighted images. CONCLUSION: Because cyst fluid of Rathke's cleft cysts shows variable intensities on MR images, the specific diagnosis is often difficult when based on MR signal intensity values alone. The presence of an intracystic nodule with characteristic signal intensities on MR images may be indicative of the diagnosis of Rathke's cleft cyst.

Adolescent↗

PubMed2000

Visceral ischemia after cardiopulmonary bypass.

The diagnosis and management of gastrointestinal complications associated with cardiopulmonary bypass is often hindered by a complicated clinical picture and equivocal examination. To better define the incidence, risk factors, and mortality, we reviewed the records of all patients undergoing cardiopulmonary bypass from 1988 through 1996. The database for this study comprised 14,521 patients who underwent cardiac surgery. The patients (543) with gastrointestinal complications were identified, and those with major complications (166) were individually reviewed. Major complications included pancreatitis, gastritis, laparotomy, gastric ulcer, cholecystitis, colonic perforation, gastrointestinal bleeding, diverticulitis, bowel obstruction, perforation, and visceral ischemia. Our results were the following. 1) Gastrointestinal complications were noted in 3.7 per cent (543) of patients with major complications occurring in 1.2 per cent. In 166 patients, 187 major complications were noted. 2) Visceral ischemia, an infrequent but usually fatal (71%) complication, occurred in 24 (0.17%). 3) Of the ischemic events, 83 per cent (20 of 24) affected the bowel; with the colon involved 80 per cent of the time (16 of 20). 4) Patients with visceral ischemia were more likely to be female (relative risk 2.1), have longer pump times (92.2 versus 74.2), have cardiac procedures other than coronary artery bypass graft (relative risk 2.6), and have end-stage renal disease (relative risk 16.7). We conclude that, given the incidence and mortality related to visceral ischemia, especially to the colon, patients with risk factors (end-stage renal disease, female sex, non-coronary artery bypass graft, and longer pump times) should undergo routine endoscopic examination of the colon early after bypass and when clinically indicated thereafter.

Aged↗

PubMed2000

Effects of age and dietary restriction on lifespan and oxidative stress of SAMP8 mice with learning and memory impairments.

This study was to evaluate the effect of dietary restriction (DR) on lifespan and oxidative stress of dementia mouse model SAMP8 with impaired learning and memory. SAMP8 female mice were fed either ad libitum (AL) or fed 60% of food intake of AL. Results showed that basal metabolic rates (BMR) were significantly lower (15 to 22%) in DR with increased median and maximum lifespans, suggesting feed and gross efficiencies were significantly lower in DR than in AL. Grading score of senescence resulted in a marked improvement about 2-fold by DR compared with AL. The amounts of lipofuscin at 12 months were significantly lowered 16% in DR than that of AL. Median and maximal lifespans significantly increased (28.5% and 16.4%, respectively) by DR, and also lowered superoxide radical about 15 approximately 45% in DR compared with AL at 4, 8 and 12 months of age. On the other hand, superoxide dismutase (SOD) activities were higher (about 15 approximately 30%) in DR than those in AL group of SAMP8 except for 4 months of age. Our results suggest that 40% calorie restricted SAMP8 can effectively suppress dementia-related abnormalities during aging.

Aging↗

PubMed1999

Formation of indigo by recombinant mammalian cytochrome P450.

The development of bicistronic systems for coexpression of recombinant human cytochrome P450 enzymes (P450s) with their redox partner, NADPH-cytochrome P450 reductase (NPR), has enabled P450 activity to be reconstituted within bacterial cells. During expression of recombinant P450 2E1 and some other forms, we observed the formation of a blue pigment in bacterial cultures. The pigment was extracted from cultures and shown to comigrate with standard indigo on TLC. UV-visible spectroscopy and mass spectrometric analysis provided further support for identification of the pigment as indigo. Indigo is known to form following the spontaneous oxidation of 3-hydroxyindole. Accordingly, we speculated that indole, formed as a breakdown product of tryptophan in bacteria, was hydroxylated by the P450 system, leading to indigo formation. Bacterial membranes containing recombinant P450 2E1 and human NPR were incubated in vitro with indole and shown to catalyze formation of a blue pigment in a time- and cofactor-dependent manner. These studies suggest potential applications of mammalian P450 enzymes in industrial indigo production or in the development of novel colorimetric assays based on indole hydroxylation.

