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31.
Summary Gap junctional coupling was studied in pairs of murine pancreatic acinar cells using the double whole-cell patch-clamp technique. During stable electrical coupling, addition of OAG (1-oleoyl-2-acetyl-sn-glycerol) induced a progressive reduction of the junctional conductance to the detectable limit (3 pS). Prior to complete electrical uncoupling, varius discrete single channel conductances between 20 and 100 pS could be observed. Polymyxin B, a potent inhibitor of the protein kinase C (PKC) system, completely suppressed OAG-stimulated electrical uncoupling. Dialysis of cell pairs with solutions containing PKC. isolated from rat brain, also caused electrical uncoupling. The presence of 0.1mm dibutyryl cyclic AMP and 5mm ATP in the pipette solution, which serves to stabilize the junctional conductance, did not suppress the effects of OAG or isolated PKC. We conclude that an increase of protein kinase C activity leads to the closure of gap junction channels, presumably via a PKC-dependent phosphorylation of the junctional peptide, and that this mechanism is dominant over cAMP-dependent upregulatory effects in the experimental time range (1 hr). A correlation of the observed single channel conductances with the appearance of channel subconductance states or various channel populations is discussed. 相似文献
32.
Summary We have isolated a new small heat shock gene, HSP12, from Saccharomyces cerevisiae. It encodes a polypeptide of predicted Mr 12 kDa, with structural similarity to other small heat shock proteins. HSP12 gene expression is induced several hundred-fold by heat shock and on entry into stationary phase. HSP12 mRNA is undetectable during exponential growth in rich medium, but low levels are present when cells are grown in minimal medium. Analysis of HSP12 expression in mutants affected in cAMP-dependent protein phosphorylation suggests that the gene is regulated by cAMP as well as heat shock. A disruption of the HSP12 coding region results in the loss of an abundant 14.4 kDa protein present in heat shocked and stationary phase cells. It also leads to the induction of the heat shock response under conditions normally associated with low-level HSP12 expression. The HSP12 disruption has no observable effect on growth at various temperatures, nor on the ability to acquire thermotolerance. 相似文献
33.
Techel D. Gebauer G. Kohler W. Braumann T. Jastorff B. Rensing L. 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1990,159(6):695-706
Summary Pulses of some Ca2+ channel blockers (dantrolene, Co2+, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5–9 h) phase shifts of the circadian conidiation rhythm ofNeurospora crassa. Pulses of high Ca2+, or of low Ca2+, a Ca2+ ionophore (A23187) together with Ca2+, and other Ca2+ channel blockers (La3+, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca2+ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca2+ (Cornelius et al., 1989).Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6–9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180° out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0–12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0.Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with35S-thio -ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca2+ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42° C) temperatures.Altogether, the results indicate that Ca2+-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism ofNeurospora. Ca2+ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism. 相似文献
34.
A rat complementary DNA (cDNA) for the RI beta isoform of type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase regulatory subunit was cloned and sequenced and was found to contain the entire protein coding and 3'-untranslated regions, with a single (ATTAAA) poly-adenylation site. The largest open reading frame was preceded by a short out-of-phase open reading frame, which is not seen in the corresponding mouse RI beta cDNA due to a single base substitution. The rat RI beta cDNA clone was 2,374 bases long and detected a rat mRNA of approximately 2.8 kilobases. Rat RI beta mRNA was abundant in adult rat brain and testis but was undetectable in other rat tissues. The rat RI beta cDNA also detected RI beta mRNA in mouse brain, but not mouse testis, from 10-week-old BALB/c or 10- and 6-week-old Swiss Webster mice. Thus, despite a 96% nucleotide identity in the coding region of RI beta in rat vs. mouse, there are at least two differences in these closely related species. First, there is a short open reading frame, which precedes the coding region in the rat but not the mouse. Second, unlike the mouse testis, the rat testis contains abundant levels of RI beta mRNA. 相似文献
35.
