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Background Prevention of acute hemolytic transfusion reactions is a worldwide concern. The objective of this study was to develop a simple rat model of complement-mediated acute intravascular hemolysis. Study Design and Methods Human AB red blood cells (RBCs) were incubated with complement-sufficient or complement-deficient Wistar rat serum (WRS) in the presence and absence of human RBC antibody in vitro to elucidate the mechanism of hemolysis. To study the role of complement in acute intravascular hemolysis in vivo, Wistar rats were treated either with or without cobra venom factor (CVF) to deplete complement activity. Human AB RBCs were then injected into both groups of rats, followed by serial blood draws up to 2 hours. Venous blood clearance and lysis of transfused RBCs at each time point were measured by flow cytometry and spectrophotometry. RBC sequestration was determined in the liver, spleen, and kidney by immunohistochemistry. Results In vitro incubation of human RBCs with WRS demonstrated that RBC lysis was mediated via the classical complement pathway and that hemolysis was antibody dependent. Transfusion of human RBCs into rats showed significantly less hemolysis in the CVF group versus untreated group. RBC sequestration in the spleen and liver 2 hours posttransfusion were not quantitatively different between the two groups. Conclusions Given the much higher degree of similarity for rat and human complement compared to mice, this simple rat model is ideal for testing novel inhibitors of classical pathway activation for the prevention and treatment of acute intravascular hemolysis. more...

Objective The aim of this study was to compare the endometrial expression of milk fat globule-EGF factor 8 (MFG-E8), its receptor integrin αvβ3, and leukemia inhibitory factor (LIF) in patients with endometriosis and infertility and in healthy fertile patients during the window of implantation. Methods Five patients with peritoneal endometriosis and infertility (case group) and four healthy fertile patients (control group) were recruited. All patients were either diagnosed with or ruled out for endometriosis by laparoscopic surgery; the case group underwent surgery for infertility investigation and the control group for tubal ligation. Endometrial biopsies were performed in all patients during the window of implantation (LH+8 to LH+10), and then the samples were analyzed by immunochemistry for MFG-E8, integrin αvβ3, and LIF. Results In patients with endometriosis and infertility, expression of MFG-E8 was significantly increased in the glandular epithelium when compared to healthy fertile patients (p more...

Therapeutic hypothermia (HT) is the only intervention that improves outcomes in neonatal hypoxic-ischemic encephalopathy (HIE). However, the multifactorial mechanisms by which HT impacts HIE are incompletely understood. The complement system plays a major role in the pathogenesis of ischemia-reperfusion injuries such as HIE. We have previously demonstrated that HT modulates complement activity in vitro. Term equivalent rat pups were subjected to unilateral carotid ligation followed by hypoxia (8% O2) for 45 min to simulate HIE. A subset of animals was subjected to HT (31–32°C for 6 h). Plasma and brain levels of C3a and C5a were measured. Receptors for C3a (C3aR) and C5a (C5aR) along with C1q, C3, and C9 were characterized in neurons, astrocytes, and microglia. We found that HT increased systemic expression of C3a and decreased expression of C5a after HIE. In the brain, C3aR and C5aR are predominantly expressed on microglia after HIE. HT increased local expression of C3aR and decreased expression on C5aR after HIE. Furthermore, HT decreased local expression of C1q, C3-products, and C9 in the brain. HT is associated with significant alteration of complement effectors and their cognate receptors. Complement modulation may improve outcomes in neonatal HIE. more...

