ORAI1 inhibition as an efficient preclinical therapy for tubular aggregate myopathy and Stormorken syndrome.

Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) are clinically overlapping disorders characterized by childhood-onset muscle weakness and a variable occurrence of multisystemic signs, including short stature, thrombocytopenia, and hyposplenism. TAM/STRMK is caused by gain-of-function mutations in the Ca2+ sensor STIM1 or the Ca2+ channel ORAI1, both of which regulate Ca2+ homeostasis through the ubiquitous store-operated Ca2+ entry (SOCE) mechanism. Functional experiments in cells have demonstrated that the TAM/STRMK mutations induce SOCE overactivation, resulting in excessive influx of extracellular Ca2+. There is currently no treatment for TAM/STRMK, but SOCE is amenable to manipulation. Here, we crossed Stim1R304W/+ mice harboring the most common TAM/STRMK mutation with Orai1R93W/+ mice carrying an ORAI1 mutation partially obstructing Ca2+ influx. Compared with Stim1R304W/+ littermates, Stim1R304W/+Orai1R93W/+ offspring showed a normalization of bone architecture, spleen histology, and muscle morphology; an increase of thrombocytes; and improved muscle contraction and relaxation kinetics. Accordingly, comparative RNA-Seq detected more than 1,200 dysregulated genes in Stim1R304W/+ muscle and revealed a major restoration of gene expression in Stim1R304W/+Orai1R93W/+ mice. Altogether, we provide physiological, morphological, functional, and molecular data highlighting the therapeutic potential of ORAI1 inhibition to rescue the multisystemic TAM/STRMK signs, and we identified myostatin as a promising biomarker for TAM/STRMK in humans and mice.

Expanding the clinical and genetic spectrum of pathogenic variants in STIM1.

INTRODUCTION/AIMS: Stromal interaction molecule 1 (STIM1) is a reticular Ca2+ sensor composed of a luminal and a cytosolic domain. Autosomal dominant mutations in STIM1 cause tubular aggregate myopathy and Stormorken syndrome or its variant York platelet syndrome. In this study we aimed to expand the features related to new variants in STIM1. METHODS: We performed a cross-sectional study of individuals harboring monoallelic STIM1 variants recruited at five tertiary centers involved in a study of inherited myopathies analyzed with a multigene-targeted panel. RESULTS: We identified seven individuals (age range, 26-57 years) harboring variants in STIM1, including five novel changes: three located in the EF-hand domain, one in the sterile α motif (SAM) domain, and one in the cytoplasmatic region of the protein. Functional evaluation of the pathogenic variants using a heterologous expression system and measuring store-operated calcium entry demonstrated their causative role and suggested a link of new variants with the clinical phenotype. Muscle contractures, found in three individuals, showed variability in body distribution and in the number of joints involved. Three patients showed cardiac and respiratory involvement. Short stature, hyposplenism, sensorineural hearing loss, hypothyroidism, and Gilbert syndrome were variably observed among the patients. Laboratory tests revealed hyperCKemia in six patients, thrombocytopenia in two patients, and hypocalcemia in one patient. Muscle biopsy showed the presence of tubular aggregates in three patients, type I fiber atrophy in one patient, and nonspecific myopathic changes in two patients. DISCUSSION: Our clinical, histological, and molecular data expand the genetic and clinical spectrum of STIM1-related diseases.

Functional analyses of STIM1 mutations reveal a common pathomechanism for tubular aggregate myopathy and Stormorken syndrome.

Tubular aggregate myopathy (TAM) is a progressive disorder characterized by muscle weakness, cramps, and myalgia. TAM clinically overlaps with Stormorken syndrome (STRMK), combining TAM with miosis, thrombocytopenia, hyposplenism, ichthyosis, short stature, and dyslexia. TAM and STRMK arise from gain-of-function mutations in STIM1 (stromal interaction molecule 1) or ORAI1, both encoding key regulators of Ca2+ homeostasis, and mutations in either gene result in excessive extracellular Ca2+ entry. The pathomechanistic similarities and differences between TAM and STRMK are only partially understood. Here we provide functional in vitro experiments demonstrating that STIM1 harboring the TAM D84G or the STRMK R304W mutation similarly cluster and exert a dominant effect on the wild-type protein. Both mutants recruit ORAI1 to the clusters, increase cytosolic Ca2+ levels, promote major nuclear import of the Ca2+ -dependent transcription factor NFAT (nuclear factor of activated T cells), and trigger the formation of circular membrane stacks. In conclusion, the analyzed TAM and STRMK mutations have a comparable impact on STIM1 protein function and downstream effects of excessive Ca2+ entry, highlighting that TAM and STRMK involve a common pathomechanism.

