Activation of protein kinase C by myristate and its requirements of Ca2+ and phospholipid

Myristate (C14:0) was found to significantly activate partially purified rat brain Ca(2+)- and phospholipid-dependent protein kinase (PKC). The Ka value, the concentration needed for half maximum activation, for C14:0 in the presence of 1 microM Ca2+ and 20 microM phosphatidylserine (PS) was 20 microM. This activation required Ca2+ and acidic phospholipid and was associated with a decreased Ka for Ca2+ of the enzyme to 10 microM in an analogous fashion as dioleoylglycerol (DO) or phorbol myristate acetate (PMA). The phospholipid requirement for the activation was concentration dependent and was inhibited by 1-(5-isoquinolinesulfonyl)-methylpiperazine dihydrochloride (H-7), a inhibitor of this enzyme. The concentration of H-7 required for half inhibition of the enzyme was about 15 microM and maximum inhibition was about 75%. The concentration profile of cytoplasmic proteins phosphorylated by C14:0-activated PKC was similar to that by PMA-activated PKC. The 47 kDa protein of guinea pig neutrophil was also phosphorylated by the C14:0-activated PKC. It is further discussed whether PKC can function as signal transduction for stimulus-mediated generation of superoxide in neutrophils.     

Effects of selective protein kinase c isozymes in prostaglandin2alpha-induced Ca2+ signaling and luteinizing hormone-induced progesterone accumulation in the mid-phase bovine corpus luteum

A single-cell approach for measuring the concentration of cytoplasmic calcium ions ([Ca(2+)](i)) and a protein kinase C-epsilon (PKCepsilon)-specific inhibitor were used to investigate the developmental role of PKCepsilon in the prostaglandin F(2alpha)(PGF(2alpha))-induced rise in [Ca(2+)](i) and the induced decline in progesterone accumulation in cultures of cells isolated from the bovine corpus luteum. PGF(2alpha) increased [Ca(2+)](i) in Day 4 large luteal cells (LLCs), but the response was significantly lower than in Day 10 LLCs (4.3 +/- 0.6, n = 116 vs. 21.3 +/- 2.3, n = 110). Similarly, the fold increase in the PGF(2alpha)-induced rise in [Ca(2+)](i) in Day 4 small luteal cells (SLCs) was lower than in Day 10 SLCs (1.6 +/- 0.2, n = 198 vs. 2.7 +/- 0.1, n = 95). A PKCepsilon inhibitor reduced the PGF(2alpha)-elicited calcium responses in both Day 10 LLCs and SLCs to 3.5 +/- 0.3 (n = 217) and 1.3 +/- 0.1 (n = 205), respectively. PGF(2alpha) inhibited LH-stimulated progesterone (P(4)) accumulation only in the incubation medium of Day 10 luteal cells. Both conventional and PKCepsilon-specific inhibitors reversed the ability of PGF(2alpha) to decrease LH-stimulated P(4) accumulation, and the PKCepsilon inhibitor was more effective at this than the conventional PKC inhibitor. In conclusion, the evidence indicates that PKCepsilon, an isozyme expressed in corpora lutea with acquired PGF(2alpha) luteolytic capacity, has a regulatory role in the PGF(2alpha)-induced Ca(2+) signaling in luteal steroidogenic cells, and that this in turn may have consequences (at least in part) on the ability of PGF(2alpha) to inhibit LH-stimulated P(4) synthesis at this developmental stage.     

Activation of protein kinase C by fatty acids and its dependency on Ca2+ and phospholipid

Ca2+- and phospholipid-dependent protein kinase (PKC) was activated by arachidonic and myristic acids. This activation by both fatty acids required the calcium ion. Acidic phospholipid was also required for the activation by myristic acid, while that by arachidonic acid was inhibited by phospholipid.     

Activation of SAD kinase by Ca2+/calmodulin-dependent protein kinase kinase

To search for the downstream target protein kinases of Ca (2+)/calmodulin-dependent protein kinase kinase (CaMKK), we performed affinity chromatography purification of a rat brain extract using a GST-fused CaMKKalpha catalytic domain (residues 126-434) as the affinity ligand. Proteomic analysis was then carried out to identify the CaMKK-interacting protein kinases. In addition to identifying the catalytic subunit of 5'-AMP-activated protein kinase, we identified SAD-B as interacting. A phosphorylation assay and mass spectrometry analysis revealed that SAD-B was phosphorylated in vitro by CaMKK at Thr (189) in the activation loop. Phosphorylation of Thr (189) by CaMKKalpha induced SAD-B kinase activity by over 60-fold. In transfected COS-7 cells, kinase activity and Thr (189) phosphorylation of overexpressed SAD-B were significantly enhanced by coexpression of constitutively active CaMKKalpha (residues 1-434) in a manner similar to that observed with coexpression of LKB1, STRAD, and MO25. Taken together, these results indicate that CaMKKalpha is capable of activating SAD-B through phosphorylation of Thr (189) both in vitro and in vivo and demonstrate for the first time that CaMKK may be an alternative activating kinase for SAD-B.     

