Non-invasive measurement of brain temperature using radiometric thermometry: experimental validation and clinical observations in asphyxiated newborns.

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.

Chronic topical administration of WIN-55-212-2 maintains a reduction in IOP in a rat glaucoma model without adverse effects.

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.

Do injections of 5-fluorouracil after trabeculectomy have toxic effects on the anterior segment?

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.

Comparison of topical steroids for acute anterior uveitis.

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.

The birth of ocular pharmacology in the 20th century.

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.

Structural analysis of murine zona pellucida glycans. Evidence for the expression of core 2-type O-glycans and the Sd(a) antigen.

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.

Memory of previous receptor activation induces a delay in Ca2+ mobilization and decreases the [Ca2+]i sensitivity of arterial contractions.

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.

Memory of arterial receptor activation involves reduced [Ca2+]i and desensitization of cross bridges to [Ca2+]i.

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.

Ketamine relaxes rabbit femoral arteries by reducing [Ca2+]i and phospholipase C activity.

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.

L-type Ca2+ channel desensitization by F- reduces PhE-induced increase in [Ca2+]i but not stress.

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.

Cell surface localization of a novel non-genomic progesterone receptor on the head of human sperm.

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.

Myosin phosphorylation and calcium in tonic and phasic contractions of colonic smooth muscle.

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.

The effect of pH on rate constants, ion selectivity and thermodynamic properties of fluorescent calcium and magnesium indicators.

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.

The effects of pH and temperature on fluorescent calcium indicators as determined with Chelex-100 and EDTA buffer systems.

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.

The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes.

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.

Alterations in intracellular calcium activity and contractility of isolated perfused rabbit hearts by ionophores and adrenergic agents.

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.

A functional identification of cardiac junctional sarcoplasmic reticulum.

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.

Comparative study of the effects of salinomycin and monensin on electrophysiologic and contractile properties of canine myocardium.

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.

Subcellular fractions of the adrenal medulla. Comparison by two-dimensional polyacrylamide gel electrophoresis.

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.

Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium.

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.