Automated Direct Selective Laser Trabeculoplasty: First Prospective Clinical Trial.

Purpose: Direct selective laser trabeculoplasty (DSLT) is a rapid, noncontact automated procedure performed directly through the limbus without gonioscopy. In this first nonrandomized clinical trial we assessed its safety and ability to reduce intraocular pressure (IOP). Methods: Fifteen patients (15 eyes: 10 with open-angle glaucoma [OAG], 4 with ocular hypertension, and 1 with pseudoexfoliation glaucoma), naive or after medication washout, with an IOP ≥22 mm Hg, underwent DSLT by irradiation with 100 or 120 sequential noncontact 532-nm, Q-switched laser shots (0.8-1.4 mJ) automatically applied during 1.5 or 2.3 seconds on the limbus, guided by image analysis and eye tracking. Results were assessed at 1 and 3 hours, 1 day, 1 week, and 1, 3, and 6 months. Results: The mean ± standard deviation baseline IOP (mm Hg) in all eyes was 26.7 ± 2.3. At 1, 3, and 6 months, this value was significantly reduced to 21.7 ± 4.2 (by 18.1%), to 20.8 ± 2.5 (by 21.4%), and to 21.5 ± 4.1 (by 18.8%), respectively. In six patients treated with 1.4 mJ/shot, the mean IOP at 6 months decreased from 26.7 ± 3.2 to 19.3 ± 2.0 (27.1%, P = 0.03). There was a significant reduction in hypotensive medications (from 1.6 ± 1.0 to 0.4 ± 0.7, P = 0.03). No serious adverse events occurred. Conclusions: Automated DSLT appears to be an effective and safe noncontact, rapid modality for reducing IOP in patients with OAG. Higher energy usage led to better results. Translational Relevance: Studying laser transmission through sclera enabled laser irradiation of the trabeculum without gonioscopy.

Non-contact direct selective laser trabeculoplasty: light propagation analysis.

Selective laser trabeculoplasty (SLT), used to treat glaucoma and ocular hypertension, requires the use of a gonioscope placed on the cornea to visualize and irradiate the trabecular meshwork (TM). Alternatively, non-contact direct SLT (DSLT) irradiates the TM through the overlying tissues. Here we analyze this innovative procedure using analytical modeling and Monte Carlo simulations to quantify the laser energy reaching the TM through the overlying tissues. Compared with energy launched from the laser, DSLT energy transmission to the TM is 2.8 times less than SLT, which verifies the efficacy of non-contact DSLT given the lowest reported effective SLT energies.

Differential roles of PKC isoforms (PKCs) in GnRH stimulation of MAPK phosphorylation in gonadotrope derived cells.

The role of protein kinase C (PKC) isoforms (PKCs) in GnRH-stimulated MAPK [ERK1/2, JNK1/2 and p38) phosphorylation was examined in gonadotrope derived cells. GnRH induced a protracted activation of ERK1/2 and a slower and more transient activation of JNK1/2 and p38MAPK. Gonadotropes express conventional PKCα and PKCßII, novel PKCδ, PKCε and PKCθ, and atypical PKC-ι/λ. The use of green fluorescent protein (GFP)-PKCs constructs revealed that GnRH induced rapid translocation of PKCα and PKCßII to the plasma membrane, followed by their redistribution to the cytosol. PKCδ and PKCε localized to the cytoplasm and Golgi, followed by the rapid redistribution by GnRH of PKCδ to the perinuclear zone and of PKCε to the plasma membrane. The use of dominant negatives for PKCs and peptide inhibitors for the receptors for activated C kinase (RACKs) has revealed differential role for PKCα, PKCßII, PKCδ and PKCε in ERK1/2, JNK1/2 and p38MAPK phosphorylation in a ligand-and cell context-dependent manner. The paradoxical findings that PKCs activated by GnRH and PMA play a differential role in MAPKs phosphorylation may be explained by persistent vs. transient redistribution of selected PKCs or redistribution of a given PKC to the perinuclear zone vs. the plasma membrane. Thus, we have identified the PKCs involved in GnRH stimulated MAPKs phosphorylation in gonadotrope derived cells. Once activated, the MAPKs will mediate the transcription of the gonadotropin subunits and GnRH receptor genes.

