Effects of long-acting GnRH antagonist, degarelix acetate, on plasma insulin-like peptide 3, testosterone and luteinizing hormone concentrations, and scrotal circumference in male goats.

We recently reported that plasma insulin-like peptide 3 (INSL3) concentrations increased soon after endogenous and exogenous stimulations of LH in male goats and bulls. However, the effects of LH suppression on INSL3 secretion are unknown in domestic animals. Here, we examined the effects of a long-acting GnRH antagonist (degarelix acetate; 4 mg/kg) on the secretions of plasma INSL3 and testosterone in two phases, an immediate and a long-term phase in male goats (n = 6; aged, 13-16 months). During the immediate phase, blood was taken at 15-minute intervals for 8 hours on Days -5, 0, and 3. The GnRH antagonist was administered after 2-hour sampling of Day 0. Moreover, a daily blood sample was taken from Day 0 to Day 7, followed by twice a week until 9 weeks and finally at week 10. The scrotal circumference was recorded before treatment and continued biweekly until week 10. Concentrations of LH, INSL3, and testosterone in plasma were determined by EIA and the pulsatile nature of secretion analyzed using pulse XP software. The mean concentrations, pulse frequency (per hour), and pulse amplitude (peak-nadir) of plasma LH and testosterone reduced from pretreatment to posttreatment Day 0 and Day 3 (P < 0.05). A decline in mean concentrations, pulse frequency, and pulse amplitude of INSL3 was exhibited on posttreatment Day 3 compared with pretreatment (P < 0.01). During long-term sampling, a decline (P < 0.01) in plasma testosterone and INSL3 concentrations was observed 1 day after treatment and remained lower until 8.5 weeks after treatment, and thereafter returned to pretreatment levels. A reduction in scrotal circumference was recorded 4 weeks after treatment and remained lower until 10 weeks after treatment (P < 0.05). In conclusion, the acute regulation of INSL3 by LH was confirmed by reduction of plasma INSL3 levels within 3 days after GnRH antagonist treatment in male goats. Although the onset of suppression of testosterone was more rapid than that of INSL3, the low levels persisted for 8.5 weeks for both hormones, and subsequently the concentrations returned to pretreatment levels. A significant reduction in testicular size was also observed. The quick, long-lasting, and transient suppression of testosterone and INSL3 after a single injection implies a potential application of this antagonist in reversible long-term chemical castration in male goats.

Acute regulation of plasma insulin-like peptide 3 concentrations by luteinizing hormone in male goats.

Recently, it was reported that in bulls secretion of insulin-like peptide 3 (INSL3) in blood occurred in a pulsatile manner and was acutely regulated by LH. In the present study, the acute regulation of plasma INSL3 and its temporal relationships with LH and testosterone were examined in six sexually matured male goats using the following experimental design. (1) After stimulating LH release by administering a GnRH analogue, blood levels of LH, INSL3, and testosterone were monitored at 15-minute intervals for 2 hours followed by hourly intervals up to 8 hours. (2) After activation of the LH receptor by hCG blood levels of INSL3 and testosterone were determine at 15-minute intervals for 2 hours, followed by hourly intervals up to 8 hours, daily intervals up to Day 8, and finally on Day 12. (3) The release of LH, INSL3, and testosterone in normal physiology was established at 15-minute intervals for an 8-hour session. Concentrations of LH, INSL3, and testosterone in plasma were measured by enzyme-immunoassays. After GnRH treatment, mean plasma concentrations of all three hormones increased (P < 0.05) dramatically from 30 minutes and remained high until 120 minutes (LH), 75 minutes (INSL3), and 4 hours (testosterone) after treatment. After hCG treatment, mean plasma INSL3 concentrations increased (P < 0.05) from 30 minutes and remained elevated until the end of sampling on Day 12. An increase (P < 0.05) in mean plasma testosterone concentrations occurred from 15 minutes and remained high until Day 6. The mean increase (maximum per pretreatment concentration) of INSL3 concentrations after administration of GnRH and hCG was lower (P < 0.01) than that of testosterone. The secretory pattern of LH, INSL3, and testosterone in the general circulation was pulsatile with a frequency of 5.5 ± 0.6, 4.7 ± 0.5, and 2.2 ± 0.5, respectively, during the 8-hour period. Twenty out of 28 (71%) of these INSL3 pulses peaked within 1 hour after a peak of an LH pulse. The mean increase (peak per basal concentration) of INSL3 pulses (2.1 ± 0.1 fold, n = 28) was lower (P < 0.01) than that of testosterone pulses (4.3 ± 2.2 fold, n = 13). In conclusion, secretion of INSL3 in blood occurred, like in bulls, in a pulsatile manner soon after LH pulses in male goats. The absolute concentrations of INSL3 in male goats were higher than that reported in other mammals. Insulin-like peptide 3 concentrations were acutely increased by endogenous and exogenous LH in male goats, but the rise of INSL3 was lower than that of testosterone.