Objective— Prediabetic states are associated with accelerated atherosclerosis, but the availability of mouse models to study connections between these diseases has been limited. The aim of this study was to test the selective role of impaired insulin receptor/insulin receptor substrate-1 signaling on atherogenesis. Methods and Results— To address the effects of impaired insulin signaling associated with hyperinsulinemia on atherosclerosis in the absence of obesity and hyperglycemia, we generated insulin receptor ( Insr )/insulin receptor substrate-1 ( Insr1 ) double heterozygous apolipoprotein ( Apoe )-knockout mice ( Insr +/− Irs1 +/− Apoe −/− ) mice. Insr +/− Irs1 +/− Apoe −/− mice fed a Western diet for 15 weeks showed elevated levels of fasting insulin compared to Insr +/+ Irs1 +/+ Apoe −/− mice. There were no significant differences in glucose, triglyceride, HDL, VLDL, cholesterol levels or free fatty acid in the plasma of Insr +/− Irs1 +/− Apoe −/− and Insr +/+ Irs1 +/+ Apoe −/− mice. Atherosclerotic lesions were increased in male (brachiocephalic artery) and female (aortic tree) Insr +/− Irs1 +/− Apoe −/− compared to Insr +/+ Irs1 +/+ Apoe −/− mice. Bone marrow transfer experiments demonstrated that nonhematopoietic cells have to be Insr +/− Irs1 +/− to accelerate atherosclerosis. Impaired insulin signaling resulted in decreased levels of vascular phospho-eNOS, attenuated endothelium-dependent vasorelaxation and elevated VCAM-1 expression in aortas of Insr +/− Irs1 +/− Apoe −/− mice. In addition, phospho-ERK and vascular smooth muscle cell proliferation were significantly elevated in aortas of Insr +/− Irs1 +/− Apoe −/− mice. Conclusion— These results demonstrate that defective insulin signaling is involved in accelerated atherosclerosis in Insr +/− Irs1 +/− Apoe −/− mice by promoting vascular dysfunction and inflammation. more...

3,4-Methylenedioxymethamphetamine (MDMA) is an illicit psychoactive drug with cardiovascular effects that have not been fully described. In the current study, we observed the effects of acute MDMA on rabbit left ventricular function. We also observed the effects of MDMA on nuclear factor-kappa B (NF-kB) activity in cultured rat ventricular myocytes (H9c2). In the rabbit, MDMA (2 mg/kg) alone caused a significant increase in heart rate and a significant decrease in the duration of the cardiac cycle. Inhibition of nitric oxide synthase (NOS) by pretreatment with L-NAME (10 mg/kg) alone caused significant dysfunction in heart rate, systolic pressure, diastolic pressure, duration of relaxation, duration of cardiac cycle, and mean left ventricular pressure. Pretreatment with L-NAME followed by treatment with MDMA caused significant dysfunction in additional parameters that were not abnormal upon exposure to either compound in isolation: duration of contraction, inotropy, and pulse pressure. Exposure to 1.0 mM MDMA for 6 h or 2.0 mM MDMA for 12 h caused increased nuclear localization of NF-kB in cultured H9c2 cells. The current results suggest that MDMA is acutely detrimental to heart function and that an intact cardiovascular NOS system is important to help mitigate early sequelae in some functional parameters. The delayed timing of NF-kB activation suggests that this factor may be relevant to MDMA induced cardiomyopathy of later onset. more...

Successful ovulation requires elevated follicular prostaglandin E2 (PGE2) levels. To determine which PGE2 receptors are available to mediate periovulatory events in follicles, granulosa cells and whole ovaries were collected from monkeys before (0 h) and after administration of an ovulatory dose of hCG to span the 40-h periovulatory interval. All PGE2 receptor mRNAs were present in monkey granulosa cells. As assessed by immunofluorescence, PTGER1 (EP1) protein was low/nondetectable in granulosa cells 0, 12, and 24 h after hCG but was abundant 36 h after hCG administration. PTGER2 (EP2) and PTGER3 (EP3) proteins were detected by immunofluorescence in granulosa cells throughout the periovulatory interval, and Western blotting showed an increase in PTGER2 and PTGER3 levels between 0 h and 36 h after hCG. In contrast, PTGER4 (EP4) protein was not detected in monkey granulosa cells. Granulosa cell response to PGE2 receptor agonists was examined 24 h and 36 h after hCG administration, when elevated PGE2 levels present in periovulatory follicles initiate ovulatory events. PGE2 acts via PTGER1 to increase intracellular calcium. PGE2 increased intracellular calcium in granulosa cells obtained 36 h, but not 24 h, after hCG; this effect of PGE2 was blocked by a PTGER1 antagonist. A PTGER2-specific agonist and a PTGER3-specific agonist each elevated cAMP in granulosa cells obtained 36 h, but not 24 h, after hCG. Therefore, the granulosa cells of primate periovulatory follicles express multiple receptors for PGE2. Granulosa cells respond to agonist stimulation of each of these receptors 36 h, but not 24 h, after hCG, supporting the hypothesis that granulosa cells are most sensitive to PGE2 as follicular PGE2 levels peak, leading to maximal PGE2-mediated periovulatory effects just before ovulation. more...