Differential signaling of the GnRH receptor in pituitary gonadotrope cell lines and prostate cancer cell lines.

The GnRH receptor (GnRHR) mediates the pituitary functions of GnRH, as well as its anti-proliferative effects in sex hormone-dependent cancer cells. Here we compare the signaling of GnRHR in pituitary gonadotrope cell lines vs. prostate cancer cell lines. We first noticed that the expression level of PKCα, PKCßII and PKCε is much higher in αT3-1 and LßT2 gonadotrope cell lines vs. LNCaP and DU-145 cell lines, while the opposite is seen for PKCδ. Activation of PKCα, PKCßII and PKCε by GnRH is relatively transient in αT3-1 and LßT2 gonadotrope cell lines and more prolonged in LNCaP and DU-145 cell lines. On the otherhand, the activation and re-distribution of the above PKCs by PMA was similar for both gonadotrope cell lines and prostate cancer cell lines. Activation of ERK1/2 by GnRH and PMA was robust in the gonadotrope cell lines, with a smaller effect observed in the prostate cancer cell lines. The Ca(2+) ionophore A23187 stimulated ERK1/2 in gonadotrope cell lines but not in prostate cancer cell lines. GnRH, PMA and A23187 stimulated JNK activity in gonadotrope cell lines, with a more sustained effect in prostate cancer cell lines. Sustained activation of p38 was observed for PMA and A23187 in Du-145 cells, while p38 activation by GnRH, PMA and A23187 in LßT2 cells was transient. Thus, differential expression and re-distribution of PKCs by GnRH and the transient vs. the more sustained nature of the activation of the PKC-MAPK cascade by GnRH in gonadotrope cell lines vs. prostate cancer cell lines respectively, may provide the mechanistic basis for the cell context-dependent differential biological responses observed in GnRH interaction with pituitary gonadotropes vs. prostate cancer cells.

Differential role of PKC isoforms in GnRH and phorbol 12-myristate 13-acetate activation of extracellular signal-regulated kinase and Jun N-terminal kinase.

GnRH is the first key hormone of reproduction. The role of protein kinase C (PKC) isoforms in GnRH-stimulated MAPK [ERK and Jun N-terminal kinase (JNK)] was examined in the αT3-1 and LßT2 gonadotrope cells. Incubation of the cells with GnRH resulted in a protracted activation of ERK1/2 and a slower and more transient activation of JNK1/2. Gonadotropes express conventional PKCα and conventional PKCßII, novel PKCδ, novel PKCε, and novel PKCθ, and atypical PKC-ι/λ. The use of green fluorescent protein-PKC constructs revealed that GnRH induced rapid translocation of PKCα and PKCßII to the plasma membrane, followed by their redistribution to the cytosol. PKCδ and PKCε localized to the cytoplasm and Golgi, followed by the rapid redistribution by GnRH of PKCδ to the perinuclear zone and of PKCε to the plasma membrane. Interestingly, PKCα, PKCßII, and PKCε translocation to the plasma membrane was more pronounced and more prolonged in phorbol-12-myristate-13-acetate (PMA) than in GnRH-treated cells. The use of selective inhibitors and dominant-negative plasmids for the various PKCs has revealed that PKCßII, PKCδ, and PKCε mediate ERK2 activation by GnRH, whereas PKCα, PKCßII, PKCδ, and PKCε mediate ERK2 activation by PMA. Also, PKCα, PKCßII, PKCδ, and PKCε are involved in GnRH and PMA stimulation of JNK1 in a cell-context-dependent manner. We present preliminary evidence that persistent vs. transient redistribution of selected PKCs or redistribution of a given PKC to the perinuclear zone vs. the plasma membrane may dictate its selective role in ERK or JNK activation. Thus, we have described the contribution of selective PKCs to ERK and JNK activation by GnRH.

A preformed signaling complex mediates GnRH-activated ERK phosphorylation of paxillin and FAK at focal adhesions in L beta T2 gonadotrope cells.

