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1.
J Neonatal Perinatal Med ; 7(4): 279-86, 2014.
Article in English | MEDLINE | ID: mdl-25468621

ABSTRACT

BACKGROUND: Therapeutic hypothermia (HT) has been shown to decrease death and severe disability in infants with hypoxic-ischemic encephalopathy (HIE). Rectal temperature (RT) is used to determine the temperature set-points for treatment with HT, however experimental studies have shown significant differences between RT and brain temperature during HT. Knowledge of actual brain temperature during HT might allow better determination of optimal degree of cooling and improve outcomes. OBJECTIVES: To compare measurements of brain temperature obtained by non-invasive radiometric thermometry (RadT) to direct tissue measurements in an experimental model of HT, and to use RadT in newborn infants with HIE undergoing HT. STUDY DESIGN: RadT measurements of brain temperature were compared to fiber optic (Luxtron) thermometry measurements placed at a depth of 1.5 centimeters into the brain of cooled miniswine. Following validation studies, brain RadT and RT measurements were continuously recorded in thirty infants with HIE during HT and rewarming. RESULTS: RadT and Luxtron probe temperatures were comparable in miniswine throughout a temperature range similar to therapeutic HT. RadT measurements of brain temperature were higher than RT in 60% of infants with HIE undergoing HT. Higher RadT measurements compared to RT were associated with cerebral white matter abnormalities (p = 0.01). CONCLUSIONS: RadT provides a safe, passive and non-invasive way to measure brain temperature that can be used in the clinical setting. RadT may be helpful in determining the optimal degree of cooling and identifying infants at highest risk of brain injury.


Subject(s)
Asphyxia Neonatorum/physiopathology , Body Temperature/physiology , Hypoxia-Ischemia, Brain/physiopathology , Animals , Disease Models, Animal , Humans , Hypothermia, Induced/methods , Hypoxia-Ischemia, Brain/therapy , Infant, Newborn , Magnetic Resonance Imaging/methods , Swine , Thermometry/methods
2.
Exp Eye Res ; 82(5): 753-9, 2006 May.
Article in English | MEDLINE | ID: mdl-16289049

ABSTRACT

Glaucoma, one of the leading causes of blindness, is associated with high intraocular pressure (IOP) as a risk factor. The aim of this study was to examine both local and systemic effects of chronic topical administration of the synthetic CB1/CB2 agonist, WIN-55-212-2 and its potential to sustain ocular hypotension. WIN-55-212-2 (0.5%) or Tocrisolve, the vehicle, was administered topically three times daily to rats with surgically created glaucoma for 4 weeks, followed by a 1-week washout period. IOP, blood pressure and heart rate were measured weekly along with confocal microscopy and slit lamp biomicroscopy to detect ocular toxicity. IOP decreased rapidly by up to 47% in the WIN-55-212-2 treated group from 14.1+/-0.7 to 6.6+/-0.2 mmHg. The decrease was maintained during the treatment period. After the washout period, IOP (12.3+/-0.2 mmHg) was not different from baseline. In the contralateral eye, IOP showed a downward trend. Tocrisolve alone had no effect on IOP. No changes in blood pressure, heart rate or indicators of ocular toxicity were noted within either group. Topical application of WIN-55-212-2 significantly deceased IOP for duration of treatment. The decrease was sustained without the development of tolerance. Following cessation of therapy, IOP rapidly returned to baseline. No significant cardiovascular effects or ocular toxicity were noted during chronic topical therapy with either drug or vehicle.


Subject(s)
Antihypertensive Agents/therapeutic use , Glaucoma/drug therapy , Morpholines/therapeutic use , Naphthalenes/therapeutic use , Animals , Antihypertensive Agents/adverse effects , Benzoxazines , Blood Pressure/drug effects , Cornea/drug effects , Cornea/pathology , Drug Administration Schedule , Glaucoma/physiopathology , Heart Rate/drug effects , Male , Microscopy, Confocal , Morpholines/adverse effects , Naphthalenes/adverse effects , Ophthalmic Solutions , Rats , Rats, Sprague-Dawley
3.
J Ocul Pharmacol Ther ; 21(3): 223-35, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15969640

ABSTRACT

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.