CRAC channels and disease - From human CRAC channelopathies and animal models to novel drugs.

Ca2+ release-activated Ca2+ (CRAC) channels are intimately linked with health and disease. The gene encoding the CRAC channel, ORAI1, was discovered in part by genetic analysis of patients with abolished CRAC channel function. And patients with autosomal recessive loss-of-function (LOF) mutations in ORAI1 and its activator stromal interaction molecule 1 (STIM1) that abolish CRAC channel function and store-operated Ca2+ entry (SOCE) define essential functions of CRAC channels in health and disease. Conversely, gain-of-function (GOF) mutations in ORAI1 and STIM1 are associated with tubular aggregate myopathy (TAM) and Stormorken syndrome due to constitutive CRAC channel activation. In addition, genetically engineered animal models of ORAI and STIM function have provided important insights into the physiological and pathophysiological roles of CRAC channels in cell types and organs beyond those affected in human patients. The picture emerging from this body of work shows CRAC channels as important regulators of cell function in many tissues, and as potential drug targets for the treatment of autoimmune and inflammatory disorders.

Mutagenesis of Snu114 domain IV identifies a developmental role in meiotic splicing.

Snu114, a component of the U5 snRNP, plays a key role in activation of the spliceosome. It controls the action of Brr2, an RNA-stimulated ATPase/RNA helicase that disrupts U4/U6 snRNA base-pairing prior to formation of the spliceosome's catalytic centre. Snu114 has a highly conserved domain structure that resembles that of the GTPase EF-2/EF-G in the ribosome. It has been suggested that the regulatory function of Snu114 in activation of the spliceosome is mediated by its C-terminal region, however, there has been only limited characterisation of the interactions of the C-terminal domains. We show a direct interaction between protein phosphatase PP1 and Snu114 domain 'IVa' and identify sequence 'YGVQYK' as a PP1 binding motif. Interestingly, this motif is also required for Cwc21 binding. We provide evidence for mutually exclusive interaction of Cwc21 and PP1 with Snu114 and show that the affinity of Cwc21 and PP1 for Snu114 is influenced by the different nucleotide-bound states of Snu114. Moreover, we identify a novel mutation in domain IVa that, while not affecting vegetative growth of yeast cells, causes a defect in splicing transcripts of the meiotic genes, SPO22, AMA1 and MER2, thereby inhibiting an early stage of meiosis.

Gain-of-function mutations in STIM1 and ORAI1 causing tubular aggregate myopathy and Stormorken syndrome.

Calcium (Ca2+) is a key regulator for a large number of cellular functions in all kinds of cells, and small disturbances of Ca2+ homeostasis can severely compromise normal physiology in various tissues and organs. A major mechanism controlling Ca2+ homeostasis is store-operated Ca2+ entry (SOCE), which relies on the concerted action of the reticular Ca2+ sensor STIM1 and the plasma membrane Ca2+ channel ORAI1. Gain-of-function mutations in the respective genes induce excessive Ca2+ entry, and cause tubular aggregate myopathy (TAM) and Stormorken syndrome. Both disorders are part of a clinical continuum and involve muscle weakness and additional variably pronounced features including miosis, thrombocytopenia, hyposplenism, ichthyosis, dyslexia, and short stature. Mutations in the reticular Ca2+ buffer calsequestrin (CASQ1) have moreover been associated with the mild end of the TAM/Stormorken syndrome spectrum. Here we review the clinical and histological characteristics of both disorders, provide an overview on the genetic causes, and thereby focus on the pathomechanisms leading to muscle dysfunction and the multi-systemic phenotype of tubular aggregate myopathy and Stormorken syndrome.

A dual mechanism promotes switching of the Stormorken STIM1 R304W mutant into the activated state.

