Studies on the endogenous phospholipids of chick embryo myocardium and their in vitro hydrolysis by endogenous phospholipases during embryogenesis

The phospholipid profiles of the myocardium (from 10- and 18-day old chick embryos and 13-day old chick) and their in vitro response to the endogenous lipolytic enzymes (mainly of the phospholipase group) at pH 7.4 and 38 degrees C for 60 min were analyzed by TLC technology and densitometry. Cardiolipin (CL) was shown to be one of the major phospholipids of the chick embryo myocardium and its concentration increased as the chick embryo advanced in development. Monolysocardiolipin (MLCL) was produced subsequent to in vitro incubation of whole tissue homogenates in all myocardia studied as well as a concurrent reduction in CL. This deacylation of CL increased in magnitude as the chick embryo advanced in development indicating its age relatedness. The level of phosphatidyl ethanolamine (PE) plasmalogen was also high in all myocardia studied. Lyso alkenyl PE (LPE) was produced subsequent to in vitro incubation and its level increased as the chick embryo advanced in development, indicating PLA(2) action on the sn-2 fatty acid of PE. Phosphatidyl choline (PC) plasmalogen was also present in the chick embryo myocardium and its level increased gradually as the chick embryo advanced in development. In contrast, yolk-sac membrane contains very minute amounts of CL and PE. No PC was detected and no LPE was formed following in vitro incubation. The yolk of the unfertilized chicken egg has no CL and has very minute amounts of PE, no PC and no lysophospholipids were detected following in vitro incubation in all samples analyzed.     

On the differential lipolytic capabilities of rat spleen and cardiac muscle. An in vitro incubation in conjunction with chromatographic and densitometric analysis

The phospholipid profiles of newborn, young adult and aged rat heart and spleen and their in vitro response to endogenous phospholipases at pH 7.4 and 38 degrees C for 60 min were analysed by thin layer chromatography (TLC) technology and densitometry measurement. The noticeable high level of cardiolipin (CL) and its preferential deacylation, as detected by the formation of monolysocardiolipin (MLCL) and concurrent reduction of CL level were the most prevalent lipolytic events of rat cardiac muscle (newborn, young adult and aged) but the least prevalent in rat spleen. The level of ethanolamine plasmalogen (PE) was high in both the rat spleen and cardiac muscle (newborn, young adult and aged). Following in vitro incubation, the reduction in the level of PE and the high level of lyso alkenyl PE produced were most conspicuous in rat spleen (newborn, young adult and aged) and noticeably less in rat cardiac muscle. These data clearly illustrate the differential response of the endogenous substrates (phospholipids) to the endogenous phospholipases of these two tissues, and probably are related to their physiological activities in vivo.     

Studies on the endogenous phospholipids of mammalian kidney and their in vitro hydrolysis by endogenous phospholipases: a thin layer chromatographic and densitometric study

The phosphoglycerides profile of six species of mammalian kidney (guinea pig, pig, cat, dog, mouse and rat) and their in vitro response to the endogenous phospholipases were determined by TLC technology in conjunction with densitometric measurements. Changes in their phospholipids profile subsequent to in vitro incubation of whole tissue homogenate of these kidneys for 60 min, at pH 7.4, 38 degrees C, and prior to phospholipids extraction have shown that the deacylation of the endogenous cardiolipin (CL) is the most prevalent lipolytic event of all mammalian kidneys studied. Concurrent with the deacylation of CL, there was also formation of monolysocardiolipin (MLCL) and a reduction in CL level. To a much lesser extent, lyso alkenyl phosphatidyl ethanolamine (LPE) was also produced concomitant with a decrease of the endogenous alkenyl phosphatidyl ethanolamine (PE) level. The deacylation of PE plasmalogen to its lyso form confirms the action of endogenous PLA(2) releasing sn-2 fatty acids.     