Plasma insulin-like peptide 3 concentrations are acutely regulated by luteinizing hormone in pubertal Japanese Black beef bulls.

Insulin-like peptide 3 (INSL3) is a major secretory product of testicular Leydig cells. The mechanism of acute regulation of INSL3 secretion is still unknown. The present study was undertaken in pubertal beef bulls to (1) determine the temporal relationship of pulsatile secretion among LH, INSL3, and testosterone and (2) monitor acute regulation of INSL3 secretion by LH using GnRH analogue and hCG. Blood samples were collected from Japanese Black beef bulls (N = 6) at 15-minute intervals for 8 hours. Moreover, blood samples were collected at -0.5, 0, 1, 2, 3, 4, 5, and 6 hours after GnRH treatment and -0.5, 0, 2, 4, and 8 hours on the day of treatment (Day 0), and Days 1, 2, 4, 8, and 12 after hCG treatment. Concentrations of LH, INSL3, and testosterone determined by EIAs indicated that secretion in the general circulation was pulsatile. The frequency of LH, INSL3, and testosterone pulses was 4.7 ± 0.9, 3.8 ± 0.2, and 1.0 ± 0.0, respectively, during the 8-hour period. Seventy percent of these INSL3 pulses peaked within 1 hour after a peak of an LH pulse had occurred. The mean increase (peak per basal concentration) of testosterone pulses was higher (P < 0.001) than that of INSL3 pulses. After GnRH treatment, LH concentrations increased (P < 0.01) dramatically 1 hour after treatment and remained high (P < 0.05) until the end of sampling, whereas an elevated (P < 0.05) INSL3 concentration occurred at 1, 2, 5, and 6 hours after treatment. Testosterone concentrations increased (P < 0.01) 1 hour after the treatment and remained high until the end of sampling. After hCG treatment, an increase of INSL3 concentration occurred at 2 and 4 hours, and Days 2, 4, and 8 after treatment (P < 0.05), whereas in case of testosterone, concentrations remained high (P < 0.01) until Day 8 after treatment. The increase (maximum per pretreatment concentration) of INSL3 concentrations after injecting GnRH or hCG was much lower (P < 0.001) than that of testosterone. In conclusion, secretion of INSL3 in blood of bulls occurred in a pulsatile manner. We inferred an acute regulation of INSL3 by LH in bulls because INSL3 concentrations increased immediately after endogenous and exogenous LH stimulation. The increase of INSL3 concentrations by LH was much lower than that of testosterone in bulls.

Clinical effect of hyperbaric oxygen therapy in adhesive postoperative small bowel obstruction.

