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M Wiederholt

Publications and source records attributed to M Wiederholt.

At least 19 recordsLinked to original sources
PubMed2001

Muscarinic receptors of the M2 subtype in human and bovine trabecular meshwork.

BACKGROUND: The trabecular meshwork is a tissue actively involved in the regulation of intraocular pressure via contractile mechanisms. The present study was performed to investigate the effects of muscarinic m2-receptor antagonists on trabecular meshwork contractility and to identify the m2 muscarinic receptor in human and bovine trabecular meshwork cells. METHODS: Isometric tension measurements of bovine trabecular meshwork strips were performed using a custom-made force length transducer. Western blot and immunoprecipitation analysis was used to detect the m2-receptor proteins in membrane preparations of human and bovine trabecular meshwork cells. RESULTS: Immunoblotting results showed the expression of an m2-receptor protein band at 56 kDa in both human and bovine trabecular meshwork cells. Two different m2-receptor antagonists were tested on trabecular meshwork contractility. After carbachol-induced contraction (10(-6) M set to 100% contractile force), specific m2-receptor antagonists were applied. 3 alpha-Chloroimperaline (10(-6) M) had no effect on the maximal carbachol-induced contraction in trabecular meshwork strips. Methoctramine induced a significant relaxation at concentrations of 10(-7), 10(-6) and 5 x 10(-6) M even in the presence of m1- and m3-receptor antagonists. CONCLUSION: These data indicate that in addition to the m3-receptor subtype present in the trabecular meshwork this tissue also features the m2 receptor. This receptor is partly involved in the regulation of trabecular meshwork contractility, suggesting that outflow facility might be influenced through this receptor.

Animals↗
PubMed2001

Flufenamic acid enhances current through maxi-K channels in the trabecular meshwork of the eye.

PURPOSE: Flufenamic acid relaxes trabecular meshwork, a smooth muscle-like tissue involved in the regulation of ocular outflow in the eye. In this study, we attempted to determine if ionic channels are involved in this response. METHODS: Cultured human (HTM) and bovine (BTM) trabecular meshwork cells were investigated using the patch-clamp technique. RESULTS: In trabecular meshwork, flufenamic acid (10(-5) M) reversibly stimulated outward current to 406 +/- 71% of initial outward current level in BTM (n = 10) and 294 +/- 75% of initial current level in HTM (n = 12) in all cells investigated; no significant differences emerged. The response was dosage-dependent. Replacement of potassium in all solutions eliminated the response to flufenamic acid (n = 4, BTM). Blocking K(ATP ) channels with glibenclamide (10(-5) M, n = 6) and small-conductance calcium-activated potassium channels with apamin (10(-6) M, n = 5) had no effect. A direct effect on calcium channels could also not be detected. Blockage of the large-conductance calcium-activated potassium channel (maxi-K) by iberiotoxin (10(-7) M) suppressed 87 +/- 9% (n = 6; HTM) and 91 +/- 10% (n = 6; BTM) of the response. Depleting the cells of calcium did not significantly alter the response to flufenamic acid. CONCLUSIONS: Flufenamic acid stimulates maxi-K channels in trabecular meshwork of both human and bovine origin. This should lead to hyperpolarization, closure of L-type channels and lowered cytosolic calcium levels, possibly explaining the relaxation observed in response to this substance.

Animals↗
PubMed2001

Mechanisms of action of unoprostone on trabecular meshwork contractility.

