Novel approach for evaluation of estrogenic and anti-estrogenic activities of genistein and daidzein using B16 melanoma cells and dendricity assay

The effects of soy isoflavones, genistein and daidzein, which exhibit estrogenic, anti-estrogenic and/or tyrosine kinase inhibitory activity, on the dendritic morphology of B16 mouse melanoma cells were quantitatively evaluated and compared with those of 17 beta-estradiol (Est) and tyrphostin, a tyrosine kinase inhibitor. Dendricity was significantly stimulated in the order of Est > genistein > daidzein = tyrphostin, but not by glycosides of genistein and daidzein. In competition experiments, Est counteracted the stimulatory activity of genistein and daidzein, but enhanced the activity of tyrphostin additively, suggesting that genistein and daidzein agonized Est. In addition, when the concentration ratios of genistein/Est and daidzein/Est were higher than 5000 and 50,000, respectively, genistein and daidzein agonized Est. In contrast, when the ratio of daidzein/Est was lower than 500, daidzein antagonized Est. Furthermore, genistein and daidzein competed with each other in stimulatory activity. These observations suggest that: 1) dendricity is stimulated by agonists (genistein and daidzein) of Est and tyrosine kinase inhibitors (genistein and tyrphostin), 2) the concentration ratio of isoflavone aglycone/Est is very important as one regulatory factor for estrogenic and/or anti-estrogenic activity, and 3) daidzein antagonizes not only Est but also genistein. It is concluded that a quantitative and simple dendricity assay using B16 mouse melanoma cells is available to evaluate estrogenic and anti-estrogenic activity in vitro.     

Variable effects of tyrosine kinase inhibitors on avian osteoclastic activity and reduction of bone loss in ovariectomized rats

We compared the effects of the tyrosine kinase inhibitor genistein, a naturally occurring isoflavone, to those of tyrphostin A25, tyrphostin A47, and herbimycin on avian osteoclasts in vitro. Inactive analogs daidzein and tyrphostin A1 were used to control for nonspecific effects. None of the tyrosine kinase inhibitors inhibited bone attachment. However, bone resorption was inhibited by genistein and herbimycin with ID₅₀s of 3 μM and 0.1 μM, respectively; tyrphostins and daidzein were inactive at concentrations below 30 μM, where nonspecific effects were noted. Genistein and herbimycin thus inhibit osteoclastic activity via a mechanism independent of cellular attachment, and at doses approximating those inhibiting tyrosine kinase autophosphorylation in vitro; the tyrphostins were inactive at meaningful doses. Because tyrosine kinase inhibitors vary widely in activity spectrum, effects of genistein on cellular metabolic processes were compared to herbimycin. Unlike previously reported osteoclast metabolic inhibitors which achieve a measure of selectivity by concentrating on bone, neither genistein nor herbimycin bound significantly to bone. Osteoclastic protein synthesis, measured as incorporation of ³H-leucine, was significantly inhibited at 10 μM genistein, a concentration greater than that inhibiting bone degradation, while herbimycin reduced protein synthesis at 10 nM. These data suggested that genistein may reduce osteoclastic activity at pharmacologically attainable levels, and that toxic potential was lower than that of herbimycin. To test this hypothesis in a mammalian system, bone mass was measured in 200 g ovariectomized rats treated with 44 μmol/day genistein, relative to untreated controls. During 30 d of treatment, weights of treated and control group animals were indistinguishable, indicating no toxicity, but femoral weight in the treated group was 12% greater than controls (P < 0.05). Our data indicate that the isoflavone inhibitor genistein suppresses osteoclastic activity in vitro and in vivo at concentrations consistent with its ID₅₀s on tyrosine kinases, with a low potential for toxicity. © 1996 Wiley-Liss, Inc.     

Tyrosine kinase-independent inhibition of cyclic-AMP phosphodiesterase by genistein and tyrphostin 51.