BACKGROUND: Hyperbaric oxygen (HBO) therapy is a controversial treatment for adhesive postoperative small bowel obstruction, with only a few small studies reported. The aim of this study was to assess the clinical value of HBO therapy in the treatment of adhesive postoperative small bowel obstruction. METHODS: Between April 2006 and March 2012, all patients with adhesive postoperative small bowel obstruction were treated using either decompression therapy or HBO. Patients undergoing HBO therapy were treated once a day at a pressure of 2·0 atmospheres absolute and received 100 per cent oxygen. Patients showing no clinical and radiological improvement with HBO therapy were converted to decompression therapy by means of a long tube. Medical records were reviewed and outcomes analysed. RESULTS: A total of 305 patients were treated, of whom 142 underwent tube decompression therapy during the first 3 years and the remaining 163 had HBO therapy during the last 3 years. The median number of HBO treatments was 3 (range 1-7). A total of 143 patients (87·7 per cent) were treated successfully with HBO without long-tube decompression. HBO therapy was associated with earlier resumption of oral intake (mean 4·7 versus 6·5 days; P = 0·001) and a shorter hospital stay (mean 10·3 versus 14·1 days; P = 0·001). The rate of operation was 7·4 per cent in the HBO group and 14·8 per cent in group treated by decompression alone (P = 0·037). CONCLUSION: In this study, HBO therapy was safe for the treatment of adhesive postoperative small bowel obstruction. It reduced the need for surgery and time to recovery as well as the hospital stay.

[Nongenomic mechanisms of progesterone].

Rapid, independent of transcriptional effects of progesterone have been observed in various types of cells, tissues and species. In some biological systems, these nongenomic actions and associated with them signal transduction pathways are characterized in detail at molecular level. This review summarizes findings concerning the role of progestins in the regulation of such physiological functions and processes as meiotic maturation of fish and amphibian oocytes; growth and proliferation of normal and transformed cells of mammary gland; contraction of myometrium; survival and functional activity of granulose cells; sperm capacitation, acrosome reaction and hypermotility; immune function of T lymphocytes; survival and function of brain cells. The participation of several types of receptor proteins in the nongenomic progesterone signaling is discussed. They include the classic nuclear progesterone receptor, PR, the membrane progestin receptor, mPR, the progesterone membrane receptor component, PGMRC, the oxytocin receptor, OTR, and the GABA receptor, GABA(A).

Adaptor protein Shc undergoes translocation and mediates up-regulation of the tyrosine kinase c-Src in EGF-stimulated A431 cells.

BACKGROUND: Shc is the adaptor protein that exists in three isoforms, P46, P52 and P66, and acts as a bridge between activated cell surface receptors and downstream signalling molecules which act in extracellular signal-regulated cell events such as cell cycle progression. In our previous studies, Shc was shown to be a substrate of the tyrosine kinase c-Src in vitro and in vivo. RESULTS: Using green fluorescent protein-fusion Shc (GFP-Shc), we have shown that following epidermal growth factor (EGF) stimulation of A431 cells, all Shc isoforms were rapidly recruited from the cytoplasm to the plasma membrane (within 5 min) and then redistributed to the cytoplasmic vesicle structures (in the next 10-20 min). Indirect immunofluorescent study demonstrated that all Shc isoforms co-localize with EGF receptor (EGFR) and activated c-Src in both plasma membranes and cytoplasmic vesicle structures. Our previous study has shown that EGF induces the indirect association of EGFR and c-Src and activation of c-Src in A431 cells. An immunoprecipitation study demonstrated that the EGFR-Src association and c-Src activation are augmented in cells expressing GFP-Shc P52 or P66, but not P46. In addition, P52 and P66, but not P46, are in association with EGFR-Src complex. We also found that EGFR and Shc can be dissociated from c-Src by the addition of a synthetic peptide that corresponds to the autophosphorylation site of c-Src. Interestingly, the peptide-induced dissociation of the complex was not affected by the tyrosine phosphorylation state of the peptide. CONCLUSION: These results demonstrated a dynamic subcellular movement of Shc in response to EGF, and suggested a hitherto unknown scheme whereby Shc can work not only as a substrate of c-Src but also as a mediator of the EGF-induced activation of c-Src in an isoform-specific manner.