Hypoxia regulates cell proliferation and steroidogenesis through protein kinase A signaling in bovine corpus luteum

Hypoxia is an important physiological process which ensures corpus luteum (CL) formation and development, thus playing an important role in steroidogenesis. Recent studies have shown that CL develops in an analogous to tumorigenesis by accumulation of hypoxia-inducible factor-1 alpha subunit (HIF1A) in response to hypoxia. To investigate the relationship among hypoxia, steroidogenesis, and cell proliferation during CL lifespan, histological and steroidogenic analyses of CL were performed at various CL stages in non-pregnant Holstein. Also, the hypoxia-mediated steroidogenesis and cell proliferation were studied in vitro with both primary luteal and luteinized granulosa cells. Our results showed that progesterone (P(4)) concentration increased with the upregulation of steroidogenic protein including steroidogenic acute regulatory protein (STAR) and CYP11A1 (P450scc) in the middle luteal stage. On the other hand, the cell proliferation- or hypoxia-associated proteins were upregulated in the early stage, including the proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor A (VEGFA), HIF1A, and aryl hydrocarbon receptor nuclear translocator (ARNT). In primary culture, phospho-protein kinase A (p-PKA) was downregulated, as were P(4) secretion and steroidogenic proteins both under oxygen-conditioned hypoxia in luteal cells and cobalt chloride-induced hypoxia in luteinized granulosa cells. However, under the treatment of hypoxia, PCNA, which was downregulated in luteal cells, was upregulated together with HIF1A and VEGFA in luteinized granulosa cells. Taken together, present study suggested that hypoxia downregulated steroidogenesis through PKA signaling and that the hypoxia-regulated cell proliferation could be activated during CL formation.     

FSH receptors in the bovine corpus luteum

Binding of follicle stimulating hormone (FSH) to a crude membrane fraction of bovine corpus luteum (CL) has been detected. This binding meets the usual criteria for a receptor based on specificity, time course of reaction and association constant (Ka = 8.5 x 10(10)M(-1)). Physiological studies with CL removed from heifers at specific times after estrus indicate that day-6 CL had the highest FSH binding. However, a correlation with physiological function was not obvious since some functional mid-cycle CL were high in progesterone and luteinizing hormone (LH) receptor but had nondetectable FSH receptor. Conversely, some late-cycle CL had low progesterone and LH receptor but significant quantities of FSH receptor.     

Expression and activation of protein kinase C isozymes by prostaglandin F(2alpha) in the early- and mid-luteal phase bovine corpus luteum

Western blotting was used to identify the array of protein kinase C (PKC) isozymes expressed in the early (Day 4) and midcycle (Day 10) bovine corpus luteum (CL). PCKalpha, betaI, betaII, epsilon, and micro isozymes were detected in total protein samples prepared from both Day-4 and Day-10 corpora lutea. In contrast, specific antibodies for PKCgamma, eta, lambda, and theta isozymes failed to detect protein bands in the luteal samples. PKCbetaII and epsilon isozymes were expressed differentially at these two developmental stages of the bovine CL. In the Day-4 luteal samples, PKCepsilon was barely detectable; in contrast, in the Day-10 samples, the actin-corrected ratio for PKCepsilon was 1.16 +/- 0.13. This ratio was higher than the detected ratio for PKCbetaI and micro at this developmental phase of the CL (P < 0.01), but it was comparable with the ratio detected for the PCKalpha and betaII. The amount of PKCbetaII was, although not as dramatic, also greater in the Day-10 CL (actin-corrected ratio was 0.85 +/- 0.2) than in the Day-4 CL (0.35 +/- 0.09 [P < 0.01]). The actin-corrected ratios for all other PKC isozymes, alpha (Day 4 = 0.93 +/- 0.16, Day 10 = 0.97 +/- 0.09), betaI (Day 4 = 0.54 +/- 0.073, Day 10 = 0.48 +/- 0.74), and micro (Day 4 = 0.21 +/- 0.042, Day 10 = 0.21 +/- 0.38) were not different at these 2 days of the cycle. An experiment was designed to test whether activation of specific isozymes differed between CL that do or do not regress in response to PGF(2alpha). Bovine CL from Day 4 and Day 10 of the estrous cycle were collected and 1 mm CL fragments were treated in vitro for 0, 2.5, 5, 10 or 20 min with PGF(2alpha) (0.1, 1.0, and 10 nM) or minimal essential medium-Hepes vehicle. Translocation of PKC from cytoplasm to membrane fraction was used as indication of PKC activation by PGF(2alpha). Evidence for PKC activation was observed in both Day-4 and Day-10 luteal samples treated with 10 nM PGF(2alpha). Therefore, if PKC, an intracellular mediator associated with the luteal PGF(2alpha) receptor, contributes to the lesser sensitivity of the Day-4 CL, it is likely due to the differential expression of the epsilon and betaII isozymes of PKC at this stage and not due to an inability of the PGF(2alpha) receptor to activate the isozymes expressed in the early CL.     

