scholarly journals Carbachol causes rapid phosphodiesteratic cleavage of phosphatidylinositol 4,5-bisphosphate and accumulation of inositol phosphates in rabbit iris smooth muscle; prazosin inhibits noradrenaline- and ionophore A23187-stimulated accumulation of inositol phosphates

1984 ◽  
Vol 224 (1) ◽  
pp. 291-300 ◽  
Author(s):  
R A Akhtar ◽  
A A Abdel-Latif
Keyword(s):  
Smooth Muscle ◽  
Time Course ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ionophore A23187 ◽  
Release Of Noradrenaline ◽  
Adrenergic Blocker ◽  
Rabbit Iris

Rabbit iris smooth muscle was prelabelled with myo-[3H]inositol for 90 min and the effect of carbachol on the accumulation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) was monitored with anion-exchange chromatography. Carbachol stimulated the accumulation of inositol phosphates and this was blocked by atropine, a muscarinic antagonist, and it was unaffected by 2-deoxyglucose. The data presented demonstrate that, in the iris, carbachol (50 microM) stimulates the rapid breakdown of PtdIns(4,5)P2 into [3H]inositol trisphosphate (InsP3) and diacylglycerol, measured as phosphatidate, and that the accumulation of InsP3 precedes that of [3H]inositol bisphosphate (InsP2) and [3H]inositol phosphate (InsP). This conclusion is based on the following findings. Time course experiments with myo-[3H]inositol revealed that carbachol increased the accumulation of InsP3 by 12% in 15s and by 23% in 30s; in contrast, a significant increase in InsP release was not observed until about 2 min. Time-course experiments with 32P revealed a 10% loss of radioactivity from PtdIns(4,5)P2 and a corresponding 10% increase in phosphatidate labelling by carbachol in 15s; in contrast a significant increase in PtdIns labelling occurred in 5 min. Dose-response studies revealed that 5 microM-carbachol significantly increased (16%) the accumulation of InsP3 whereas a significant increase in accumulation of InsP2 and InsP was observed only at agonist concentrations greater than 10 microM. Studies on the involvement of Ca2+ in the agonist-stimulated breakdown of PtdIns(4,5)P2 in the iris revealed the following. Marked stimulation (58-78%) of inositol phosphates accumulation by carbachol in 10 min was observed in the absence of extracellular Ca2+. Like the stimulatory effect of noradrenaline, the ionophore A23187-stimulated accumulation of InsP3 was inhibited by prazosin, an alpha 1-adrenergic blocker, thus suggesting that the ionophore stimulation of PtdIns(4,5)P2 breakdown we reported previously [Akhtar & Abdel-Latif (1978) J. Pharmacol. Exp. Ther. 204, 655-688; Akhtar & Abdel-Latif (1980) Biochem. J. 192, 783-791] was secondary to the release of noradrenaline by the ionophore. The carbachol-stimulated accumulation of inositol phosphates was inhibited by EGTA (0.25 mM) and this inhibition was reversed by excess Ca2+ (1.5 mM), suggesting that EGTA treatment of the tissue chelates extracellular Ca2+ required for polyphosphoinositide phosphodiesterase activity. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not change the level of InsP3.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals The labelling of polyphosphoinositides with [32P]Pi and the accumulation of inositol phosphates in vasopressin-stimulated hepatocytes

1986 ◽  
Vol 238 (2) ◽  
pp. 491-499 ◽  
Author(s):  
S Palmer ◽  
P T Hawkins ◽  
R H Michell ◽  
C J Kirk
Keyword(s):  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Water Soluble ◽  
Ionophore A23187 ◽  
Inositol Tetrakisphosphate ◽  
Inositol Phosphate Accumulation ◽  
Phosphatidylinositol 4 ◽  
Phosphate Groups