Subject(s)
Anterior Eye Segment/drug effects , Antimetabolites/toxicity , Fluorouracil/toxicity , Intraocular Pressure/drug effects , Trabeculectomy , Animals , Anterior Eye Segment/metabolism , Anterior Eye Segment/ultrastructure , Antimetabolites/administration & dosage , Antimetabolites/pharmacokinetics , Antimetabolites/therapeutic use , Aqueous Humor/metabolism , Female , Fluorouracil/administration & dosage , Fluorouracil/pharmacokinetics , Fluorouracil/therapeutic use , Injections, Intralesional , Microscopy, Confocal , Microscopy, Electron, Scanning , Proteins/analysis , Rabbits , Wound Healing/drug effects
4.
J Ocul Pharmacol Ther ; 20(6): 533-47, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15684812

ABSTRACT

This study objectively compares efficacy of dexamethasone Na phosphate 0.1%, fluorometholone 0.1% (FML), loteprednol etabonate 0.5% (Lotemax [LE]; Bausch & Lomb Pharmaceuticals, Inc., Tampa, FL), prednisolone acetate 1% (Pred Forte [PRED F]; Allergan Pharmaceuticals, Irvine, CA), and generic prednisolone acetate 1% (PRED A). These steroids were administered for 24 hours or 72 hours to New Zealand white rabbits with endotoxin-induced uveitis. Intraocular pressure (IOP), slit-lamp examination, and confocal microscopy were performed daily. Internalization of the glucocorticoid receptor (GC) was assayed in iris tissue by Western blot, and protein in aqueous humor by Bradford assay. Only LE and PRED F treatments significantly internalized GC receptor after 72 hours of treatment. Only LE and PRED A reduced protein concentration between 24 hours and 72 hours of treatment. All drugs improved clinical signs after 24 hours of treatment. None of the steroids promoted return of the inflammation-induced corneal thickness to baseline. While none returned IOP to baseline, LE was most effective. Confocal microscopy indicated that only treatment with LE reverted the abnormal endothelial-cell shape to normal. In conclusion, all steroid treatments reduced uveitis to some degree but LE was consistently effective. A longer observation period may be required to document the return of IOP and corneal thickness to baseline values.


Subject(s)
Glucocorticoids/therapeutic use , Uveitis, Anterior/drug therapy , Acute Disease , Administration, Topical , Androstadienes/administration & dosage , Androstadienes/therapeutic use , Animals , Aqueous Humor/chemistry , Aqueous Humor/drug effects , Blotting, Western , Conjunctiva/blood supply , Conjunctiva/physiopathology , Corneal Stroma/chemistry , Corneal Stroma/drug effects , Corneal Stroma/pathology , Dexamethasone/administration & dosage , Dexamethasone/analogs & derivatives , Dexamethasone/therapeutic use , Endothelium, Corneal/drug effects , Endothelium, Corneal/pathology , Endothelium, Corneal/physiopathology , Eye Proteins/analysis , Fibrin/analysis , Fluorometholone/administration & dosage , Fluorometholone/therapeutic use , Glucocorticoids/administration & dosage , Intraocular Pressure/drug effects , Lipopolysaccharides/administration & dosage , Lipopolysaccharides/toxicity , Loteprednol Etabonate , Male , Particle Size , Prednisolone/administration & dosage , Prednisolone/analogs & derivatives , Prednisolone/therapeutic use , Rabbits , Receptors, Glucocorticoid/metabolism , Suspensions , Time Factors , Uveitis, Anterior/chemically induced , Uveitis, Anterior/metabolism , Vitreous Body/chemistry , Vitreous Body/drug effects , Vitreous Body/pathology
5.
J Clin Pharmacol ; 40(9): 990-1006, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10975070

ABSTRACT

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.