PURPOSE: This study was performed to clarify the possible mechanism behind the ocular hypotensive effect of unoprostone isopropyl (Rescula; Novartis Ophthalmics AG, Basel, Switzerland), a new docosanoid that has been shown to reduce intraocular pressure (IOP) in patients with ocular hypertension or primary open-angle glaucoma. To gain insight into the possible mode of action, the effects of unoprostone on ciliary muscle (CM) and trabecular meshwork (TM) contractility, intracellular calcium levels, and membrane channels were investigated. METHODS: The effects of unoprostone (M1 metabolite = free acid, 10(-5) M) and endothelin (ET)-1 (10(-9) M) on bovine TM (BTM) and ciliary muscle (CM) strips were investigated, by using a custom-made force-length transducer system. The effects of unoprostone and ET-1 (5 x 10(-8) M) on intracellular Ca(2+) mobilization in cultured human TM (HTM) were measured using fura-2AM as a fluorescent probe. Patch-clamp experiments were performed on HTM and BTM cells to investigate the unoprostone-dependent modulation of membrane currents. RESULTS: In isolated TM and CM strips, unoprostone almost completely inhibited ET-induced contractions (TM: 2.9% +/- 4.3% vs. 19.6% +/- 5.7%, P < 0.05, n = 6; CM: 1.4% +/- 1.6% vs. 30.1% +/- 5.3%, P < 0.01, n = 6; 100% = maximal carbachol-induced (10(-6) M) contraction). However, neither carbachol-induced contraction nor baseline tension was affected by unoprostone. Furthermore, unoprostone had no effect on baseline intracellular calcium levels (baseline: 126 +/- 45 nM versus unoprostone: 132 +/- 42 nM, n = 8) in HTM cells. The endothelin-induced increase (679 +/- 102 nM), however, was almost completely (P < 0.01) blocked by unoprostone (178 +/- 40 nM). In patch-clamp recordings, unoprostone could be shown to double the amplitude of outward current (HTM: 200% +/- 33%; n = 6; BTM: 179% +/- 20%; n = 8). This effect was blocked by the specific inhibitor of maxi-K channels, iberiotoxin. CONCLUSIONS: This study presents evidence for direct interaction of unoprostone with the contractility of the TM and CM. This compound may lower IOP by affecting aqueous outflow, most probably conventional outflow pathways (i.e., TM) through inhibition of ET-dependent mechanisms. In addition, unoprostone interacts with the maxi-K channel. Although primarily Ca(2+)-sensitive signal-transduction pathways seem to be involved, effects of unoprostone on Ca(2+)-independent pathways and uveoscleral outflow cannot be excluded.

Adult↗
PubMed2000

Influence of muscarinic agonists and tyrosine kinase inhibitors on L-type Ca(2+)Channels in human and bovine trabecular meshwork cells.

Trabecularmeshwork (TM), a smooth muscle-like tissue with contractile properties, is involved in the regulation of aqueous humor outflow. However, little is known about the regulation of Ca(2+)influx in trabecular meshwork cells. We investigated the influence of acetylcholine and tyrosine kinases on Ca(2+)conductances of bovine TM (BTM) and human TM (HTM) cells using the perforated-patch configuration of the patch-clamp technique and measurements of intracellular free Ca(2+)([Ca(2+)](i)). Depolarization of the cells in the presence of 10 m m Ba(2+)or Ca(2+)led to an activation of inward currents at potentials positive to -30 mV with characteristics typical of L-type Ca(2+)currents: when using 10 m m Ba(2+), maximal inward current and inactivation time constant (tau) increased; the L-type Ca(2+)channel blocker nifedipine (1 microm) reduced and the L-type Ca(2+)channel agonist BayK8644 (5 microm) enhanced maximal inward current. Acetylcholine (100 microm) and carbachol (1 microm) led to an increase in inward Ba(2+)current whereas application of the tyrosine kinase inhibitors genistein (50 microm) and lavendustin A (20 microm) resulted in a decrease in inward current. The application of daidzein (10 microm), an inactive analog of genistein had no effect. Depolarization of the cells with 135 m m K(+)or direct stimulation of L-type channels by application of BayK 8644 led to an increase in [Ca(2+)](i). Carbachol (1 microm) induced an increase in [Ca(2+)](i)which was decreased by application of the tyrosine kinase inhibitor genistein (50 microm). We conclude that HTM and BTM cells express voltage-dependent L-type Ca(2+)channels that influence intracellular Ca(2+)concentration and thus may modulate TM contractility. The activity of L-type Ca(2+)currents is influenced by muscarinic agonists and tyrosine kinases.