Comparative studies of the endogenous phospholipids and their in vitro hydrolysis by endogenous phospholipases of various tissues from 7-day-old chicks: a thin layer chromatographic and densitometric analysis

The phospholipid profiles of heart, kidney, and pectoral muscle of 7-day-old chicks and their in vitro response to the endogenous lipolytic enzymes (mainly in the phospholipase group) at pH 7.4 and 38 degrees C for 60 min were analysed by TLC technology and densitometry. The noticeable preferential deacylation of cardiolipin (CL) as detected by the formation of monolysocardiolipin (MLCL) and concurrent reduction of CL level were the most prevalent lipolytic events of chick cardiac muscle, but the least prevalent in chick pectoral muscle. Deacylation of ethanolamine plasmalogen (PE) as revealed by the formation of the corresponding lyso alkenyl derivative was also prominent in cardiac muscle, but much less so in kidney and none at all was detected in pectoral muscle. The level of sphingomyelin (SM) was much higher in kidney than heart and pectoral muscle. Following in vitro incubation, the reduction in the level of SM and the high level of ceramide (Cer) production were most conspicuous in kidney, less in cardiac muscle and least in pectoral muscle. The hydrolysis of PE and SM confirm the action of endogenous PLA(2) and endogenous sphingomyelinase on PE and SM respectively. These data clearly illustrate the differential response of the endogenous substrates (phospholipids) to the endogenous phospholipases of the tissues studied and are probably related to their physiological activities in vivo.     

Age-related changes of the endogenous cardiolipin and plasmalogens of guinea pig kidney and their in vitro hydrolysis by endogenous phospholipases: a thin layer chromatographic analysis in conjunction with densitometric measurement

The phosphoglycerides profile of guinea pig kidney, fetal, young adult, and aged, and their in vitro response to the endogenous lipolytic enzymes, mainly in the phospholipase group were determined by TLC technology in conjunction with densitometric measurement. Changes in phosphoglycerides profile subsequent to in vitro incubation of these tissues at pH 7.4, and 38 degrees C for 45 min and prior to phospholipid extraction has provided evidence relating to their respective lipolytic enzymes capabilities and age. These changes are mainly related to endogenous cardiolipin (CL), alkenyl phospholipids (phosphatidyl ethanolamine and phosphatidyl choline) and their endogenous deacylation to their respective lyso derivatives monolysocardiolipin (MLCL), lyso alkenyl phosphatidyl ethanolamine (LPE), and lyso alkenyl phosphatidyl choline (LPC) by endogenous phospholipases. The hydrolysis of the plasmalogen confirms the action of endogenous PLA(2) on sn-2 fatty acids of these compounds.     

Taenia taeniaeformis: inhibition of rat testosterone production by excretory-secretory product of the cultured metacestode

In 3- to 5-month-old male Sprague-Dawley rats infected with the hepatic metacestode, Taenia taeniaeformis, the serum testosterone level was significantly lower than in comparable uninfected controls. By transmission electron microscopy, testicular Leydig cells of infected rats had less smooth endoplasmic reticulum than control Leydig cells. Cultured metacestodes isolated from the hepatic cysts secreted or excreted substances into the incubation medium. The effect of the excretory-secretory product on testosterone concentration in the sera and testes of 15-day-old rats was examined. Subcutaneous injection of 50-200 micrograms of excretory-secretory product/0.1 ml saline/rat for 2 days significantly reduced human chorionic gonadotropin-stimulated serum and testicular testosterone concentrations. Furthermore, the effect of the excretory-secretory product on isolated rat Leydig cell testosterone production was examined. Rat Leydig cells produced testosterone in vitro and, in the presence of 50 IU human chorionic gonadotropin/ml incubation medium, they responded with approximately 100% increase in testosterone production. Addition of 2-10 micrograms excretory-secretory product protein/ml of culture medium significantly reduced the testosterone production by rat Leydig cells in vitro. These results indicate that excretory-secretory product of cultured T. taeniaeformis metacestodes has a direct inhibitory effect on Leydig cell testosterone production under stimulation with human chorionic gonadotropin.     

Monolysocardiolipin in cultured fibroblasts is a sensitive and specific marker for Barth Syndrome

Barth Syndrome (BTHS) is an X-linked recessive disorder that results in abnormal metabolism of the mitochondrial phospholipid cardiolipin (CL). CLs are decreased and monolysocardiolipins (MLCLs), intermediates in CL metabolism, are increased in a variety of tissues. Measurement of decreased CL levels in skin fibroblasts has previously been proposed as a diagnostic test for BTHS. We investigated whether elevated MLCL is specific for BTHS and whether the MLCL-to-CL ratio is a more sensitive and specific marker for BTHS. We measured CLs and MLCLs in skin fibroblasts from 5 BTHS patients, 8 controls, and 14 patients with biochemical and clinical findings similar to those in BTHS (group D), using high performance liquid chromatography-mass spectrometry. Our results showed a clear decrease of CL in combination with a marked increase of MLCL in fibroblasts from BTHS patients when compared with controls. MLCL/CL ratios ranged from 0.03-0.12 in control fibroblasts and from 5.41-13.83 in BTHS fibroblasts. In group D, the MLCL/CL ratio range was 0.02-0.06. We therefore conclude that elevations of MLCLs are specific for BTHS and that the MLCL/CL ratio in fibroblasts is a better diagnostic marker than CL alone. We also report the finding of two novel mutations in the TAZ gene that cause BTHS.     