Subject(s)
Eye Diseases/therapy , Ophthalmology/history , Anti-Allergic Agents/therapeutic use , Anti-Infective Agents/therapeutic use , Anti-Inflammatory Agents/therapeutic use , Blindness/etiology , Blindness/prevention & control , Eye Diseases/complications , Eye Diseases/drug therapy , Eye Diseases/surgery , Glaucoma/etiology , Glaucoma/surgery , History, 20th Century , Humans , Mydriatics/administration & dosage , Mydriatics/therapeutic use , Ophthalmology/trends , Palliative Care
6.
J Biol Chem ; 275(11): 7731-42, 2000 Mar 17.
Article in English | MEDLINE | ID: mdl-10713085

ABSTRACT

Murine sperm initiate fertilization by binding to specific oligosaccharides linked to the zona pellucida, the specialized matrix coating the egg. Biophysical analyses have revealed the presence of both high mannose and complex-type N-glycans in murine zona pellucida. The predominant high mannose-type glycan had the composition Man(5)GlcNAc(2), but larger oligosaccharides of this type were also detected. Biantennary, triantennary, and tetraantennary complex-type N-glycans were found to be terminated with the following antennae: Galbeta1-4GlcNAc, NeuAcalpha2-3Galbeta1-4GlcNAc, NeuGcalpha2-3Galbeta1-4GlcNAc, the Sd(a) antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc, NeuGcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc), and terminal GlcNAc. Polylactosamine-type sequence was also detected on a subset of the antennae. Analysis of the O-glycans indicated that the majority were core 2-type (Galbeta1-4GlcNAcbeta1-6[Galbeta1-3]GalNAc). The beta1-6-linked branches attached to these O-glycans were terminated with the same sequences as the N-glycans, except for terminal GlcNAc. Glycans bearing Galbeta1-4GlcNAcbeta1-6 branches have previously been suggested to mediate initial murine gamete binding. Oligosaccharides terminated with GalNAcbeta1-4Gal have been implicated in the secondary binding interaction that occurs following the acrosome reaction. The significant implications of these observations are discussed.


Subject(s)
Glycoside Hydrolases , Oligosaccharides/chemistry , Polysaccharides/chemistry , Zona Pellucida/chemistry , Animals , Carbohydrate Sequence , Female , Gas Chromatography-Mass Spectrometry , Glycopeptides/chemistry , Lectins/metabolism , Mannosidases/metabolism , Methylation , Mice , Models, Chemical , Models, Molecular , Molecular Sequence Data , Neuraminidase/metabolism , Protein Binding , Sequence Analysis , Spectrometry, Mass, Fast Atom Bombardment , alpha-Mannosidase , beta-Galactosidase/metabolism , beta-N-Acetylhexosaminidases/metabolism
7.
J Vasc Res ; 33(6): 489-98, 1996.
Article in English | MEDLINE | ID: mdl-8998198

ABSTRACT

alpha 1-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 microM PE 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 microM 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 alpha 1-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 alpha-adrenoceptor activation in rabbit femoral arteries.


Subject(s)
Calcium/physiology , Vascular Resistance/drug effects , Animals , Arteries/drug effects , Caffeine/pharmacology , Dose-Response Relationship, Drug , Muscle Contraction , Phenylephrine/pharmacology , Potassium Chloride/pharmacology , Rabbits , Time Factors , Vasoconstriction/drug effects
8.
Am J Physiol ; 269(6 Pt 1): C1402-7, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8572169

ABSTRACT

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.


Subject(s)
Arteries/physiology , Calcium/metabolism , Intracellular Membranes/metabolism , Mechanoreceptors/physiology , Vasoconstriction/physiology , Animals , Homeostasis , In Vitro Techniques , Isometric Contraction/physiology , Myosin Light Chains/metabolism , Osmolar Concentration , Phenylephrine/pharmacology , Phosphorylation/drug effects , Potassium Chloride/pharmacology , Rabbits
9.
Eur J Pharmacol ; 236(3): 433-41, 1993 Jun 04.
Article in English | MEDLINE | ID: mdl-7689465

ABSTRACT

The effects of the short-acting anesthetic, ketamine, on intracellular free Ca2+ concentrations, ([Ca2+]i), inositol phosphate levels and force produced by contractile agonists were investigated in strips of rabbit femoral artery. In concentration-response curves, ketamine produced an insurmountable inhibition of contractions produced by KCl and the L-type Ca2+ channel agonist, Bay k 8644. However, in K(+)-depolarized tissues, high concentrations of CaCl2 could overcome the inhibition produced by ketamine, suggesting that ketamine may have competed with Ca2+ in activated L-type Ca2+ channels. In support of the contention that it inhibits L-type Ca2+ channels, ketamine was found to concomitantly reduce the levels of force and [Ca2+]i produced by 50 mM KCl. Ketamine reduced the potency, but not the maximum force, produced by phenylephrine. However, this surmountable inhibition may have been due to activation of 'spare' alpha-adrenoceptors rather than to competition of receptor binding because, after phenoxybenzamine pretreatment to reduce alpha-adrenoceptor numbers, phenylephrine concentration-response curves in the presence of ketamine were insurmountable. Ketamine at 0.32 mM reduced the transient contractions produced in a Ca(2+)-free solution and the increase in phospholipase C activity (estimated by measuring inositol phosphate production in the presence of Li+) produced by 1 but not 10 microM phenylephrine. These data suggest that ketamine inhibited contractions produced in rabbit femoral artery by decreasing Ca2+ channel activity and by reducing phospholipase C activation.


