Smartphone electrocardiogram for QT interval monitoring in Coronavirus Disease 2019 (COVID-19) patients treated with Hydroxychloroquine.

INTRODUCTION: The global pandemic of Corona Virus Disease 2019 (COVID-19) has led to the re-purposing of medications, such as hydroxychloroquine and lopinavir-ritonavir in the treatment of the earlier phase of COVID-19 before the recognized benefit of steroids and antiviral. We aim to explore the corrected QT (QTc) interval and 'torsadogenic' potential of hydroxychloroquine and lopinavir-ritonavir utilising a combination of smartphone electrocardiogram and 12-lead electrocardiogram monitoring. MATERIALS AND METHODS: Between 16-April-2020 to 30-April- 2020, patients with suspected or confirmed for COVID-19 indicated for in-patient treatment with hydroxychloroquine with or without lopinavir-ritonavir to the Sarawak General Hospital were monitored with KardiaMobile smartphone electrocardiogram (AliveCor®, Mountain View, CA) or standard 12-lead electrocardiogram. The baseline and serial QTc intervals were monitored till the last dose of medications or until the normalization of the QTc interval. RESULTS: Thirty patients were treated with hydroxychloroquine, and 20 (66.7%) patients received a combination of hydroxychloroquine and lopinavir-ritonavir therapy. The maximum QTc interval was significantly prolonged compared to baseline (434.6±28.2msec vs. 458.6±47.1msec, p=0.001). The maximum QTc interval (456.1±45.7msec vs. 464.6±45.2msec, p=0.635) and the delta QTc (32.6±38.5msec vs. 26.3±35.8msec, p=0.658) were not significantly different between patients on hydroxychloroquine or a combination of hydroxychloroquine and lopinavir-ritonavir. Five (16.7%) patients had QTc of 500msec or more. Four (13.3%) patients required discontinuation of hydroxychloroquine and 3 (10.0%) patients required discontinuation of lopinavirritonavir due to QTc prolongation. However, no torsade de pointes was observed. CONCLUSIONS: QTc monitoring using smartphone electrocardiogram was feasible in COVID-19 patients treated with hydroxychloroquine with or without lopinavir-ritonavir. The usage of hydroxychloroquine and lopinavir-ritonavir resulted in QTc prolongation, but no torsade de pointes or arrhythmogenic death was observed.

A novel frameshift deletion in the COL1A1 gene identified in a Chinese family with osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a genetically heterogeneous group of disorders, characterized by abnormal bone fragility, blue sclera, deafness, joint laxity, and soft-tissue dysplasia. The purpose of this study was to elucidate the genetic or molecular basis for OI type IA in a Chinese family. We evaluated the members of a family, in which six individuals are affected with increased bone fragility and blue sclera. Results of exome sequencing revealed a novel 1-bp deletion (c.2329delG, p.A777fs) in exon 33 of the COL1A1 gene in two affected individuals, but not in a control family member without OI. The variation co-segregated with the disease in all the OI patients but not in the unaffected family members. The mutation caused a frameshift alteration after codon 777, leading to premature termination of the COL1A1 protein. Thus, our findings identified a novel frameshift deletion c.2329delG (p.A777fs) in the COL1A1 gene, which is associated with OI type IA in a Chinese family.

Neuritin 1 promotes retinal ganglion cell survival and axonal regeneration following optic nerve crush.

Neuritin 1 (Nrn1) is an extracellular glycophosphatidylinositol-linked protein that stimulates axonal plasticity, dendritic arborization and synapse maturation in the central nervous system (CNS). The purpose of this study was to evaluate the neuroprotective and axogenic properties of Nrn1 on axotomized retinal ganglion cells (RGCs) in vitro and on the in vivo optic nerve crush (ONC) mouse model. Axotomized cultured RGCs treated with recombinant hNRN1 significantly increased survival of RGCs by 21% (n=6-7, P<0.01) and neurite outgrowth in RGCs by 141% compared to controls (n=15, P<0.05). RGC transduction with AAV2-CAG-hNRN1 prior to ONC promoted RGC survival (450%, n=3-7, P<0.05) and significantly preserved RGC function by 70% until 28 days post crush (dpc) (n=6, P<0.05) compared with the control AAV2-CAG-green fluorescent protein transduction group. Significantly elevated levels of RGC marker, RNA binding protein with multiple splicing (Rbpms; 73%, n=5-8, P<0.001) and growth cone marker, growth-associated protein 43 (Gap43; 36%, n=3, P<0.01) were observed 28 dpc in the retinas of the treatment group compared with the control group. Significant increase in Gap43 (100%, n=5-6, P<0.05) expression was observed within the optic nerves of the AAV2-hNRN1 group compared to controls. In conclusion, Nrn1 exhibited neuroprotective, regenerative effects and preserved RGC function on axotomized RGCs in vitro and after axonal injury in vivo. Nrn1 is a potential therapeutic target for CNS neurodegenerative diseases.