Cytochrome P-450 CYP2E1↗

PubMed1999

Comparative study of the inhibition of alpha-glucosidase, alpha-amylase, and cyclomaltodextrin glucanosyltransferase by acarbose, isoacarbose, and acarviosine-glucose.

Bacillus stearothermophilus maltogenic amylase hydrolyzes the first glycosidic linkage of acarbose to give acarviosine-glucose. In the presence of carbohydrate acceptors, acarviosine-glucose is primarily transferred to the C-6 position of the acceptor. When d-glucose is the acceptor, isoacarbose is formed. Acarbose, acarviosine-glucose, and isoacarbose were compared as inhibitors of alpha-glucosidase, alpha-amylase, and cyclomaltodextrin glucanosyltransferase. The three inhibitors were found to be competitive inhibitors for alpha-glucosidase and mixed noncompetitive inhibitors for alpha-amylase and cyclomaltodextrin glucanosyltransferase. The K(i) values were dependent on the type of enzyme and their source. Acarviosine-glucose was a potent inhibitor for baker's yeast alpha-glucosidase, inhibiting 430 times more than acarbose, and was an excellent inhibitor for cyclomaltodextrin glucanosyltransferase, inhibiting 6 times more than acarbose. Isoacarbose was the most effective inhibitor of alpha-amylase and cyclomaltodextrin glucanosyltransferase, inhibiting 15.2 and 2.0 times more than acarbose, respectively.

Acarbose↗

PubMed1999

Promotion of skeletal muscle differentiation by K252a with tyrosine phosphorylation of focal adhesion: a possible involvement of small GTPase Rho.

K252a, a protein kinase inhibitor, acts as a neurotrophic factor in several neuronal cells. In this study we show that K252a enhanced the differentiation of C2C12 myoblasts as well as tyrosine phosphorylation of several focal adhesion-associated proteins including p130(Cas), focal adhesion kinase, and paxillin. The tyrosine phosphorylation of these proteins, reaching a maximum at 30 min after K252a treatment, closely correlated with the colocalization of these proteins in focal adhesion complexes and the coimmunoprecipitation of these proteins with p130(Cas). In addition, K252a stimulated longitudinal development of stress fiber-like structures and cell-matrix interaction in postmitotic myoblasts and eventually formation of well-developed myofibrils in multinucleated myotubes. Herbimycin A, a potent inhibitor of Src family kinases, and cytochalasin D, a selective disrupting-agent of actin filament, completely inhibited K252a-induced tyrosine phosphorylation as well as myoblast differentiation. Similar inhibitory effect was observed in the cells scrape loaded with a Rho inhibitor, C3 transferase, and the treatment of K252a induced a rapid translocation of Rho. These results are consistent with the model that Rho-dependent tyrosine phosphorylation of focal adhesion-associated proteins plays an important role in skeletal muscle differentiation.

Animals↗

PubMed1999

Proteasome inhibitors lactacystin and MG132 inhibit the dephosphorylation of HSF1 after heat shock and suppress thermal induction of heat shock gene expression.

Recently, we have shown that two proteasome inhibitors, MG132 and lactacystin, induce hyperphosphorylation and trimerization of HSF1, and transactivate heat shock genes at 37 degrees C. Here, we examined the effects of these proteasome inhibitors and, in addition, a phosphatase inhibitor calyculin A (CCA) on the activation of HSF1 upon heat shock and during post-heat-shock recovery, with emphasis on HSF1 hyperphosphorylation and the ability of HSF1 to transactivate heat shock genes. When lactacystin, MG132, or CCA was present after heat shock, HSF1 remained hyperphosphorylated during post-heat-shock recovery at 37 degrees C. Failure of HSF1 to recover to its preheated dephosphorylated state correlated well with the suppression of the heat-induced hsp70 expression. In vitro, HSF1 from heat-shocked cells, when dephosphorylated, showed an increase in HSE-binding affinity. Taken together, these data suggest that phosphorylation of HSF1 plays an important role in the negative regulation of heat-shock response. Specifically, during post-heat-shock recovery phase, prolonged hyperphosphorylation of HSF1 suppresses heat-induced expression of heat shock genes.