Subject(s)
Calcium/metabolism , Ketamine/pharmacology , Muscle, Smooth, Vascular/drug effects , Type C Phospholipases/metabolism , 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology , Animals , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Femoral Artery/drug effects , In Vitro Techniques , Ketamine/administration & dosage , Male , Muscle Contraction/drug effects , Muscle Relaxation/drug effects , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/physiology , Phenylephrine/pharmacology , Phosphatidylinositols/metabolism , Rabbits
10.
Am J Physiol ; 262(3 Pt 1): C621-7, 1992 Mar.
Article in English | MEDLINE | ID: mdl-1372479

ABSTRACT

F- (10 mM sodium fluoride plus deferoxamine to chelate contaminating aluminum) causes arterial contractions primarily by activating L-type Ca2+ channels. Results from the present study indicate that, although F(-)-induced contractions could be completely relaxed by washing out the F- with fresh buffer, a long-lasting effect of F- pretreatment was to produce L-type Ca2+ channel desensitization. Pretreatment of arteries for 4 h with F- (followed by washout of F-) resulted in much reduced increases in stress and [Ca2+]i produced by the subsequent addition of 110 mM KCl, such that steady-state values were, respectively, only 9 and 15% of the control values. However, a 4-h F- pretreatment caused a reduction only in the rate of stress development, but not the steady-state level of stress, produced by maximum concentrations of receptor agonists. In tissues that were pretreated with F- and then stimulated with the alpha-adrenoceptor agonist, phenylephrine, steady-state stress was still 104% of the control value, while the increase in [Ca2+]i was only 10% of the control value. F- is known to inhibit protein phosphatases, and similar reductions in the ability of KCl to produce contractions and increase [Ca2+]i were seen after pretreatment with the protein phosphatase inhibitor, okadaic acid. These data suggest that L-type Ca2+ channel desensitization by F- pretreatment was caused by increased protein phosphorylation. In addition, they suggest that much of the contribution made by L-type Ca2+ channels to increase [Ca2+]i during receptor stimulation may not be necessary for the maintenance of maximum stress at steady state.


Subject(s)
Calcium Channels/physiology , Calcium/metabolism , Fluorides/pharmacology , Muscle Contraction/physiology , Muscle, Smooth, Vascular/physiology , Phenylephrine/pharmacology , 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology , Animals , Calcium Channels/drug effects , Deferoxamine/pharmacology , Dinoprost/pharmacology , Female , Femoral Artery/physiology , In Vitro Techniques , Male , Muscle Contraction/drug effects , Muscle, Smooth, Vascular/drug effects , Potassium Chloride/pharmacology , Rabbits , Stress, Mechanical , Time Factors
11.
Biochem Biophys Res Commun ; 181(1): 331-6, 1991 Nov 27.
Article in English | MEDLINE | ID: mdl-1958202

ABSTRACT

Cell surface receptors for progesterone were visualized in human sperm using fluorescein isothiocyanate-progesterone 3-(O-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 Ca2+ into the underlying cytoplasm and or acrosome and induce the acrosome reaction and facilitate fertilization.