The phosphodiesterase activity in the HT4.7 neural cell line was pharmacologically characterized, and phosphodiesterase isozyme 4 (PDE4) was found to be the predominant isozyme. The Km for cAMP was 1-2 microM, indicative of a "low Km" phosphodiesterase, and the activity was inhibited by PDE4-selective inhibitors rolipram and Ro20-1724, but not PDE3- or PDE2-selective inhibitors. Calcium, calmodulin, and cGMP, regulators of PDE1, PDE2, and PDE3, had no effect on cAMP hydrolysis. The protein tyrosine kinase inhibitor, genistein, inhibited HT4.7 cAMP phosphodiesterase activity by 85-95% with an IC50 of 4 microM; whereas daidzein, an inactive structural analog of genistein, had little effect on phosphodiesterase activity. This is a common pharmacological criterion used to implicate the regulation by a tyrosine kinase. However, genistein still inhibited phosphodiesterase activity with a mixed pattern of inhibition even when ion-exchange chromatography was used to partially purify phosphodiesterase away from the tyrosine kinase activity. Moreover, tyrphostin 51, another tyrosine kinase inhibitor, was found to also inhibit partially purified phosphodiesterase activity noncompetitively. These data suggest that HT4.7 phosphodiesterase activity is dominated by PDE4 and can be regulated by genistein and tyrphostin 51 by a tyrosine kinase-independent mechanism.     

Oxysterol Regulation of Estrogen Receptor α-Mediated Gene Expression in a Transcriptional Activation Assay System Using HeLa Cells

In order to test the estrogenic activity of sterol oxidation products from cholesterol and phytosterols, an estrogen-dependent gene expression assay was performed in estrogen receptor α-stably transformed HeLa cells. The ranking of the estrogenic potency of these compounds was different: 17β-estradiol >> genistein >> β-epoxycholesterol = daidzein = cholestanetriol = 22(R)-hydroxycholesterol = 20(S)-hydroxycholesterol = sitostanetriol > campestanetriol = β-epoxysitosterol = 7β-hydroxycholesterol. These compounds were not estrogenic in estrogen receptor-negative HeLa cells.     

Tyrosine Kinases Are Required for Catecholamine Secretion and Mitogen-Activated Protein Kinase Activation in Bovine Adrenal Chromaffin Cells

Abstract: Nicotine-induced catecholamine secretion in bovine adrenomedullary chromaffin cells is accompanied by rapid tyrosine phosphorylation of multiple cellular proteins, most notably the mitogen-activated protein kinases (MAPKs). The requirement for activation of tyrosine kinases and MAPKs in chromaffin cell exocytosis was investigated using a panel of tyrosine kinase inhibitors. Genistein and tyrphostin 23, two compounds that inhibit tyrosine kinases by distinct mechanisms, were found to inhibit secretion by >90% in cells stimulated by nicotine, 55 m M KCI, or the Ca²⁺ ionophore A23187. Inhibition of secretion induced by all three secretagogues correlated with a block in both protein tyrosine phosphorylation and activation of the MAPKs and their activators (MEKs) in situ. However, neither genistein nor tyrphostin 23 inhibited the activities of the MAPKs or MEKs in vitro. These results indicate that the target(s) of inhibition lie down-stream of Ca²⁺ influx and upstream of MEK activation. This Ca²⁺-activated tyrosine kinase activity could not be accounted for entirely by c-Src or Fyn (two nonreceptor tyrosine kinases that are expressed abundantly in chromaffin cells), because their in vitro kinase activities were not inhibited by tyrphostin 23 and only partially inhibited by genistein. These results demonstrate that an unidentified Ca²⁺-activated tyrosine kinase(s) is required for MAPK activation and exocytosis in chromaffin cells and suggest that MAPK participates in the regulation of secretion.     