Acetylcysteine↗

PubMed1999

Isolation and characterization of a thermophilic lipase from bacillus thermoleovorans ID-1.

A thermophilic microorganism, Bacillus thermoleovorans ID-1, isolated from hot springs in Indonesia, showed extracellular lipase activity and high growth rates on lipid substrates at elevated temperatures. On olive oil (1.5%, w/v) as the sole carbon source, the isolate ID-1 grew very rapidly at 65 degrees C with its specific growth rate (2.50 h(-1)) and its lipase activity reached the maximum value of 520 U l(-1) during the late exponential phase and then decreased. In addition to this, isolate ID-1 could grow on a variety of lipid substrates such as oils (olive oil, soybean oil and mineral oil), triglycerides (triolein, tributyrin) and emulsifiers (Tween 20, 40). The excreted lipase of ID-1 was purified 223-fold to homogeneity by ammonium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography and Sephacryl S-200 gel filtration chromatography. As a result, the relative molecular mass of the lipase was determined to be 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme showed optimal activity at 70-75 degrees C and pH 7.5 and exhibited 50% of its original activity after 1 h incubation at 60 degrees C and 30 min at 70 degrees C and its catalytic function was activated in the presence of Ca(2+) or Zn(2+).

Bacillus↗

PubMed1999

Cloning and characterization of a nuclear S6 kinase, S6 kinase-related kinase (SRK); a novel nuclear target of Akt.

Akt is stimulated by several growth factors, and mediates their cell survival signals. Recent studies have shown that Akt may play an intermediate role between phosphatidylinositol 3-kinase (PI3K) and p70 S6 kinase (p70S6K). Here we show that a novel nuclear p70S6K-related kinase (SRK) exists and that its in vivo function is also augmented by over-expression of Akt. Conceptual translation of the SRK cDNA revealed that the catalytic domain of SRK was highly homologous to that of p70S6K, and that the treatment of wortmannin or rapamycin strongly inhibited the phosphorylation and the activation of SRK, as in p70S6K. However, the N- and C-terminal domains of SRK were quite different from those of p70S6K. In immunolocalization analyses, we demonstrated a constitutive nuclear localization of SRK and the presence of a nuclear localization signal in its C-terminus. In vitro S6 phosphotransferase activities of SRK were stimulated with a slower kinetics by a variety of agonists to p70S6K. Interestingly, over-expression of the proto-oncogene Akt resulted in EGF-independent activation of SRK, while over-expression of kinase-dead Akt actually had an inhibitory effect. This relationship between Akt and SRK suggests that SRK may be a novel target of Akt and perhaps an important downstream component in the nuclear function of Akt.

Catalytic Domain↗

PubMed1999

Sequence of neurodegeneration and accumulation of phosphorylated tau in cultured neurons after okadaic acid treatment.

Within neurofibrillary tangles and dystrophic neurites of Alzheimer's disease (AD), the cytoskeletal protein tau is abnormally hyperphosphorylated. In the present study, we examined the effect of okadaic acid (OA), a protein phosphatase inhibitor, in rat cultured neurons. Low concentrations of OA induce degeneration of neurites, rounding of cell bodies, detachment from the substratum, and eventual neuronal death. During OA-induced degeneration, SMI-31 immunoreactivity became punctate in neurites at 6 h after OA treatment, and over time, accumulated in cell bodies and dystrophic neurites. Hyperphosphorylation of tau and marked loss of MAP-2-positive dendrites occurred after 6 h of treatment with OA. Thereafter, AT-8 and PHF-1 immunoreactivity accumulated in cell bodies and subsequently appeared in distal axon-like neurites. These results demonstrate that OA treatment induced hyperphosphorylation of tau and preferential dendritic damage, with subsequent accumulation of phosphorylated tau in cell bodies and dystrophic axon-like neurites. OA-induced neurodegeneration may provide a useful model to study AD.