STIM1 and Orai1 are key components of the Ca2+-release activated Ca2+ (CRAC) current. Orai1, which represents the subunit forming the CRAC channel complex, is activated by the ER resident Ca2+ sensor STIM1. The genetically inherited Stormorken syndrome disease has been associated with the STIM1 single point R304W mutant. The resulting constitutive activation of Orai1 mainly involves the CRAC-activating domain CAD/SOAR of STIM1, the exposure of which is regulated by the molecular interplay between three cytosolic STIM1 coiled-coil (CC) domains. Here we present a dual mechanism by which STIM1 R304W attains the pathophysiological, constitutive activity eliciting the Stormorken syndrome. The R304W mutation induces a helical elongation within the CC1 domain, which together with an increased CC1 homomerization, destabilize the resting state of STIM1. This culminates, even in the absence of store depletion, in structural extension and CAD/SOAR exposure of STIM1 R304W leading to constitutive CRAC channel activation and Stormorken disease.

Choroideremia: melanopsin-mediated postillumination pupil relaxation is abnormally slow.

PURPOSE: To investigate the rod-cone and melanopsin pupillary light response (PLR) pathways in choroideremia. METHODS: Eight patients with choroideremia and 18 healthy age-matched controls underwent chromatic pupillometry by applying blue (463 nm) and red light (643 nm) at 100 lux intensity to the right eye while recording pupil diameters. Absolute baseline pupil size (mm), normalized maximal pupil constriction and the early and late postillumination pupillary dilation, from 0 to 10 seconds and 10 to 30 seconds after the end of illumination, respectively, were determined. Postillumination responses to blue light were considered to be primarily driven by melanopsin activation of the intrinsic photosensitive retinal ganglion cells. RESULTS: Baseline pupil diameters were comparable in patients with choroideremia and control subjects (p = 0.48). The maximum pupil constriction in patients with choroideremia was severely weakened in red light but only mildly weakened in blue light (p < 0.05). Postillumination dilation of the pupil was normal after red illumination but extremely protracted after blue illumination. Also, in contrast to healthy subjects, no abrupt change in the dilation curve was seen in the patients after the end of blue illumination, the early-phase dilation being completely abolished (p < 0.01). CONCLUSION: Rod-cone-driven pupil responses were decreased as expected in an outer retinal degeneration, and near-normal pupil constriction in blue light supports that the melanopsin system is normal. In contrast, the lack of brisk early-phase dilation after blue illumination in choroideremia is remarkable and may be interpreted to mean that the absence of photoreceptor inhibition promotes a tonic contraction of the pupil.

ORAI1 Mutations with Distinct Channel Gating Defects in Tubular Aggregate Myopathy.

Calcium (Ca2+ ) is a physiological key factor, and the precise modulation of free cytosolic Ca2+ levels regulates multiple cellular functions. Store-operated Ca2+ entry (SOCE) is a major mechanism controlling Ca2+ homeostasis, and is mediated by the concerted activity of the Ca2+ sensor STIM1 and the Ca2+ channel ORAI1. Dominant gain-of-function mutations in STIM1 or ORAI1 cause tubular aggregate myopathy (TAM) or Stormorken syndrome, whereas recessive loss-of-function mutations are associated with immunodeficiency. Here, we report the identification and functional characterization of novel ORAI1 mutations in TAM patients. We assess basal activity and SOCE of the mutant ORAI1 channels, and we demonstrate that the G98S and V107M mutations generate constitutively permeable ORAI1 channels, whereas T184M alters the channel permeability only in the presence of STIM1. These data indicate a mutation-dependent pathomechanism and a genotype/phenotype correlation, as the ORAI1 mutations associated with the most severe symptoms induce the strongest functional cellular effect. Examination of the non-muscle features of our patients strongly suggests that TAM and Stormorken syndrome are spectra of the same disease. Overall, our results emphasize the importance of SOCE in skeletal muscle physiology, and provide new insights in the pathomechanisms involving aberrant Ca2+ homeostasis and leading to muscle dysfunction.

Effect of defocus on response time in different age groups: A pilot study / Efecto del desenfoque en el tiempo de respuesta en diferentes grupos de edad: estudio piloto