Intercellular communication in the bovine corpus luteum

There is a growing body of evidence that intercellular communication is important in the regulation of luteal function. Although the nature of the interactions between small and large luteal cells are not yet clear, it seems likely that they do exist. Many of the substances to which luteal cells respond, such as prostaglandins, growth factors, oxytocin and progesterone, are produced locally. These substances may act as paracrine factors to modulate the response of luteal cells to hormonal signals. Endothelial cells also produce factors that can modify steroidogenesis, and luteal cell-stimulation of endothelial cell proliferation is necessary for the extensive angiogenesis that occurs during luteinization Finally, bidirectional intercellular communication likely occurs between luteal cells and resident immune cells. Immune cells produce cytokines that can modify progesterone and prostaglandin synthesis by luteal cells. Cytokines may also have direct cytotoxic effects on luteal cells, and dead cells are then phagocytized by resident macrophages. Also, factors secreted by luteal cells can serve as chemoattractants for immune cells, and can enhance or suppress immune cell functions. There is little doubt that intercellular communication within the corpus luteum is very complex. One must remember, however, that nearly all evidence collected thus far is based on in vitro studies. Eventually, technology will allow for study of these interactions in vivo, and may lead to new methods for control of luteal function.     

Production of enkephalins by the human and bovine corpus luteum

In search of early pregnancy factors, we detected by radioimmunoassay the presence of enkephalin in bovine and human corpus luteum. In vitro met-enkephalin release by bovine corpus luteum is about 0.5 to 1 pmole/mg of fresh tissue/24 hrs. The content of the fresh tissue is between 0.7 and 1.9 pmoles per gram of human tissue, and 0.9 pmoles for bovine tissue. Furthermore, we determined the presence of leu-enkephalin and met-enkephalin Arg-Gly-Leu to. The ratios observed confirm a pro-enkephalin A expression in the ovary. Opiates or opioid-like peptides are present in the female genitalia at the time of early embryo development. The roles of these opioid peptides is discussed in term of ovum transport, granulosa cell physiology and early pregnancy factors.     

Isozymes of cAMP-dependent protein kinase present in the rat corpus luteum.

Regulatory (R) subunits and their association with catalytic subunits to form cAMP-dependent protein kinase holoenzymes were investigated in corpora lutea of pregnant rats. Following separation by DEAE-cellulose chromatography, R subunits were identified by labeling with 8-N3[32P]cAMP and autophosphorylation on one and two-dimensional gel electrophoresis and by reactivity with antisera. DEAE-cellulose elution of R subunits with catalytic subunits as holoenzymes or without catalytic subunits was determined by sedimentation characteristics on sucrose density gradient centrifugation and by cAMP-stimulated kinase activation characteristics on Eadie-Scatchard analysis. We identified the presence of a type I holoenzyme containing RI alpha (Mr 47,000) subunits, a prominent type II holoenzyme containing RII beta (Mr 52,000) subunits, and a second more acidic type II holoenzyme peak containing both RII beta and RII alpha (Mr 54,000) subunits. However, the majority of total R subunit activity was associated with a catalytic subunit-free peak of RI alpha protein which on elution from DEAE-cellulose was associated with cAMP. This report establishes the more basic elution position from DEAE-cellulose of the prominent rat luteal RII beta holoenzyme in very close proximity to free RI alpha and presents one of the few reports of a normal tissue containing a large percentage of catalytic subunit-free RI alpha.     

