Composite polarization systems for independent controlling polarization of two beams with different wavelengths.

The usage of independent and simultaneous control of the state of light polarization at different wavelengths can expand the capabilities of polarization methods for biomedical application. Unfortunately, all known methods of polarization conversion cannot convert the state of light polarization at different wavelengths independently. We propose a method and device for independent and simultaneous control of the polarization state at two wavelengths. We have theoretically proved the possibility of maintaining the phase shift at the first wavelength unchanged while simultaneously and independently changing the phase shift at the second wavelength from 0 to 180 degrees. The capabilities of the method were for the first time demonstrated for radiation with wavelengths λ = 632.8 nm and λ = 488 nm. At the wavelength λ = 632.8 nm, the phase shift remained equal to 180° whereas at the wavelength λ = 488 nm, it varied in the range from 121° to 136°.

Collagenase-2 and -3 mediate epidermal growth factor receptor transactivation by bradykinin B2 receptor in kidney cells.

We have previously shown that stimulation of extracellular signal-regulated protein kinase (ERK) by bradykinin (BK) in murine inner medullary collecting duct (mIMCD)-3 cells is mediated by epidermal growth factor receptor (EGFR) transactivation. The mechanism of EGFR transactivation seemed to be novel, because it does not require phospholipase C, Ca(2+), calmodulin, protein kinase C, G alpha(i) subunits, or EGFR-B(2) receptor heterodimerization. In this study, we demonstrated the involvement of matrix metalloproteinases (MMPs) in B(2) receptor-induced EGFR transactivation using their broad-spectrum inhibitors batimastat and N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (Galardin) (GM-6001). Selective inhibitors for collagenase-2 and -3 (MMP-8 and MMP-13, respectively) blocked BK-induced EGFR phosphorylation and ERK activation, whereas inhibitors for MMP-1, -2, -3, -7, or -9 were without effect. Transfection of mIMCD-3 cells with MMP-8 small interfering RNA (siRNA) resulted in approximately 50% decrease of BK-induced ERK activation. A neutralizing antibody against MMP-13 as well as transfection with MMP-13 siRNA produced a similar effect. Inhibition of both collagenases resulted in approximately 65% decrease of BK-induced ERK activation, supporting roles for both enzymes. Stimulation of mIMCD-3 cells with 10 nM BK increased the activity of collagenases in concentrated culture media within 10 min. Moreover, recombinant MMP-13 and MMP-8, when applied to mIMCD-3 cells for 10 min without BK, stimulated tyrosine phosphorylation of EGFR and caused approximately 250% increase over basal ERK phosphorylation comparable with BK-induced ERK activation. Collagenases-induced ERK activation was inhibited by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478) and thus dependent on EGFR tyrosine kinase activity. This study demonstrates a novel role for collagenase-2 and -3 in signaling of the G(q)-coupled BK B(2) receptor in mIMCD-3 cells.

Mitogen-induced rapid phosphorylation of serine 795 of the retinoblastoma gene product in vascular smooth muscle cells involves ERK activation.

We examined the relationship between mitogen-activated MEK (mitogen and extracellular signal-regulated protein kinase kinase) and phosphorylation of the gene product encoded by retinoblastoma (hereafter referred to as Rb) in vascular smooth muscle cells. Brief treatment of the cells with 100 nm angiotensin II or 1 microm serotonin resulted in serine phosphorylation of Rb that was equal in magnitude to that induced by treating cells for 20 h with 10% fetal bovine serum ( approximately 3 x basal). There was no detectable rapid phosphorylation of two close cousins of Rb, p107 and p130. Phosphorylation state-specific antisera demonstrated that the rapid phosphorylation occurred on Ser(795), but not on Ser(249), Thr(252), Thr(373), Ser(780), Ser(807), or Ser(811). Phosphorylation of Rb Ser(795) peaked at 10 min, lagging behind phosphorylation of MEK and ERK (extracellular signal-regulated protein kinase). Rb Ser(795) phosphorylation could be blocked by PD98059, a MEK inhibitor, and greatly attenuated by apigenin, an inhibitor of the Ras --> Raf --> MEK --> ERK pathway. The effect also appears to be mediated by CDK4. Immunoprecipitation/immunoblot studies revealed that serotonin and angiotensin II induced complex formation between CDK4, cyclin D1, and phosphorylated ERK. These studies show a rapid, novel, and selective phosphorylation of Rb Ser(795) by mitogens and demonstrate an unexpected rapid linkage between the actions of the Ras --> Raf --> MEK --> ERK pathway and the phosphorylation state of Rb.

Evidence for multiple P2Y receptors in trabecular meshwork cells.