Purpose: To assess the response time associated with visual performance (VP) tasks in the presence of defocus in different presbyopic populations. Methods: 58 eyes between the ages of 35 and 50 years were studied. Subjects were categorized as pre-presbyopic (35-39 years), early-presbyopic (40-45 years), and mid-presbyopic (46-50 years). VP measurements obtained monocularly included distance and near high contrast (HC) and low contrast (LC) optotype recognition, and contrast threshold at 12cpd for different defocus magnitudes between 0D and 3D in 1D steps. Response time defined as the time taken to recognize and verbalize an optotype, was compared among different presbyopic age groups. Results: From 58 eyes, mean (SD) response time for high contrast distance visual acuity for 0D through 3D ranged between 1.48 (0.23) and 1.87 (0.31)s, whereas low contrast distance visual acuity ranged between 1.5 (0.22) and 2.09 (0.49)s. Mean response time for high contrast near visual acuity for 0D through 3D ranged between 1.56 (0.19) and 2.23 (0.45)s. However, for low contrast near visual acuity it ranged between 1.75 (0.32) and 2.71 (0.94)s. Mean (SD) response time for 12cpd ranged between 2.11 (0.50) and 5.72 (1.09)s. ANOVA revealed a significant difference in response time for distance, near visual acuity and contrast sensitivity as a function of defocus for different age groups. Conclusions: Response time is increased in the presence of increasing defocus for both distance and near visual acuity and could impact on performance for critical tasks. Full correction of visual acuity at distance and near in presbyopes is warranted always (AU)

Objetivo: Evaluar el tiempo de respuesta asociado a las tareas del desempeño visual (DV) en presencia de desenfoque, en diferentes poblaciones présbitas. Métodos: Se estudiaron 58 ojos de personas en edades comprendidas entre 35 y 50 años. Se clasificó a los sujetos conforme a las siguientes categorías: pre-presbicia (35-39 años), presbicia temprana (40-45 años), y presbicia media (46-50 años). Las mediciones del desempeño visual obtenidas de forma monocular incluyeron el reconocimiento de optotipos cercanos y lejanos de alto y bajo contraste y el umbral de contraste a 12cpd para las diferentes magnitudes de desenfoque, entre 0D y 3D, a intervalos de 1D. El tiempo de respuesta es el tiempo empleado en reconocer y verbalizar un optotipo, y se comparó entre los diferentes grupos de edad de los individuos présbitas. Resultados: De los 58 ojos, el tiempo de respuesta media (DE) para la agudeza visual de la distancia a alto contraste, entre 0D y 3D, osciló entre 1,48 (0,23) y 1,87 (0,31) segundos, mientras que la agudeza visual de la distancia a bajo contraste osciló entre 1,5 (0,22) y 2,09 (0,49) segundos. El tiempo de respuesta media para la agudeza visual cercana de alto contraste entre 0D y 3D osciló entre 1,56 (0,19) y 2,23 (0,45) segundos. Sin embargo para la agudeza visual cercana de bajo contraste osciló entre 1,75 (0,32) y 2,71 (0,94) segundos. El tiempo de respuesta media (DE) para 12cpd osciló entre 2,11 (0,50) y 5,72 (1,09) segundos. ANOVA reveló una diferencia significativa en cuanto al tiempo de respuesta para la distancia, agudeza visual cercana y sensibilidad de contraste como función del desenfoque para los diferentes grupos de edad. Conclusiones: El tiempo de respuesta se eleva al incrementarse el desenfoque en la agudeza visual lejana y cercana, pudiendo repercutir sobre el desempeño de ciertas tareas esenciales. La corrección plena de la agudeza visual cercana y lejana en individuos présbitas debe de ser siempre garantizada (AU)

Anillo de Malyugin para miosis intraoperatoria en femtofacovitrectomía / Malyugin ring for intraoperative miosis in femtosecond laser phacovitrectomy

OBJETIVO: Evaluar la utilidad del anillo de Malyugin transoperatorio en escasa dilatación pupilar durante facoemulsificación asistida con láser de Fs combinado con vitrectomía pars plana 23 G (femtofacovitrectomía). MÉTODO: Se presenta una mujer de 57 años con catarata y hemorragia vítrea, dilatación pupilar de 5,5 mm. Se realiza facoemulsificación asistida con láser de femtosegundo, colocando anillo de Malyugin posterior a extracción de cápsula anterior, seguido de vitrectomía pars plana y retirándolo al final. RESULTADOS: Se logró una dilatación pupilar transoperatoria adecuada, sin complicaciones, con una agudeza visual posoperatoria de 20/40. CONCLUSIONES: El uso del anillo de Malyugin puede ser una alternativa útil en casos con escasa dilatación pupilar en femtofacovitrectomía, conservando la integridad anatómica-funcional pupilar

OBJECTIVE: To evaluate the usefulness of the Malyugin ring in poor pupil dilation during phacoemulsification assisted with femtosecond laser with 23 gauge pars plana vitrectomy. METHOD: A 57-year-old female with cataract and vitreous hemorrhage, and poor pupil dilation (5.5 mm). The phacoemulsification assisted with femtosecond laser, using Malyugin ring after capsulorrhexis, followed by pars plana vitrectomy, and removing at the end without complications. RESULTS: A successfull intraoperative pupil dilation was achieved without complications, with a final BCVA of 20/40. CONCLUSIONS: The Malyugin ring is an effective alternative in cases with poor pupil dilation in femtophacovitrectomy, preserving the anatomical and functional integrity

York platelet syndrome is a CRAC channelopathy due to gain-of-function mutations in STIM1.