Subject(s)
Receptors, Progesterone/physiology , Sperm Head/physiology , Calcium/metabolism , Cell Membrane/physiology , Fluorescein-5-isothiocyanate , Humans , Male , Progesterone/pharmacology , Receptors, Progesterone/drug effects , Receptors, Progesterone/metabolism , Sperm Head/metabolism , Sperm Head/ultrastructure , Sperm Motility
12.
Am J Physiol ; 260(6 Pt 1): G958-64, 1991 Jun.
Article in English | MEDLINE | ID: mdl-1905487

ABSTRACT

The time dependence of lightly loaded shortening velocity, myosin phosphorylation, and changes in myoplasmic Ca2+ concentration ([Ca2+]i) were measured during tonic and phasic contractions of circular smooth muscle from the proximal colon of the dog. Shortening velocity was measured by quick release to a 10% afterload. Myosin phosphorylation was measured by an immunoblot method, and changes in [Ca2+]i were estimated by measuring fluorescence intensity at 550 nm in muscle strips loaded with fluo-3. During tonic contractions induced by 60 mM K+, phosphorylation increased monotonically from 0.11 +/- 0.011 to 0.29 +/- 0.015 mol Pi/mol light chain at 10 min. In contrast, lightly loaded shortening velocity increased rapidly within 10 s to 0.042 +/- 0.003 lengths/s and decreased exponentially to 0.013 +/- 0.001 lengths/s at 15 min. During transient contractions induced by 100 microM acetylcholine, phosphorylation increased from 0.16 +/- 0.03 to 0.30 +/- 0.06 mol Pi/mol light chain at 19 s. In contrast, shortening velocity increased to 0.068 +/- 0.015 lengths/s within 2.4 s and decreased significantly to 0.027 +/- 0.009 lengths/s at 22 s. Fluo-3 fluorescence increased in parallel with force during both tonic and transient contractions. In a smooth muscle that is able to contract both tonically and phasically we observed transient increases in shortening velocity without concurrent phosphorylation or [Ca2+]i transients. Therefore, there are factors in addition to myosin phosphorylation or changes in [Ca2+]i that regulate cross-bridge cycling rates in both tonic and phasic contractions.


Subject(s)
Calcium/metabolism , Colon/physiology , Isometric Contraction , Muscle, Smooth/physiology , Myosins/metabolism , Acetylcholine/pharmacology , Aniline Compounds , Animals , Calcium Chloride/pharmacology , Colon/drug effects , Dogs , Egtazic Acid/pharmacology , Female , Fluorescent Dyes , Immunoblotting , In Vitro Techniques , Isometric Contraction/drug effects , Kinetics , Male , Muscle, Smooth/drug effects , Myosins/isolation & purification , Phosphorylation , Potassium/pharmacology , Sodium Fluoride/pharmacology , Spectrometry, Fluorescence , Xanthenes
13.
Biochem Biophys Res Commun ; 177(1): 184-91, 1991 May 31.
Article in English | MEDLINE | ID: mdl-2043105

ABSTRACT

Fluorescent calcium indicators fluo-3, fura-2 and indo-1, and fluorescent magnesium indicators mag-fura-2 (FURAPTRA) and mag-indo-1 were evaluated for the effects of pH on their association and dissociation rates, ion selectivity and thermodynamic properties. Calcium indicator affinities for Ca and Mg were reduced and the discrimination between Ca and Mg decreased in fura-2 and indo-1 at acidic pH. Alterations in apparent dissociation constants were caused primarily by reduced association rates. Magnesium indicators did not show these changes. The enthalphies of the calcium indicators' Ca complex were 1-3 kcal/mole and magnesium indicators' Mg complex were 7-9 kcal/mole. The potential effects of a biexponential dissociation rate of fluo-3 and of Ca interactions with magnesium indicators were examined.


Subject(s)
Calcium/analysis , Fluorescent Dyes , Magnesium/analysis , Aniline Compounds , Benzofurans , Fura-2 , Hydrogen-Ion Concentration , Indoles , Oxazoles , Spectrometry, Fluorescence/methods , Thermodynamics , Xanthenes
14.
Biochem Biophys Res Commun ; 171(1): 102-8, 1990 Aug 31.
Article in English | MEDLINE | ID: mdl-2118341

ABSTRACT

A novel method of determining the apparent dissociation constants of fluorescent calcium indicators is described which utilizes Chelex-100 ion exchange resin and 45Ca. The affinity for calcium of indicators fluo-3, fura-2 and indo-1 measured at either 22 degrees or 37 degrees C decreases as pH is decreased from 7.4 to 5.5. These measurements agree with determinations made using EDTA-calcium buffers. The 1:1 calcium:indicator complex is maintained under all conditions. The necessity to correct dissociation constants during intracellular acidification to properly interpret fluorescence measurements is illustrated by indo-1 measurements in the ischemic rat heart.