Acetylcholine↗
PubMed2000

The regulation of trabecular meshwork and ciliary muscle contractility.

Current models of aqueous humor outflow no longer treat trabecular meshwork (TM) as an inert tissue passively distended by the ciliary muscle (CM). Instead, ample evidence supports the theory that trabecular meshwork possess smooth muscle-like properties and is actively involved in the regulation of aqueous humor outflow and intraocular pressure. In this model, trabecular meshwork and ciliary muscle appear as functional antagonists, with ciliary muscle contraction leading to a distension of trabecular meshwork with subsequent reduction in outflow. and with trabecular meshwork contraction leading to the opposite effect. Smooth-muscle relaxing substances would therefore appear to be ideal candidates for glaucoma therapy with the dual goal of reducing intraocular pressure via the trabecular meshwork and of improving vascular perfusion of the optic nerve head. However, for such substances to effectively lower intraocular pressure, the effect on the ciliary muscle would have to he minimal. For this reason, more information is needed on the signalling processes involved in regulating trabecular meshwork and ciliary muscle contractility. This review attempts to outline current knowledge of signal transduction pathways leading to relaxation and contraction of ciliary muscle and trabecular meshwork. Pathways can be classified as involving or not involving changes of membrane voltage and of requiring or not requiring external calcium: possibly, other pathways exist. These different pathways involve different ion channels and isoforms of PKC and are expressed to a differing degree in ciliary muscle and trabecular meshwork, leading to differential responses when exposed to relaxing or contracting pharmacological agents. Some of these agents. like tyrosine kinase inhibitors and inhibitors of PKC. have been shown to relax trabecular meshwork while leaving ciliary muscle comparatively unaffected. This profile makes these substances appear as ideal drugs for simultaneously improving ocular outflow and retinal circulation, parameters that determine the time course of visual deterioration in glaucoma.

Animals↗
PubMed2000

Mediation of calcium-independent contraction in trabecular meshwork through protein kinase C and rho-A.

PURPOSE: Inhibition of protein kinase C (PKC) and rho-kinase (ROCK) may represent a new way of influencing outflow facility through isolated relaxation of the trabecular meshwork (TM). This work was performed to investigate the existence of calcium-independent contraction in this smooth-muscle-like tissue and its modulation by targeting the rho-guanosine triphosphatase (GTPase)-mediated pathway. METHODS: Isometric tension measurements of bovine TM and ciliary muscle (CM) were performed. Intra- and extracellular calcium buffering was accomplished with EGTA and 1, 2-bis(2-aminophenoxy)-ethane-N,N:,N:,N:',N:'-tetra-acetic acid tetrakis/acetoxymethhyl ester (BAPTA-AM) followed by stimulation of PKC with phorbolester (PMA) or 4alpha-phorbol. Calcium-independent contraction was blocked using the highly specific ROCK inhibitor Y-27632. Western blot analysis and immunoprecipitation was performed using human TM cells. RESULTS: In TM, carbachol induced partial contraction under conditions of extracellular calcium depletion (22. 1% +/- 2.3% versus 100%, n = 9). The membrane-permeable calcium chelator BAPTA-AM completely blocked this response (1.1% +/- 1.4% versus 100%, n = 9). When calcium was completely blocked, PMA induced contraction in TM (16.7% +/- 5.9% versus 100%, n = 9) but not in CM (1.8% +/- 2.5% versus 100%, n = 6). The inactive PMA analogue 4alpha-phorbol did not induce contraction, indicating that activation of PKC is involved in this contractile response. The ROCK inhibitor Y-27632 completely blocked the calcium-independent PMA-induced contraction in TM. Western blot analysis and immunoprecipitation revealed the expression of the rho-A protein in human TM cells. CONCLUSIONS: The data indicate that contrary to CM, the TM features calcium-independent contractile mechanisms linked to rho-A and PKC isoforms that do not require calcium for activation. ROCK inhibitors may allow specific modulation of the TM to enhance outflow facility, thus lowering intraocular pressure.