Most receptor tyrosine kinases and G protein-coupled receptors (GPCRs) operate via a limited number of MAPK cascades but still exert diverse functions, and therefore signal specificity remains an enigma. Also, most GPCR ligands utilize families of receptors for mediation of diverse biological actions; however, the mammalian type I GnRH receptor (GnRHR) seems to be the sole receptor mediating GnRH-induced gonadotropin synthesis and release. Signaling complexes associated with GPCRs may thus provide the means for signal specificity. Here we describe a signaling complex associated with the GnRHR, which is a unique GPCR lacking a C-terminal tail. Unlike other GPCRs, this signaling complex is preformed, and exposure of L beta T2 gonadotropes to GnRH induces its dynamic rearrangement. The signaling complex includes c-Src, protein kinase C delta, -epsilon, and -alpha, Ras, MAPK kinase 1/2, ERK1/2, tubulin, focal adhesion kinase (FAK), paxillin, vinculin, caveolin-1, kinase suppressor of Ras-1, and the GnRHR. Exposure to GnRH (5 min) causes MAPK kinase 1/2, ERK1/2, tubulin, vinculin, and the GnRHR to detach from c-Src, but they reassociate within 30 min. On the other hand, FAK, paxillin, the protein kinase Cs, and caveolin-1 stay bound to c-Src, whereas kinase suppressor of Ras-1 appears in the complex only 30 min after GnRH stimulation. GnRH was found to activate ERK1/2 in the complex in a c-Src-dependent manner, and the activated ERK1/2 subsequently phosphorylates FAK and paxillin. In parallel, caveolin-1, FAK, vinculin, and paxillin are phosphorylated on Tyr residues apparently by GnRH-activated c-Src. Receptor tyrosine kinases and GPCRs translocate ERK1/2 to the nucleus to phosphorylate and activate transcription factors. We therefore propose that the role of the multiprotein signaling complex is to sequester a cytosolic pool of activated ERK1/2 to phosphorylate FAK and paxillin at focal adhesions.

Identification of extracellular signal-regulated kinase 1/2 and p38 MAPK as regulators of human sperm motility and acrosome reaction and as predictors of poor spermatozoan quality.

Mature spermatozoa acquire progressive motility only after ejaculation. Their journey in the female reproductive tract also includes suppression of progressive motility, reactivation, capacitation, and hyperactivation of motility (whiplash), the mechanisms of which are obscure. MAPKs are key regulatory enzymes in cell signaling, participating in diverse cellular functions such as growth, differentiation, stress, and apoptosis. Here we report that ERK1/2 and p38 MAPK are primarily localized to the tail of mature human spermatozoa. Surprisingly, c-Jun N-terminal kinase 1/2, which is thought to be ubiquitously expressed, could not be detected in mature human spermatozoa. ERK1/2 stimulation is downstream to protein kinase C (PKC) activation, which is also present in the human sperm tail (PKCbetaI and PKCepsilon). ERK1/2 stimulates and p38 inhibits forward and hyperactivated motility, respectively. Both ERK1/2 and p38 MAPK are involved in the acrosome reaction. Using a proteomic approach, we identified ARHGAP6, a RhoGAP, as an ERK substrate in PMA-stimulated human spermatozoa. Inverse correlation was obtained between the relative expression level of ERK1 or the relative activation level of p38 and sperm motility, forward progression motility, sperm morphology, and viability. Therefore, increased expression of ERK1 and activated p38 can predict poor human sperm quality.

Activation of mitogen-activated protein kinase (MAPK) by GnRH is cell-context dependent.

The interaction of GnRH with its cognate receptor (GnRHR) in pituitary gonadotropes includes activation of Gq/G11 and phospholipase Cbeta (PLCbeta), which generates the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which are required for Ca2+ mobilization and PKC isoforms activation. Activation of PKC in pituitary gonadotropes leads to the activation of the major members of the mitogen-activated protein kinase superfamily (MAPK), namely: extracellular signal-regulated kinase (ERK), jun-N-terminal Kinase (JNK) and p38MAPK. The above pathways mediate GnRH-induced gonadotropin release and synthesis. Here we summarise the diverse mechanisms utilized by GnRH to activate the MAPK members and show that they depend on "cell-context".