When hepatocytes were incubated with [32P]Pi, the kinetics for the labelling of the monoester phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were similar to each other and slightly slower than that for the labelling of the gamma-phosphate of ATP. Analysis of the water-soluble 3H-labelled materials derived from [3H]inositol-labelled hepatocytes revealed that, in addition to inositol and its mono-, bis- and tris-phosphates (Ins, InsP, InsP2 and InsP3), these cells contained two unidentified radioactive compounds which co-eluted with InsP on anion-exchange chromatography. When [3H]inositol-labelled hepatocytes were stimulated with 0.23 microM-vasopressin in the presence of 10 mM-Li+, there was an accumulation of radioactivity in InsP, InsP2 and InsP3 but not in Ins or the two unidentified compounds. Further analysis of these inositol phosphates by h.p.l.c. revealed that vasopressin also stimulates the accumulation of inositol tetrakisphosphate (InsP4) in these cells. Vasopressin-stimulated InsP and InsP2 accumulations were maximal in the presence of 1-10 mM-Li+ but InsP3 accumulation continued to increase up to 50 mM-Li+. Accumulated inositol phosphates were retained within the cell. Li+ from 1 to 50 mM did not influence the extent of vasopressin-stimulated inositol lipid degradation in hepatocytes. In the absence of Li+, radioactivity in vasopressin-stimulated hepatocytes accumulated almost entirely in free inositol. The vasopressin-stimulated accumulation of inositol phosphates in the presence of 10 mM-Li+ was abolished by a V1-vasopressin antagonist. Inositol phosphate accumulation was not influenced by ionophore A23187, dimethyl sulphoxide or indomethacin.

scholarly journals The quantitative spectrum of inositol phosphate metabolites in avian erythrocytes, analysed by proton n.m.r. and h.p.l.c. with direct isomer detection

1989 ◽  
Vol 264 (2) ◽  
pp. 323-333 ◽  
Author(s):  
T Radenberg ◽  
P Scholz ◽  
G Bergmann ◽  
G W Mayr
Keyword(s):  
Mammalian Cells ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Detection System ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Phosphate Metabolism ◽  
Qualitative And Quantitative ◽  
Inositol Phosphate Metabolism ◽  
Avian Erythrocytes

The spectrum of inositol phosphate isomers present in avian erythrocytes was investigated in qualitative and quantitative terms. Inositol phosphates were isolated in micromolar quantities from turkey blood by anion-exchange chromatography on Q-Sepharose and subjected to proton n.m.r. and h.p.l.c. analysis. We employed a h.p.l.c. technique with a novel, recently described complexometric post-column detection system, called ‘metal-dye detection’ [Mayr (1988) Biochem. J. 254, 585-591], which enabled us to identify non-radioactively labelled inositol phosphate isomers and to determine their masses. The results indicate that avian erythrocytes contain the same inositol phosphate isomers as mammalian cells. Denoted by the ‘lowest-locant rule’ [NC-IUB Recommendations (1988) Biochem. J. 258, 1-2] irrespective of true enantiomerism, these are Ins(1,4)P2, Ins(1,6)P2, Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, Ins(1,3,4,5,6)P5, and InsP6. Furthermore, we identified two inositol trisphosphate isomers hitherto not described for mammalian cells, namely Ins(1,5,6)P3 and Ins(2,4,5)P3. The possible position of these two isomers in inositol phosphate metabolism and implications resulting from absolute abundances of inositol phosphates are discussed.

scholarly journals Guanosine 5′-[γ-thio]triphosphate-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate in HL-60 granulocytes. Evidence that the guanine nucleotide acts by relieving phospholipase C from an inhibitory constraint

1990 ◽  
Vol 271 (3) ◽  
pp. 743-748 ◽  
Author(s):  
M Camps ◽  
C F Hou ◽  
K H Jakobs ◽  
P Gierschik
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Anion Exchange Chromatography ◽  
Major Product ◽  
Exchange Chromatography ◽  
High Protein ◽  
High Protein Concentration ◽  
Inositol Phosphate Accumulation ◽  
Stimulation Of

