Calcium-dependent protein kinases (CDPKs) belong to a unique family of enzymes containing a single polypeptide chain with a kinase domain at the amino terminus and a putative calcium-binding EF hands structure at the carboxyl terminus. From Arabidopsis thaliana, we have cloned three distinct cDNA sequences encoding CDPKs, which were designated as atcdpk6, atcdpk9 and atcdpk19. The full-length cDNA sequences for atcdpk6, atcdpk9 and atcdpk19 encode proteins with a molecular weight of 59343, 55376 and 59947, respectively. Recombinant atCDPK6 and atCDPK9 proteins were fully active as kinases whose activities were induced by Ca2+. Biochemical studies suggested the presence of an autoinhibitory domain in the junction between the kinase domain and the EF hands structure. Serial deletion of the four EF hands of atCDPK6 demonstrated that the integrity of the four EF hands was crucial to the Ca2+ response. All the three atcdpk genes were ubiquitiously expressed in the plant as demonstrated by RNA gel blot experiments. Comparison of the genomic sequences suggested that the three cdpk genes have evolved differently. Using antibodies against atCDPK6 and atCDPK9 for immunohistochemical experiments, CDPKs were found to be expressed in specific cell types in a temporally and developmentally regulated manner.

Amino Acid Sequence↗

PubMed1996

The Ba2+ block of the ATP-sensitive K+ current of mouse pancreatic beta-cells.

We studied the block of whole-cell ATP-sensitive K+ (KATP) currents in mouse pancreatic beta-cells produced by external Ba2+. Ba2+ produced a time-and voltage-dependent block of KATP currents, both the rate and extent of the block increasing with hyperpolarization. With 5.6 mM [K+]o, the relationship between the steady-state KATP current and [Ba2+]o was fit by the Hill equation with a Kd of 12.5 +/- 2.8 microM at -123 mV and of 0.18 +/- 0.02 mM at -62 mV. The Hill coefficient (n) was close to 1 at all potentials indicating that binding of a single Ba2+ ion is sufficient to block the channel. When [K+]o was raised to 28 mM the Kd was little changed (12.4 +/- 4.1 microM at -123 mV, 0.27 +/- 0.05 mM at -62 mV) and n was unaffected, suggesting that K+ does not interact with the Ba2+ binding site. The kinetics of Ba2+ block were slow, 10 microM Ba2+ blocking the KATP current with a time constant of 20 ms at -123 mV in 28 mM [K+]o. The blocking rate constant was calculated as 1.7 mM-1ms-1 and the unblocking rate as 0.02 ms-1, at -123 mV. The data are discussed in terms of a model in which Ba2+ binds to a site at the external mouth of the channel to inhibit the KATP channel.

Adenosine Triphosphate↗

PubMed1996

Induction of hydrotropism in clinorotated seedling roots of Alaska pea, Pisum sativum L.

Roots of the agravitropic pea (Pisum sativum L.) mutant ageotropum show positive hydrotopism, whereas roots of Alaska peas are hydrotropically almost non-responsive. When the gravitropic response was nullified by rotation on clinostats, however, roots of Alaska peas showed unequivocal positive hydrotropism in response to a water potential gradient. These results suggest that roots of Alaska peas possess normal ability to respond hydrotropicallly and their weak hydrotropic response results from a counteracting effect of gravitropism.

Gravitation↗

PubMed1996

Clinical application of three-dimensional myocardial imaging: evaluation of efficacy of medical treatment on myocardial perfusion.

To investigate the clinical applicability of the three-dimensional (3D) myocardial imaging using a newly developed system (the Application Visualization System-Medical Viewer), thallium-201 myocardial single photon emission computed tomography was performed in 19 patients with previous myocardial infarction before and after treatment with nisoldipine. We have developed a new method for automatically reconstructing 3D imaging for the stereoscopic evaluation of myocardial perfusion. The left ventricular myocardial volume with a radioisotope count > or = 50% of maximum was calculated by using the conventional surface rendering method. With these images, the effect of nisoldipine on myocardial perfusion was assessed and the myocardial volume with a radioisotope count > or = 50% of maximum was compared. In fifteen (88%) of 19 patients, myocardial perfusion increased in the infarct areas after nisoldipine treatment. Nisoldipine significantly increased the myocardial volume with a radioisotope count > or = 50% of maximum from 141 +/- 17 to 153 +/- 18 ml on the stress 3D imagings. These findings indicate that nisoldipine improved myocardial perfusion during exercise. 3D imaging provided stereoscopic assessment of the changes in myocardial perfusion following treatment with nisoldipine and also detected transient enlargement of the left ventricular lumen induced by exercise.

