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M S Shearman

Publications and source records attributed to M S Shearman.

54 records · Page 3Linked to original sources
PubMed1991

Synaptosomal protein kinase C subspecies: B. Down-regulation promoted by phorbol ester and its effect on evoked norepinephrine release.

The effect of phorbol esters was investigated on the down-regulation of protein kinase C (PKC) and on the release of [3H]norepinephrine (NE) in synaptosomes from the rat cerebrum. Treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) promoted the translocation of PKC activity in a P2 fraction from the cytosol to the membrane fraction and then its down-regulation, in a dose-dependent manner. TPA induced a rapid down-regulation of the type II(beta) and type III(alpha) subspecies, but did not change the activity of the type I(gamma) subspecies in the cytosolic fraction for at least 15 min. The gamma-subspecies was subsequently decreased at a slower rate. In the synaptosomes thus having only the gamma-subspecies, a subsequent dose of TPA could not enhance K(+)-evoked NE release, although, in the original synaptosomes, TPA was able to enhance K(+)-evoked NE release. Pretreatment with TPA did not alter the K(+)-evoked NE release itself. TPA was also found to enhance the K(+)-evoked NE release from synaptosomes prepared from both hippocampus, which express the gamma-subspecies of PKC at a negligible level, and cerebral cortex, which have a significant level of the gamma-subspecies, to the same degree. These results suggest that the gamma-subspecies of PKC does not participate in the TPA-enhanced K(+)-evoked NE release from synaptosomes.

Animals↗
PubMed1990

Protein kinase C activation enhances the delayed rectifier potassium current in guinea-pig heart cells.

The possible involvement of protein kinase C in modulating membrane currents was investigated in isolated guinea-pig ventricular cells. In a Na(+)-and K(+)-free external solution, the delayed rectifier K+ current (IK) was increased by the activator of protein kinase C (PKC), 12-O-tetradecanoylphorbol-13-acetate (TPA). The amplitude of the IK tail elicited by a return from a depolarizing pulse for 3 s at + 50 mV to a holding potential of -30 mV was increased by 32 +/- 4% (mean +/- S.E., (n = 6) after the external application of 1 nM TPA, and by 60 +/- 17% (n = 5) after 10 nM. The increase in IK produced by 1 nM TPA was abolished by the inhibitor of PKC, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 microM). In addition, the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG, 125 microM) also increased IK (58 +/- 9%, n = 3). PKC purified from bovine brain remarkably increased IK (151 +/- 101%, n = 5) in the presence of 1 nM TPA when it was internally applied using the cell dialysis method. The concentration-response curve of IK for the intracellular concentration of Ca2+ was shifted to the left by 1 nM TPA, suggesting a Ca2(+)-dependent action of PKC and/or altered Ca2(+)-sensitivity of IK channels by phosphorylation. On the other hand, 1 nM TPA had no substantial influence on the Ca2+ current (decreased by 7 +/- 4%, n = 5) or the inward-rectifier K+ current (decreased by 5 +/- 5% in outward component, and 3 +/- 8% in inward component, n = 6). Therefore, the action of PKC was to specifically increase IK without affecting the other two currents.

Animals↗
PubMed1989

Selective activation of the gamma-subspecies of protein kinase C from bovine cerebellum by arachidonic acid and its lipoxygenase metabolites.

The gamma-subspecies of protein kinase C (PKC) apparently is expressed only in central nervous tissues, and at a high level in the cerebellum and hippocampus. gamma-PKC from bovine cerebellum, but not the alpha- or beta I/beta II-subspecies, is activated by micromolar concentrations of arachidonic acid (AA), in the absence of both phospholipid and diacylglycerol. A significant component of this activation is also calcium independent. Other unsaturated fatty acids are much less active in this respect. Among the AA metabolites tested, lipoxin A (5(S),6(R),15(S)-11-cis-isomer) was a potent, selective activator of the gamma-subspecies, and also, to a lesser extent, 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid could support activation. These results raise the possibility that AA and some of its lipoxygenase metabolites may function as messenger molecules in neurones to activate the gamma-subspecies of PKC.