Myeloid differentiated human leukaemia (HL-60) cells contain a soluble phospholipase C that hydrolysed phosphatidylinositol 4.5-bisphosphate and was markedly stimulated by the metabolically stable GTP analogue guanosine 5′-[gamma-thio]triphosphate (GTP[S]). Half-maximal and maximal (up to 5-fold) stimulation of inositol phosphate formation by GTP[S] occurred at 1.5 microM and 30 microM respectively. Other nucleotides (GTP, GDP, GMP, guanosine 5′-[beta-thio]diphosphate. ATP, adenosine 5′-[gamma-thio]triphosphate, UTP) did not affect phospholipase C activity, GTP[S] stimulation of inositol phosphate accumulation was inhibited by excess GDP, but not by ADP. The effect of GTP[S] on inositol phosphate formation was absolutely dependent on and markedly stimulated by free Ca2+ (median effective concn. approximately 100 nM). Analysis of inositol phosphates by anion-exchange chromatography revealed InsP3 as the major product of GTP[S]-stimulated phospholipase C activity. In the absence of GTP[S], specific phospholipase C activity was markedly decreased when tested at high protein concentrations, whereas GTP[S] stimulation of the enzyme was markedly enhanced under these conditions. As both basal and GTP[S]-stimulated inositol phosphate formation were linear with time whether studied at low or high protein concentration, these results suggest that (a) phospholipase C is under an inhibitory constraint and (b) GTP[S] relieves this inhibition, most likely by activating a soluble GTP-binding protein.

Rapid increase in inositol phosphate levels in norepinephrine-stimulated vascular smooth muscle

1991 ◽  
Vol 261 (1) ◽  
pp. C17-C22 ◽  
Author(s):  
H. Gu ◽  
H. Martin ◽  
R. J. Barsotti ◽  
E. F. LaBelle
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Time Course ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Receptor Activation ◽  
Force Development ◽  
Tissue Extracts ◽  
Treatment Analysis

We examined the correlation between agonist-stimulated increases in inositol phosphates and force development in vascular smooth muscle. Segments of rat tail artery were preincubated with [3H]inositol and treated with norepinephrine (10(-5) M) for 3-10 s. Tissue levels of inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) were measured. IP and IP2 increased significantly after 3 s of norepinephrine treatment. IP3 increased significantly after 5 s of norepinephrine treatment. Analysis of tissue extracts by high-pressure liquid chromatography demonstrated that the only isomer of IP3 present in any tissue extract was the 1,4,5-isomer [Ins(1,4,5)P3]. Contractile response to norepinephrine stimulation showed that the increase in inositol phosphates coincides well with the time course of force development. This is the first report demonstrating such an early increase in Ins(1,4,5)P3 in agonist-stimulated vascular smooth muscle. These results are consistent with the hypothetical role of Ins(1,4,5)P3 as a mediator linking agonist-receptor activation to increased intracellular calcium and force development in norepinephrine-stimulated vascular smooth muscle.

scholarly journals Requirement for calcium ions in acetylcholine-stimulated phosphodiesteratic cleavage of phosphatidyl-myo-inositol 4,5-bisphosphate in rabbit iris smooth muscle

1980 ◽  
Vol 192 (3) ◽  
pp. 783-791 ◽  
Author(s):  
Rashid A. Akhtar ◽  
Ata A. Abdel-Latif
Keyword(s):  
Smooth Muscle ◽  
Specific Activity ◽  
Inositol Phosphates ◽  
Inositol Trisphosphate ◽  
Water Soluble ◽  
Ionophore A23187 ◽  
Experimental Conditions ◽  
Phosphatidylinositol Bisphosphate ◽  
Low Concentrations ◽  
Rabbit Iris