Amides↗
PubMed1999

Involvement of protein tyrosine kinase in the InsP3-induced activation of Ca2+-dependent Cl- currents in cultured cells of the rat retinal pigment epithelium.

This combined study of patch-clamp and intracellular Ca2+ ([Ca2+]i) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca2+-dependent Cl- channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 microM) led to an increase in [Ca2+]i and activation of Cl- currents. In contrast, intracellular application of Ca2+ (10 microM) only induced transient activation of Cl- currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca2+ and to the blocker of ICRAC, La3+ (10 microM), despite the fact that both maneuvers led to a decline in [Ca2+]i. The InsP3-induced rise in Cl- conductance could be prevented either by thapsigargin-induced (1 microM) depletion of intracellular Ca2+ stores or by removal of Ca2+ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca2+-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 microM) had no effect. Inhibition of Ca2+/calmodulin kinase (KN-63, KN-92; 5 microM) reduced Cl--conductance in 50% of the cells investigated without affecting [Ca2+]i. Inhibition of protein tyrosine kinase (50 microM tyrphostin 51, 5 microM genistein, 5 microM lavendustin) reduced an increase in [Ca2+]i and Cl- conductance. In summary, elevation of [Ca]i by InsP3 leads to activation of Cl- channels involving cytosolic Ca2+ stores and Ca2+ influx from extracellular space. Tyrosine kinases are essential for the Ca2+-independent maintenance of this conductance.

Animals↗
PubMed1999

Stimulation of maxi-K channels in trabecular meshwork by tyrosine kinase inhibitors.

PURPOSE: Muscarinic agonists contract and tyrosine kinase inhibitors relax precontracted trabecular meshwork, a smooth muscle-like tissue involved in the regulation of aqueous humor outflow. The effect of tyrosine kinase inhibitors on membrane currents of cells stimulated by acetylcholine was examined. METHODS: Cells from bovine trabecular meshwork were studied using both the perforated patch-clamp technique with nystatin and the single-channel technique. RESULTS: Application of the tyrosine kinase inhibitor genistein (5 x 10(-5) M) on trabecular meshwork cells stimulated with acetylcholine resulted in a reversible increase in outward current to 578%+/-154% (n = 16) of the initial current level. The effect of genistein was dose dependent. Reversal potential was hyperpolarized by 15+/-3 mV (n = 9). Tyrphostin 51, a synthetic inhibitor of tyrosine kinases, had the same effect (433%+/-46%; n = 7). Daidzein, a nonactive structural analogue of genistein, had no effect (n = 4). The stimulation of outward current by tyrosine kinase inhibitors was blocked by substitution of tetraethylammonium (TEA+) for potassium, whereas the potassium channel blockers glibenclamide (K-ATP) and apamin (low-conductance calcium-activated potassium channel) had no effect. Blockage of the high-conductance calcium-activated potassium channel (maxi-K) by charybdotoxin or iberiotoxin (10(7) M) suppressed 86%+/-18% (n = 4) of the response. Depleting the cells of calcium did not have an effect on the current stimulated by genistein. In the excised inside-out configuration, open probability increased to 417%+/-39% (n = 3) after exposure to genistein. CONCLUSIONS: In trabecular meshwork, tyrosine kinase inhibitors activate maxi-K (K(Ca)) channels. Hyperpolarization caused by efflux of potassium could lead to the relaxation of trabecular meshwork by tyrosine kinase inhibitors.