Deregulation of mitogen-activated protein kinase at low pH due to a structural rearrangement of activation segment.

Autophosphorylation of recombinant mitogen-activated protein kinase (MAPK) on Tyr was found to be several-fold stimulated at weakly acidic pH (5.5-6.0), whereas the phosphorylation of a protein substrate, myelin basic protein, was greatly inhibited at pH below 6. 0. In contrast to phosphorylation at pH 8.0, both MAPK autophosphorylation and MAPK phosphorylation with upstream MAPK kinase at low pH failed to stimulate essentially its kinase activity towards the exogenous protein substrate. Immunoprecipitation and ELISA with an activation segment-specific antibody, kinetic analysis, and reversible phosphorylation assay revealed a difference in the folding of MAPK activation segment at pH 5.5 and 8.0. The data suggest that a rearrangement of the activation segment at low pH promotes a stable low-activity conformation of the enzyme which is favorable for intramolecular autophosphorylation. In this conformation, the phosphorylation of the exogenous protein substrate is inhibited due to persistent blocking of the enzyme catalytic center by the activation segment.

Peptide inhibitors of the mitogen-activated protein kinase pathway: a structure -mimetic peptide corresponding to the conserved inter-DFG-APE region in the kinase domain.

The signal transduction pathway mediated by mitogen-activated protein kinases is an attractive target for the design of pharmacologically effective inhibitors. Two specific cell-permeant small molecule inhibitors of this pathway have been reported. However, under certain circumstances, nonpermeable inhibitors, such as neutralizing antibodies and peptide inhibitors, are also useful. We present here a novel approach for such peptide inhibitor design. The procedure is based on the synthesis of a structure-mimetic peptide corresponding to a short peptide segment in the target molecule. The results obtained so far show that a peptide designed in such a way is an effective inhibitor of the pathway. The possible application of such peptides and antipeptide antibodies as probes for protein kinase regulation mechanisms is also evaluated.

Identification of the catalytic subunit of cAMP-dependent protein kinase from the photosynthetic flagellate, Euglena gracilis Z.

A gene named epk2 that encodes the amino acid sequence of a protein kinase was identified from the photosynthetic flagellate, Euglena gracilis Z. Homology search and phylogenetic analysis revealed that the deduced amino acid sequence of epk2 is most similar to that of the catalytic subunit of cAMP-dependent protein kinase (PKA). Northern blot analysis showed that Euglena cells express a 1.4-kb transcript of this gene. When the EPK2 protein was coexpressed with the rat regulatory subunit of PKA in cultured mammalian cells, these two proteins were coimmunoprecipitated. The association of EPK2 and the rat regulatory subunit of PKA was not detected in the cell lysate incubated with cAMP. EPK2 immunoprecipitated from the transfected cells phosphorylated Kemptide, a synthetic peptide substrate for PKA, and the phosphorylation was inhibited by PKI, a PKA-selective protein kinase inhibitor. These results indicate that EPK2 is a PKA homologue in the photosynthetic flagellate, and this is the first evidence for the occurrence of the PKA catalytic subunit in photosynthetic organisms.

Evidence for the involvement of a Src-related tyrosine kinase in Xenopus egg activation.