Changes in prostaglandin secretion by the regressing bovine corpus luteum

Secretion of prostaglandins (PGs) by the regressing corpus luteum (CL) was investigated in the cow. Six cows were implanted with microcapillary dialysis membranes of a microdialysis system (MDS) into the CL during Days 8-9 (Day 0 = estrus), and a prostaglandin (PG) F2alpha analogue (Estrumate) was injected intramuscularly (i.m.) to induce luteolysis. Acute increases in intraluteal release of PGF2alpha and PGE2 were observed during the first 4 h, followed by decreases over the next 8 h. Intraluteal release of both PGs gradually increased again during the period 48-72 h. Concentrations of PGF2alpha in ovarian venous plasma (OVP) were 4-13 times higher than those of jugular venous plasma (JVP) (P < 0.001) during the period of the experiment, and increased from 24 h after treatment with Estrumate (P < 0.05). Cyclooxygenase (COX)-2 mRNA expression increased (P < 0.05) at 2 and 24 h after treatment with Estrumate. The results indicated that local release of PGF2alpha and PGE2, and COX-2 mRNA expression were increased by Estrumate in the regressing CL at the later stages of luteolysis. Thus, luteal secretion of PGs may be involved in the local mechanism for structural rather than functional luteolysis.     

Activation of Mg-ATPase (spectrin-dependent ATPase) by Ca2+.

The dependence of saponin-stimulated Mg-ATPase activity in the erythrocyte membrane on Ca2+ concentration was studied. In the membrane of freshly sampled human erythrocytes we found for this enzyme and Ca2+ an apparent dissociation constant of 0.611 mumol/l (SE +/- 0.106 mumol/l) and Hill coefficient of 0.93 (SE +/- 0.05). The enzyme is in most probability identical with Ca,Mg-ATPase of high affinity to Ca2+ described also as spectrin-dependent Ca,Mg-ATPase.     

Biosynthesis of retinal in bovine corpus luteum

Bovine corpus luteum tissue was sliced and incubated with beta-[15,15'-(3)H]carotene. The conversion of radioactive beta-carotene into radioactive retinal was substantiated utilizing column chromatography, thin-layer chromatography, high-speed liquid chromatography, and a derivative formation. Lowering of the incubation temperature to 20 degrees C or boiling the tissue eliminated the conversion of beta-carotene to retinal. In addition, other carotenoids and possible oxidation products of beta-carotene in the corpus luteum were investigated. Our results indicate that the bovine corpus luteum possesses the ability to synthesize retinal in situ, which may play a role in reproductive functions.     

Luteotropic and luteolytic mechanisms in the bovine corpus luteum

The function of the corpus luteum (CL) is a key element in many reproductive processes including ovulation, length of the estrous cycle, recognition of pregnancy and embryo survival in all mammalian species. The main function of the CL is to produce progesterone which acts on its tissues to prepare them for successful pregnancy. The CL is controlled by numerous biological compounds which provide luteotropic support during the estrous cycle and pregnancy and for inducing luteolysis at the end of the cycle The purpose of this paper is to review the mechansims responsible for controlling the endocrine function of this tissue in the bovine ovary.     

Regulation of prostaglandin synthesis by progesterone in the bovine corpus luteum

The objective of the present study was to investigate the influence of progesterone on prostaglandin synthesis by the corpus luteum (CL). Corpora lutea were obtained from dairy cows on days 4, 6, 10, and 18 of the estrous cycle, dissociated, and placed in serum-free culture. The addition of luteinizing hormone (LH) resulted in a slight, but non-significant (p greater than 0.05), increase in levels of 6-keto-PGF1 alpha, and had no effect on PGF2 alpha. Progesterone treatment caused a significant, dose-dependent decrease in both PGF2 alpha and 6-keto-PGF1 alpha in 6-day and 10-day corpora lutea, but not in 4-day or 18-day corpora lutea. In the 6- and 10-day corpora lutea, progesterone treatment resulted in a greater inhibition of PGF2 alpha than 6-keto-PGF1 alpha production. Therefore, progesterone treatment brought about an increase in the 6-keto-PGF1 alpha to PGF2 alpha ratio in these cells (12.9 vs. 21.3). It is concluded from these studies that progesterone can modulate luteal prostacyclin and PGF2 alpha synthesis, suggesting an interaction of progesterone and prostaglandin production within the corpus luteum.     

Activation of ryanodine receptors by flash photolysis of caged Ca2+.

Flash photolysis of DM-nitrophen generates an extremely large [Ca2+] transient ("Ca2+ spike") at the start of each Ca2+ "step." The Ca2+ spike greatly increases the speed of activation of the ryanodine receptor channel ("supercharging") and could be responsible for apparent channel adaptation.