Store-operated Ca(2+) entry is the major route of replenishment of intracellular Ca(2+) in animal cells in response to the depletion of Ca(2+) stores in the endoplasmic reticulum. It is primarily mediated by the Ca(2+)-selective release-activated Ca(2+) (CRAC) channel, which consists of the pore-forming subunits ORAI1-3 and the Ca(2+) sensors, STIM1 and STIM2. Recessive loss-of-function mutations in STIM1 or ORAI1 result in immune deficiency and nonprogressive myopathy. Heterozygous gain-of-function mutations in STIM1 cause non-syndromic myopathies as well as syndromic forms of miosis and myopathy with tubular aggregates and Stormorken syndrome; some of these syndromic forms are associated with thrombocytopenia. Increased concentration of Ca(2+) as a result of store-operated Ca(2+) entry is essential for platelet activation. The York Platelet syndrome (YPS) is characterized by thrombocytopenia, striking ultrastructural platelet abnormalities including giant electron-opaque organelles and massive, multilayered target bodies and deficiency of platelet Ca(2+) storage in delta granules. We present clinical and molecular findings in 7 YPS patients from 4 families, demonstrating that YPS patients have a chronic myopathy associated with rimmed vacuoles and heterozygous gain-of-function STIM1 mutations. These findings expand the phenotypic spectrum of STIM1-related human disorders and define the molecular basis of YPS.

Safety, tolerability, pharmacokinetics, and pharmacodynamic effects of naloxegol at peripheral and central nervous system receptors in healthy male subjects: A single ascending-dose study.

This randomized, double-blind, placebo-controlled, ascending-dose, crossover study evaluated single oral doses of naloxegol (NKTR-118; 8, 15, 30, 60, 125, 250, 500, and 1000 mg), a PEGylated derivative of naloxone, for safety and tolerability, antagonism of peripheral and central nervous system (CNS) effects of intravenous morphine, and pharmacokinetics. Healthy men were randomized 1:1 to naloxegol or naloxegol-matching placebo administered with morphine and lactulose in a 2-period crossover design. Periods were separated by a 5- to 7-day washout. Assessments included safety, tolerability, orocecal transit time (OCTT), pupillary miosis, and pharmacokinetics. The study was completed by 46 subjects. The most common adverse events were somnolence, dizziness, headache, and nausea. Greater reversal of morphine-induced delay in OCTT occurred with increasing naloxegol dose, demonstrating dose-ordered antagonism of morphine's peripheral gastrointestinal effects. Forty-four subjects showed no reversal of pupillary miosis; 2 showed potential partial reversal at 250 and 1000 mg, indicating negligible antagonism of morphine's CNS effects at doses ≤ 125 mg. Rapid absorption, linear pharmacokinetics up to 1000 mg, and low to moderate between-subject pharmacokinetic variability was observed. The pharmacokinetics of morphine or its glucuronide metabolites were unaltered by concurrent naloxegol administration. Naloxegol was generally safe and well tolerated at single doses up to 1000 mg.

Gain-of-Function Mutation in STIM1 (P.R304W) Is Associated with Stormorken Syndrome.

Stormorken syndrome is a rare autosomal dominant disorder characterized by a phenotype that includes miosis, thrombocytopenia/thrombocytopathy with bleeding time diathesis, intellectual disability, mild hypocalcemia, muscle fatigue, asplenia, and ichthyosis. Using targeted sequencing and whole-exome sequencing, we identified the c.910C > T transition in a STIM1 allele (p.R304W) only in patients and not in their unaffected family members. STIM1 encodes stromal interaction molecule 1 protein (STIM1), which is a finely tuned endoplasmic reticulum Ca(2+) sensor. The effect of the mutation on the structure of STIM1 was investigated by molecular modeling, and its effect on function was explored by calcium imaging experiments. Results obtained from calcium imaging experiments using transfected cells together with fibroblasts from one patient are in agreement with impairment of calcium homeostasis. We show that the STIM1 p.R304W variant may affect the conformation of the inhibitory helix and unlock the inhibitory state of STIM1. The p.R304W mutation causes a gain of function effect associated with an increase in both resting Ca(2+) levels and store-operated calcium entry. Our study provides evidence that Stormorken syndrome may result from a single-gene defect, which is consistent with Mendelian-dominant inheritance.