The FEN1 E359K germline mutation disrupts the FEN1-WRN interaction and FEN1 GEN activity, causing aneuploidy-associated cancers.

Polymorphisms and somatic mutations in Flap Endonuclease 1 (FEN1), an essential enzyme involved in DNA replication and repair, can lead to functional deficiencies of the FEN1 protein and a predisposition to cancer. We identified a FEN1 germline mutation that changed residue E359 to K in a patient whose family had a history of breast cancer. We determined that the E359K mutation, which is in the protein-protein domain of FEN1, abolished the interaction of FEN1 with Werner syndrome protein (WRN), an interaction that is critical for resolving stalled DNA replication forks. Furthermore, although the flap endonuclease activity of FEN1 E359K was unaffected, it failed to resolve bubble structures, which require the FEN1 gap-dependent endonuclease activity. To determine the etiological significance of E359K, we established a mouse model containing this mutation. E359K mouse embryonic fibroblasts (MEF) were more sensitive to DNA crosslinking agents that cause replication forks to stall. Cytological analysis suggested that the FEN1-WRN interaction was also required for telomere stability; mutant cell lines had fragile telomeres, increased numbers of spontaneous chromosomal anomalies and higher frequencies of transformation. Moreover, the incidence of cancer was significantly higher in mice homozygous for FEN1 E359K than in wild-type mice, suggesting that the FEN1 E359K mutation is oncogenic.

Characterization of a transformed rat retinal ganglion cell line.

The purpose of the present study was to establish a rat retinal ganglion cell line by transformation of rat retinal cells. For this investigation, retinal cells were isolated from postnatal day 1 (PN1) rats and transformed with the psi2 E1A virus. In order to isolate retinal ganglion cells (RGC), single cell clones were chosen at random from the transformed cells. Expression of Thy-1 (a marker for RGC), glial fibrillary acidic protein (GFAP, a positive marker for Muller cells), HPC-1/syntaxin (a marker for amacrine cells), 8A1 (a marker for horizontal and ganglion cells) and neurotrophins was studied using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunocytochemistry. One of the retinal cell clones, designated RGC-5, was positive for Thy-1, Brn-3C, Neuritin, NMDA receptor, GABA-B receptor, and synaptophysin expression and negative for GFAP, HPC-1, and 8A1, suggesting that it represented a putative RGC clone. The results of RT-PCR analysis were confirmed by immunocytochemistry for Thy-1 and GFAP. Upon further characterization by immunoblotting, the RGC-5 clone was positive for Thy-1, negative for GFAP, 8A1 and syntaxin. RGC 5 cells were also positive for the expression of neurotrophins and their cognate receptors. To establish the physiological relevance of RGC-5, the effects of serum/trophic factor deprivation and glutamate toxicity were analyzed to determine if these cells would undergo apoptosis. The protective effects of neurotrophins on RGC-5 after serum deprivation was also investigated. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated fluoresceinated dUTP nick end labeling (TUNEL). Serum deprivation resulted in apoptosis and supplementation with both BDNF and NT-4 in the growth media, protected the RGC-5 cells from undergoing apoptosis. On differentiation with succinyl concanavalin A (sConA), RGC-5 cells became sensitive to glutamate toxicity, which could be reversed by inclusion of ciplizone (MK801). In conclusion, a transformed rat retinal cell line, RGC-5, has certain characteristics of retinal ganglion cells based on Thy-1 and Brn-3C expression and its sensitivity to glutamate excitotoxicity and neurotrophin withdrawal. These cells may be valuable in understanding of retinal ganglion cell biology and physiology including in vitro manipulations in experimental models of glaucoma.