Differential effects of tyrosine kinase inhibitors on volume-sensitive chloride current in human atrial myocytes: evidence for dual regulation by Src and EGFR kinases

To determine whether protein tyrosine kinase (PTK) modulates volume-sensitive chloride current (I(Cl.vol)) in human atrial myocytes and to identify the PTKs involved, we studied the effects of broad-spectrum and selective PTK inhibitors and the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (VO(4)(-3)). I(Cl.vol) evoked by hyposmotic bath solution (0.6-times isosmotic, 0.6T) was enhanced by genistein, a broad-spectrum PTK inhibitor, in a concentration-dependent manner (EC(50) = 22.4 microM); 100 microM genistein stimulated I(Cl.vol) by 122.4 +/- 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 150 microM) and tamoxifen (20 microM), blockers of I(Cl.vol). Moreover, the current augmented by genistein was volume dependent; it was abolished by hyperosmotic shrinkage in 1.4T, and genistein did not activate Cl(-) current in 1T. In contrast to the stimulatory effects of genistein, 100 microM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited I(Cl.vol) by 38.2 +/- 4.9% and 40.9 +/- 3.4%, respectively. The inactive analogs, daidzein and tyrphostin A63 (AG 43), did not alter I(Cl.vol). In addition, the PTP inhibitor VO(4)(-3) (1 mM) reduced I(Cl.vol) by 53.5 +/- 4.5% (IC(50) = 249.6 microM). Pretreatment with VO(4)(-3) antagonized genistein-induced augmentation and A23- or A25-induced suppression of I(Cl.vol). Furthermore, the selective Src-family PTK inhibitor PP2 (5 microM) stimulated I(Cl.vol), mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 microM) reduced I(Cl.vol), mimicking A23 and A25. The effects of both PP2 and B56 also were substantially antagonized by pretreatment with VO(4)(-3). The results suggest that I(Cl.vol) is regulated in part by the balance between PTK and PTP activity. Regulation is complex, however. Src and EGFR kinases, distinct soluble and receptor-mediated PTK families, have opposing effects on I(Cl.vol), and multiple target proteins are likely to be involved.     

Genistein inhibits the inward rectifying potassium current in guinea pig ventricular myocytes

Genistein is an isoflavone with potent inhibitory activity on protein tyrosine kinase. Previous studies have shown that genistein has additional effects, among which the direct blocking effects on various ionic channels have recently been disclosed. Using whole-cell voltage clamp and current clamp techniques, we demonstrate that micromolar concentrations of genistein dose-dependently and reversibly inhibit the inward rectifying K(+) current, and depolarize the resting membrane potential, resulting in abnormal automaticity in guinea pig ventricular myocytes. Interestingly, another potent tyrosine kinase inhibitor, tyrphostin 51, did not produce the same inhibitory effect, while the inactive analogue of genistein, daidzein, had a similar blocking effect. We suggest that genistein directly blocks the inward rectifying K(+) current in ventricular myocytes, and one should be cautious of its pro-arrhythmic effect in clinical use.     

Potentiation of Agonist-Stimulated Cyclic AMP Accumulation by Tyrosine Kinase Inhibitors in Rat Pinealocytes

Abstract: To study cross-talk mechanisms in rat pinealocytes, the role of tyrosine kinase or kinases in the regulation of adrenergic-stimulated cyclic AMP production was investigated. Both norepinephrine- and isoproterenol-stimulated cyclic AMP accumulation were increased by two distinct tyrosine kinase inhibitors, genistein or erbstatin, in a concentration-dependent manner. A similar increase was observed with two other inhibitors, tyrphostin B44 and herbimycin. In contrast, daidzein, an inactive analogue of genistein, was ineffective; whereas vanadate, a phosphotyrosine phosphatase inhibitor, reduced the adrenergic-stimulated cyclic AMP accumulation. The tyrosine kinase inhibitors were effective in potentiating the cholera toxin-or forskolin-stimulated cyclic AMP accumulation, indicating that their sites of action are at the postreceptor level. Neither an activator nor inhibitors of protein kinase C influenced the potentiation of the cyclic AMP responses by genistein, suggesting that the potentiation effect by tyrosine kinase inhibitors does not involve the phospholipase C/protein kinase C pathway. However, when the phosphodiesterase was inhibited by isobutylmethylxanthine, genistein failed to potentiate and vanadate did not inhibit the adrenergic-stimulated cyclic AMP accumulation, indicating that the phosphodiesterase is a probable site of action for these inhibitors. These results suggest that cyclic AMP metabolism in the pinealocytes is tonically inhibited by tyrosine kinase acting on the cyclic AMP phosphodiesterase.     