Acetylcholine↗
PubMed1999

The effects of protein kinase C on trabecular meshwork and ciliary muscle contractility.

PURPOSE: The possible role of protein kinase C (PKC) inhibitors in novel pressure-lowering drugs is currently under investigation. To gain further insight into regulation of contractility by PKC in trabecular meshwork (TM) and ciliary muscle (CM), the effects of various PKC inhibitors and activators were tested. METHODS: Isometric tension measurements of bovine TM and CM strips were performed. PKC was stimulated by phorbol ester and by the diacylglycerol analogue diC8. PKC blockade was accomplished using H7 and myristoilated PKC substrate (mPKC). Western blot analysis was used to identify specific PKC isoforms in human trabecular meshwork (HTM), human ciliary muscle (HCM), and bovine TM and CM. RESULTS: In tissues precontracted by carbachol PKC antagonist H7 led to a relaxation of TM (25+/-7.2 versus 100%; n = 8) with no effect on CM. mPKC substrate selectively blocks PKC. This substance led to relaxation of TM (32.8+/-7.4 versus 100%, n = 7), whereas CM was not affected. PMA at concentrations of 10(-6) M led to a slow contraction of both tissues that was more marked in TM. DiC8 and 4alpha-phorbol had no effect on contractility. Western blot analysis revealed expression of calcium-dependent PKC-alpha and calcium-independent PKC-epsilon isoforms in HTM and HCM. PKC-epsilon expression was more pronounced in HTM than in HCM. Similar PKC isoform expression was found in native bovine tissue. CONCLUSIONS: PKC isoforms show different tissue distributions in human and bovine TM and CM. Contractility differences exist in both tissues in response to PKC antagonists and agonists. The data indicate that PKC may be involved in regulation of aqueous humor outflow by the TM. Thus, inhibition of PKC may represent a new way of influencing outflow facility through isolated relaxation of TM.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1998

Activation of a Cl--conductance by protein kinase-dependent phosphorylation in cultured rat retinal pigment epithelial cells.

While chloride conductances are involved in signals of the electroretinogram generated by the retinal pigment epithelium (RPE), patch-clamp experiments of freshly isolated or cultured RPE cells have shown that potassium conductances predominate. The purpose of this study was to investigate mechanisms which activate Cl--conductances in RPE cells. Membrane currents of cultured rat RPE cells were measured using the whole-cell configuration of the patch-clamp technique under extra- and intracellular K+-free conditions. The bath solution was hyperosmolal to the pipette solution to prevent hypoosmotic swelling. Exchange of the physiological intracellular fluid by a pipette solution with physiological levels of ATP (2 mm) induced a continuous increase of membrane conductance. Conductance was blocked by DIDS (1 mm), and showed a reversal potential close to the Nernst potential for Cl-. When the experiments were carried out under conditions in which all cations, and not only potassium, were replaced by NMDG, the same responses could be observed. Current activation was independent of extracellular calcium. Chloride currents were also induced when ATPgammaS or AMP-PNP were used instead of ATP. In the presence of AMP-PNP currents were 10 times smaller than in the presence of ATP or ATPgammaS. In cells preincubated with staurosporine or chelerythrine no currents were induced. Establishing the whole-cell configuration with ATP and with myristoylated PKC substrate in addition, no voltage-dependent currents were activated. We conclude that ATP hydrolysis leads to activation of chloride currents via PKC in the whole-cell configuration. The perforated patch configuration, with the intracellular compartment intact, no currents were induced under otherwise identical experimental conditions. Inhibition of phosphatase by calyculin (10 nm) in the perforated-patch configuration did not change membrane conductance. In the intact cell, chloride conductance is possibly inhibited by a cytosolic factor which is washed out when the whole-cell configuration is established.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Altered regulation of L-type channels by protein kinase C and protein tyrosine kinases as a pathophysiologic effect in retinal degeneration.