Aged↗

PubMed1996

Fibroblasts synthesize kininogen in response to inflammatory mediators.

Mouse fibroblasts in vitro secret kininogen (KGN). Rat fibroblasts also synthesized and secreted T-KGN in vitro. KGN production by these fibroblasts is greatly stimulated by dibutyryl cAMP, prostaglandin E2 and tumor necrosis factor. Human fibroblast WI-38 cells also express the L-KGN gene, which was stimulated by dibutyryl cAMP and prostaglandin E2. These results demonstrate that fibroblasts express the KGN gene, and suggest that the expression is regulated by inflammatory mediators. RT-PCR, using specific primers for the T-KGN and L-KGN genes, reveals that the rat hind-paw express both T- and L-KGN mRNAs, and the expression of both KGN mRNAs was increased in the hind-paw 24 h after inducing inflammation by injecting Freund's complete adjuvant into the paw. Thus, it is suggested that local connective tissues express the KGN gene, and that the expression is enhanced under pathological conditions, such as inflammation.

Animals↗

PubMed1996

Modulation of organic cation transport and lipid fluidity by benzyl alcohol in rat renal brush-border membranes.

PURPOSE: Organic cations are actively transported in renal brush-border membranes (BBM) by the H+/organic cation antiport system. In the present study, we investigated the relationship between membrane fluidity and organic cation transport in the BBM. METHODS: The effects of benzyl alcohol, a membrane fluidizing agent, on the organic cation tetraethylammonium (TEA) uptake were studied using renal BBM vesicles isolated from rat kidney. BBM fluidity was assessed by fluorescence polarization technique. RESULTS: H+ gradient-dependent uptake of TEA in BBM vesicles was inhibited by benzyl alcohol in a dose-dependent manner, with an apparent half inhibitory concentration of 18mM. The decrease in fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene in BBM, which represents the increase in membrane fluidity, was correlated with the decrease in TEA transport activity. The dissipation rate of H+ gradient, a driving force for organic cation transport in BBM, was increased by benzyl alcohol. In addition, H+ gradient-independent TEA-TEA exchange was also inhibited by benzyl alcohol. These findings indicate that benzyl alcohol inhibits the uptake of TEA by affecting the intrinsic activity of the organic cation transporter and the H+ gradient dissipation rate. CONCLUSIONS: The membrane fluidity should be an important determinant for organic cation transport in renal BBM.

Animals↗

PubMed1996

In-vivo clearance study of vancomycin in rats.

The renal handling of vancomycin in rats and the effects of various drugs (probenecid, cimetidine and quinidine) on urinary excretion of the antibiotic were investigated by in-vivo clearance. The vancomycin-to-inulin excretion ratio (ER) was greater than unity at various infusion rates of vancomycin. Quinidine, co-administered with vancomycin, significantly decreased the total, renal and net secretory clearance of the antibiotic. Cimetidine also decreased, though not significantly, the secretory clearance of vancomycin by about 20%, but probenecid did not. These results suggested that vancomycin is secreted in renal tubules in rats, and that quinidine decreases the total clearance of vancomycin partly by inhibiting its tubular secretion in the kidney.

Analysis of Variance↗

PubMed1996

Regulation of p-aminohippurate transport by protein kinase C in OK kidney epithelial cells.