Changes in prostaglandin levels in patients undergoing femtosecond laser-assisted cataract surgery.

PURPOSE: To investigate the intraocular prostaglandin concentrations after femtosecond laser treatment and the potential relationship to miosis. METHODS: Aqueous humor was collected from patients after femtosecond laser pretreatment and at the beginning of routine cataract surgery. The total prostaglandin and the prostaglandin E2 concentrations were measured in two independent studies using an enzyme-linked immunoassay. RESULTS: A significantly higher level of prostaglandins was noted in the aqueous humor of patients immediately after femtosecond laser treatment. This could be confirmed in two studies consisting of independent patient populations (study I: control = 17.3 ± 4.0 pg/mL [n = 22], femtosecond laser [femto] = 182.1 ± 38.1 pg/mL [n = 22], P = .0001, and study II: control = 17.5 ± 1.4 pg/mL [n = 37], femto: 377.1 ± 83.6 pg/ mL [n = 35], P = .00004). A significant increase of prostaglandin E2 was noted in two measurements (study III: control = 4.5 ± 1.9 pg/mL [n = 13], femto: 19.2 ± 2.5 pg/mL [n = 20], P = .0002, and study IV: control = 11.3 ± 1.6 pg/mL [n = 35], femto: 60.3 ± 16.1 pg/mL [n = 36], P = .004). No correlation was noted between age or cataract density and prostaglandin/prostaglandin E2 level or between corneal incision, suction time, or laser time in the femto groups and prostaglandin/prostaglandin E2 level. CONCLUSIONS: Prostaglandins rise immediately after femtosecond laser treatment. Future patients should perhaps be treated with non-steroidal anti-inflammatory drugs to maintain mydriasis before undergoing femto-second laser treatment for cataract surgery.

Muscarinic receptor dysfunction induced by exposure to low levels of soman vapor.

In the eye, it has been previously reported that exposure to a cholinesterase inhibitor results in a reduced miotic response following prolonged exposure and a decreased miotic response to the cholinergic agonists. However, no studies exist that characterize the effect of a single low-level vapor exposure to a nerve agent on parasympathetic function in the eye or determine the threshold dose for such an effect. The present study investigated the hypotheses that a single low-level exposure to soman vapor would result in dysfunction of the parasympathetic pathway mediating the pupillary light reflex resulting from a loss of muscarinic receptor function on the pupillary sphincter muscle. Adult male rats were exposed to soman vapor in a whole-body dynamic airflow exposure chamber. Rats exposed to low levels of soman vapor dose-dependently developed miosis (threshold dose between 4.1 and 6.1 mg-min/m3). Pupil size returned to preexposure levels within 48 h due to desensitization of pupillary muscarinic receptors, as assessed by the pupillary response to the muscarinic agonist oxotremorine. An attenuated pupillary light reflex was also present in miotic animals (threshold dose near 6.1 mg-min/m3). While pupil size recovers within 48 h, other measures of pupillary function, including the light reflex, acetylcholinesterase activity, and muscarinic receptor responsiveness, did not return to normal for up to 10 days postexposure. Recovery of the light reflex coincided with the recovery of pupillary muscarinic receptor function, suggesting that the attenuation of the light reflex was due to receptor desensitization.

Possible involvement of nitric oxide in morphine-induced miosis and reduction of intraocular pressure in rabbits.