Recently, we have purified a Src-related tyrosine kinase, named Xenopus tyrosine kinase (Xyk), from oocytes of Xenopus laevis and found that the enzyme is activated within 1 min following fertilization [Sato et al. (1996) J. Biol. Chem. 271, 13250-13257]. A concomitant translocation of a part of the activated enzyme from the membrane fraction to the cytosolic fraction was also observed. In the present study, we show that parthenogenetic egg activation by a synthetic RGDS peptide [Y. Iwao and T. Fujimura, T. (1996) Dev. Biol. 177, 558-567], an integrin-interacting peptide, but not by electrical shock or the calcium ionophore A23187 causes the kinase activation, tyrosine phosphorylation, and translocation of Xyk. A synthetic tyrosine kinase-specific inhibitor peptide was employed to analyze the importance of the Xyk activity in egg activation. We found that the peptide inhibits the kinase activity of purified Xyk at IC50 of 8 microM. Further, egg activation induced by sperm or RGDS peptide but not by A23187 was inhibited by microinjection of the peptide. In the peptide-microinjected eggs, penetration of the sperm nucleus into the egg cytoplasm and meiotic resumption in the egg were blocked. Indirect immunofluorescence study demonstrates that Xyk is exclusively localized to the cortex of Xenopus eggs, indicating that Xyk can function in close proximity to the sperm-egg or RGDS peptide-egg interaction site. Taken together, these data suggest that the tyrosine kinase Xyk plays an important role in the early events of Xenopus egg activation in a manner independent or upstream of calcium signaling.

Inhibition of MAPK pathway by a synthetic peptide corresponding to the activation segment of MAPK.

Mitogen-activated protein kinase (MAPK) is activated by phosphorylation within its activation segment. Upon phosphorylation, the activation segment refolds to provide the active conformation of the enzyme. We reported previously that a phosphorylation-sensitive secondary structure could be formed in a 26-amino-acid long synthetic peptide corresponding to the activation segment of Xenopus MAPK, termed IDA (Inter-DFG-APE) MAPK peptide (Tokmakov, A. A., et al. 1997, Biochem. Biophys. Res. Commun. 236, 243-247). Here, we show that unphosphorylated IDA MAPK peptide can inhibit in vitro both MAPK and MAPK kinase activities with the inhibition constants of 82 and 18 microM, respectively. Phosphorylated forms of the peptide were of little effect. IDA MAPK peptide did not inhibit significantly the activity of some other protein kinases, including MAPK homologue p38 kinase, suggesting the specificity for MAPK and MAPK kinase. Microinjection of unphosphorylated IDA MAPK peptide into immature Xenopus oocytes significantly suppressed progesterone-induced oocyte maturation by inhibiting activation of both MAPK and maturation promoting factor. Similar inhibition of maturation was registered upon oocyte treatment with another specific inhibitor of MAPK pathway, PD098059. These results depict IDA MAPK peptide as a selective inhibitor of the MAPK pathway that can be used for the investigations of MAPK-mediated signaling.

c-Src and phosphatidylinositol 3-kinase are involved in NGF-dependent tyrosine phosphorylation of Shc in PC12 cells.

The adaptor protein Shc exists in three isoforms; p46, p52, and p66, and is a key regulator of a variety of biological processes. Our previous studies have shown that the tyrosine kinase c-Src phosphorylates Shc in a phosphatidylinositol (PtdIns) 4,5-bisphosphate-dependent manner. Here we demonstrate that PtdIns 3,4,5-trisphosphate stimulates phosphorylation of Shc by c-Src. The phosphorylation is blocked by a glutathione S-transferase fusion protein containing Shc phosphotyrosine binding (PTB) domain or a phosphotyrosine-containing Shc PTB domain-binding peptide. In rat pheochromocytoma cell line PC12, nerve growth factor (NGF) stimulates tyrosine phosphorylation of both Triton-soluble and -insoluble Shc which was maximal at 2-5 min after NGF treatment. We find that pretreatment of PC12 cells with the PtdIns 3-kinase inhibitor wortmannin or LY294002 results in almost half inhibition of the NGF-dependent tyrosine phosphorylation of only Triton-insoluble Shc. Similar inhibitory effect is observed with tyrosine kinase inhibitors genistein and PP1. Upon NGF stimulation, c-Src also becomes tyrosine-phosphorylated and accumulates in the Triton-insoluble fraction. The c-Src events are insensitive to wortmannin but sensitive to genistein. These results suggest that coordinate action of PtdIns 3-kinase and/or PtdIns 3,4,5-trisphosphate and c-Src can function as positive regulator in tyrosine phosphorylation of Shc in vitro and in vivo.