We studied the effect of phorbol 12-myristate 13-acetate (PMA), a phorbol ester which activates protein kinase C, on p-aminohippurate (PAH) transport in OK cells. PMA (10(-7) M) almost completely inhibited the transcellular transport of PAH across OK cell monolayers from the basal to the apical side, as well as the accumulation of PAH in the cells. The uptake of PAH across the basolateral membrane of OK cells was inhibited by PMA in a time-and dose-dependent fashion. Exposing the cells with other protein kinase C activators such as active phorbol esters and diacylglycerols also resulted in a significant inhibition of basolateral PAH uptake, but the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, had no effect. The inhibition of basolateral PAH uptake by PMA was blocked by staurosporine, an inhibitor of protein kinase C. Cycloheximide, actinomycin D, colchicine, and cytochalasin D did not affect the inhibitory effect of PMA on basolateral PAH uptake. These results suggested that the PAH transport system in OK cells is under the regulatory control of protein kinase C.

Animals↗

PubMed1996

Effect of CYP2C polymorphisms on the pharmacokinetics of phenytoin in Japanese patients with epilepsy.

We examined the effect of CYP2C9/19 polymorphisms on the pharmacokinetics of phenytoin in 17 Japanese patients with epilepsy. The maximal elimination rate (Vmax) of phenytoin was slightly decreased (up to 14%) in patients with CYP2C19 mutations for the defective allele. The Vmax values in patients with a CYP2C9 mutation for the heterozygous Ile/Leu359 allele were 40% lower than those in patients with wild-type CYP2C9 for the homozygous Ile359 allele. These findings suggested that the genetic polymorphism of CYP2C isoenzymes plays an important role in the pharmacokinetic variability of phenytoin, and that the mutation in CYP2C9 proteins is a determinant of impaired metabolism of the drug.

Anticonvulsants↗

PubMed1996

Population pharmacokinetics of phenytoin in Japanese patients with epilepsy: analysis with a dose-dependent clearance model.

The population pharmacokinetic parameters of phenytoin were estimated using routine therapeutic drug monitoring data from 116 epileptic patients. The 531 serum concentration values at steady-state after repetitive oral administration were analyzed using a nonlinear mixed effects model (NONMEM) program designed for estimation of population pharmacokinetic parameters. A one-compartment model with dose-dependent clearance was used for the pharmacokinetic analysis of phenytoin. The volume of distribution (V) was estimated to be 1.231/kg in a typical 42-kg patient, assuming that the bioavailability of orally administered phenytoin is 100%. The maximal elimination rate (V(max)) and the Michaelis-Menten constant (K(m)) were 9.80 mg/d/kg and 9.19 micrograms/ml, respectively. The parameter of power function of weight to adjust V and V(max) was estimated to be 0.463. In addition, K(m) for phenytoin appeared to be 16% increased in patients receiving zonisamide concurrently. The population pharmacokinetic parameters of phenytoin will be useful for designing dosage regimens in epileptic patients.

Adolescent↗

PubMed1996

Expression of low-molecular-weight kininogen mRNA in human fibroblast WI38 cells.

Expression of the low-molecular-weight kininogen (L-kininogen) mRNA in the human fibroblast cell line WI38 was examined by means of the reverse-transcription polymerase chain reaction and Southern blotting using human L-kininogen cDNA as a probe. The results demonstrated that WI38 fibroblasts expressed L-kininogen mRNA and that the expression was stimulated by 1 mM dibutyryl cAMP or 10 microM prostaglandin E2. Dexamethasone (1 microM) inhibited the stimulatory effect of prostaglandin E2. These results suggest that human fibroblasts supply L-kininogen, a protein precursor of the inflammatory mediator kinins, to connective tissues in response to inflammatory stimuli and that glucocorticoids may exert the antiinflammatory effect in part by inhibiting the local production of L-kininogen.

Cells, Cultured↗

PubMed1996

Cloning and functional expression of the rat brain KIR6.2 channel.

We obtained cDNA encoding KIR6.2 channel from the rat brain cDNA library. The reconstituted K+ channel with KIR6.2 and SUR1 genes retained properties similar to those reported in the native ATP-sensitive K+ channel including run-down and recovery of channel activity. When KIR6.2 was co-expressed with CFTR or MDR1, no K+ channel activity was recorded.

Amino Acid Sequence↗