1. The mechanism of acetylcholine-stimulated breakdown of phosphatidyl-myo-inositol 4,5-bisphosphate and its dependence on extracellular Ca2+ was investigated in the rabbit iris smooth muscle. 2. Acetylcholine (50μm) increased the breakdown of phosphatidylinositol bisphosphate in [3H]inositol-labelled muscle by 28% and the labelling of phosphatidylinositol by 24% of that of the control. Under the same experimental conditions there was a 33 and 48% increase in the production of 3H-labelled inositol trisphosphate and inositol monophosphate respectively. Similarly carbamoylcholine and ionophore A23187 increased the production of these water-soluble inositol phosphates. Little change was observed in the 3H radioactivity of inositol bisphosphate. 3. Both inositol trisphosphatase and inositol monophosphatase were demonstrated in subcellular fractions of this tissue and the specific activity of the former was severalfold higher than that of the latter. 4. The acetylcholine-stimulated production of inositol trisphosphate and inositol monophosphate was inhibited by atropine (20μm), but not tubocurarine (100μm); and it was abolished by depletion of extracellular Ca2+ with EGTA, but restored on addition of low concentrations of Ca2+ (20μm). 5. Calcium-antagonistic agents, such as verapamil (20μm), dibenamine (20μm) or La3+ (2mm), also abolished the production of the water-soluble inositol phosphates in response to acetylcholine. 6. Release of inositol trisphosphate from exogenous phosphatidylinositol bisphosphate by iris muscle microsomal fraction (‘microsomes’) was stimulated by 43% in the presence of 50μm-Ca2+. 7. The results indicate that increased Ca2+ influx into the iris smooth muscle by acetylcholine and ionophore A23187 markedly activates phosphatidylinositol bisphosphate phosphodiesterase and subsequently increases the production of inositol trisphosphate and its hydrolytic product inositol monophosphate. The marked increase observed in the production of inositol monophosphate could also result from Ca2+ activation of phosphatidylinositol phosphodiesterase. However, there was no concomitant decrease in the 3H radioactivity of this phospholipid.

scholarly journals Protein kinase C isoenzymes in airway smooth muscle

1997 ◽  
Vol 324 (1) ◽  
pp. 167-175 ◽  
Author(s):  
Benjamin L. J. WEBB ◽  
Mark A. LINDSAY ◽  
Peter J. BARNES ◽  
Mark A. GIEMBYCZ
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Oligonucleotide Primer ◽  
Mrna Levels ◽  
Kinase C ◽  
Gf 109203X ◽  
Bona Fide

The protein kinase C (PKC) isoenzymes expressed by bovine tracheal smooth muscle (BTSM) were identified at the protein and mRNA levels. Western immunoblot analyses reliably identified PKCα, PKCβI and PKCβII. In some experiments immunoreactive bands corresponding to PKCδ, PKCϵ and PKCθ were also labelled, whereas the γ, η and ζ isoforms of PKC were never detected. Reverse transcriptase PCR of RNA extracted from BTSM using oligonucleotide primer pairs designed to recognize unique sequences in the PKC genes for which protein was absent or not reproducibly identified by immunoblotting, amplified cDNA fragments that corresponded to the predicted sizes of PKCδ, PKCϵ and PKCζ, which was confirmed by Southern blotting. Anion-exchange chromatography of the soluble fraction of BTSM following homogenization in Ca2+-free buffer resolved two major peaks of activity. Using ϵ-peptide as the substrate, the first peak of activity was dependent upon Ca2+ and 4β-PDBu (PDBu = phorbol 12,13-dibutyrate), and represented a mixture of PKCs α, βI and βII. In contrast, the second peak of activity, which eluted at much higher ionic strength, also appeared to comprise a combination of conventional PKCs that were arbitrarily denoted PKCα′, PKCβI′ and PKCβII′. However, these novel enzymes were cofactor-independent and did not bind [3H]PDBu, but were equally sensitive to the PKC inhibitor GF 109203X compared with bona fide conventional PKCs, and migrated on SDS/polyacrylamide gels as 81 kDa polypeptides. Taken together, these data suggest that PKCs α′, βI′ and βII′ represent modified, but not proteolysed, forms of their respective native enzymes that retain antibody immunoreactivity and sensitivity to PKC inhibitors, but have lost their sensitivity to Ca2+ and PDBu when ϵ-peptide is used as the substrate.