Mitochondrial monolysocardiolipin acyltransferase is elevated in the surviving population of H9c2 cardiac myoblast cells exposed to 2-deoxyglucose-induced apoptosis.

Cardiolipin (CL) is a major mitochondrial membrane phospholipid in the mammalian heart and the remodeling of CL is essential to maintain its unique unsaturated fatty acyl composition. We examined CL de novo biosynthesis and remodeling in the surviving population of H9c2 cardiac myoblast cells exposed to 2-deoxyglucose (2-DG). H9c2 cells were incubated in the absence or presence of 2-DG for 16 h with [1,3-3H]glycerol or [1-14C]linoleic acid (bound to albumin in a 1:1 molar ratio). Dead cells were removed and radioactivity was incorporated into CL. Its pool size, fatty acid composition, and the activities of the CL biosynthesis and remodeling enzymes were determined. The CL pool size, its fatty acid composition, and [1,3-3H]glycerol or [1-14C]linoleic acid incorporated into CL were unaltered in the surviving population of 2-DG-treated cells compared with controls. In addition, the activities of the CL de novo biosynthetic enzymes were unaltered. Cleaved caspase-3 and poly(ADP-ribose) polymerase were slightly elevated in the surviving population of 2-DG-treated cells compared with controls, indicating that apoptosis induction was occurring in these cells. Mitochondrial phospholipase A2 and monolysocardiolipin acyltransferase (MLCL AT) activities increased 33% (p < 0.05) and 63% (p < 0.05), respectively, in 2-deoxyglucose-treated cells compared with controls. In contrast, the activity of ALCAT1, an endoplasmic reticulum MLCL AT, decreased 77% (p < 0.05), but this was not due to a reduction in ALCAT1 mRNA expression. The mRNA expression of the Barth syndrome gene TAZ, encoding a mitochondrial CL transacylase, was unaltered in 2-DG treated cells. The increase in mitochondrial MLCL AT activity was due to an elevated expression in MLCL AT protein. Thus, an increase in MLCL AT activity and expression occurs to maintain the CL pool in the surviving population of H9c2 cells as a compensatory mechanism for the elevated phospholipase A2 activity seen in 2-DG-induced apoptosis. We hypothesize that increased mitochondrial MLCL AT activity and its expression, and hence, elevated CL resynthesis, may be a protective mechanism against monolysocardiolipin-mediated apoptosis.     

Monolysocardiolipin: improved preparation with high yield

A simple, high-yielding preparation of monolysocardiolipin (MLCL) by phospholipase A2 hydrolysis of cardiolipin (CL) in methanol on a semi-preparative scale is described. In methanol, phospholipase A2 preferentially hydrolyzes CL to MLCL. This selectivity results in ～80% yield of MLCL. The synthesized MLCL and dilysocardiolipin were characterized by NMR and ESI-MS/MS. Only the sn-2 position of CL was hydrolyzed by phospholipase A2 in methanol.     

In vivo synthesis of monolysocardiolipin and cardiolipin by Acinetobacter baumannii phospholipase D and effect on cationic antimicrobial peptide resistance

Acinetobacter baumannii is an opportunistic pathogen, which has become a rising threat in healthcare facilities worldwide due to increasing antibiotic resistances and optimal adaptation to clinical environments and the human host. We reported in a former publication on the identification of three phopholipases of the phospholipase D (PLD) superfamily in A. baumannii ATCC 19606^T acting in concerted manner as virulence factors in Galleria mellonella infection and lung epithelial cell invasion. This study focussed on the function of the three PLDs. A Δpld1-3 mutant was defect in biosynthesis of the phospholipids cardiolipin (CL) and monolysocardiolipin (MLCL), whereas the deletion of pld2 and pld3 abolished the production of MLCL. Complementation of the Δpld1-3 mutant with pld1 restored CL biosynthesis demonstrating that the PLD1 is implicated in CL biosynthesis. Complementation of the Δpld1-3 mutant with either pld2 or pld3 restored MLCL and CL production leading to the conclusion that PLD2 and PLD3 are implicated in CL and MLCL production. Mutant studies revealed that two catalytic motifs are essential for the PLD3-mediated biosynthesis of CL and MLCL. The Δpld1-3 mutant exhibited a decreased colistin and polymyxin B resistance indicating a role of CL in cationic antimicrobial peptides (CAMPs) resistance.     