Characterization of differentiated syrian golden hamster pancreatic duct cells maintained in extended monolayer culture 总被引:4,自引:0,他引:4
S. Hubchak M. M. Mangino M. K. Reddy D. G. Scarpelli 《In vitro cellular & developmental biology. Plant》1990,26(9):889-897
Summary Epithelial cells isolated from fragments of hamster pancreas interlobular ducts were freed of fibroblast contamination by
plating them on air-dried collagen, maintaining them in serum-free Dulbecco's modified Eagle's (DME):F12 medium suppleneted
with growth factors, and selecting fibroblast-free aggregates of duct cells with cloning cylinders. Duct epithelial cells
plated on rat type I collagen gel and maintained in DME:F12 supplemented with Nu Serum IV, bovine pituitary extract, epidermal
growth factor, 3,3′, 5-triodothyronine, dexamethasone, and insulin, transferrin, selenium, and linoleic acid conjugated to
bovine serum albumin (ITS+), showed optimal growth as monolayers with a doubling time of about 20 h and were propagated for as long as 26 wk. Early
passage cells consisted of cuboidal cells with microvilli on their apical surface, complex basolateral membranes, numerous
elongated mitochondria, and both free and membrane-bound ribosomes. Cell grown as monolayers for 3 mo. were more flattened
and contained fewer apical microvilli, mitochondria, and profiles of rough surfaced endoplasmic reticulum; in addition, there
were numerous autophagic vacuoles. Functional characteristics of differentiated pancreatic duct cells which were maintained
during extended monolayer culture included intracellular levels of carbonic anhydrase and their capacity to generate cyclic
AMP (cAMP) after stimulation by 1×10−6
M secretin. From 5 to 7 wk in culture, levels of carbonic anhydrase remained stable but after 25 to 26 wk decreased by 1.9-fold.
At 5 to 7 wk of culture, cyclic AMP increased 8.7-fold over basal levels after secretin stimulation. Although pancreatic duct
cells cultured for 25 to 26 wk showed lower basal levels of cAMP, they were still capable of generating significant levels
of cAMP after exposure to serretin with a 7.0-fold increase, indicating that secretin receptors and the adenyl cyclase system
were both present and functional. These experiments document that pancreatic duct monolayer cultures can be maintained in
a differentiated state for up to 6 mo. and suggest that this culture system may be useful for in vitro physiologic and pathologic
studies.
This research was supported by grant CA34051 from the National Cancer Institute, Bethesda, MD. 相似文献
36.
Mitsuru Iwata Shoko Iwata Mark A. Everett Bryan B. Fuller 《In vitro cellular & developmental biology. Plant》1990,26(6):554-560
Summary A human foreskin organ culture system has been developed to study the response of human skin to hormonal stimulation. Foreskins
are maintained in culture on floating plastic supports which allows the epidermal surface to be exposed to air while the dermis
is bathed in nutrient medium. Both black and white human foreskins can be maintained in organ culture for at least 1 wk with
no change in the tissue structure or cell viability as determined by histochemical staining and by dopa reaction staining.
Tyrosinase activity in both black and white human foreskin cultures decays markedly during the first 2 d of culture to a new
steady state level which remains stable throughout the culture period. Both black and white foreskin cultures consistently
demonstrate 2- to 10-fold increases in tyrosinase activity when treated with theophylline (1 mM). Approximately 90% of all skin cultures examined showed an increase in enzyme activity when treated with this phosphodiesterase
inhibitor. Dibutyryl cAMP (0.1 mM) and [Nle4, D-phe7]-alpha MSH (10−8
M), were also found to markedly stimulate tyrosinase activity in some skin cultures, whereas alpha-MSH and prostaglandin E1 produced only an inconsistent and small increase in the activity of the enzyme. Histamine (1 μM), vitamin D3 (1 μM), and retinoic acid (1μM) failed to stimulate tyrosinase activity in either white or black foreskin cultures. This hormone-responsive organ culture
system can be utilized to characterize the molecular processes responsible for the regulation of tyrosinase and pigmentation
in human skin.
This work was supported by a research contract from the Oklahoma Center for the Advancement of Science and Technology (OCAST)
and by a research grant from the Presbyterian Health Foundation. 相似文献
37.
Mark Phillippe Trevania Saunders Shrikar Bangalore 《In vitro cellular & developmental biology. Plant》1990,26(4):369-378
Summary The following studies were undertaken to develop a cultured uterine myocyte model which would allow further clarification
of the adrenergic signal transduction mechanisms utilized by these myocytes. After mechanical removal of the endometrium,
rabbit uterine myoctes were isolated by an overnight enzymatic disaggregation using collagenase and DNase I. The isolated
myocytes were maintained in culture in 75-cm2 flasks containing Waymouth's MB 751/1 medium-10% fetal bovine serum along with 10−8
M estradiol, penicillin, streptomycin, and Fungizone. The phase contrast and electron micrographic appearance of these cells
was consistent with that previously reported for smooth muscle myocytes in culture. Immunocytochemical studies utilizing monoclonal
anti-alpha-smooth muscle actin antibodies confirmed the presence of smooth muscle actin in these cultured myocytes. Western
blot studies similarly confirmed the presence of alpha-smooth muscle actin in rabbit myometrial tissue and the cultured myocytes,
both the primary and F1 generation. After prelabeling the myocytes with [3H]inositol, adrenergic stimulation experiments demonstrated alpha-1 receptor mediated stimulation of inositol phosphates.