Functional characterization of peanut serine/threonine/tyrosine protein kinase: molecular docking and inhibition kinetics with tyrosine kinase inhibitors

Serine/threonine/tyrosine (STY) protein kinase from peanut is developmentally regulated and is induced by abiotic stresses. In addition, STY protein kinase activity is regulated by tyrosine phosphorylation. Kinetic mechanism of plant dual specificity protein kinases is not studied so far. Recombinant STY protein kinase occurs as a monomer in solution as shown by gel filtration chromatography. The relative phosphorylation rate of kinase against increasing enzyme concentrations follows a first-order kinetics indicating an intramolecular phosphorylation mechanism. Moreover, the active recombinant STY protein kinase could not transphosphorylate a kinase-deficient mutant of STY protein kinase. Molecular docking studies revealed that the tyrosine kinase inhibitors bind the protein kinase at the same region as ATP. STY protein kinase activity was inhibited by the tyrosine kinase inhibitors, and the inhibitor potency series against the recombinant STY protein kinase was tyrphostin > genistein > staurosporine. The inhibition constant (K(i)), and the IC(50) value of STY protein kinase for tyrosine kinase inhibitors with ATP and histone are discussed. All the inhibitors competed with ATP. Genistein was an uncompetitive inhibitor with histone, whereas staurosporine and tyrphostin were linear mixed type noncompetitive inhibitors with histone. Molecular docking and kinetic analysis revealed that Y148F mutant of the "ATP-binding loop" and Y297F mutant of the "activation loop" showed a dramatic increase in K(i) values for genistein and tyrphostin with respect to wild-type STY protein kinase. Data presented here provide the direct evidence on the mechanism of inhibition of plant protein kinases by tyrosine kinase inhibitors. This study also suggests that tyrosine kinase inhibitors may be useful in unraveling the plant tyrosine phosphorylation signaling cascades.     

Tumor necrosis factor alpha-induced activation of downstream NF-kappaB site of the promoter mediates epithelial ICAM-1 expression and monocyte adhesion. Involvement of PKCalpha, tyrosine kinase, and IKK2, but not MAPKs, pathway

TNF-alpha induced an increase in intercellular adhesion molecule-1 (ICAM-1) expression in human A549 epithelial cells and immunofluorescence staining confirmed this result. The enhanced ICAM-1 expression was shown to increase the adhesion of U937 cells to A549 cells. Tyrosine kinase inhibitors (genistein or tyrphostin 23) or phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D 609) attenuated TNF-alpha-induced ICAM-1 expression. TNF-alpha produced an increase in protein kinase C (PKC) activity and this effect was inhibited by D 609. PKC inhibitors (staurosporine, Ro 31-8220, calphostin C, or Go 6976) also inhibited TNF-alpha-induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression, this effect was inhibited by genistein or tyrphostin 23. Treatment of cells with TNF-alpha resulted in stimulation of p44/42 MAPK, p38, and JNK. However, TNF-alpha-induced ICAM-1 expression was not affected by either MEK inhibitor, PD 98059, or p38 inhibitor, SB 203580. A cell-permeable ceramide analog, C(2) ceramide, also stimulated the activation of these three MAPKs, but had no effect on ICAM-1 expression. NF-kappaB DNA-protein binding and ICAM-1 promoter activity were enhanced by TNF-alpha and these effects were inhibited by D 609, calphostin C, or tyrphostin 23, but not by PD 98059 or SB 203580. TPA also stimulated NF-kappaB DNA-protein binding and ICAM-1 promoter activity, these effects being inhibited by genistein or tyrphostin 23. TNF-alpha- or TPA-induced ICAM-1 promoter activity was inhibited by dominant negative PKCalpha or IKK2, but not IKK1 mutant. IKK activity was stimulated by both TNF-alpha and TPA, and these effects were inhibited by Ro 31-8220 or tyrphostin 23. These data suggest that, in A549 cells, TNF-alpha activates PC-PLC to induce activation of PKCalpha and protein tyrosine kinase, resulting in the stimulation of IKK2, and NF-kappaB in the ICAM-1 promoter, then initiation of ICAM-1 expression and neutrophil adhesion. However, activation of p44/42 MAPK, p38, and JNK is not involved in this event.     