Cardiolipin-depleted bovine heart cytochrome c oxidase: binding stoichiometry and affinity for cardiolipin derivatives

Detergent-solubilized bovine heart cytochrome c oxidase requires 2 mol of tightly bound cardiolipin (CL) per mole of monomeric complex for functional activity. Four lines of evidence support this conclusion: (1) Phospholipid depletion shows that two tightly bound CL's must remain associated with cytochrome c oxidase in order to maintain full electron transport activity. (2) Removal of the two tightly bound CL's correlates with decreased activity that is restored by reassociation of 2 mol of exogenous CL. (3) CL-depleted cytochrome c oxidase has two high-affinity binding sites for 2-[14C]acetylcardiolipin (AcCL), Kd,app less than 0.1 microM, that are not present in enzyme containing endogenous CL. An additional 2-3 lower affinity AcCL binding sites, Kd,app = 4 microM, are present in the CL-depleted complex, but these sites are also present in enzyme containing endogenous CL. (4) CL, monolysocardiolipin (MLCL), and dilysocardiolipin (DLCL) compete for AcCL binding with approximately the same relative affinities as those measured by the restoration of electron transport activity (MLCL competes much better than DLCL). However, MLCL and DLCL are only 60% and 15% as effective as CL in restoring maximum activity when they are bound to the high-affinity sites. The binding specificity of CL, MLCL, DLCL, and some of their acylated derivatives indicates that the apolar tails are most important for binding, not the polar head group. The presence or absence of hydroxyl groups in CL, MLCL, or DLCL also has little effect upon binding affinities. Binding specificity clearly favors CL since phosphatidylglycerol, phosphatidic acid, and phosphatidylcholine each have very low affinity for the CL binding sites (Kd,app greater than 20 microM). We, therefore, conclude that restoration of activity to CL-depleted cytochrome c oxidase is highly specific and requires the reassociation of CL, or structurally similar compounds, with two high-affinity binding sites.     

Human Trifunctional Protein Alpha Links Cardiolipin Remodeling to Beta-Oxidation

Cardiolipin (CL) is a mitochondrial membrane phospholipid which plays a key role in apoptosis and supports mitochondrial respiratory chain complexes involved in the generation of ATP. In order to facilitate its role CL must be remodeled with appropriate fatty acids. We previously identified a human monolysocardiolipin acyltransferase activity which remodels CL via acylation of monolysocardiolipin (MLCL) to CL and was identical to the alpha subunit of trifunctional protein (αTFP) lacking the first 227 amino acids. Full length αTFP is an enzyme that plays a prominent role in mitochondrial β-oxidation, and in this study we assessed the role, if any, which this metabolic enzyme plays in the remodeling of CL. Purified human recombinant αTFP exhibited acyl-CoA acyltransferase activity in the acylation of MLCL to CL with linoleoyl-CoA, oleoyl-CoA and palmitoyl-CoA as substrates. Expression of αTFP increased radioactive linoleate or oleate or palmitate incorporation into CL in HeLa cells. Expression of αTFP in Barth Syndrome lymphoblasts, which exhibit reduced tetralinoleoyl-CL, elevated linoleoyl-CoA acylation of MLCL to CL in vitro, increased mitochondrial respiratory Complex proteins and increased linoleate-containing species of CL. Knock down of αTFP in Barth Syndrome lymphoblasts resulted in greater accumulation of MLCL than those with normal αTFP levels. The results clearly indicate that the human αTFP exhibits MLCL acyltransferase activity for the resynthesis of CL from MLCL and directly links an enzyme of mitochondrial β-oxidation to CL remodeling.     

Silicic acid HPLC of cardiolipin, mono- and dilysocardiolipin, and several of their chemical derivatives

A silicic acid HPLC system in hexane-2-propanol-1 mM H3PO4 50:50:3.5 (v/v/v) is described for the analysis and/or purification of cardiolipin (CL), monolysocardiolipin (MLCL), dilysocardiolipin (DLCL), and several of their chemical derivatives. Derivatives that have been successfully analyzed include CL that is acetylated, succinylated, or tetrahydropyranylated at the 2-hydroxyl; MLCL acetylated at the 2 and 2'-hydroxyls; DLCL acetylated at the 2-hydroxyl and both 2'-hydroxyls; and MLCL tetrahydropyranylated at only the 2-hydroxyl. Water can replace 1 mM H3PO4 in the eluting solvent, but prior conditioning of the silicic acid column with the phosphoric acid solvent is necessary for acceptable chromatography. The most significant factor affecting the elution times of these compounds is the percentage of aqueous component, i.e., water or 1 mM H3PO4.     