Animals↗

PubMed1999

Involvement of c-Jun NH(2)-terminal kinase pathway in differential regulation of heat shock proteins by anticancer drugs.

In the present study, we examined the modulation of heat shock factor 1 (HSF1) activity and expression of heat shock proteins (HSPs) after exposure to anticancer drugs. Anticancer drugs induced HSF1 DNA-binding activity, and this was followed by an increase of mitochondrial HSP75 and HSP60 levels and concurrent decrease of cytoplasmic HSP70 levels. Unlike heat shock-induced full phosphorylation, HSF1 was partially phosphorylated after exposure to vincristine, and this result was tightly correlated with the kinetics of JNK/SAPK activation, and up-regulation of mitochondrial HSP75 level and concurrent down-regulation of HSP70. Furthermore, the dominant-negative mutant of SEK1 blocked the phosphorylation of HSF1 and up-regulation of mitochondrial HSP75 in response to vincristine or vinblastine. These data suggest that anticancer drugs regulate the HSF1 transcriptional activity differently from heat shock, and JNK/SAPK pathway appears to be involved in anticancer drug-induced HSF1 phosphorylation and consequently differential regulation of mitochondrial HSP75 and HSP60 and cytoplasmic HSP70.

Animals↗

PubMed1999

Lipopolysaccharide bilayer structure: effect of chemotype, core mutations, divalent cations, and temperature.

Lipopolysaccharide (LPS), the primary lipid on the surface of Gram-negative bacteria, is thought to act as a protective and permeability barrier. X-ray diffraction analysis of osmotically stressed LPS multilayers was used to determine the structure and interactive properties of LPSs from strains containing the minimum number of sugars necessary for bacterial survival (Re chemotype) to the maximum number of sugars found in rough bacteria (Ra chemotype). At 20 degrees C in the absence of divalent cations, LPS suspensions gave a sharp wide-angle reflection at 4.23 A and a broad low-angle band centered at 50-68 A depending on the chemotype, indicating the presence of gel phase bilayers separated by large fluid spaces. As osmotic pressure was applied, the apposing bilayers were squeezed together and lamellar diffraction at 6 A resolution was obtained. At low applied pressures (<10(6) dyn/cm2), the total repulsive pressure between bilayers could be explained by electrostatic double layer theory. At higher applied pressures, there was a sharp upward break in each pressure-distance relation, indicating the presence of a hydrophilic steric barrier whose range depended strongly on the LPS chemotype. The positions of these upward breaks, along with electron density profiles, showed that the sugar core width systematically increased from 10 A for the Re chemotype to 27 A for the Ra chemotype. In excess buffer, the addition of divalent cations brought the bilayers into steric contact. Electron density profiles were used to determine the locations of cation binding sites and polar substituents on the LPS oligosaccharide core. The area per hydrocarbon chain was approximately 26 A2 in liquid-crystalline LPS bilayers, an indication of an acyl chain packing that is much tighter than that found in bilayers composed of typical membrane lipids. This unusually tight packing could be a critical factor in the permeability barrier provided by LPS.

Barium↗

PubMed1999

Ku autoantigen affects the susceptibility to anticancer drugs.