Tyrosine kinase inhibitors and the contractile action of epidermal growth factor-urogastrone and other agonists in gastric smooth muscle.

We examined the effects of the tyrosine kinase (TK) inhibitors, genistein, and tyrphostin (RG-50864) on the contractile action of epidermal growth factor - urogastrone (EGF-URO), transforming growth factor-alpha (TGF-alpha), and other agonists in two smooth muscle bioassay systems (guinea pig gastric longitudinal muscle, LM, and circular muscle, CM). We also studied the inhibition by tyrphostin of EGF-URO stimulated protein phosphorylation in identical smooth muscle strips. The selective inhibition by genistein and tyrphostin of EGF-URO and TGF-alpha induced contraction, but not of carbachol- and bradykinin-mediated contraction, occurred at much lower concentrations (genistein, less than 7.4 microM (2 micrograms/mL); tyrphostin, less than 20 microM (4 micrograms/mL)) than those used in previously published studies with these TK inhibitors. In LM tissue, the IC50 values were for genistein 1.1 +/- 0.1 microM (0.30 micrograms/mL; mean +/- SEM) and 3.6 +/- 0.5 microM (0.74 micrograms/mL) for tyrphostin, yielding a molar potency ratio (GS: TP) of 1:3 in the longitudinal preparation. In CM tissue, the IC50 values were 3.0 +/- 0.3 microM (0.81 micrograms/mL) for genistein and 2.4 +/- 0.2 microM (0.49 micrograms/mL) for tyrphostin, yielding a molar potency ratio (GS:TP) of 1.0:0.8 in the circular strips. The inhibition by genistein and tyrphostin of EGF-URO and TGF-alpha mediated contraction was rapid (beginning within minutes) and was reversible upon washing the preparations free from the enzyme inhibitors. In intact tissue strips studied under bioassay conditions, tyrphostin (40 microM) also blocked EGF-URO triggered phosphorylation of substrates detected on Western blots using monoclonal antiphosphotyrosine antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of domestic cat spermatozoa acrosome reaction and zona pellucida penetration by tyrosine kinase inhibitors

Spermatozoa from teratospermic domestic cats (>60% morphologically abnormal spermatozoa per ejaculate) consistently exhibit lower levels of oocyte penetration in vitro than their normospermic (<40% abnormal spermatozoa per ejaculate) counterparts. This could be caused by structural abnormalities or intracellular defects resulting in disruption of normal cellular functions. Spermatozoa from teratospermic cats also are compromised in the ability to capacitate and undergo the acrosome reaction (AR) in vitro. Further, we recently identified two tyrosine phosphorylated proteins (95- and 160-kDa) localized over the acrosome region in domestic cat spermatozoa. Phosphorylation of these proteins is reduced in teratospermic compared with normospermic ejaculates. To begin to understand the relationship between tyrosine phosphorylation and sperm function, we examined the effects of two protein tyrosine kinase inhibitors (tyrphostin RG-50864 and genistein) on (1) sperm motility; (2) protein tyrosine phosphorylation; (3) the ionophore A23187-induced AR; (4) the spontaneous and zona pellucida (ZP)–induced AR, and (5) the ability of spermatozoa from normospermic cats to penetrate conspecific ZP-intact oocytes. Over a wide range of concentrations, neither inhibitor affected sperm percentage motility during incubation (P > 0.05). Preincubation with either inhibitor reduced tyrosine phosphorylation of both (95- and 160-kDa) sperm proteins. Although both inhibitors blocked the ZP-induced AR, neither influenced the spontaneous AR nor the A23187-induced AR, suggesting that tyrosine phosphorylation may be involved in physiologic AR. No differences (P > 0.05) were observed in the ability of control or inhibitor-treated spermatozoa to bind to or penetrate the outer ZP layer. However, percentages of oocytes with treated spermatozoa in the inner ZP (tyrphostin, 8.7%; genistein, 20.4%) and perivitelline space (tyrphostin, 0%; genistein, 2.3%) were less (P < 0.001) than untreated controls (inner ZP, 62.7%; perivitelline space, 10.2%). These results (1) demonstrate that ZP-induced acrosomal exocytosis in domestic cat spermatozoa is regulated via a tyrosine kinase–dependent pathway and (2) suggest that defects in these signaling pathways may represent one of the causes for compromised sperm function in teratospermic males. Mol. Reprod. Dev. 49:48–57, 1998. © 1998 Wiley-Liss, Inc.     