Ocular hypotensive and aqueous outflow-enhancing effects of AL-3037A (sodium ferri ethylenediaminetetraacetate).

AL-3037A (Sodium ferri ethylenediaminetetraacetate), a novel compound shown to stimulate the degradation of glycosaminoglycans, was evaluated for its effects on aqueous humor outflow and intraocular pressure (IOP) in four experimental models. Its effect on outflow facility was assessed in bovine and human ocular perfusion organ cultures. Its IOP effect was tested in normotensive and dexamethasone-induced ocular hypertensive rabbits. In bovine eyes, perfusion with AL-3037A (0.1% w/v, 2.3 m M) significantly increased the outflow facility well above the normal 'wash-out' effect. At 30 min after perfusion, the outflow facility of drug-treated eyes increased by 26.0+/-2.8% (mean +/- S.E.(M.), n = 8), significantly higher than the 12.1 +/- 2.8% increase in vehicle-treated eyes. This difference sustained throughout the study period (2 hr). The compound also enhanced aqueous outflow in perfused human anterior segments. In non-glaucomatous eyes, it produced a small decrease in IOP (15.4 +/- 4.6%, n = 17), but in tissues derived from glaucoma patients, bolus administration of 3 mg (7 micromol) of AL-3037A lowered the IOP by 52-68% (n = 2) lasting for at least 3 hr. This outflow-enhancing effect of AL-3037A in ex vivo studies was confirmed by in vivo results. In normotensive rabbits, oral (50 mg kg(-1)), intravenous (10 mg kg(-1)), or topical (2 mg; 50 microl of 4% w/v solution) administration of AL-3037A produced maximum reduction of IOP, when compared to vehicle-treated animals, by 34.7+/-3.5% (n = 10), 22.0 +/- 4.6% (n = 10), and 21.6 +/-4.5% (n = 10), respectively. In dexamethasone induced ocular hypertensive rabbits, topical application of the compound (0.5 mg; 25 microl of 2% w/v solution) reduced IOP significantly by 19.2+/- 0.4% (n = 7) at 3 hr after dosing. Importantly, the IOP lowering effect of AL-3037A did not diminish even after repeated treatments in consecutive days. Thus, in the four study models across three animal species, AL-3037A was demonstrated to be an efficacious ocular hypotensive compound whose effect is most likely mediated by augmentation of the aqueous outflow. Its proposed action on the metabolism of glycosaminoglycans may provide a new and unique mechanism for the treatment of glaucoma.

Protection by eliprodil against excitotoxicity in cultured rat retinal ganglion cells.

PURPOSE: To test whether eliprodil (SL 82.0715), a unique antagonist for the N-methyl-D-aspartate (NMDA) receptor, is protective in the glutamate-induced cytotoxicity model in cultured rat retinal ganglion cells (RGCs). METHODS: Two to four days after a fluorescent dye, Di-I, was injected near the superior colliculi, neonatal rats were killed, and retinal cells were dissociated and cultured. Survival of RGCs after drug treatment was assayed by counting Di-I fluorescent cells. RESULTS: In rat RGCs, glutamate-induced toxicity with a mean EC50 of 10.7 microM. Only 47% of RGCs survived after a 3-day treatment with 100 microM glutamate. Studies using selective agonists and antagonists indicated that the glutamate-induced toxicity was mediated largely by the NMDA receptor. Pretreatment with eliprodil protected against such toxicity. Eliprodil exhibited a mean IC50 of 1.0 nM (log [IC50] = -9.00 +/- 0.01, mean +/- SEM, n = 3; against cell death produced by 100 microM glutamate). At 1 microM, eliprodil was maximally protective; cell survival in the presence of 100 microM glutamate challenge was 100% +/- 5% (n = 3). This protective effect of eliprodil may be related to its reduction (by 78%) of NMDA-induced currents recorded under patch-clamp recording in these cells. CONCLUSIONS: Eliprodil is protective against glutamate cytotoxicity in retinal neurons. It may be a useful novel compound for the treatment of retinopathies including glaucoma in which excitotoxicity has been implicated.