The role of mu3 opioid receptors in morphine-induced intraocular pressure (IOP) lowering effect and miosis was evaluated in conscious, dark-adapted New Zealand white (NZW) rabbits using a masked-design study. IOP and pupil diameter (PD) measurements were taken at just before and 0.5, 1, 2, 4, 6 h after monolateral instillation of morphine (10, 50 and 100 microg/30 microl) as compared to vehicle administered in the contralateral eye. Morphine-induced ocular effects were challenged by a pre-treatment with the non-selective opioid receptor antagonist, naloxone (100 microg/30 microl), the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME, 1%, 30 microl), or the non-selective mu3 opioid receptor inhibitor, reduced L-glutathione (GSH, 1%, 30 microl). Morphine induced a dose-dependent decrease in IOP and PD. Pre-treatment with naloxone totally prevented morphine-induced decrease in IOP and miosis. Ocular administration of L-NAME or GSH alone failed to affect IOP or PD of NZW rabbits. However, pre-treatment with either drugs significantly reduced, but not totally prevented ocular effects of morphine. These results suggest that biochemical mechanisms related to nitric oxide release are involved, at least in part, in morphine effects on the eye. Since the mu3 opioid receptor subtype is able to release nitric oxide and is sensitive to inactivation by GSH, it may be possible that mu3 opioid receptors are involved in morphine-induced miosis and reduction in IOP.

Effects of meiosis-inhibiting agents and equine chorionic gonadotropin on nuclear maturation of canine oocytes.

Experiments were conducted to determine the effects of meiosis-inhibiting-agents and gonadotropins on nuclear maturation of canine oocytes. The culture medium was TCM199 + 10 ng/ml epidermal growth factor supplemented with 25 microM beta-mercaptoethanol, 0.25 mM pyruvate, and 1.0 mM L-glutamine (Basal TCM). Initially, oocytes were cultured in Basal TCM alone or in Basal TCM + dibutylryl cyclic adenosine monophosphate (0.5, 1, 5, or 10 mM dbcAMP) for 24 hr. Dibutylryl cAMP inhibited resumption of meiosis in a dose-dependent manner; 60% of oocytes remained at the germinal vesicle (GV) stage after being cultured for 24 hr in 5 mM dbcAMP. The meiosis-inhibitory effect of dbcAMP appeared to be reversible, as the oocytes resumed meiosis and completed nuclear maturation after being cultured for an additional 48 hr in its absence. Oocytes were then cultured in Basal TCM alone or in Basal TCM + roscovitine (12.5, 25, or 50 microM) for 24 hr. Although approximately 60% of oocytes cultured in 25 microM roscovitine remained at the GV stage, this percentage was not significantly different from the 48% that also remained at the GV stage when cultured in its absence. Oocytes were cultured in Basal TCM + 25 microM roscovitine for 17 hr, exposed briefly to equine chorionic gonadotropin (eCG), and then cultured in Basal TCM for 48 hr. Short exposure of oocytes to eCG was beneficial, as it significantly increased the proportion of oocytes developing beyond germinal vesicle breakdown (P < 0.05) with approximately 20-30% of these were metaphase I (MI) oocytes. Study of the kinetics of nuclear maturation demonstrated that large numbers of oocytes remained at MI even after being cultured for 52 hr following brief exposure to eCG. This study showed that in vitro maturation of canine oocytes can be somewhat improved by short exposure of oocytes to eCG. However, further studies are still required to derive effective methods to mature canine oocytes in vitro.

Effect of pituitary adenylate cyclase-activating peptide on isolated rabbit iris sphincter and dilator muscles.

PURPOSE: Pituitary adenylate cyclase-activating peptide (PACAP) is a sensory neuropeptide in the eye that is released by noxious stimuli and considered to be a mediator of the neurogenic ocular injury response, including miosis. The purpose of this study was to clarify the functional role of PACAP in iris sphincter and dilator muscles. METHODS: Iris sphincter and dilator muscles were isolated from rabbit eyes, and the effect of PACAP on mechanical responses of these muscles using isometric tension-recording methods was investigated. RESULTS: The iris sphincter responded to electric field stimulation with contractions composed of fast twitch and subsequent slow components. Both PACAP 27 and PACAP 38 enhanced the twitch response, but neither had an effect on the slow response. The effect of both PACAPs on the twitch response was dose dependent. Neither PACAP had an effect on the amplitude of contraction evoked by exogenously applied Ach. For the iris dilator muscle, PACAP 27 inhibited the contractions induced by field stimulation or phenylephrine, whereas PACAP 38 had no effect. CONCLUSIONS: Both PACAP 27 and PACAP 38 enhance cholinergic transmission in sphincter muscle. The PACAP 27 induces relaxation of the dilator muscle by a direct effect on the muscle itself. The PACAP released during an ocular inflammatory response may induce miosis by the enhancement of cholinergic stimulation of the iris sphincter and by direct relaxation of the dilator muscles.