Effects of genistein and lavendustin on reproductive processes in domestic animals in vitro

The aim of our experiments was to study the influence of genistein [tyrosine kinase (TK) inhibitor with estrogenic activity] and lavendustin A (TK inhibitor without estrogenic activity) on female reproductive processes in domestic animals in vitro. It was found that genistein (0.001–1 μg/ml) increased IGF-I release by cultured bovine and porcine granulosa cells, but decreased its secretion by rabbit granulosa cells (0.01–10 μg/ml). Genistein stimulated progesterone secretion by bovine and rabbit granulosa cells (at 0.01–10 μg/ml), estradiol output by rabbit granulosa cells (at 1 μg/ml) and porcine ovarian follicles (at 10 μg/ml), as well as cAMP production by bovine (at 0.001–1 μg/ml) and rabbit (at 1 μg/ml) granulosa cells. No effects of genistein (at 10 μg/ml) on PGF-2 alpha and progesterone release by porcine ovarian follicles were observed. Genistein significantly (P < 0.05) stimulated the reinitiation and completion of nuclear maturation of porcine oocytes (at 5 μg/ml), as well as the preimplantation development of rabbit zygotes (at 1 μg/ml). Lavendustin A (0.001–1 μg/ml) increased IGF-I release by bovine (but not by porcine) granulosa cells, cAMP release by bovine granulosa cells, and PGF-2 alpha output by porcine ovarian follicles (at 10 μg/ml). Lavendustin (at 1 μg/ml) had no significant effect on IGF-I release by porcine granulosa cells, on estradiol and cAMP output by rabbit granulosa cells, or on progesterone secretion by porcine follicles (at 10 μg/ml). Inhibitory actions of lavendustin (at 10 μg/ml) on estradiol secretion by porcine follicles were also found. Furthermore, lavendustin, like genistein, promoted the reinitiation and completion of meiosis in porcine oocytes. The present study demonstrates a predominantly stimulatory effect of TK inhibition on endocrine and generative processes in domestic animals. The majority of these effects are similar for both compounds, indirectly suggesting that their action is due to tyrosine kinase inhibition and protein kinase A-stimulation, rather than estrogenic activity.     

Inhibition of ovulation by tyrosine kinase inhibitors in the in vitro perfused rat ovary