Pharmacological characterization of an FP prostaglandin receptor on rat vascular smooth muscle cells (A7r5) coupled to phosphoinositide turnover and intracellular calcium mobilization.

An FP prostaglandin (PG) receptor on the A7r5 rat aorta smooth muscle cell line has been characterized by assays of phosphoinositide (PI) turnover and intracellular calcium mobilization stimulated by structurally diverse PGs. In the PI turnover assay, cloprostenol was the most potent PG tested, with a potency (EC50) of 0.84 +/- 0.06 nM (mean +/- S.E.M., n = 34), and was a full agonist. Other known FP receptor agonists tested in this assay had efficacies > or = 85% of the cloprostenol value and high potencies: 16-phenoxy PGF2 alpha (2.05 +/- 0.19 nM), 17-phenyl PGF2 alpha (2.80 +/- 0.59 nM), fluprostenol (4.45 +/- 0.19 nM), PGF2 alpha (30.9 +/- 2.82 nM) and PhXA85 (43.5 +/- 11.4 nM). Other classes of PGs evaluated (PGD2, enprostil, 17-phenyl PGE2, PGE2, sulprostone and U-46619) were less potent and less efficacious than the FP receptor agonists, or were inactive. For a large group of standard PGs evaluated in the PI turnover assay, both potencies and efficacies correlated well with those reported for the FP receptor of Swiss mouse 3T3 fibroblasts. The potencies of fluprostenol and PGF2 alpha as stimuli of intracellular calcium mobilization matched well their potencies in the PI turnover assay, but fluprostenol had twice the efficacy of PGF2 alpha. Both signaling responses stimulated by fluprostenol were significantly inhibited by U73122, a selective inhibitor of phosphoinositide turnover (IC50 = 1.25 +/- 0.16 microM for PI turnover), and by chelation of calcium in the medium. Together with the PI turnover data, these studies of intracellular calcium mobilization linked to activation of the FP receptor, provide additional characterization of the pharmacological properties of this receptor.

Activation of phospholipase C and guanylyl cyclase by endothelins in human trabecular meshwork cells.

PURPOSE: To characterize effects of endothelins on activities of phospholipase C (PLC) and nucleotide cyclases in human trabecular meshwork (TM) cells. METHODS: Cultured simian virus 40-transformed human TM (HTM-3) or non-transformed (HTM-16) cells were used. Changes in the PLC activity were determined by assaying the production of [3H] inositol phosphates. Accumulation of cyclic GMP or cyclic AMP in cell lysate was measured by radioimmunoassay. RESULTS: Endothelin-1 (ET-1; 1 microM) stimulated PLC in HTM-16 cells, but Sarafotoxin S6c (SRTX), an ET(B) receptor subtype-selective agonist (1 microM), did not. Similar results were obtained in HTM-3 cells: ET-1, but not ET-3 or SRTX, activated PLC in a dose-dependent manner, with a calculated EC50 of 646 pM. The peptide also stimulated the accumulation of cGMP in a concentration-dependent manner with an EC50 of 37.2 pM. ET-3 or SRTX was not effective except at much higher concentrations. Both the PLC and guanylyl cyclase stimulation induced by ET-1 (10 nM) were completely inhibited by pretreating the cells with BQ-123 (<10 microM), an ET(A) receptor selective antagonist, but not by BQ-788 (10 microM), an ET(B) receptor subtype-specific antagonist. Neither ET-1 nor ET-3 stimulated adenylyl cyclase activity in HTM-3 cells at concentration as high as 1 microM. CONCLUSION: ET-1 activates PLC and guanylyl cyclase in TM cells. Potency profiles of ET receptor agonists and antagonists suggest that the ET(A) receptor subtype is involved in both actions of ET-1. The effects of the ET peptides in TM cells are interesting and could be part of the mechanism of their IOP-lowering effect.

Histamine induced contraction of human ciliary muscle cells.