PURPOSE Current adjunctive therapies to glaucoma surgery have unreliable effects, are toxic, and have numerous late complications associated with their use. This study examined whether topical cyclosporin (CsA) prolongs bleb survival after glaucoma filtration surgery. METHODS Anesthetized white New Zealand rabbits underwent glaucoma filtration surgery with a drainage tube. Cyclosporin (2%), applied intraoperatively or as topical treatment following glaucoma filtration surgery, was compared with intraoperative mitomycin C (MMC) and an untreated control group. RESULTS The bleb remained elevated for 15.1 +/- 3.2 days in the untreated control group, 12.2 +/- 2.1 days after intraoperative cyclosporin, and 27.5 +/- 1.7 days after intraoperative mitomycin C (P < 0.001). When topical treatment with cyclosporin followed intraoperative mitomycin C, bleb survival significantly decreased to 19.2 +/- 4.6 days (P = 0.003). Intraocular pressure (IOP) remained significantly reduced in the mitomycin C-treated group longer than in either the control or cyclosporine-treated groups. CONCLUSIONS In comparison with mitomycin C, neither intraoperative nor postoperative treatment with cyclosporin was associated with a decrease in intraocular pressure or prolonged bleb survival. Contrary to the initial hypothesis, topical treatment with cyclosporin actually mitigated the beneficial effects of mitomycin C on bleb survival. Clinical implications of these findings for patients with functioning blebs deserve further study. more...

To discourage fibrosis of the filtering bleb, 5 fluorouracil (5-FU) may be injected after trabeculectomy. 5-FU is an antimetabolite that also can damage extraocular tissues at concentrations as low as 0.5%. This study ascertained whether repeated injection of 5-FU has toxic effects on intraocular structures.After unilateral trabeculectomy in anesthetized New Zealand rabbits, 5-FU (5.0 mg/0.1 mL) was injected at the trabeculectomy site every 5 days for 15 days. Evaluation included slit-lamp examination, confocal microscopy, and intraocular pressure (IOP). After sacrifice, aqueous humor (AH) was drawn and eyes excised for scanning electron microscopy (SEM) and light microscopy.The 5-FU injection not decrease IOP beyond trabeculectomy alone. Bleb height remained constant, thickness increased, and vascularity decreased. No changes in cornea or anterior segment were observed. No inflammation was observed in the bleb or surrounding tissues by slit-lamp or histologic examination. Protein in AH increased from 0.6 +/- 0.5 microg/mL at baseline to 19.8 +/- 4.4 microg/mL after trabeculectomy but only to 0.9 +/- 0.6 microg/mL after trabeculectomy plus 5-FU. Both in vivo confocal microscopy and SEM revealed deleterious effects on corneal epithelial and endothelial cells with a minor shift toward smaller cells.In this study 5-FU did not provoke an intraocular inflammatory response and had minimal effect on extraocular structures. Changes in corneal epithelium and endothelium detectable by confocal microscopy suggest a small toxic effect. These in vivo measurements by confocal microscopy were confirmed by SEM. Repeated administration did not cause additional cumulative toxic effects in the anterior segment. Therefore, multiple injections of 5- FU into the filtering bleb pose minimal risk to intraocular structures. more...

* BACKGROUND AND OBJECTIVE: Almost all patients develop iritis following argon laser peripheral iridoplasty. Numerous adverse effects, particularly elevated intraocular pressure (IOP) and reduced microbial resistance, complicate therapy with topical corticosteroids. An immunomodulator, such as cyclosporin A (CsA), avoids these undesirable effects, yet may suppress ocular inflammation. * MATERIALS AND METHODS: Argon laser peripheral iridoplasty was performed on anesthetized rabbits with pigmented iris epithelium. Rabbits were randomly assigned to the untreated control, CsA (2%), or dexamethasone (0.1%) groups. Postoperative inflammation was documented by digital photography, IOP, and protein in aqueous humor. * RESULTS: Iris injection, aqueous flare, and fibrin decreased most rapidly in the control group, as did protein in aqueous humor. Decreases in IOP of 49% to 58% were similar in all three groups. There were no differences in conjunctival congestion between the groups. * CONCLUSION: Neither treatment with anti-inflammatory drugs that inhibit phagocytosis (eg, topical steroids) nor treatment with anti-inflammatory drugs that suppress T-lymphocytes (eg, topical CsA) significantly attenuated inflammation following iridoplasty. [Ophthalmic Surg Lasers Imaging 2004;35:475-481.] more...