Fatty acid inhibition of angiotensin II-stimulated inositol phosphates in smooth muscle cells

1993 ◽  
Vol 264 (2) ◽  
pp. H595-H603
Author(s):  
M. E. Ullian
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Smooth Muscle Cells ◽  
Receptor Binding ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Muscle Cells ◽  
Ang Ii ◽  
Guanine Nucleotide Binding Protein ◽  
Cellular Incorporation

Inositol phosphate (InsP) responses to angiotensin II (ANG II) stimulation were measured in cultured rat vascular smooth muscle cells (VSMC) incubated with and without fatty acids (FA). VSMC were washed after 24 h of FA incubation to achieve cellular incorporation of FA yet eliminate ambient FA. Incubation with eicosapentaenoic acid (EPA)-supplemented medium resulted in concentration-dependent incorporation of EPA and depletion of arachidonic acid in VSMC membranes. Incubation with EPA, but not other FA, resulted in inhibition of ANG II-stimulated InsP formation (29% inhibition with 100 microM EPA). In contrast, InsP formation in response to guanine nucleotide-binding protein stimulation was not affected by EPA. ANG II receptor binding to membranes prepared from EPA-loaded VSMC was 18% lower than binding in membranes from sham-loaded cells. In other studies, VSMC were exposed acutely to FA to avoid cellular incorporation. Exposure to all FA resulted in concentration-dependent reductions in ANG II binding and ANG II-stimulated InsP formation; binding affinity was reduced without changes in receptor density. We conclude that ANG II-stimulated InsP formation is modestly and selectively inhibited by EPA incorporation and more profoundly inhibited by acute exposure to many FA via interference with ANG II receptor binding.

Stored calcium modulates inositol phosphate synthesis in cultured smooth muscle cells

1992 ◽  
Vol 263 (2) ◽  
pp. C535-C539 ◽  
Author(s):  
D. M. Berman ◽  
W. F. Goldman
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Smooth Muscle Cells ◽  
Inverse Relationship ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Muscle Cells ◽  
Inhibitory Effect ◽  
A7r5 Cells ◽  
Cultured Smooth Muscle Cells

Cytosolic Ca2+ concentrations ([Ca2+]cyt) and [3H]inositol phosphates ([3H]InsP) were correlated while varying the Ca2+ content of the sarcoplasmic reticulum (SR) in cultured A7r5 cells at rest and during activation with [Arg8]-vasopressin (AVP). Thapsigargin (TG) raised and superfusion with 0 Ca2+ lowered [Ca2+]cyt, but both treatments decreased SR Ca2+ and AVP-evoked Ca2+ transients. Neither TG nor 0 Ca2+ affected basal [3H]InsP, but both treatments increased AVP-evoked synthesis of [3H]InsP. Exposure for several minutes to 40 mM K+ solution, BAY K 8644, or low-Na+ solution all elevated [Ca2+]cyt and, thereby, increased SR Ca2+, as manifested by augmented AVP-evoked Ca2+ transients. In all three cases, AVP-evoked, but not basal, [3H]InsP were reduced. The inhibitory effect of 40 mM K+ on AVP-evoked [3H]InsP synthesis was blocked when SR Ca2+ uptake was prevented by TG. Brief (30-s) exposures to 40 mM K+, which elevated [Ca2+]cyt but not SR Ca2+ loading, did not modify AVP-evoked [3H]InsP synthesis or Ca2+ transients. These results demonstrate an inverse relationship between SR Ca2+ content and evoked [3H]-InsP synthesis. Moreover, they suggest that SR Ca2+ may serve as a signal that modulates sarcolemmal [3H]InsP formation.

