scholarly journals Protein phosphatase composition in the smooth muscle of guinea-pig ileum studied with okadaic acid and inhibitor 2

1989 ◽  
Vol 262 (2) ◽  
pp. 617-623 ◽  
Author(s):  
A Takai ◽  
M Troschka ◽  
G Mieskes ◽  
A V Somlyo
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Okadaic Acid ◽  
Total Activity ◽  
Guinea Pig Ileum ◽  
Control Activity ◽  
Triton X ◽  
Type 1 Phosphatase ◽  
Intracellular Structure

Using okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases, and inhibitor 2, an intrinsic inhibitory factor of type 1 phosphatase, we characterized the phosphorylated myosin light-chain (PMLC) phosphatase activity in the smooth-muscle extracts of guinea-pig ileum. In the intact fibres the control activity was 254 +/- 13 nmol of Pi/min per g wet wt. (n = 15) against 32P-labelled PMLC (4 microM) from chicken gizzard. The following phosphatase fractions were identified: an inhibitor-2-sensitive (type 1) fraction (fractional activity = 35%), a Mg2+-dependent and okadaic acid-insensitive (type 2C) fraction (17%), and two type 2A-like fractions that had different susceptibility to okadaic acid. The type 2A-like fraction with lower affinity to okadaic acid accounted for 30% of the control activity. After the cell membrane was permeabilized by Triton X-100, more than 60% of this fraction remained and accounted for about 90% of the total activity, whereas the other fractions were nearly abolished. The type 2A-like fraction may be bound to some intracellular structure such as contractile proteins.

Polyamines increase Ca2+ sensitivity in permeabilized smooth muscle of guinea pig ileum

1994 ◽  
Vol 266 (6) ◽  
pp. C1754-C1763 ◽  
Author(s):  
K. Sward ◽  
B. O. Nilsson ◽  
P. Hellstrand
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Aminoglycoside Antibiotic ◽  
Free Medium ◽  
Guinea Pig Ileum ◽  
High Concentration ◽  
Increased Sensitivity ◽  
Tissue Contents ◽  
Triton X ◽  
Gamma S

The effects of polyamines were investigated in strips of smooth muscle from guinea pig ileum permeabilized with beta-escin (0.005%). Spermine (1 mM) inhibited transient contractions induced in Ca(2+)-free medium by carbachol (0.1 mM) and GTP gamma S (0.1 mM) but potentiated responses to caffeine (20 mM) and D-myo-inositol 1,4,5-trisphosphate (40 microM). At high ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid concentration (10 mM) and in the presence of A-23187 (10 microM), force at optimal and suboptimal Ca2+ concentrations was increased both by spermine and by carbachol. Spermine did not potentiate contraction in Ca(2+)-free medium or after full thiophosphorylation of the regulatory 20-kDa myosin light chains but slightly potentiated contractions produced by partial thiophosphorylation. Also, spermidine and putrescine, as well as the aminoglycoside antibiotic neomycin, increased sensitivity to Ca2+, with potency correlating with number of positive charges. After permeabilization by a high concentration (0.1%) of beta-escin, the sensitivity to Ca2+ was increased by spermine but not by GTP gamma S. In preparations permeabilized by Triton X-100, spermine slightly increased Ca2+ sensitivity but not maximal force. Tissue contents of putrescine, spermidine, and spermine in intact ileum muscle were 8, 98, and 184 nmol/g, respectively. Permeabilization by 0.005 and 0.1% beta-escin reduced spermine contents by 40 and 53%, respectively. Effects of added polyamines in permeabilized preparations may thus reflect physiological effects of endogenous polyamines modulating contraction in the intact tissue.