Protein tyrosine kinase activity, leading to tyrosine phosphorylation of the intracellular domains of receptors or non-receptor proteins, is an important feature of downstream signalling after receptor binding of a variety factors, such as growth factors and cytokines. Since several members of these classes of paracrine-autocrine mediator may be involved in the intraovarian events of ovulation, the present study was designed to evaluate the effect of protein tyrosine kinase inhibition on the in vitro perfused rat ovary. Immature rats were primed with 20 iu pregnant mares' serum gonadotrophin 48 h before surgical isolation of the right ovary with connecting vasculature. The ovary was placed in a perfusion system for either 10 h, to examine ovarian concentrations of the established ovulatory mediators plasminogen activator, prostaglandins E2 and F2 alpha, or for 20 h, enabling a complete ovulatory process to occur in vitro. Ovulation was induced by ovine LH (0.2 microgram ml-1) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2 mmol l-1) and the effects of two different protein tyrosine kinase inhibitors, genistein and tyrphostin A25, were studied. Unstimulated control ovaries did not ovulate and showed low secretion of progesterone and oestradiol. Addition of LH + 3-isobutyl-1-methylxanthine resulted in a marked stimulation of steroid release, and ovulations occurred in all ovaries (9.0 +/- 0.9; mean +/- SEM). The protein tyrosine kinase inhibitors, genistein and tyrphostin A25, significantly inhibited ovulation at the higher concentrations tested (3.0 +/- 0.3 at 100 mumol genistein l-1; 5.8 +/- 1.0 at 500 mumol tyrphostin A25 l-1) but no effect was seen at lower concentrations. The presence of genistein and tyrphostin A25 at any concentration used did not significantly decrease the LH + 3-isobutyl-1-methylxanthine-induced progesterone or oestradiol concentrations. The intraovarian concentrations of plasminogen activator activity, and prostaglandin E2 and F2 alpha were not altered by the presence genistein (100 mumol l-1). In conclusion, the results of the present study indicate that protein tyrosine kinase signalling pathways are integral parts of the mammalian ovulatory process but do not involve actions on the synthesis of steroids, plasminogen activator or prostaglandins.     

Tyrphostin AG556 increases the activity of large conductance Ca2+‐activated K+ channels by inhibiting epidermal growth factor receptor tyrosine kinase

The present study was designed to investigate whether large conductance Ca²⁺‐activated K⁺ (BK) channels were regulated by epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase. BK current and channel tyrosine phosphorylation level were measured in BK‐HEK 293 cells expressing both functional α‐subunits and the auxiliary β1‐subunits using electrophysiology, immunoprecipitation and Western blotting approaches, respectively, and the function of rat cerebral basilar arteries was determined with a wire myography system. We found that BK current in BK‐HEK 293 cells was increased by the broad spectrum protein tyrosine kinase (PTK) inhibitor genistein and the selective EGFR tyrosine kinase inhibitor AG556, one of the known tyrphostin. The effect of genistein or AG556 was antagonized by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. On the other hand, orthovanadate or EGF decreased BK current, and the effect was counteracted by AG556. The tyrosine phosphorylation level of BK channels (α‐ and β1‐subunits) was increased by EGF and orthovanadate, while decreased by genistein and AG556, and the reduced tyrosine phosphorylation of BK channels by genistein or AG556 was reversed by orthovanadate. Interestingly, AG556 induced a remarkable enhancement of BK current in rat cerebral artery smooth muscle cells and relaxation of pre‐contracted rat cerebral basilar arteries with denuded endothelium, and these effects were antagonized by the BK channel blocker paxilline or orthovanadate. These results demonstrate that tyrosine phosphorylation of BK channels by EGFR kinase decreases the channel activity, and inhibition of EGFR kinase by AG556 enhances the channel activity and dilates rat cerebral basilar arteries.     

Mechanisms of ADRF release from rat aortic adventitial adipose tissue

Blood vessels are surrounded by variable amounts of adipose tissue. We showed earlier that adventitial adipose tissue inhibits rat aortic contraction by release of a transferable factor, adventitium-derived relaxing factor (ADRF), which activates smooth muscle K(+) channels. However, little is known about the mechanisms of ADRF release. Using isolated rat aortic rings and isometric contraction measurements, we show that ADRF release depends on extracellular [Ca(2+)] (EC(50) approximately 4.7 mM). ADRF effects do not involve neuronal presynaptic N-type Ca(2+) and Na(+) channels or vanilloid, cannabinoid, and CGRP receptors. ADRF release is strongly inhibited by the protein tyrosine kinase inhibitors genistein and tyrphostin A25. In contrast, daidzein, an inactive genistein analog, and the protein tyrosine kinase inhibitor ST638 had no effect. Protein kinase A inhibition by H89 also inhibited ADRF release, whereas the protein kinase G inhibitor KT-5823 had no effect. We propose that ADRF release is Ca(2+) dependent and is regulated by intracellular signaling pathways involving tyrosine kinase and protein kinase A. Furthermore, ADRF release does not depend on perivascular nerve endings.     