Nineteenth century ophthalmology, characterized by significant gains in diagnostic techniques, provided the basis for great advancements in treatment during the 20th century. Drug therapy at the turn of the century was empiric, palliative, and often toxic. The development of ocular pharmacology during the 20th century provided the basis for a rational therapeutic approach to ocular disease. Foremost among the therapeutic developments were antibiotics, due to their potential to cure conditions that frequently resulted in blindness. Second, other therapeutic classes provided palliative therapy for chronic diseases, and thus decreased morbidity. For example, drugs specifically targeting many different aspects of glaucoma have had remarkable success controlling intraocular pressure and forestalling development of blindness. In addition, other new approaches provided palliative therapy for nonblinding conditions and effective adjuncts to surgical procedures. Antiallergy and anti-inflammatory drugs greatly increased patient comfort and facilitated treatment of allergic and inflammatory reactions. Local anesthetics and analgesia reduced patient discomfort during surgery. Other adjunct drugs improved surgical outcomes by reducing inflammation and infectious complications. The 21st century will undoubtedly provide novel approaches to address many of today's therapeutic dilemmas. Photodynamic therapy, growth factors, antisense technology, and genetic-based therapies all show great promise. Many of the conditions that are only treated palliatively today will be curable in the next century using many of these pharmacological advances. more...

Purpose To report the early, rapid diagnosis of the Chandler variant of the iridocorneal endothelial (ICE) syndrome using confocal light microscopy. Methods A 62-year-old man with a long history of unilateral glaucoma reported progressively blurred vision in his right eye. Examination of both eyes included visual acuity, slit-lamp examination, pneumotonometry, visual field, gonioscopy, and confocal microscopy. Results On examination, visual acuity was 20/80 and 20/20 and the IOPs were 26 and 12. The anterior segment OD revealed 1+ inferior and axial corneal edema, while the OS was normal to biomicroscopy and posterior segment. Chandler syndrome or Fuchs endothelial dystrophy was suspected. In the affected eye, confocal light microscopy clearly showed an "epithelium-like" transformation of the corneal endothelium with irregularly shaped cells and hyperreflective nuclei, establishing the diagnosis of Chandler syndrome. Conclusions In the presence of corneal edema or haze, corneal endothelium can be clearly visualized by confocal microscopy. "Epithelium-like" endothelial cells with highly reflective nuclei characteristic of Chandler syndrome were easily identified by confocal light microscopy to establish the diagnosis, despite the presence of corneal edema. Thus, confocal microscopy is a sensitive tool for the rapid, early diagnosis of ICE syndrome and may help distinguish among its variants. more...

α1-Adrenoceptor agonists not only contract rabbit femoral arteries, but also desensitize them so that the strength of subsequent contractions induced by 110 mM KCl is reduced. To determine the mechanisms by which this postreceptor desensitization occurs, tissues loaded with fura-2/AM were pretreated with phenylephrine (PE), washed, then activated with submaximum (23 mM) and maximum (30 mM) KCl concentrations. Pretreatment of tissues with 1 µMPE for 1–30 min resulted in reductions compared to control in the ability of 30 mM KCl to increase stress (force/tissue cross-sectional area). Pretreatment durations of 20 or 30 min with 10 µM PE also introduced delays between addition of KCl and commencement of both contraction (9.8 ± 0.8 and 2.4 ± 1.4 min when stimulated with, respectively, 23 and 30 mM KCl) and an increase in [Ca2+]i. At the end of the delay period, both [Ca2+]i and stress spontaneously increased, but although [Ca2+]i increased to control levels, stress did not. These data support the hypothesis that at least two postreceptor desensitizing mechanisms are activated by prior αi-adrenoceptor stimulation: (1) short-term inhibition of stimulus-induced increases in [Ca2+]i and (2) reductions in the sensitivity of the contractile response to [Ca2+]i. Interestingly, caffeine pretreatment mimicked the actions of PE pretreatment, implying that the superficial buffer barrier function of the sarcoplasmic reticulum or increases in cyclic nucleotide levels may have played a role in memory of α-adrenoceptor activation in rabbit femoral arteries. more...