The purpose of this study was to determine whether functional purinergic P2 receptors are present in trabecular meshwork cells. The human trabecular cell line HTM-3 and cultured bovine trabecular cells were used to assess the effects of P2 agonists on intracellular Ca(2+) levels, extracellular signal-regulated kinase (ERK1/2) activation, and P2Y receptor expression. ATP, UTP, ADP, and 2-methyl-thio-adenosine triphosphate (2-MeS-ATP) each produced a concentration-dependent increase in intracellular Ca(2+) in bovine trabecular cells and the HTM-3 cell line. The addition of UDP did not produce any detectable rise in intracellular Ca(2+). Pretreatment with the P2Y(1) receptor antagonist 2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate (MRS-2179) blocked the ADP- and 2-MeS-ATP-induced rise in intracellular Ca(2+). However, the ATP- or UTP-induced rise in intracellular Ca(2+) was not inhibited by MRS-2179 pretreatment. The addition of ADP, 2-MeS-ATP, ATP, or UTP were also found to activate the ERK1/2 signaling pathway. This activation of ERK1/2 was blocked by pretreatment with the mitogen-activated protein kinase kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene (U-0126) or the protein kinase C inhibitor chelerythrine chloride, but not by MRS-2179. Analysis of mRNA from HTM-3 cells by reverse transcription-polymerase chain reaction revealed the expression of P2Y(1), P2Y(4), and P2Y(11) receptor subtypes. These data demonstrate that multiple P2Y receptors are present in trabecular cells. Our results are consistent with the idea that the mobilization of intracellular Ca(2+)results from the activation of P2Y(1) and P2Y(4) receptors, whereas the activation of the ERK1/2 pathway results from the activation of P2Y(4) receptors alone. However, a role for the P2Y(11) receptors in mobilization of Ca(2+), or activation of the ERK1/2 pathway, cannot be discounted.

ERK is regulated by sodium-proton exchanger in rat aortic vascular smooth muscle cells.

The purposes of this study were to test 1) the relationship between two widely studied mitogenic effector pathways, and 2) the hypothesis that sodium-proton exchanger type 1 (NHE-1) is a regulator of extracellular signal-regulated protein kinase (ERK) activation in rat aortic smooth muscle (RASM) cells. Angiotensin II (Ang II) and 5-hydroxytryptamine (5-HT) stimulated both ERK and NHE-1 activities, with activation of NHE-1 preceding that of ERK. The concentration-response curves for 5-HT and Ang II were superimposable for both processes. Inhibition of NHE-1 with pharmacological agents or by isotonic replacement of sodium in the perfusate with choline or tetramethylammonium greatly attenuated ERK activation by 5-HT or Ang II. Similar maneuvers significantly attenuated 5-HT- or Ang II-mediated activation of MEK and Ras but not transphosphorylation of the epidermal growth factor (EGF) receptor. EGF receptor blockade attenuated ERK activation, but not NHE-1 activation by 5-HT and Ang II, suggesting that the EGF receptor and NHE-1 work in parallel to stimulate ERK activity in RASM cells, converging distal to the EGF receptor but at or above the level of Ras in the Ras-MEK-ERK pathway. Receptor-independent activation of NHE-1 by acute acid loading of RASM cells resulted in the rapid phosphorylation of ERK, which could be blocked by pharmacological inhibitors of NHE-1 or by isotonic replacement of sodium, closely linking the proton transport function of NHE-1 to ERK activation. These studies identify NHE as a new regulator of ERK activity in RASM cells.

Mitogen-induced activation of Na+/H+ exchange in vascular smooth muscle cells involves janus kinase 2 and Ca2+/calmodulin.

The sodium/proton exchanger type 1 (NHE-1) plays an important role in the proliferation of vascular smooth muscle cells (VSMC). We have examined the regulation of NHE-1 by two potent mitogens, serotonin (5-HT, 5-hydroxytryptamine) and angiotensin II (Ang II), in cultured VSMC derived from rat aorta. 5-HT and Ang II rapidly activated NHE-1 via their G protein-coupled receptors (5-HT(2A) and AT(1)) as assessed by proton microphysiometry of quiescent cells and by measurements of intracellular pH on a FLIPR (fluorometric imaging plate reader). Activation of NHE-1 was blocked by inhibitors of phospholipase C, CaM, and Jak2 but not by pertussis toxin or inhibitors of protein kinase C. Immunoprecipitation/immunoblot studies showed that 5-HT and Ang II induce phosphorylation of Jak2 and induce the formation of signal transduction complexes that included Jak2, CaM, and NHE-1. The cell-permeable Ca(2+) chelator BAPTA-AM blocked activation of Jak2, complex formation between Jak2 and CaM, and tyrosine phosphorylation of CaM, demonstrating that elevated intracellular Ca(2+) is essential for those events. Thus, mitogen-induced activation of NHE-1 in VSMC is dependent upon elevated intracellular Ca(2+) and is mediated by the Jak2-dependent tyrosine phosphorylation of CaM and subsequent increased binding of CaM to NHE-1, similar to the pathway previously described for the bradykinin B(2) receptor in inner medullary collecting duct cells of the kidney [Mukhin, Y. V., et al. (2001) J. Biol. Chem. 276, 17339-17346]. We propose that this pathway represents a fundamental mechanism for the rapid regulation of NHE-1 by G(q/11) protein-coupled receptors in multiple cell types.