Calcium influx induced by activation of tyrosine kinase receptors in cultured bovine aortic endothelial cells

We studied the ionic currents activated by basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I) in cultured bovine aortic endothelial cells (BAE-1) by using patch-clamp and single-cell fluorimetric calcium measurements. In whole-cell, voltage-clamp experiments at V(h) = -50 mV, the addition of either bFGF (20 ng/ml) or IGF-I (50 ng/ml) induced an inward current with similar amplitude, time course, and permeation properties. The response was dependent on receptor occupancy and showed a desensitisation in the continued presence of the factors. Ionic substitutions in whole-cell experiments indicated that the current barely discriminated among Na(+), Ca(+), and K(+) ions. Accordingly, stimulation with bFGF or IGF-I induced a dose-dependent [Ca(2+)](i) elevation completely due to entry from the extracellular medium, whereas no detectable release from internal stores was observed. Calcium influx was dependent on protein tyrosine kinase (PTK) activity; it was significantly inhibited by treatment with genistein or tyrphostin 47, two PTK inhibitors, and not affected by inactive analogues, daidzein, and tyrphostin 1. Moreover, addition of 200 microM Na(3)VO(4), an inhibitor of protein tyrosine phosphatase (PTP) activity, evoked the responses to the factors both in patch-clamp and in fluorimetric measurements. Cell-attached recordings using 100 mM CaCl(2) in the pipette showed that bFGF and IGF-I activate calcium-permeable channels with similar properties. These results provide evidence for a calcium influx induced by two factors that bind to tyrosine kinase receptors (RTK) in endothelial cells.     

Inhibition of Kv4.3 by genistein via a tyrosine phosphorylation-independent mechanism

The effects of genistein, a protein tyrosine kinase (PTK) inhibitor, on voltage-dependent K(+) (Kv) 4.3 channel were examined using the whole cell patch-clamp techniques. Genistein inhibited Kv4.3 in a reversible, concentration-dependent manner with an IC(50) of 124.78 μM. Other PTK inhibitors (tyrphostin 23, tyrphostin 25, lavendustin A) had no effect on genistein-induced inhibition of Kv4.3. Orthovanadate, an inhibitor of protein phosphatases, did not reverse the inhibition of Kv4.3 by genistein. We also tested the effects of two inactive structural analogs: genistin and daidzein. Whereas Kv4.3 was unaffected by genistin, daidzein inhibited Kv4.3, albeit with a lower potency. Genistein did not affect the activation and inactivation kinetics of Kv4.3. Genistein-induced inhibition of Kv4.3 was voltage dependent with a steep increase over the channel opening voltage range. In the full-activation voltage range positive to +20 mV, no voltage-dependent inhibition was found. Genistein had no significant effect on steady-state activation, but shifted the voltage dependence of the steady-state inactivation of Kv4.3 in the hyperpolarizing direction in a concentration-dependent manner. The K(i) for the interaction between genistein and the inactivated state of Kv4.3, which was estimated from the concentration-dependent shift in the steady-state inactivation curve, was 1.17 μM. Under control conditions, closed-state inactivation was fitted to a single exponential function, and genistein accelerated closed-state inactivation. Genistein induced a weak use-dependent inhibition. These results suggest that genistein directly inhibits Kv4.3 by interacting with the closed-inactivated state of Kv4.3 channels. This effect is not mediated via inhibition of the PTK activity, because other types of PTK inhibitors could not prevent the inhibitory action of genistein.