Animals↗
PubMed1989

Modulation of ion channel activity: a key function of the protein kinase C enzyme family.

The considerable volume of data now available strongly implicates the modulation of ion channel activity as a key function of the PKC enzyme family. In some systems, such as the PC 12, RINm5F, and NG108-15 clonal cell lines, the action of the enzyme is becoming clear, whereas in others, such as cardiac and smooth muscle cells, contradictory evidence exists, and clarification of the role of PKC in these tissues must await further analysis. Few general statements can be made concerning the regulation of ion conductance through the various classes of channel, or through one particular species of channel that is expressed in and subject to regulation in different cell types. Rather, it seems that many different patterns of modulation can occur in different cells, and so, elucidation of the molecular mechanisms that determine these diverse patterns represents a major challenge. To summarize, we will focus on three aspects of the modulatory action of PKC.

Animals↗
PubMed1988

Guanine nucleotide effects on agonist binding to serotonin 5HT2 receptors in rat frontal cortex.

Specific [3H]ketanserin binding to serotonin 5-HT2 receptors of rat frontal cortex tissue is of high affinity, saturable and unaffected by guanine nucleotides. Antagonists displace [3H]ketanserin from a single recognition site (pseudo-Hill coefficients close to unity), which is also unaffected by guanine nucleotides. Agonist displacement of either [3H]ketanserin or [3H]spiperone from three different membrane preparations showed pseudo-Hill coefficients less than one, and may be described in terms of two agonist binding sites with differing agonist affinities. In the presence of guanine nucleotides, overall agonist affinity was lowered slightly, with little or no change in pseudo-Hill coefficient.

Animals↗
PubMed1988

The heterogeneity and differential expression of protein kinase C in nervous tissues.

Protein kinase C exists as a large family of multiple subspecies with subtle individual characteristics. This heterogeneity comes from different genes as well as from different splicings of a single RNA transcript. The members of this family have closely related structures with a high degree of homology. Biochemical studies have shown that their mode of activation and kinetic and catalytic properties differ slightly from one another. By using a combination of biochemical and immunocytochemical techniques, their differential regional and cellular expression have been shown in the nervous tissues. Each member of this enzyme family may have a specialized function in transducing various physiological and pathological signals into different cell types.

Amino Acid Sequence↗
PubMed1988

Isolation of protein kinase C subspecies from a preparation of human T lymphocytes.

Using a preparation of purified human T lymphocytes, we were able to resolve a partially purified protein kinase C (PKC) enzyme fraction into two distinct subspecies, of approximately equal activity. Biochemical and immunocytochemical analysis revealed that these fractions closely resembled the type II (beta) and type III(alpha) PKC subspecies previously identified and characterised from brain tissue. These results provide valuable information for further studies on the role of individual PKC subspecies in T lymphocyte proliferation.

Calcium Chloride↗
PubMed1988

Activation of a brain-specific protein kinase C subspecies in the presence of phosphatidylethanol.

Protein kinase C (PKC) is normally activated by diacylglycerol in the presence of Ca2+ and phosphatidylserine. At physiological concentrations of Ca2+, however, phosphatidylethanol, a product of the phospholipase D-catalyzed transphosphatidylation reaction between membrane phospholipids and ethanol, can replace phosphatidylserine, and activate PKC. This mode of activation is most effective for the gamma-subspecies, which is expressed only in central nervous tissue. Phosphatidylmethanol is also effective to some extent. Consideration of these results suggests the possibility that ethanol may exert some effect on signal transduction in this tissue via changes in protein phosphorylation.

Animals↗
PubMed1988

Calcium-independent activation of hypothalamic type I protein kinase C by unsaturated fatty acids.