The identification of acetyl-l-carnitylcholine in rat brain extracts and the comparison of its cholinomimetic properties with acetylcholine

1970 ◽  
Vol 48 (10) ◽  
pp. 709-722 ◽  
Author(s):  
E. A. Hosein ◽  
A. Kato ◽  
E. Vine ◽  
A. M. Hill
Keyword(s):  
Guinea Pig ◽  
Rat Brain ◽  
Total Activity ◽  
Guinea Pig Ileum ◽  
Molar Basis ◽  
Choline Esters ◽  
Brain Extracts

Acetyl-l-carnitylcholine (l-ACCh) was identified in rat brain extracts on paper chromatograms developed in butanol–water for 138 h. l-ACCh was also identified in brain extracts fractionated on t.l.c. plates and on Sephadex G-10 columns. In every instance l-ACCh was separated from the acetylcholine (ACh) present and the ACh-like activity of l-ACCh was about 20% of the total activity in the extract. Both l-ACCh and ACh were found to be inseparable in a variety of chromatographic systems including electrophoresis. Treatment of these choline esters with cholinesterases showed that while true acetylcholinesterase hydrolyzed both l-ACCh and ACh, pseudocholinesterase destroyed only ACh. On a molar basis, the ACh-like activity of ACCh is one-half that of ACh on both the guinea pig ileum and frog rectus preparations. Like ACh, the ratio of the nicotinic to muscarinic potency of l-ACCh is unity. Mixtures of l-ACCh and ACh show summation of ACh-like activity on both the guinea pig ileum and frog rectus preparations.

Regulation of flagellar dynein by an axonemal type-1 phosphatase in Chlamydomonas

1996 ◽  
Vol 109 (7) ◽  
pp. 1899-1907 ◽  
Author(s):  
G. Habermacher ◽  
W.S. Sale
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Double Mutant ◽  
Sliding Velocity ◽  
Wild Type ◽  
Phosphatase Inhibitors ◽  
Radial Spokes ◽  
Gamma S ◽  
Type 1 Phosphatase

Physiological studies have demonstrated that flagellar radial spokes regulate inner arm dynein activity in Chlamydomonas and that an axonemal cAMP-dependent kinase inhibits dynein activity in radial spoke defective axonemes. These studies also suggested that an axonemal protein phosphatase is required for activation of flagellar dynein. We tested whether inhibitors of protein phosphatases would prevent activation of dynein by the kinase inhibitor PKI in Chlamydomonas axonemes lacking radial spokes. As predicted, preincubation of spoke defective axonemes (pf14 and pf17) with ATP gamma S maintained the slow dynein-driven microtubule sliding characteristic of paralyzed axonemes lacking spokes, and blocked activation of dynein-driven microtubule sliding by subsequent addition of PKI. Preincubation of spoke defective axonemes with the phosphatase inhibitors okadaic acid, microcystin-LR or inhibitor-2 also potently blocked PKI-induced activation of microtubule sliding velocity: the non-inhibitory okadaic acid analog, 1-norokadaone, did not. ATP gamma S or the phosphatase inhibitors blocked activation of dynein in a double mutant lacking the radial spokes and the outer dynein arms (pf14pf28). We concluded that the axoneme contains a type-1 phosphatase required for activation of inner arm dynein. We postulated that the radial spokes regulate dynein through the activity of the type-1 protein phosphatase. To test this, we performed in vitro reconstitution experiments using inner arm dynein from the double mutant pf14pf28 and dynein-depleted axonemes containing wild-type radial spokes (pf28). As described previously, microtubule sliding velocity was increased from approximately 2 microns/second to approximately 7 microns/second when inner arm dynein from pf14pf28 axonemes ws reconstituted with axonemes containing wild-type spokes. In contrast, pretreatment of inner arm dynein from pf14pf28 axonemes with ATP gamma S, or reconstitution in the presence of microcystin-LR, blocked increased velocity following reconstitution, despite the presence of wild-type radial spokes. We conclude that the radial spokes, through the activity of an axonemal type-1 phosphatase, activate inner arm dynein by dephosphorylation of a critical dynein component. Wild-type radial spokes also operate to inhibit the axonemal cAMP-dependent kinase, which would otherwise inhibit axonemal dynein and motility.

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