In cultured human ciliary muscle cells we previously showed that histamine, via an H1 receptor, stimulates the production of inositol phosphates and mobilization of intracellular calcium. We further investigated in this study whether histamine would cause contraction of human ciliary muscle cells. Photomicrographs were taken of the ciliary muscle cells before and after exposure to histamine. Cross sectional surface area of the cells was quantified using image analysis software. A decrease in cross sectional surface area was interpreted as an indication of cell contraction. The results of this study indicated that histamine (10(-6) M-10(-4) M) caused contraction of human ciliary muscle cells in a concentration-dependent fashion. The effect of histamine was mediated by the H1 receptor subtype since the histamine effect was antagonized by 10(-6) M chlorphentramine (an H1 receptor subtype selective antagonist) but not by 10(-6) M cimetidine (H2 antagonist) or thioperamide (H3 antagonist). The phospholipase C (PLC) inhibitor, U73122 (10(-6) M) and the intracellular calcium store depleting agent thapsigargin (10(-6) M) both prevented the histamine induced contraction, demonstrating that the activation of PLC and the intracellular calcium release were the key steps necessary for contraction. Our data indicate that in ciliary muscle cells, histamine, via an H1 receptor, activates PLC and increases intracellular calcium, which subsequently causes contraction of the cells.

Presence of functional type B natriuretic peptide receptor in human ocular cells.

PURPOSE: To study the effects of natriuretic peptides on cyclic guanosine monophosphate (cGMP) production and calcium mobilization in cultured human ocular cells. METHODS: Cultured simian virus 40-transformed (HTM-3) and nontransformed (HTM-16) human trabecular meshwork (TM) cells and nontransformed human ciliary muscle (CM) cells were used. Accumulation of cGMP in cells lysate was measured by radioimmunoassay. Intracellular calcium concentration was measured by microscope-based ratiofluorometry. RESULTS: Both atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) increased the accumulation of cGMP in HTM-3, HTM-16, and CM cells. In the nontransformed TM cells, CNP was five times more efficacious (maximal effect of CNP was 497% +/- 44% that of ANP) and 10 times more potent than ANP (ANP, log [EC50] = -6.99 +/- 0.08; CNP, log [EC50] = -7.96 +/- 0.20). Similar results were seen in HTM-3 and CM cells. Under the assay conditions used, the peptides increased only the production of cGMP without changing its degradation rate. The peptide-induced increase of cGMP in the TM and CM cells correlated with suppression of carbachol-induced calcium mobilization in the cell. CONCLUSIONS: It is known that CNP, but not ANP, selectively activates the guanylyl cyclase associated with the type B natriuretic peptide receptor (NPR-B). Thus, the data suggest that NPR-B is the primary functional NPR in the TM and CM cells. The effects on cGMP and calcium produced by the activation of this receptor are expected to alter TM and CM contractility and may affect aqueous humor hydrodynamics and intraocular pressure.

Human conjunctival epithelial cells express histamine-1 receptors coupled to phosphoinositide turnover and intracellular calcium mobilization: role in ocular allergic and inflammatory diseases.

Dispase-dissociated primary cultures of human conjunctival epithelial (HCE) cells were stimulated with histamine and the generation of inositol phosphates ([3H]IPs) from [3H]phosphoinositide (PI) hydrolysis and the mobilization of intracellular calcium ([Ca2+]i) were studied using ion exchange chromatography and Fura-2 fluorescence techniques, respectively. Histamine (100 microM) maximally stimulated PI turnover in HCE cells by 210 +/- 10% (n = 21) above basal levels and with a potency (EC50) of 3.3 microM (n = 4). Histamine (EC50 = 5.8 microM, n = 3) rapidly mobilized [Ca2+]i which peaked within 10 sec but which was still significantly elevated 20 min after stimulation. The histamine-induced [Ca2+]i responses did not desensitize upon repeated applications of histamine. The effects of histamine (100 microM) on PI turnover and [Ca2+]i were potently antagonized by the H1-antagonists, emedastine (IC50 = 1.6-2.9 nM), triprolidine (IC50 = 3.1 nM) and levocabastine (IC50 = 8 nM), but weakly by the H2-(ranitidine/cimetidine) and H3-(thioperamide) antagonists (IC50s = 10-100 microM). In conclusion, HCE cells have been shown to possess functional H1-histamine receptors that couple to inositol phosphates generation which then mobilize intracellular calcium. These intracellular signaling mechanisms may be intimately linked with the process of inflammatory cytokine secretion from the HCE cells after stimulation by histamine released from the conjunctival mast cells. The current results strongly suggest that the HCE cells are active participants in mediating, and perhaps amplifying, the pro-inflammatory and allergic effects of histamine which is released from conjunctival mast cells during ocular allergic and inflammatory reactions.