The effect of protein tyrosine kinases (PTK) on L-type calcium channels in cultured retinal pigmented epithelium (RPE) from rats with retinal dystrophy was investigated. Barium currents through Bay K 8644 (10(-6) M) sensitive L-type channels were measured using the patch-clamp technique. The current density of L-type currents is twice as high and the inactivation time constants are much slower than in cells from nondystrophic control rats. Application of the PTK blockers genistein, lavendustin A, and herbimycin A (all 5 x 10(-6) M) led to an increase of L-type currents. Intracellular application of pp60c-src (30 U/ml) via the patch pipette led to a transient decrease of L-type currents. The protein kinase A (PKA) and PKG blocker H9 (10(-6) M) showed no effect on L-type currents. However, the protein kinase C blocker chelerythrine (10(-5) M) reduced these currents. Up-regulation of PKC by 10(-6) M 4beta-phorbol-12 myristate-13 acetate (PMA) led to a decrease of L-type currents. Additional application of genistein led to a further decrease of these currents. However, intracellular application of pp60(c-src) in PMA-treated cells led to a transient increase of L-type currents. Investigating the calcium response to bFGF application showed that RPE cells from RCS rats used different pathways than control RPE cells to increase cytosolic free calcium. This different pathway does not involve the activation of L-type channels. The present study with RPE cells from rats with retinal dystrophy shows a changed integration of PTK and PKC in channel regulation. Considering the altered response to bFGF in RCS-RPE cells, this disturbed regulation of L-type channels by tyrosine kinases is involved in the etiology of retinal degeneration in RCS rats.

Alkaloids↗
PubMed1998

Direct involvement of trabecular meshwork in the regulation of aqueous humor outflow.

Our new hypothesis for the regulation of aqueous humor outflow suggests that the trabecular meshwork is not a passive filter but an active contractile element contributing to the ciliary muscle traction affecting it. The trabecular meshwork contains contractile smooth-muscle-specific alpha-actin filaments, and its cells exhibit electrical properties typical for smooth muscle cells. Contractility measurements performed for the first time in isolated trabecular meshwork enable a functional comparison with ciliary muscle. Pharmacologic outflow regulation has been determined in isolated perfused anterior segments with intact trabecular meshwork and total absence of ciliary muscle. Substances that contracted isolated trabecular meshwork (e.g., pilocarpine) decreased the outflow rate, whereas relaxants (e.g., low-dose epinephrine) increased it. The concept of a functional antagonism between the trabecular meshwork and the ciliary muscle has to be considered.

Animals↗
PubMed1998

The Royal College of Surgeons rat: an animal model for inherited retinal degeneration with a still unknown genetic defect.

The Royal College of Surgeons (RCS) rat is the first known animal with inherited retinal degeneration. Despite the fact that the genetic defect is not known, the RCS rat is widely used for research in hereditary retinal dystrophies. This review tries to summarize observations which have been made in the RCS rat and to make an attempt to formulate candidate genes which may the cause for the retinal degeneration in this rat strain. The genetic defect in RCS rats causes the inability of the retinal pigment epithelium (RPE) to phagocytose shed photoreceptor outer segments. In normal rats or humans, this circadian process is regulated by both the cyclic adenosine monophosphate (cAMP) and the calcium/ inositol phosphate systems. The calcium/inositol phosphate system seems to be linked to the phagocytosis receptors which recognize photoreceptor outer membranes to initialize phagocytosis. The cAMP system appeared as modulator of the regulation of phagocytosis. An increase in the intracellular cAMP concentration is an 'off' signal for phagocytosis. In RPE cells from RCS rats many observations have been made which indicate a changed second messenger metabolism concerning both the cAMP and the calcium/inositol phosphate systems. The genetic defect seems to concern a protein which is involved in the initialization of a second messenger pathway. We conclude that the genes coding for the phagocytosis receptor or for proteins which are linked to receptors (for example G proteins) are good candidates for defective genes in RCS rats.