Tyrosine residues 239 and 240 of Shc are phosphatidylinositol 4,5-bisphosphate-dependent phosphorylation sites by c-Src.

In the previous study (Sato K.-I. et al. (1997) FEBS Lett. 410, 136-140), we showed that the phosphorylation of Shc protein by c-Src is dependent on the binding of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to the PTB domain of Shc. In this study, we demonstrate that, in contrast to c-Src, v-Src and epidermal growth factor (EGF) receptor can phosphorylate Shc in a PtdIns(4,5)P2-independent manner and at different phosphorylation sites. To determine the phosphorylation sites in Shc, we used mutant Shc proteins in which tyrosine residues (Y) 317 and/or 239 and 240 were replaced by phenylalanine residues (F). We found that Y317F Shc but not Y239/240F or Y239/240/317F Shc was phosphorylated by c-Src. The reaction was PtdIns(4,5)P2-dependent and inhibited by the addition of PTB domain of Shc. On the other hand, v-Src and EGF receptor were able to phosphorylate both Y317F and Y239/240F but not Y239/240/317F Shc in a PtdIns(4,5)P2-independent manner. These results highlight the difference between c-Src and v-Src or EGF receptor and suggest that c-Src can phosphorylate predominantly on Tyr239/240 of Shc only when Shc PTB domain is bound to PtdIns(4,5)P2.

Fourier transform infrared study on the primary donor P798 of Heliobacterium modesticaldum: cysteine S-H coupled to P798 and molecular interactions of carbonyl groups.

Light-induced Fourier transform infrared (FTIR) difference spectra of the primary donor P798 upon its cation formation (P798(+)/P789) were measured using the membranes and purified RC complex of Heliobacterium modesticaldum. A differential signal at 2550/2560 cm-1 was observed in the difference spectra and assigned to the S-H stretching mode of cysteine by an isotopic shift to 1854/1861 cm-1 upon deuteration. The observed frequencies indicate that this S-H forms a strong hydrogen bond and that the bond is further strengthened upon P798(+) formation. Polarized FTIR difference spectra showed that this S-H group is oriented at <40 degrees with respect to the membrane normal. It was proposed that the cysteine S-H is coupled to P798 through a hydrogen-bond network or by direct hydrogen bonding to either a P798 carbonyl or a ligand histidine. In the carbonyl stretching region, differential signals were observed at 1741/1737, 1725/1718, 1702/1693, and 1687/1666 cm-1. In a dry membrane film, the signal at 1687/1666 cm-1 was mostly lost and hence was assigned to the amide I bands arising from the protein conformational change, which was suppressed upon dehydration of the membranes. The 1702/1693 cm-1 signal was assigned to the 13(1)-keto C&dbd;O of P798, which was free from hydrogen bonding and had a nearly parallel orientation to the membrane plane. The upshift by 9 cm-1 upon P798 oxidation, which is much smaller than upshifts of monomeric (bacterio)chlorophylls [(B)Chls] in organic solution, indicates that the positive charge on P798(+) is significantly delocalized in a BChlg dimer. The signals at 1741/1737 and 1725/1718 cm-1 were assigned to a free and a hydrogen-bonded ester C=O group, respectively. The dichroism measurement showed that the C=O of 1741/1737 cm-1 was oriented nearly parallel to the membrane plane while that of 1725/1718 cm-1 was considerably tilted by <31 degrees to the membrane normal. It was proposed that one of the two ester signals arose from the 13(2)-carbomethoxy C=O of P798 while the other arose either from the 17(2)-ester C=O of P798 or from an ester C&dbd;O of adjacent BChlg or 8(1)-OH-Chla that was electrostatically influenced by oxidation of P798.

Phosphatidylinositol 4,5-bisphosphate stimulates phosphorylation of the adaptor protein Shc by c-Src.