Rapid stimulation of Ins (1,4,5)P3 production in rat aorta by NE: correlation with contractile state

1993 ◽  
Vol 264 (1) ◽  
pp. H126-H132
Author(s):  
V. Pijuan ◽  
I. Sukholutskaya ◽  
W. G. Kerrick ◽  
M. Lam ◽  
C. van Breemen ◽  
...  
Keyword(s):  
Time Course ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Rat Aorta ◽  
Release Mechanism ◽  
Maximal Increase ◽  
Contractile State ◽  
Rapid Stimulation ◽  
Relationship Of ◽  
Stimulation Of

Rapid stimulation of Ins(1,4,5)P3 production in rat aorta by NE: correlation with contractile state. Am. J. Physiol. 264 (Heart Circ. Physiol. 33): H126-H132, 1993.--The isomeric composition of inositol phosphates generated in response to norepinephrine (NE) stimulation and the relationship of inositol phosphate production to release of intracellular Ca2+ as measured by contraction were characterized in rat aorta prelabeled with [3H]inositol. NE stimulated a rapid and transient increase in labeled D-myo-inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3] levels. A maximal increase in labeled Ins(1,4,5)P3 occurred within 15 s of stimulation followed by a decline to control levels at 5 min. D-Myo-inositol 1,3,4-trisphosphate [Ins-(1,3,4)P3] and D-myo-inositol 1-monophosphate [Ins(1)P] levels also increased rapidly in response to NE. In contrast to the transient production of Ins(1,4,5)P3, Ins(1,3,4)P3 and Ins(1)P production was maintained in the presence of NE. Half-maximal stimulation of Ins(1,4,5)P3 production and Ca2+ release occurred at 0.3 microM NE, and maximal effects were obtained with 10 microM NE. The concentration-response curve and time course for production of Ins(1,4,5)P3 correlated with the neurotransmitter-induced Ca2+ release from intracellular stores, indicating that the level of Ins(1,4,5)P3 regulated the Ca(2+)-release mechanism. In the continued presence of NE, the intracellular pools did not completely refill with Ca2+ despite the return of Ins-(1,4,5)P3 levels to basal at 5 min. These results demonstrate that NE stimulates a rapid increase in Ins(1,4,5)P3 that correlates with contraction in Ca(2+)-free buffer. The reuptake of Ca2+ into intracellular stores is regulated by a mechanism that may not involve Ins(1,4,5)P3.

Molecular cloning of chicken calcyclin (S100A6) and identification of putative isoforms

1997 ◽  
Vol 75 (6) ◽  
pp. 733-738 ◽  
Author(s):  
Bruce G Allen ◽  
Jacquelyn E Andrea ◽  
Cindy Sutherland ◽  
Brett O Schönekess ◽  
Michael P Walsh
Keyword(s):  
Smooth Muscle ◽  
Amino Acid ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Chain Reaction ◽  
Proteolytic Fragment ◽  
Chicken Gizzard ◽  
Muscle Tissues ◽  
Bona Fide ◽  
Polymerase Chain

A full-length cDNA encoding smooth muscle calcyclin (S100A6) was cloned from chicken gizzard, using reverse transcription - polymerase chain reaction techniques. The deduced amino acid sequence contains 92 residues with 12 substitutions and a 2 amino acid C-terminal extension when compared with human calcyclin. Calcyclin was purified from chicken gizzard by Ca2+-dependent hydrophobic chromatography, heat treatment, and anion-exchange chromatography. N-terminal sequencing of two CNBr peptides confirmed its identity as calcyclin. Two isoforms of calcyclin (A and B), which differ with respect to the presence or absence of a C-terminal lysine, were identified and the native protein was shown to exist as noncovalently associated homodimers (AA and BB) and heterodimers (AB). Incubation of purified calcyclin AA with an extract of chicken gizzard did not result in degradation of calcyclin A or appearance of calcyclin B, suggesting that calcyclin B is a bona fide isoform rather than a proteolytic fragment generated during purification. Western blotting of chicken tissues with anti-(gizzard calcyclin) indicated abundant expression of calcyclin in smooth muscle tissues, including esophagus, large intestine, and trachea, with lower levels in lung, heart, kidney, and brain, and none detectable in liver or skeletal muscle.Key words: Ca2+-binding proteins, calcyclin, smooth muscle, cDNA cloning, isoforms.

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