Animals↗
PubMed1998

Role of protein tyrosine kinase on regulation of trabecular meshwork and ciliary muscle contractility.

PURPOSE: Trabecular meshwork and ciliary muscle express properties of smooth muscle cells. The contractility of trabecular meshwork and ciliary muscle is differently modulated by various agents. To reveal contractile regulatory processes, the effects of activation and inhibition of protein tyrosine kinases (PTKs) and their interaction with other protein kinases on contractility were measured. METHODS: Measurements of isometric tension were performed on isolated bovine trabecular meshwork and ciliary muscle strips using a custom-built, electromagnetic, force-length transducer. Protein tyrosine kinase (PTK) was stimulated by epidermal growth factor (EGF) and was inhibited by genistein or tyrphostin 51. Protein kinase C (PKC) was inhibited by chelerythrine or NPC-15437 and protein kinases A and G (PKA-PKG) by H8. RESULTS: Isolated strips were precontracted by applying carbachol 10(-6) M for 30 minutes (100% carbachol maximum contraction). Inhibition of PTK evoked a maximum relaxation of 79.2+/-4.2% in trabecular meshwork and of 38.1+/-3.1% in ciliary muscle (n=8). Inhibition of PKC or PKA-PKG induced relaxations only in trabecular meshwork. When PTK and PKC or PKA-PKG were inhibited, the relaxation induced by inhibition of PTK was additive to inhibition of the other protein kinases. Stimulation of a receptor with PTK activity by EGF induced a relaxation in trabecular meshwork and a contraction in ciliary muscle precontracted by carbachol. When trabecular meshwork and ciliary muscle were activated by EGF, inhibition of PTK by genistein relaxed the cell preparations. CONCLUSIONS: Inhibition of PTK induces more prominent relaxation in trabecular meshwork than in ciliary muscle. The effects of inhibition of PTK on relaxation are independent of inhibition of PKC and PKA-PKG. The signaling cascade after activation of a tyrosine kinase receptor by EGF is differently modulated in trabecular meshwork and ciliary muscle. The effect of genistein on relaxation is probably not directly related to the EGF receptor. PTK inhibitors are possible agents for the development of novel antiglaucoma drugs.

Animals↗
PubMed1997

Prostaglandin effects on the contractility of bovine trabecular meshwork and ciliary muscle.

The ocular hypotensive activity of prostaglandins (PGs) has previously been demonstrated in various species including man. The underlying mechanism of action of prostanoids other than PGF2 alpha remains contentious. Because the trabecular meshwork and ciliary muscle are believed to have a role in the regulation of aqueous humor outflow, the aim of this study was to identify the PG-receptor subtypes present in these tissues using receptor-selective agonists. Contractions of isolated strips of bovine trabecular meshwork and ciliary muscle were recorded isometrically in continuously perfused tissue chambers. Contractile activity of PGs was determined relative to a maximally effective concentration of carbachol (1 microM) as a standard agonist. The following prostanoids were employed: PGF2 alpha, 17-phenyl PGF2 alpha (FP-receptor agonists), sulprostone (EP3 > EP1-agonist), AH13205 (EP2-agonist), 11-deoxy PGE1 (non-selective EP-agonist), and U-46619 (TP-agonist). The thromboxane-mimetic U-46619 elicited a strong contraction of the trabecular meshwork with the highest concentration (1 microM) being almost twice as efficacious (186.6%) as the maximal carbachol concentration, whereas the effect on the ciliary muscle was small. The U-46619 induced trabecular meshwork contraction could be blocked with a potent and selective TP-receptor antagonist, 1 microM SQ29548, indicating the involvement of TP-receptors. The other PG-analogs studied had either no or a small but statistically significant effect. Thus, 17-phenyl PGF2 alpha (1 microM) weakly contracted the ciliary muscle (4.8%), sulprostone (1 microM) the trabecular meshwork (10.1%), 11-deoxy PGE1 (1 microM) and AH13205 (10 microM) elicited relaxations in both tissue precontracted with carbachol (1 microM). The relaxant effects were more pronounced in trabecular meshwork (15.6% for 11-deoxy PG1 and 21.4% for AH13205) than ciliary muscle (6.8 and 7.4% respectively). PGF2 alpha did not elicit a significant response in either tissue. Our studies suggest the existence of TP- and EP2-receptors in the bovine trabecular meshwork and potentially FP- and EP2-receptors in the ciliary muscle. In conclusion, thromboxane-mimetics and EP2-agonists have opposing activities on contractile elements in the meshwork and may modulate trabecular outflow in a functionally antagonistic manner. Prostanoid effects on ciliary muscle appear rather modest compared to parasympathomimetic drugs. It is conceivable that TP-agonists may substantially affect trabecular outflow.