The adaptor protein Shc was prepared as glutathione S-transferase fusion proteins (GST-Shc) and used as in vitro substrate for c-Src. Since phosphotyrosine-binding domain of Shc has been shown to bind phosphatidyl-inositol 4,5-bisphosphate (PtdIns(4,5)P2) [Zhou et al. (1995) Nature 378, 584-592], effect of PtdIns(4,5)P2 on the phosphorylation of GST-Shc by c-Src was examined. PtdIns(4,5)P2 stimulated the phosphorylation of GST-Shc without any effect on the c-Src activity as judged by both its autophosphorylation and phosphorylation of exogenous substrate, Cdc2 peptide. On the other hand, phosphatidylserine, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol 4-phosphate but not phosphatidylcholine stimulated the c-Src activity itself. Km for GST-Shc in the presence of 1 microM PtdIns(4,5)P2 was calculated to be 90 nM. The PtdIns(4,5)P2-dependent phosphorylation of GST-Shc was inhibited by a GST-fusion protein containing the phosphotyrosine-binding domain of Shc. These results suggest that PtdIns(4,5)P2 can act as a regulator of phosphorylation of Shc by c-Src through its binding to Shc.

Role of K+ channels in EDHF-dependent relaxation induced by acetylcholine in canine coronary artery.

To identify the K+ channels responsible for endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation, we studied the effects of various K+ channel blockers on acetylcholine-induced relaxation, which persists even in the presence of both an inhibitor of nitric oxide synthase and that of cyclooxygenase, in canine coronary artery rings. A nonselective K+ channel blocker, tetrabutylammonium (TBA), a large and intermediate conductance Ca2+-activated K+ channel blocker, charybdotoxin (CTX), and a voltage-dependent K+ channel blocker, 4-aminopyridine (4-AP), significantly inhibited this residual relaxation. A combined treatment with CTX and 4-AP almost completely blocked the relaxation. Neither a large (iberiotoxin) nor a small (apamin) conductance Ca2+-activated K+ channel blocker blocked the relaxation. We also investigated effects of K+ channel blockers on basal tone to determine whether or not EDHF is involved in regulating basal tone. TBA and CTX substantially raised basal tone to a greater degree in endothelium-intact preparations than in endothelium-denuded preparations. These results indicate that EDHF may exert its relaxing action through intermediate conductance Ca2+-activated and voltage-dependent K+ channels in canine coronary arteries. In addition, EDHF may play a role in maintaining basal vascular tone.

Purification and characterization of a Src-related p57 protein-tyrosine kinase from Xenopus oocytes. Isolation of an inactive form of the enzyme and its activation and translocation upon fertilization.

In the previous study (Fukami, Y., Sato, K.-I., Ikeda, K., Kamisango, K., Koizumi, K., and Matsuno, T. (1993) J. Biol. Chem. 268, 1132-1140), we found that an antibody termed anti-pepY antibody causes a severalfold activation of bovine brain c-Src. The anti-pepY antibody was raised against a synthetic peptide corresponding to residues 410-428 of chicken c-Src, one of the most conserved regions among the Src family protein-tyrosine kinases. In this study, we have used this antibody as an in vitro activator and purified a c-Src-related protein-tyrosine kinase from the particulate fraction of Xenopus laevis oocytes. A synthetic peptide corresponding to residues 7-26 of fission yeast Cdc2 was used as substrate. Immunoreactivity toward the antibody was also monitored during the purification. The purified kinase displayed a single polypeptide of 57 kDa on SDS-gel electrophoresis and showed a specific activity of 2.37 and 20.1 nmol/min/mg protein in the absence and the presence of the anti-pepY antibody, respectively. The purified enzyme underwent autophosphorylation and phosphorylated actin and the Cdc2 peptide exclusively on tyrosine residues. Specific antibodies against c-Src, Fyn, c-Yes, c-Fgr, Lck, Lyn, Hck, and Blk proteins did not recognize the p57 Xenopus tyrosine kinase. The kinase activity of the Xenopus enzyme was not affected by oocyte maturation but was found to be elevated severalfold upon fertilization. Fertilization also caused a translocation of the activated enzyme from the particulate fraction to the cytosolic fraction. The activation and translocation was observed within 1 min after fertilization. These results suggest a possible involvement of the p57 Xenopus tyrosine kinase in the signal transduction of fertilization.