Serine and ethanolamine incorporation into different plasmalogen pools: Subcellular analyses of phosphoglyceride synthesis in cultured glioma cells

In cultured glioma cells, plasma membrane (PM) is enriched in phosphatidylserine (PtdSer) and plasmalogens (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine). Serine can be a precursor of headgroups of both ptdSer and ethanolamine phosphoglycerides (PE) including plasmalogens and non-plasmalogen PE (NP-PE). Synthesis of phospholipids was investigated at the subcellular level using established fractionation procedures and incorporation of [³H(G)]L-serine and [1,2-¹⁴C]ethanolamine. Specific radioactivity of PtdSer from [³H]serine was 2-fold greater in PM than in microsomes, reaching maximum by 2–4 h. Labeled plasmalogen from [³H]serine appeared in PM by 4 h and increased to 48 h, whereas almost no plasmalogen accumulated in microsomes within 12 h. In contrast, labeled plasmalogen from [1,2-¹⁴C]ethanolamine appeared in both PM and microsomes at early incubation times and became enriched in PM beyond 12 h. Thus, in glioma cells: (1) greater and faster accumulation of labeled PtdSer in PM may reflect direct synthesis from serine within PM; (2) PM is a major source of PtdSer for decarboxylation and PE synthesis; (3) NP-PE in both PM and microsome provides headgroup for synthesis of plasmalogen; and, (4) plasmalogen synthesis may involve different intracellular pools depending on headgroup origin.Abbreviations NP-PE nonplasmenylethanolamine phosphoglycerides including both diacyl and alkylacyl species - PE total ethanolamine phosphoglycerides: plasmalogen-plasmenylethanolamine or alkenylacyl ethanolamine phosphoglyceride (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) - PL phospholipid - PM plasma membrane - PtdCho phosphatidylcholine - PtdSer phosphatidylserine     

Chlorinated fatty acid distribution in Mycobacterium convolutum phospholipids after growth on 1-chlorohexadecane

The composition of phospholipids from Mycobacterium convolutum R22 was determined after growth at two temperatures (20 and 30 degrees C) with 1-chlorohexadecane as the substrate. Comparisons were made with the phospholipids of cells grown on n-hexadecane. Phosphatidylinositolmannosides and phosphatidylethanolamine (PE) were the major phospholipids in n-hexadecane-grown cells. In 1-chlorohexadecane-grown cells, phosphatidylinositolmannosides were approximately half of the total phospholipids, with lesser amounts of PE and cardiolipin (CL). The relative level of PE was greater at 20 degrees C (versus that at 30 degrees C) after growth on either substrate. A determination was made of structure and positional distribution of constituent fatty acid in both CL and PE. The relative amount of unsaturated fatty acid was higher at 20 degrees C. There were two C16:1 fatty acids (C16:1 delta 9 and C16:1 delta 11), and these had positional preferences in both CL and PE. The positional sites of chlorinated fatty acids differed in both CL and PE at the two temperatures. The results confirm that microorganisms can specifically distribute chlorinated fatty acids into cellular phospholipids.     

Chlorinated fatty acid distribution in Mycobacterium convolutum phospholipids after growth on 1-chlorohexadecane.

The composition of phospholipids from Mycobacterium convolutum R22 was determined after growth at two temperatures (20 and 30 degrees C) with 1-chlorohexadecane as the substrate. Comparisons were made with the phospholipids of cells grown on n-hexadecane. Phosphatidylinositolmannosides and phosphatidylethanolamine (PE) were the major phospholipids in n-hexadecane-grown cells. In 1-chlorohexadecane-grown cells, phosphatidylinositolmannosides were approximately half of the total phospholipids, with lesser amounts of PE and cardiolipin (CL). The relative level of PE was greater at 20 degrees C (versus that at 30 degrees C) after growth on either substrate. A determination was made of structure and positional distribution of constituent fatty acid in both CL and PE. The relative amount of unsaturated fatty acid was higher at 20 degrees C. There were two C16:1 fatty acids (C16:1 delta 9 and C16:1 delta 11), and these had positional preferences in both CL and PE. The positional sites of chlorinated fatty acids differed in both CL and PE at the two temperatures. The results confirm that microorganisms can specifically distribute chlorinated fatty acids into cellular phospholipids.     