Recent studies (e.g. Blackmore, P. F., Beebe, S. J., Danforth, D. R., and Alexander, N.) (1990) J. Biol. Chem. 265, 1376-1380) have shown that in human sperm, progesterone produces a rapid increase in intracellular free calcium ([Ca2+]i) and an induction of the acrosome reaction (e.g. Osman, R. A., Andria, M. L., Jones, A. D., and Meizel, S. (1989) Biochem, Biophys. Res. Commun. 160, 828-833). In this study, the location of progesterone receptors on the cell surface of human sperm was identified using progesterone immobilized on bovine serum albumin (BSA) (progesterone 3-(O-carboxymethyl)oxime:BSA) as well as progesterone and its 3-O-carboxymethyloxime derivative. Using fluorescence microscopy, BSA-fluorescein isothiocyanate was shown to be excluded from intact sperm, thus validating the use of progesterone 3-(O-carboxymethyl)oxime:BSA to identify cell surface-binding sites for progesterone. The immobilized progesterone and the 3-O-carboxymethyloxime derivative rapidly increased [Ca2+]i and were full agonists, although they were approximately 1.5 orders of magnitude less potent than progesterone. They also displayed an identical time course to increase [Ca2+]i as free progesterone, and the entire increase in [Ca2+]i was due to the influx of Ca2+. This progesterone-mediated response displayed different steroid receptor characteristics since the very potent inhibitors of genomic progesterone responses, RU38486 and ZK98.299, were very ineffective at inhibiting the progesterone-mediated increase in [Ca2+]i. Also the synthetic progestins megestrol, medroxyprogesterone acetate, norgestrel, norethynodrel, norethindrone, R5020, and cyproterone acetate did not mimic the effects of progesterone to increase [Ca2+]i. It is proposed that a distinct nongenomic cell surface receptor for progesterone exists in human sperm. more...

To determine the cardiovascular molecular events associated with acute exposure to cocaine, the present study utilized in vivo analysis of left-ventricular heart function in adult rabbits, fluorescence confocal microscopy of fluo-2, rhod-2, (5-(and-6) carboxy 2',7' dichlorodihydrofluores-cein diacetate (carboxy-H2DCFDA), and JC-1 in H9C2 cells and gene expression microarray technology for analysis of gene activation in both rabbit ventricular tissue and H9C2 cells. In the rabbit, acute cocaine exposure (2 mg/kg) caused left-ventricular dysfunction and 0.1-10 mM cocaine increased cytosolic and mitochondrial calcium activity and mitochondrial membrane depolarization in H9C2 cells. A 3-min pretreatment of H9C2 cells by 10 microM verapamil, nifedipine, or nadolol inhibited calcium increases, but only 1 mM N-acetylcysteine (NAC) or 1 mM glutathione blocked mitochondrial membrane depolarization. Cocaine induced activation of genes in the rabbit heart and H9C2 cells including angiotensinogen, ADRB1, and c-reactive protein (CRP). In H9C2 cells, NAC pretreatment blocked cocaine-mediated increases in CRP, FAS, FAS ligand, and cytokine receptor-like factor1 (CRLF1) expression. Collectively, these data suggest that acute cocaine administration initiates cellular and genetic changes that, if chronically manifested, could cause cardiac deficits similar to those seen in heart failure and ischemia, such as ventricular dysfunction, cardiac arrhythmias, and cardiac remodeling. more...