Among multiple subspecies of the protein kinase C (PKC) family, type I PKC from the hypothalamus, having the structure related to the gamma-sequence, responds to low concentrations of arachidonic acid to exhibit marked enzymatic activity. This mode of activation does not require elevated Ca2+ levels, nor does it depend on diacylglycerol and phospholipid. Type I PKC is expressed only in limited regions of central nervous tissues, such as the hypothalamus. This PKC subspecies is not detected in the pituitary gland. The results suggest that the activation of type I PKC may not always be directly associated with inositol phospholipid hydrolysis, and that this subspecies may play a role in the modulation of specialized functions of the hypothalamus.

Animals↗
PubMed1988

Distinct cellular expression of beta I- and beta II-subspecies of protein kinase C in rat cerebellum.

Immunohistochemical and biochemical studies with subspecies-specific antibodies have revealed that beta I- and beta II-subspecies of protein kinase C, which result from alternative splicing of a single RNA transcript, show different regional expression in rat CNS. In the cerebellar cortex, beta I-subspecies is localized mainly in the granular layer, whereas beta II-subspecies is found predominantly in the molecular layer, most apparently in the presynaptic nerve endings that terminate at Purkinje cells. These distribution patterns are in sharp contrast to that of gamma-subspecies, which is most abundant within the Purkinje cells. The different patterns of expression imply that the multiple subspecies of protein kinase C may each have a specific function in modulating the neuronal activity of particular cell types.

Amino Acid Sequence↗
PubMed1987

The common structure and activities of four subspecies of rat brain protein kinase C family.

Elucidation of the complete sequences of four cDNA clones (alpha, beta I, beta II, and gamma) of the rat brain protein kinase C family has revealed their common structure composed of a single polypeptide chain with four constant (C1-C4) and five variable (V1-V5) regions. Although these sequences are highly homologous and closely related to one another V3-, V4-, and V5-regions of gamma-subspecies are slightly bigger than the corresponding regions of the other three subspecies. The first constant region, C1, contains a tandem repeat of cysteine-rich sequence (6, total 12 cysteine residues). The third constant region, C3, has an ATP-binding sequence which is found in many protein kinases. In adult rat whole brain, the relative activities of alpha-, beta I-, beta II-, and gamma-subspecies are roughly 16, 8, 55, and 21%, respectively. gamma-Subspecies is expressed after birth apparently only in the central nervous tissue, implying its role in the regulation of specific neuronal functions.

Amino Acid Sequence↗
PubMed1987

Differential expression of multiple protein kinase C subspecies in rat central nervous tissue.

Protein kinase C from a number of areas of rat central nervous tissue was resolved into three distinct fractions upon hydroxyapatite column chromatography. One of the enzyme fractions, designated type II, could be further distinguished into two subspecies with polyclonal antisera, which were raised against synthetic peptides specific for the predicted amino acid sequences of two alternative cDNA clones encoding this enzyme type. Using a combination of these biochemical and immunological techniques, the relative activity of the multiple subspecies of protein kinase C was assessed for each brain area. A distinct regional pattern of expression was found, which per se may be an important factor in determining the response of different neuronal cell types to extracellular stimuli.

Animals↗
PubMed1984

The concentration of the mitochondrial pyruvate carrier in rat liver and heart mitochondria determined with alpha-cyano-beta-(1-phenylindol-3-yl)acrylate.

alpha-Cyano-beta-(1-phenylindol-3-yl)acrylate inhibited pyruvate transport into both liver and heart mitochondria approximately linearly with respect to its concentration until 65% inhibition was achieved. The extent of inhibition was dependent on the mitochondrial protein concentration. By extrapolation of plots of inhibition versus inhibitor concentration to total inhibition, or by mathematical analysis of the plots, the concentration of pyruvate transporter molecules per mg of protein was calculated to be approximately 100 pmol/mg for both heart and liver mitochondria, and the Ki about 7 nM. The data also suggest that pyruvate transport is rate-limiting for pyruvate oxidation by heart mitochondria in State 3, but not by liver mitochondria.

Acrylates↗