31P MRS analysis of the phospholipid composition of normal human peripheral blood mononuclear cells (PBMC)

The aim of this investigation was to characterize the phospholipid composition of normal human blood mononuclear cells using 31P NMR spectroscopy. Mononuclear cells of peripheral blood were obtained from 10 volunteers. Phospholipid extracts were prepared from 60x10(6) cells according to modified Folch's method. An AMX 300 Bruker spectrometer 7.05 T was used. The 31P spectrum of phospholipid extracts from normal human PBMC consisted of 9 peaks, with one each for phosphatidylcholine (PC), plasmalogen of phosphatidylcholine (CPLAS), lysophosphatidylcholine (LPC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL), and another one due to the external reference substance, methylenediphosphonic acid (MDPA). The concentrations of these phospholipids (PL), based on the integral intensities, were as follows: 0.398 +/- 0.078 mmole/l for PC; 0.033 +/- 0.019 mmole/l for CPLAS; 0.155 +/- 0.043 mmole/l for SM; 0.266 +/- 0.104 mmole/l for PI+PE; 0.101 +/- 0.040 mmole/l for PS, and 0.026 +/- 0.033 mmole/l for CL. The results of this study confirmed that 31P MRS is a convenient tool for measuring the phospholipid concentrations of biological samples.     

Turnover of nonessential fatty acids in cardiolipin from the rat heart

Cardiolipin (CL) is a unique phospholipid (PL) found in the mitochondria of mammalian cells. CL remodeling is accompanied by turnover of its fatty acid acyl groups. Abnormalities in CL remodeling have been found in Barth's syndrome, diabetes, and obesity. The objective of this study was to determine nonessential fatty acid turnover in CL and phosphatidylethanolamine (PE) in the rat heart in vivo. Sprague-Dawley rats were fed either a regular chow or a high-fat diet for 15 weeks, and consumed 6% deuterium-enriched drinking water as a tracer for 14 days. CL and PE were extracted from cardiac tissue and isolated by TLC. Fatty acids from CL, PE, and plasma were analyzed by GC/MS for deuterium incorporation. Results showed oleate and vaccenate turnover were the highest in CL whereas palmitate and stearate turnover were low. Among the nonessential fatty acids in PE, turnover of stearate and vaccenate were the highest. The high turnover rate in vaccenate was unexpected, because vaccenate previously had no known metabolic or physiologic function. In conclusion, the similarly high turnover rates of both oleate and vaccenate readily suggest that remodeling is an important functional aspect of PL metabolism in CL.     

Involvement of calcium-dependent mechanisms in T3-induced phosphorylation of vimentin of immature rat testis

Thyroid hormones have been shown to act at extra nuclear sites, inducing target cell responses by several mechanisms, frequently involving intracellular calcium concentration. It has also been reported that cytoskeletal proteins are a target for thyroid and steroid hormones and cytoskeletal rearrangements are observed during hormone-induced differentiation and development of rat testes. However, little is known about the effect of 3,5,3'-triiodo-L-thyronine (T3) on the intermediate filament (IF) vimentin in rat testes. In this study we investigated the immunocontent and in vitro phosphorylation of vimentin in the cytoskeletal fraction of immature rat testes after a short-term in vitro treatment with T3. Gonads were incubated with or without T3 and 32P orthophosphate for 30 min and the intermediate filament-enriched cytoskeletal fraction was extracted in a high salt Triton-containing buffer. Vimentin immunoreactivity was analyzed by immunoblotting and the in vitro 32P incorporation into this protein was measured. Results showed that 1 microM T3 was able to increase the vimentin immunoreactivity and in vitro phosphorylation in the cytoskeletal fraction without altering total vimentin immunocontent in immature rat testes. Besides, these effects were independent of active protein synthesis. The involvement of Ca2+-mediated mechanisms in vimentin phosphorylation was evident when specific channel blockers (verapamil and nifedipine) or chelating agents (EGTA and BAPTA) were added during pre-incubation and incubation of the testes with T3. The effect of T3 was prevented when Ca2+ influx was blocked or intracellular Ca2+ was chelated. These results demonstrate a rapid nongenomic Ca2+-dependent action of T3 in phosphorylating vimentin in immature rat testes.