The Ku70/80 autoantigens (Ku) are the DNA-binding components of a DNA-dependent protein kinase (PK) involved in DNA double strand breaks repairing a V(D)J recombination. Because apoptosis is associated with DNA fragmentation and, consequently, creation of double strand breaks, and a variety of DNA-damaging drugs kill tumor cells by apoptosis, we tested the impact of Ku deficiency on the sensitivity of anticancer drugs. Ku-null mutant cell lines Ku70-/- and Ku80-/- were highly sensitive to anticancer drugs, compared with their wild-type cells. Ku-deficient cells were more sensitive to bleomycin-induced DNA fragmentation and exhibited a higher level of c-jun NH2-kinase/stress-activated PK activity than wild-type cells, whereas R7080-6 cells overexpressing both human Ku70 and Ku80 were resistant to bleomycin-induced apoptosis and exhibited a lower level of c-jun NH2-kinase/stress-activated PK activity. The Ku-protein level and Ku DNA binding activity were decreased after treatment with bleomycin, adriamycin, or vincristine, and the decreases were blocked by the treatment of z-DEVD-fmk, a specific inhibitor of caspase-3, suggesting that loss of Ku DNA binding is, in part, due to a caspase-mediated decrease in Ku protein levels. By contrast, HSF1 DNA-binding activity was increased by the treatment of these anticancer drugs and, subsequently, mitochondrial heat shock protein HSP75 was specifically induced. Our data suggest that Ku can affect the susceptibility to anticancer drug-induced apoptosis.

Animals↗

PubMed1999

Direct agonists for serotonin receptors enhance locomotor function in rats that received neural transplants after neonatal spinal transection.

We analyzed whether acute treatment with serotonergic agonists would improve motor function in rats with transected spinal cords (spinal rats) and in rats that received transplants of fetal spinal cord into the transection site (transplant rats). Neonates received midthoracic spinal transections within 48 hr of birth; transplant rats received fetal (embryonic day 14) spinal cord grafts at the time of transection. At 3 weeks, rats began 1-2 months of training in treadmill locomotion. Rats in the transplant group developed better weight-supported stepping than spinal rats. Systemic administration of two directly acting agonists for serotonergic 5-HT(2) receptor subtypes, quipazine and (+/-)-1-[2, 5]-dimethoxy-4-iodophenyl-2-aminopropane), further increased weight-supported stepping in transplant rats. The improvement was dose-dependent and greatest in rats with poor to moderate baseline weight support. In contrast, indirectly acting serotonergic agonists, which block reuptake of 5-HT (sertraline) or release 5-HT and block its reuptake (D-fenfluramine), failed to enhance motor function. Neither direct nor indirect agonists significantly improved locomotion in spinal rats as a group, despite equivalent upregulation of 5-HT(2) receptors in the lumbar ventral horn of lesioned rats with and without transplants. The distribution of immunoreactive serotonergic fibers within and caudal to the transplant did not appear to correspond to restoration of motor function. Our results confirm our previous demonstration that transplants improve motor performance in spinal rats. Additional stimulation with agonists at subtypes of 5-HT receptors produces a beneficial interaction with transplants that further improves motor competence.

Amphetamines↗

PubMed1999

Transplants of fibroblasts genetically modified to express BDNF promote regeneration of adult rat rubrospinal axons and recovery of forelimb function.

Adult mammalian CNS neurons do not normally regenerate their severed axons. This failure has been attributed to scar tissue and inhibitory molecules at the injury site that block the regenerating axons, a lack of trophic support for the axotomized neurons, and intrinsic neuronal changes that follow axotomy, including cell atrophy and death. We studied whether transplants of fibroblasts genetically engineered to produce brain-derived neurotrophic factor (BDNF) would promote rubrospinal tract (RST) regeneration in adult rats. Primary fibroblasts were modified by retroviral-mediated transfer of a DNA construct encoding the human BDNF gene, an internal ribosomal entry site, and a fusion gene of lacZ and neomycin resistance genes. The modified fibroblasts produce biologically active BDNF in vitro. These cells were grafted into a partial cervical hemisection cavity that completely interrupted one RST. One and two months after lesion and transplantation, RST regeneration was demonstrated with retrograde and anterograde tracing techniques. Retrograde tracing with fluorogold showed that approximately 7% of RST neurons regenerated axons at least three to four segments caudal to the transplants. Anterograde tracing with biotinylated dextran amine revealed that the RST axons regenerated through and around the transplants, grew for long distances within white matter caudal to the transplant, and terminated in spinal cord gray matter regions that are the normal targets of RST axons. Transplants of unmodified primary fibroblasts or Gelfoam alone did not elicit regeneration. Behavioral tests demonstrated that recipients of BDNF-producing fibroblasts showed significant recovery of forelimb usage, which was abolished by a second lesion that transected the regenerated axons.