Long-term evaluation of dihematoporphyrin ether (DHE) safety and efficacy as photodynamic therapy (PDT) for patients with corneal neovascularization (KNV).Prospective multi-center interventional case series.Seven patients were enrolled after Institutional Review Board approval and a detailed informed consent were obtained. Eligible patients presented with clinically stable KNV without active vessel progression or inflammation. All patients with severe hypertension, history of renal or hepatic disease, or sensitivity to porphyrins, and those with active keratitis or uncontrolled ocular surface disease were excluded. DHE was administered as an intravenous bolus (2 mg/kg). Seventy-two hours later, PDT was carried out using argon green laser (514 nm). The main outcome measure, extent of vascular thrombosis, was estimated during postoperative follow-up examinations performed at day 1, 1 week, 6 months, and up to 12 years postoperative.All patients obtained an immediate reduction in measurable corneal vascularization. With at least 6 months of follow-up, six of seven patients maintained a significant reduction (52.5% +/- 19.6%, P.01) in KNV. The mean length of followup was 5.4 years (Range = 6 months to 12 years) during which time there were no other ocular changes attributable to laser treatment. Three patients suffered significant systemic short-term phototoxicity reactions.Intravenous DHE followed by photodynamic treatment in humans is effective for the reduction of inactive, established KNV. However, the significant short-term adverse effects related to systemic administration of this drug are of particular concern and warrant further investigation. more...

The purpose of this study was to determine (1) if the acute in vivo administration of cocaine and the cocaine metabolite ecgonine methyl ester (EME) activates the renin-angiotensin system (RAS) and alters cardiovascular function during pregnancy and (2) whether heart function is decreased in rabbits chronically exposed to cocaine or EME in vivo. The acute in vivo effects on plasma renin activity (PRA) were examined when cocaine (2 mg/kg) or EME (2 mg/kg) was administered intravenously. Arterial blood samples were withdrawn for hormonal analysis. Arterial pressure was measured using a COBE CDX III transducer and a Micro-Med 100 blood pressure analyzer. Hematocrit was measured using a microcapillary technique and serum protein was analyzed using refractometry. In chronic cocaine and EME studies, cardiovascular function was monitored in rabbits that were chronically exposed to cocaine or EME via Alzet osmotic pumps (1.66 microg/h for 14 d) implanted subcutaneously. On d 14, the animal was euthanized, and the heart was removed and exposed to 15 min of global ischemia using the Langendorff method. Coronary flow and left ventricular pressure (LVP) were assessed. The acute administration of cocaine or EME increased PRA and mean arterial pressure (MAP) in pregnant rabbits within 5 min. Coronary flow and LVP were significantly lower in the cocaine and EME-treated rabbits than in vehicle female rabbits. more...

Rabbit femoral arteries retain a memory of previous maximum receptor activation for up to 3-4 h after complete cessation of the stimulus, as reflected by a reduction in the steady-state contraction produced by a subsequent exposure to KCl. The present study examined the hypothesis that this modulatory effect involves alterations in postreceptor signal transduction. To quantify the degree of cellular downregulation induced by an episode of alpha 1-adrenoceptor stimulation, tissues were pretreated for 30 min with 10(-5) M phenylephrine (PE), washed for 10 min to cause complete relaxation, and activated with increasing concentrations of KCl. Pretreatment of tissues with PE resulted in a large reduction compared with control tissues in the ability of 20-60 mM KCl to increase stress and myosin light-chain phosphorylation. However, only at low (20 and 26 mM), not high (> 26 mM), KCl concentrations did PE pretreatment reduce the ability of KCl to increase intracellular free Ca2+ concentration ([Ca2+]i). These data support the hypothesis that memory of receptor activation involves reductions in both Ca2+ mobilization and the sensitivity of contractile proteins to [Ca2+]i. more...