Biochemical evidence for the interaction of regulatory subunit of cAMP-dependent protein kinase with IDA (Inter-DFG-APE) region of catalytic subunit.

To explore the structural basis required for the holoenzyme formation of cAMP-dependent protein kinase, we have prepared rabbit anti-peptide antibodies that can block the holoenzyme formation without affecting the catalytic activity of the enzyme. The antibodies were raised against a specific site in the catalytic (C)-subunit, termed IDA (Inter-DFG-APE) region, which lies between the kinase subdomains VII and VIII. Although the C-subunit immunoprecipitated with anti-IDA antibodies could not form a stable complex with regulatory (R)-subunit, it was still susceptible to inhibition by the R-subunit or by PKI, a specific inhibitor peptide containing a pseudosubstrate site. These results indicate that there exists an IDA region-mediated interaction between the R- and C-subunits, which is distinct from that mediated through the substrate site and substrate binding site. In accordance with this idea, association of synthetic IDA peptides with the R-subunit was directly demonstrated by resonance mirror analysis. The calculated association constants of IDA peptides were high enough to suggest a possible involvement of the IDA region in the initial step of holoenzyme formation.

c-Src phosphorylates epidermal growth factor receptor on tyrosine 845.

In the previous study [Sato et al. (1995) Biochem. Biophys. Res. Commun. 210, 844-851], we found that c-Src was associated with epidermal growth factor (EGF) receptor and activated upon EGF treatment in A431 cells. In the present study, we investigated the phosphorylation of EGF receptor by c-Src in the c-Src-EGF receptor complex. We have focused our attention to tyrosine residue 845 (Y845) of EGF receptor as a candidate for the phosphorylation site. A synthetic peptide containing Y845, named Y845 peptide, which corresponds to residue 837 to 856 of EGF receptor, was found to be phosphorylated by c-Src and used to provide the standard phosphopeptide. In addition to the autophosphorylated peptide of 25 kDa, a phosphopeptide of 7 kDa was detected in the cyanogen bromide-digested fragments of the c-Src-associated EGF receptor phosphorylated in vitro in an EGF-dependent manner. In phosphopeptide mapping, tryptic digest of the 7-kDa phosphopeptide was shown to co-migrate with that of the phosphorylated Y845 peptide. The 7-kDa phosphopeptide was found to be phosphorylated exclusively on tyrosine. These results suggest that c-Src can phosphorylate EGF receptor on Y845 in an EGF-dependent manner. Furthermore, we confirmed that the same site of the c-Src-associated EGF receptor was phosphorylated in EGF-treated A431 cells.

Site-specific association of c-Src with epidermal growth factor receptor in A431 cells.

We have examined the interaction between c-Src and epidermal growth factor (EGF) receptor in A431 cells. c-Src was found exclusively in the Triton X-100-solubilized particulate fraction and activated up to 3-fold within 1 min after EGF treatment of the cells. Association between c-Src and EGF receptor was detected by immunoprecipitation of c-Src followed by immunoblotting with anti-EGF receptor antibody. The c-Src-EGF receptor complex was found in both EGF-treated and untreated cells, but an augmented complex formation was observed in EGF-treated cells. We have isolated the complex by DEAE-cellulose column chromatography and found that a site-specific anti-c-Src antibody, which was raised against a synthetic peptide corresponding to residues 413 to 431 of human c-Src, did not recognize the c-Src protein in the complex, while other c-Src-specific antibodies tested did. Incubation of the complex with this synthetic peptide resulted in a partial dissociation of the complex. These results suggest that the specific region of c-Src is involved in the association with EGF receptor.