Animals↗

PubMed1999

Neuregulin stimulates myogenic differentiation in an autocrine manner.

During myogenesis, mononucleated myoblasts form multinucleated myotubes by membrane fusion. Efficiency of this intercellular process can be maximized by a simultaneous progress, with a time window, of other neighboring myoblasts in the differentiation program. This phenomenon has been described as the community effect. It proposes the existence of a molecule that acts as a differentiation-inducing signal to a group of identical cells. Here, we show that neuregulin is a strong candidate for this molecule in myoblast differentiation. The expression of neuregulin increased rapidly but transiently at early stage of differentiation of rat L6 cells. Neuregulin showed a potent differentiation-promoting activity in membrane fusion and expression of myosin heavy chain. The antibodies raised against neuregulin and its cognate receptor ErbB3, which were capable of neutralizing the signal pathway, inhibited myotube formation and expression of myosin heavy chain in both L6 cells and primary rat myoblasts. The progress of differentiation was mostly halted after the expression of myogenin and cell cycle arrest. These results suggest that the activation of an autocrine signaling of neuregulin may provide a basic mechanism for the community effect observed in the differentiation of the embryonic muscle cells.

Animals↗

PubMed1999

Modulation of rat atrial G protein-coupled K+ channel function by phospholipids.

1. G protein-gated K+ channels (KACh channels) in the heart and brain are activated by the betagamma subunit of inhibitory G protein. Phosphatidylinositol-4,5-bisphosphate (PIP2) has recently been reported to directly activate KACh channels (GIRK) expressed in oocytes, as well as to support activation by the betagamma subunit in the presence of Na+. We examined the effect of Na+, PIP2 and other phospholipids on the KACh channel to understand better their role in KACh channel activation and modulation. 2. In atrial membrane patches, none of the phospholipids tested including PIP2 caused activation of the KACh channel in either the presence or the absence of 30 mM Na+. PIP2 (3 microM) and other phospholipids (30 microM) blocked acetylcholine-induced activation of the KACh channel. 3. When KACh channels were first activated with GTPgammaS, however, all phospholipids (100 microM) tested augmented the KACh channel activity 1.5- to 2-fold. Phosphatidylinositol-4-phosphate (PIP) and PIP2 were an order of magnitude more potent than other phospholipids. The increase in KACh channel activity was the result of a shift in the gating mode of the channel from a short-lived to a longer-lived open state. Such a modulatory effect was qualitatively similar to that produced by intracellular ATP. Trypsin blocked the ATP effect but not the phospholipid effect on the KACh channel kinetics. 4. The phosphate group linked to the glycerol backbone was important for KACh channel modulation by phospholipids. The higher potency of PIP and PIP2 was due to the presence of inositol phosphates. 5. Intracellular Na+ (30 mM) increased the frequency of KACh channel opening approximately 2-fold if the channels were already active, but did not affect modulation by phospholipids. The effects of Na+ and phospholipids on KACh channel activity were additive. 6. A low concentration of ATP (20 microM), which had no effect on the KACh channel by itself, potentiated the stimulatory action of phospholipids, indicating that ATP and phospholipids interacted to modulate KACh channel function. 7. We conclude that exogenously applied PIP2 and other phospholipids block agonist-mediated KACh channel activation. However, if the KACh channel is already activated with GTPgammaS, phospholipids augment the existing activity by increasing the number of longer-lived channel openings. The evidence for and against the role of PIP and PIP2 in the stimulatory effect of ATP on the KACh channel is presented and discussed.

Acetylcholine↗