Effect of histamine on phosphoinositide turnover and intracellular calcium in human ciliary muscle cells.

This report describes the effect of histamine on phospholipase C (PLC) activity and calcium mobilization in cultured human ciliary muscle cells. PLC activity was assessed by measuring the production of inositol phosphates and intracellular calcium mobilization was assessed by Fura 2 ratio fluorometry. The stimulation of PLC by histamine was concentration dependent with an EC50 of 0.96 microM. The H1 antagonist chlorpheniramine blocked the response with an IC50 of 0.53 microM. Calcium fluorometry experiments indicated a mean basal calcium concentration of 36 nM with a 10(-4) M histamine induced mean peak value of 1132 nM followed by a gradually declining plateau phase. EC50 and IC50 (chlorpheniramine) values from histamine induced peak calcium concentrations agreed with the PLC results. Pretreatment of the cells with the PLC inhibitor U73122 at 10(-6) M completely blocked histamine induced calcium mobilization. Removal of extracellular calcium eliminated the plateau phase but not the initial calcium peak indicating that both intra and extracellular calcium sources are required for a normal response. The calcium ATPase inhibitor thapsigargin caused depletion of intracellular calcium stores and prevented a subsequent normal calcium mobilization response to histamine. Ryanodine, a release inhibitor of certain intracellular calcium stores, had no effect on the histamine induced response. The results of these experiments indicate that histamine, via an H1 receptor, activated the PLC second messenger pathway, and caused a multi-phasic mobilization of both intracellular and extracellular calcium. The entry of the extracellular calcium was shown to be dependent upon release of calcium from a ryanodine insensitive intracellular store.

Endothelin-induced changes of second messengers in cultured human ciliary muscle cells.

PURPOSE: To characterize the pharmacology of endothelin-induced changes in phospholipase C (PLC) activity, intracellular calcium concentration ([Ca2+]i) and cyclic adenosine monophosphate (cAMP) in cultured human ciliary muscle (HCM) cells. METHODS: Changes in PLC activity of HCM cells were determined by production of [3H] inositol phosphates. [Ca2+]i was determined by single-cell dynamic fluorescence ratio imaging. Radioimmunoassays were used to determine cAMP and prostaglandin E2 (PGE2) concentrations. RESULTS: Endothelin-1 (ET-1) stimulated PLC (mean EC50 = 335 pM) and activated calcium mobilization in HCM cells. These effects were mediated by the endothelin ETA receptor subtype because at ETB receptor-selective agonist, sarafotoxin S6c, was ineffective. Additionally, effects of ET-1 were inhibited by pretreatment with a selective ETA agonist, BQ610 (mean pKi = 9.96 for PLC). ET-1 also stimulated the production of PGE2 (mean EC50 = 12.0 nM) and cAMP (mean EC50 = 5.2 nM) by these cells. PGE2 appeared to mediate the stimulatory effect of ET-1 on adenylyl cyclase because blockade of ET-1-induced PGE2 production by 10 microM indomethacin also completely blocked the ET-1-activated cAMP production. CONCLUSIONS: ET-1 stimulated PLC and increased [Ca2+]i in HCM calls by the ETA receptor subtype. ET-1 also increased cAMP production, an effect likely mediated by the enhanced production of prostaglandins.

Effects of muscarinic agents on cultured human trabecular meshwork cells.