Hypertonicity activates Na+/H+ exchange through Janus kinase 2 and calmodulin.

The type 1 sodium-hydrogen exchanger (NHE-1) is a ubiquitous electroneutral membrane transporter that is activated by hypertonicity in many cells. NHE-1 may be an important pathway for Na(+) entry during volume restoration, yet the molecular mechanisms underlying the osmotic regulation of NHE-1 are poorly understood. In the present study we conducted a screen for important signaling molecules that could be involved in hypertonicity-induced activation of NHE-1 in CHO-K1 cells. Hypertonicity rapidly activated NHE-1 in a concentration-dependent manner as assessed by proton microphysiometry and by measurements of intracellular pH on a FLIPR (fluorometric imaging plate reader). Inhibitors of Ca(2+)/calmodulin (CaM) and Janus kinase 2 (Jak2) attenuated this activation, whereas neither calcium chelation nor inhibitors of protein kinase C, the Ras-ERK1/2 pathway, Src kinase, and Ca(2+)/calmodulin-dependent enzymes had significant effects. Hypertonicity also resulted in the rapid tyrosine phosphorylation of Jak2 and STAT3 (the major substrate of Jak2) and CaM. Phosphorylation of Jak2 and CaM were blocked by AG490, an inhibitor of Jak2. Immunoprecipitation studies showed that hypertonicity stimulates the assembly of a signaling complex that includes CaM, Jak2, and NHE-1. Formation of the complex could be blocked by AG490. Thus, we propose that hypertonicity induces activation of NHE-1 in CHO-K1 cells in large part through the following pathway: hypertonicity --> Jak2 phosphorylation and activation --> tyrosine phosphorylation of CaM --> association of CaM with NHE-1 --> NHE-1 activation.

Bradykinin enhancement of PGE2 signalling in bovine trabecular meshwork cells.

Kinins and prostaglandins activate signalling pathways in cells of the trabecular meshwork and have opposing effects on outflow resistance to aqueous humor. Consequently, interactions between these pathways may be important in the regulation of intraocular pressure. In the present study, the influence of bradykinin on PGE(2) signalling was examined in primary cultures of bovine trabecular meshwork cells. Incubation of cells with bradykinin produced a concentration-dependent (EC(50)=3.6+/-0.7 nM) elevation of intracellular free Ca(2+). At a maximal concentration of 100 nM, the increase in Ca(2+) was rapid, peaking in 30 sec, and then slowly returned to baseline. This effect was completely inhibited in cells pretreated with the selective B(2) kinin receptor antagonist, Hoe-140. Treatment of trabecular meshwork cells with PGE(2), in comparison, had no effect on cellular Ca(2+) but produced a concentration-dependent increase in adenosine 3', 5'-cyclic monophosphate (cAMP) formation. Bradykinin had no effect on basal cAMP. However, incubation of cells with PGE(2) in combination with bradykinin resulted in a 3- to 5-fold enhancement of PGE(2)-stimulated cAMP production. Bradykinin enhancement of cAMP stimulation was concentration-dependent with an EC(50) of 3.6+/-1.8 nM. Treatment of cells with bradykinin increased the response maximum for PGE(2) signalling, while the EC(50) for PGE(2) was not changed. This action of bradykinin was again blocked in cells pretreated with Hoe-140. Bradykinin also produced a 2- to 3-fold increase in isoproterenol and cholera toxin-stimulated cAMP accumulation. However, when adenylyl cyclase was stimulated directly with forskolin, bradykinin failed to alter cAMP production. These results indicate that bradykinin activates B(2) kinin receptors in trabecular meshwork cells to amplify PGE(2)-stimulated cAMP formation by facilitating the interaction between activated G(s) and the catalytic unit of adenylyl cyclase.

Bradykinin B2 receptor activates extracellular signal-regulated protein kinase in mIMCD-3 cells via epidermal growth factor receptor transactivation.