Beta receptor stimulation experiments confirmed cAMP production in these cultured myocytes, and the ability of clonidine,
an alpha-2 agonist, to inhibit forskolin stimulated cAMP production confirmed the presence of functional alpha-2 adrenergic
receptors in these myocytes. In conclusion, these cultured rabbit uterine myocytes have provided an in vitro model which can
be utilized to further clarify the adrenergic receptor signal transduction mechanisms in genital tract smooth muscle.
This research was supported by grant HD-22063 from the National Institutes of Health, Bethesda, MD. 相似文献
38.
T.-H. Hwang D.-J. Suh H.-R. Bae S.-H. Lee J.-S. Jung 《The Journal of membrane biology》1996,154(3):251-257
To study K+ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on
permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 μg/ml)
in the symmetrical high K+ (145 mm) Ringer solution. During measurement of the macroscopic K+ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types
of K+ currents: one is an inwardly rectifying K+ current and the other is a slowly activating outwardly rectifying K+ current. The inwardly rectifying K+ current is inhibited by Ba2+. The slowly activating K+ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristae 13-acetate (PMA) and lowering temperature.
This is consistent with the biophysical characteristics of I
SK channel. RT-PCR analysis revealed the presence of I
SK cDNA in the rat trachea epithelia. Although 0.1 mm Ba2+ only had minimal affect on short-circuit current (I
sc) induced by cAMP in intact epithelia, 0.1 mm clofilium strongly inhibited it. These results indicate that I
SK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia.
Received: 1 March 1996/Revised: 5 August 1996 相似文献
39.
The Ca2+-activated maxi K+ channel is predominant in the basolateral membrane of the surface cells in the distal colon. It may play a role in the regulation
of the aldosterone-stimulated Na+ reabsorption from the intestinal lumen. Previous measurements of these basolateral K+ channels in planar lipid bilayers and in plasma membrane vesicles have shown a very high sensitivity to Ca2+ with a K
0.5 ranging from 20 nm to 300 nm, whereas other studies have a much lower sensitivity to Ca2+. To investigate whether this difference could be due to modulation by second messenger systems, the effect of phosphorylation
and dephosphorylation was examined. After addition of phosphatase, the K+ channels lost their high sensitivity to Ca2+, yet they could still be activated by high concentrations of Ca2+ (10 μm). Furthermore, the high sensitivity to Ca2+ could be restored after phosphorylation catalyzed by a cAMP dependent protein kinase. There was no effect of addition of
protein kinase C. In agreement with the involvement of enzymatic processes, lag periods of 30–120 sec for dephosphorylation
and of 10–280 sec for phosphorylation were observed. The phosphorylation state of the channel did not influence the single
channel conductance. The results demonstrate that the high sensitivity to Ca2+ of the maxi K+ channel from rabbit distal colon is a property of the phosphorylated form of the channel protein, and that the difference
in Ca2+ sensitivity between the dephosphorylated and phosphorylated forms of the channel protein is more than one order of magnitude.
The variety in Ca2+ sensitivities for maxi K+ channels from tissue to tissue and from different studies on the same tissue could be due to modification by second messenger
systems.
Received: 28 February 1995/Revised: 22 December 1995 相似文献
40.
A. D. Hartley S. Bogaerts S. Garrett S. Garrett 《Molecular & general genetics : MGG》1996,251(5):556-564
Biochemical and physiological studies have implicated cAMP and cAMP-dependent protein kinase (PKA) in a plethora of essential cellular processes. Here we show that yeast cells partially depleted of PKA activity (due to atpk
w
mutation) and bearing a lesion in a Golgi-localized Ca2+ pump (Pmr1), arrest division with a small bud. The bud morphology of the arrestedtpk1
w
pmr1 mutant cells is characteristic of cells in S phase; however, the terminal phenotype of processes such as DNA replication and nuclear division suggests arrest at the G2/M boundary. This small bud, G2-arrest phenotype is similar to that of strains with a defect in cell wall biosynthesis (pkc1) or membrane biogenesis (och1); however, the biochemical defect may be different since thetpk1
w
pmr1 double mutants retain viability. The growth defect of thetpk1
w
pmr1 mutant can be alleviated by preventing the increase in cellular cAMP levels that is known to be associated with a decrease in PKA activity, or by supplementing the medium with millimolar amounts of Ca2+. Although the biochemical consequences of this increase in cAMP concentration are not known, the small-bud phenotype of the double mutant and the known protein processing defect of thepmr1 lesion suggest that the localization or function of some membrane component might be compromised and susceptible to perturbations in cellular cAMP levels. One candidate for such a protein is the cAMP-binding membrane ectoprotein recently described in yeast. 相似文献