Intracellular calcium measurements were performed in cultured human trabecular meshwork cells preloaded with the cell permeant dye fura 2-AM. Fluctuations in calcium levels were then monitored with microscope-based ratio fluorometry. Carbachol increased intracellular calcium in a dose-dependent manner; as did oxotremorine-M, aceclidine, and pilocarpine. Carbachol's effect was blocked by the non-selective muscarinic antagonist atropine, as well as by muscarinic receptor subtype-selective antagonists such as pirenzepine (M1-selective), p-fHHSiD (M3-selective), and 4-DAMP (M1, M3 subtypes). Rank order of potencies for the antagonists' effects was atropine = 4-DAMP > p-fHHSiD > pirenzepine, a profile suggesting that the M3 receptor subtype is essential in the carbachol effect. Phospholipase C activity was estimated via measurement of total production of inositol phosphates in cultured human trabecular meshwork cells pre-exposed to 3H-myoinositol. In these cells, carbachol also stimulated phosphoinositide production in a dose-dependent manner, and an antagonist profile similar to that seen for calcium response was obtained when carbachol was used as the effector. The data indicate that muscarinic effects on cultured human trabecular meshwork calcium mobilization and phospholipase C activity are mediated by an M3-like receptor subtype. Therefore, the muscarinic M3 receptor may play a role in trabecular meshwork cell function(s).

Bonding characteristics of low-fusing porcelain bonded to pure titanium and palladium-copper alloy.

This study compared bond strengths among palladium-copper alloy/VMK 68 porcelain, cast titanium/Duceratin porcelain, and machine-milled titanium/Procera porcelain combinations and investigated the mode of bond failure of these combinations. The effect of multiple firings on the bond strength of porcelain bonded to machine-milled pure titanium was then examined. A uniform thickness of 1 mm of porcelain was applied along an 8 mm length in the central portion of metal specimens measured 25 x 3 x 0.5 mm. The specimens were subjected to a three-point bending test on a load-testing machine with a span distance of 20 mm, and the load of bond failure was recorded and statistically analyzed. Two completely debonded specimens and two longitudinally sectioned specimens of each group were studied with a scanning electron microscope to determine the mode of bond failure. The bond strength of Pd-Cu/VMK 68 porcelain was significantly greater than two titanium/porcelain combinations. There was no significant difference in the bond strengths of porcelain bonded to machine-milled pure titanium among the five porcelain firing schedules.

A study of the surface hardness and dimensional stability of several intermaxillary registration materials.

This study compared the surface hardness, the effect of time on surface hardness, and the dimensional stability of various intermaxillary relationship registration materials. The Shore hardness values of one zinc oxide-eugenol material, one polyether, and seven poly(vinyl siloxane) materials were obtained at 30 minutes and 24 hours after the start of mixing. The dimensional stability of one polyether and seven poly(vinyl siloxane) materials was measured using a standard mold as described in American Dental Association (ADA) Specification No. 19. All materials exhibited relatively high surface hardness despite some statistical differences among them. Four materials possessed higher surface hardness at 24 hours than at 30 minutes. Although the polyether showed significantly lower dimensional stability than the other materials, all materials satisfied the minimum requirement for Type I elastomeric impression material.

Muscarinic effects on cellular functions in cultured human ciliary muscle cells.

PURPOSE: To characterize the pharmacology of the carbachol-induced changes of phospholipase C (PLC) activity and intracellular calcium concentration ([Ca2+]i) in cultured human ciliary muscle cells. METHODS: Changes in PLC activity of cultured human ciliary muscle cells were determined by production of inositol phosphates. Single-cell dynamic fluorescence ratio imaging was used to determine [Ca2+]i. RESULTS: Carbachol, oxotremorine-M, aceclidine, and pilocarpine stimulated PLC with mean EC50s of 20, 8, 17, and 2 microM, respectively. The effect of carbachol on PLC was partially suppressed by extracellular Ca2+ depletion. This muscarinic effect was blocked by muscarinic antagonists, such as atropine (apparent pKi = 9.12, nonselective for muscarinic receptor subtypes), pirenzepine (pKi = 6.76, selective for the M1 receptor subtype), 4DAMP (pKi = 9.25, selective for the M1 and M3 subtypes), and fHHSiD (pKi = 7.77, selective for the M3 subtype). In [Ca2+]i experiments, carbachol increased [Ca2+]i transients in human ciliary muscle cells in a dose-dependent manner with a mean EC50 of 7 microM. 4DAMP was approximately 100 times more potent than pirenzepine in the inhibition of the carbachol-induced [Ca2+]i increase. [Ca2+]i oscillations were observed after carbachol stimulation and persisted after extracellular Ca2+ depletion. CONCLUSIONS: Muscarinic agonists activate PLC and increase [Ca2+]i in cultured human ciliary muscle cells through an M3-like muscarinic receptor subtype.