Subject(s)
Calcium/analysis , Hydrogen-Ion Concentration , Temperature , Aniline Compounds , Benzofurans , Chelating Agents , Edetic Acid , Fura-2 , Indoles , Resins, Synthetic , Xanthenes
15.
J Biol Chem ; 262(6): 2711-8, 1987 Feb 25.
Article in English | MEDLINE | ID: mdl-2434490

ABSTRACT

Ryanodine at concentrations of 0.01-10 microM increased, while greater concentrations of 10-300 microM decreased the calcium permeability of both rabbit fast twitch skeletal muscle junctional and canine cardiac sarcoplasmic reticulum membranes. Ryanodine did not alter calcium binding by either sarcoplasmic reticulum membranes or the calcium binding protein, calsequestrin. Therefore, the effects by this agent appear to involve only changes in membrane permeability, and the characteristics of the calcium permeability pathway affected by ryanodine were those of the calcium release channel. Consistent with this, the actions by ryanodine were localized to junctional sarcoplasmic reticulum membranes and were not observed with either longitudinal sarcoplasmic reticulum or transverse tubular membranes. In addition, passage of the junctional sarcoplasmic reticulum membranes through a French press did not diminish the effects of ryanodine indicating that intact triads were not required. Under the conditions used for the permeability studies, the binding of [3H]ryanodine to skeletal junctional sarcoplasmic reticulum membranes was specific and saturable, and Scatchard analyses indicated the presence of a single binding site with a Kd of 150-200 nM and a maximum capacity of 10.1-18.9 pmol/mg protein. [3H]ryanodine binding to this site and the increase in membrane calcium permeability caused by low concentrations of ryanodine had similar characteristics suggesting that actions at this site produce this effect. Depending on the assay conditions used, ryanodine (100-300 microM) could either increase or decrease ATP-dependent calcium accumulation by skeletal muscle junctional sarcoplasmic reticulum membranes indicating that the alterations of sarcoplasmic reticulum membrane calcium permeability caused by this agent can be determined in part by the experimental environment.


Subject(s)
Alkaloids/pharmacology , Calcium/metabolism , Ryanodine/pharmacology , Sarcoplasmic Reticulum/metabolism , Action Potentials , Adenosine Triphosphate/metabolism , Animals , Cell Membrane Permeability , Dose-Response Relationship, Drug , Kinetics , Magnesium/pharmacology , Male , Membranes/metabolism , Rabbits , Ruthenium Red/pharmacology
16.
Biochem Biophys Res Commun ; 139(2): 816-21, 1986 Sep 14.
Article in English | MEDLINE | ID: mdl-3767987

ABSTRACT

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.


Subject(s)
Calcium/metabolism , Ionophores/pharmacology , Myocardial Contraction/drug effects , Sympathomimetics/pharmacology , Aminoquinolines/metabolism , Animals , Heart Rate/drug effects , Mathematics , Monensin/pharmacology , Nadolol/pharmacology , Norepinephrine/pharmacology , Phentolamine/pharmacology , Pyrans/pharmacology , Rabbits
17.
Biochem Biophys Res Commun ; 128(2): 739-45, 1985 Apr 30.
Article in English | MEDLINE | ID: mdl-2581567

ABSTRACT

Junctional sarcoplasmic reticulum (SR) has been identified in microsomes from canine ventricular muscle by the presence of calsequestrin and ryanodine-sensitive Ca2+ release channels. These properties, however, are not common to cardiac cells from all species. Seiler et al (1) have recently described a high Mr polypeptide in canine junctional SR similar to the spanning protein subunits of skeletal muscle triads. We now report the existence of a polypeptide with the same mobility in SR from rabbit ventricular muscle and show that those cardiac membranes can associate with transverse (T-) tubules from rabbit skeletal muscle in K cacodylate medium. We propose that this polypeptide and the reaction with T-tubules be considered as criteria for the identification of cardiac junctional SR.


Subject(s)
Myocardium/ultrastructure , Sarcoplasmic Reticulum/ultrastructure , Animals , Calcium/metabolism , Calsequestrin/analysis , Electrophoresis, Polyacrylamide Gel , Ion Channels/analysis , Microscopy, Electron , Molecular Weight , Rabbits , Ryanodine/analysis
18.
Eur J Pharmacol ; 100(3-4): 321-7, 1984 May 04.
Article in English | MEDLINE | ID: mdl-6734723

ABSTRACT

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.