Lacritin is a novel human tear glycoprotein that promotes basal tear peroxidase secretion by rat lacrimal acinar cells in vitro. This study investigates whether lacritin is prosecretory when added topically to the ocular surface of normal living rabbits, and if so, what is its efficacy and tolerability versus cyclosporine and artificial tears.Purified recombinant human lacritin (1, 10, 50, or 100 μg/mL), inactive lacritin truncation mutant C-25 (10 μg/mL), cyclosporine (0.05%), or artificial tears were topically administered to eyes of normal New Zealand White rabbits either as a single dose or three times daily for 14 days with monitoring of basal tear production. Basal tearing under proparacaine anesthesia was repeatedly assessed throughout and 1 week after chronic treatment ceased. Eyes were examined weekly by slit-lamp biomicroscopy.Lacritin acutely increased basal tearing to 30% over vehicle at 240 minutes. Three times daily treatment with 10-100 μg/mL lacritin was well tolerated. Basal tearing became progressively elevated 4, 7, and 14 days later and was 50% over baseline (50 μg/mL lacritin) 1 week after treatment had ceased. Cyclosporine elevated tearing to a similar level on days 4 and 7 but had little or no effect on day 14 and had returned to baseline 1 week after ending treatment. C-25 and artificial tears had no effect.Lacritin acutely stimulates basal tear flow that is sustained for at least 240 minutes. Two weeks of lacritin treatment three times daily was well tolerated and progressively elevated the basal tear flow. One week after treatment ended, basal tearing was still 50% over baseline. In contrast, cyclosporine triggered mild to moderate corneal irritation and a temporary elevation in tearing. more...

Summary Cell surface receptors for progesterone were visualized in human sperm using fluorescein isothiocyanate-progesterone 3-(0-carboxymethyl) oxime-bovine serum albumin (FITC prog CMO BSA). The receptors were confined to the head and not the midpiece or tail. FITC prog CMO BSA was also an effective stimulus to elevate intracellular free calcium in human sperm as detected by fura-2 fluorescence. The elevation of intracellular free calcium is a stimulus for the acrosome reaction, a process which is necessary to occur for sperm to fertilize the egg. It is proposed that progesterone, which is present in the female reproductive tract, can bind to progesterone receptors located in the plasma membrane of the sperm head and elicit an influx of Ca 2+ into the underlying cytoplasm and or acrosome and induce the acrosome reaction and facilitate fertilization. more...

The effects of the monocarboxylic ionophore, salinomycin (K+-selective), on isometric twitches, high K+-induced contracture and transmembrane action potentials were compared with those of the monocarboxylic ionophore, monensin (Na+-selective), in isolated canine right ventricular muscle. In a concentration (5 X 10(-6) M) which did not produce changes in resting force, salinomycin increased peak active force (Po, + 170 +/- 36%, mean % change from control +/- S.D., P less than 0.01), and maximal rates of force development (dP/dt max, + 123 +/- 33%, P less than 0.01) and relaxation (-dP/dt max, + 180 +/- 40%, P less than 0.01) of the isometric twitch. A similar response pattern was found for 5 X 10(-6) M monensin (Po, + 90 +/- 24%, P less than 0.01; dP/dt max, + 137 +/- 19%, P less than 0.01; -dP/dt max, + 145 +/- 20%, P less than 0.01). In contrast to their effects on isometric twitches, salinomycin reduced peak K+ contracture force (Pc, -35 +/- 14%, P less than 0.01) whereas monensin increased it (Pc, + 30 +/- 12%, P less than 0.02). Ventricular muscle action potential duration was shortened similarly by the ionophores. beta-Adrenergic receptor blockade with nadolol diminished salinomycin's effects on the isometric twitch and K+ contracture, but not its effect to shorten the action potential. Monensin's actions were unaffected by nadolol. These results suggest that salinomycin's effects arise from both a direct modulation of K+ movement and the release of endogenous catecholamine. In contrast, monensin may act to alter intracellular Na+ which in turn leads to Na+-Ca2+ exchange and Ca2+-mediated modulation of K+ movement. more...

The fluorescent calcium indicator quin2 has been used for the first continuous measurement of the effects of pharmacological agents on intracellular calcium activity in isolated, perfused rabbit hearts. The average intracellular calcium activity was elevated after the infusion of norepinephrine, concurrent with increases in left ventricular pressure and heart rate. These changes were abolished by pretreatment of the heart with phentolamine and nadolol, alpha and beta adrenergic receptor antagonists, respectively. Pretreatment with phentolamine and nadolol did not eliminate the increases in left ventricular pressure and intracellular calcium activity caused by the infusion of the monovalent carboxylic ionophores monensin and salinomycin. It is concluded that the ionophores cause these effects by elevating intracellular sodium activity, which then raises the intracellular calcium activity of the myocardium through intracellular displacement and/or transcellular exchange. It is suggested that the use of fluorescent calcium indicators in intact organs could be useful in evaluating the role of calcium in a variety of pathological states. more...