Animals↗
PubMed1997

Endothelin-like immunoreactivity in aqueous humor of patients with primary open-angle glaucoma and cataract.

BACKGROUND: Experimental evidence suggests a role of endothelin-1 (ET) in the regulation of intraocular pressure (IOP). METHOD: Therefore, in patients undergoing cataract surgery, ET-like immunoreactivity (ETIR) was measured by radioimmunoassay in pooled samples of aqueous humor of eyes with primary open-angle glaucoma (POAG) and normotensive eyes with cataract only. RESULTS: ETIR was significantly (P < 0.05) higher in patients with cataract and POAG (20.5 +/- 1.8 pg/ml, n = 12; preoperative IOP 21.4 +/- 1.1 mmHg, n = 33) than in patients with cataract only (15.8 +/- 1.6 pg/ml, n = 15; preoperative IOP 16.0 +/- 0.6 mmHg, n = 77). CONCLUSION: This finding may indicate a role of ET in POAG or ocular antihypertensive treatment, and its relevance should be further investigated.

Administration, Topical↗
PubMed1997

Regulation of L-type calcium channels by protein tyrosine kinase and protein kinase C in cultured rat and human retinal pigment epithelial cells.

The effect of protein tyrosine kinases (PTKs) on L-type calcium channel currents was studied in cultured rat and human retinal pigment epithelial cells. Barium currents through L-type channels were measured in the perforated patch-clamp technique and identified by using the L-type calcium channel opener Bay K8644 (10(-6) M). Application of the PTK blockers genistein (5 x 10(-6) M) or lavendustin A (5 x 10(-6) M) led to a decrease of L-type currents. The inactive genistein analog daidzein (10(-5) M) showed no effect on calcium channels. Intracellular application of pp60(c-src) (30 U/ml) via the patch-pipette during the conventional whole-cell configuration led to an increase of L-type currents. The protein kinase A and protein kinase G blocker H9 (10(-6) M) showed no effect on L-type currents; genistein reduced the current in the presence of H9. The protein kinase C (PKC) blocker chelerythrine (10(-5) M) reduced the L-type current; additional inhibition of PTK by lavendustin showed an additional reduction of currents. Intracellular application of myristoylated PKC substrate (5 x 10(-5) M) for PKC inhibition led to a fast rundown of L-type current amplitudes. Intracellularly applied myristoylated PKC substrate (10(-4) M) together with pp60(c-src) showed no effect on L-type current. Up-regulation of PKC by 10(-6) M phorbol-12-myristate-13-acetate (PMA) had no effect on the L-type current amplitude. However, genistein in cells pretreated with PMA led to an increase of the L-type currents. Intracellular application of pp60(c-src) in PMA-treated cells led to a reduction of L-type currents. We conclude that in the resting cell, PTK and PKC regulate L-type calcium channels in an additive manner. L-type channels appeared as a site of integration of PTK activation and of PKC-dependent pathways. The activity of PKC determines whether PTK decreases or increases L-type channel activity.

Animals↗