Subject(s)
Furans/pharmacology , Heart/drug effects , Ionophores/pharmacology , Monensin/pharmacology , Action Potentials/drug effects , Animals , Catecholamines/metabolism , Dogs , Electrophysiology , In Vitro Techniques , Isometric Contraction , Muscle Contraction/drug effects , Myocardial Contraction/drug effects , Potassium/pharmacology , Pyrans/pharmacology
19.
Biochim Biophys Acta ; 707(2): 226-35, 1982 Oct 05.
Article in English | MEDLINE | ID: mdl-7138885

ABSTRACT

Subfractions of adrenal medullary homogenates were analyzed in two-dimensional polyacrylamide gels to assess the extent of protein homology. Chromaffin granule proteins were highly acidic, with the exception of the soluble form of the enzyme dopamine beta-hydroxylase (EC 1.14.17.1). The purified granule membrane proteins were more heterogeneous, but still predominantly acidic. The soluble and membrane forms of dopamine beta-hydroxylase behaved identically in this gel system. Lactoperoxidase-catalyzed iodination of intact granules revealed that most, but not all, granule membrane proteins are accessible at the cytoplasmic face. Prominent proteins of the purified adrenal medullary mitochondria showed little if any homology with purified granule membranes. The crude microsome fraction showed significant homology with purified granule membranes despite low levels of cross-contamination between the two fractions in marker enzyme analysis. Among proteins that could be identified, dopamine beta-hydroxylase was at a low level in the microsomes, while the granule membrane protein cytochrome b-561 appeared to be in both fractions. The pattern obtained from primary cultures of adrenal chromaffin cells was very complex, but prominent proteins from the subcellular fractions were seen without difficulty. Actin and tubulin were very prominent in the whole cell pattern. Radioiodination of the whole cells resulted in a number of spots being labelled, although the majority of the label appeared to be in only two proteins of molecular weight 70000 and isoelectric point 5.7.


Subject(s)
Adrenal Medulla/ultrastructure , Proteins/isolation & purification , Animals , Cattle , Cell Fractionation/methods , Chromaffin Granules/ultrastructure , Dopamine beta-Hydroxylase/analysis , Electrophoresis, Polyacrylamide Gel/methods , Microsomes/ultrastructure , Molecular Weight , Subcellular Fractions/ultrastructure
20.
J Cell Biol ; 94(3): 531-9, 1982 Sep.
Article in English | MEDLINE | ID: mdl-7130269

ABSTRACT

The significance of intracellular Na+ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K+ for extracellular Na+, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secretory granule numbers appear to be qualitatively reduced after carbachol, multiple carbachol, or Ba2+ stimulation, overall granule distribution remains similar to that in untreated cells. Monensin-stimulated catecholamine release requires extracellular Na+ but not Ca2+ whereas carbachol-stimulated catecholamine release requires extracellular Ca2+ and is partially dependent on extracellular Na+. Despite its high selectivity for monovalent ions, monensin is considerably more effective in promoting catecholamine secretion than the divalent ionophores, A23187 and ionomycin, which mediate a more direct entry of extracellular Ca2+ into the cell. We propose that the monensin-stimulated increase in intracellular Na+ levels causes an increase in the availability of intracellular Ca2+ which, in turn, stimulates exocytosis. This hypothesis is supported by the comparable stimulation of catecholamine release by ouabain which inhibits the outwardly directed Na+ pump and thus permits intracellular Na+ to accumulate. The relative magnitudes of the secretion elicited by monensin, carbachol, and the calcium ionophores, are most consistent with the hypothesis that, under normal physiological conditions, Na+ acts by decreasing the propensity of Ca2+-sequestering sites to bind the Ca2+ that enters the cell as a result of acetylcholine stimulation.


Subject(s)
Calcium/physiology , Catecholamines/metabolism , Chromaffin System/metabolism , Ionophores/pharmacology , Sodium/physiology , Animals , Cattle , Cells, Cultured , Chromaffin System/ultrastructure , Microscopy, Electron , Monensin/pharmacology , Time Factors
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