Bradykinin (BK) has been implicated in the regulation of renal function. Activation of extracellular signal-regulated protein kinase (ERK1/2) has been demonstrated in several models of toxic or proliferative renal injury. We studied activation of ERK1/2 by BK in a cell model of the most distal part of the nephron, inner medullary collecting duct (mIMCD-3) cells. Exposure of mIMCD-3 cells to BK (10(-10)-10(-5) M) resulted in a concentration-dependent increase in tyrosine phosphorylation of ERK1/2, with maximal effect at 10(-8) M BK. ERK1/2 activation by BK was observed as early as 1 min, peaked at 5 min, and was sustained at least for 1 h. The effect of BK was mediated by the B(2) receptor and was pertussis toxin-independent. Inhibition of phospholipase C, protein kinase C, or phosphatidylinositol 3-kinase did not alter ERK1/2 activation by BK. BK-induced ERK1/2 activation was Ca(2+)-calmodulin-independent but was sensitive to genistein, an inhibitor of tyrosine kinase(s). AG1478, a specific inhibitor of epidermal growth factor receptor (EGFR) kinase, completely blocked the effect of BK, suggesting an essential role of EGFR in ERK1/2 activation by BK. Immunoprecipitation/Western blot studies revealed that BK stimulated tyrosine phosphorylation of EGFR, its association with an adapter molecule Grb2, and complex formation between Grb2 and the adapter protein Shc. Activation studies of monomeric G protein Ras showed that BK-induced stimulation of Ras was dependent on EGFR tyrosine kinase activity. These studies demonstrate that BK stimulates Ras-dependent activation of ERK1/2 in mIMCD-3 cells via transactivation of EGFR through a novel mechanism.

Jak2 and Ca2+/calmodulin are key intermediates for bradykinin B2 receptor-mediated activation of Na+/H+ exchange in KNRK and CHO cells.

Na(+)/H(+) exchangers are ubiquitous in mammalian cells, carrying out key functions, such as cell volume defense, acid-base homeostasis, and regulation of the cytoskeleton. We used two screening technologies (FLIPR and microphysiometry) to characterize the signal transduction pathway used by the bradykinin B(2) receptor to activate Na(+)/H(+) exchange in two cell lines, KNRK and CHO. In both cell types, B(2) receptor activation resulted in rapid increases in the rate of proton extrusion that were sodium-dependent and could be blocked by the Na(+)/H(+) exchange inhibitors EIPA and MIA or by replacing extracellular sodium with TMA. Activation of Na(+)/H(+) exchange by bradykinin was concentration-dependent and could be blocked by the selective B(2) receptor antagonist HOE140, but not by the B(1) receptor antagonist des-Arg10-HOE140. Inhibitors of Jak2 tyrosine kinase (genistein and AG490) and of CAM (W-7 and calmidazolium) attenuated bradykinin-induced activation of Na(+)/H(+) exchange. Bradykinin induced formation of a complex between CAM and Jak2, supporting a regulatory role for Jak2 and CAM in the activation of Na(+)/H(+) exchange in KNRK and CHO cells. We propose that this pathway (B(2) receptor --> Jak2 --> CAM --> Na(+)/H(+) exchanger) is a fundamental regulator of Na(+)/H(+) exchange activity.

Role for 18:1 lysophosphatidic acid as an autocrine mediator in prostate cancer cells.

Lysophosphatidic acid (LPA) is a lipid mediator that may play an important role in growth and survival of carcinomas. In this study, LPA production and response were characterized in two human prostate cancer (CaP) cell lines: PC-3 and Du145. Bombesin, a neuroendocrine peptide that is mitogenic for CaP cells, stimulated focal adhesion kinase phosphorylation and activated the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Similar responses were elicited by 18:1 LPA (oleoyl-LPA). Studies using radioisotopic labeling revealed that both PC-3 and Du145 generate LPA and that LPA production is increased by bombesin. The kinetics of bombesin-induced phospholipase D activation and LPA production were similar. Using electrospray ionization mass spectrometry, 18:1 LPA was found to be an abundant LPA species in CaP cell medium. Structure activity studies of acyl-LPAs revealed that 18:1 LPA is most efficacious for activation of extracellular signal-regulated kinase and phospholipase D in CaP cells. Incubation with 18:1 LPA caused homologous desensitization of LPA response, whereas bombesin caused heterologous desensitization. LPA was present at nanomolar levels in medium from bombesin-treated cells. LPA extracted from the medium induced calcium mobilization in CaP cells. These results demonstrate that bioactive LPA is generated by CaP cells in response to a mitogen and suggest that 18:1 LPA can act as an autocrine mediator.