Nanocarriers for optimizing the balance between interfollicular permeation and follicular uptake of topically applied clobetasol to minimize adverse effects.

The treatment of various hair disorders has become a central focus of good dermatologic patient care as it affects men and women all over the world. For many inflammatory-based scalp diseases, glucocorticoids are an essential part of treatment, even though they are known to cause systemic as well as local adverse effects when applied topically. Therefore, efficient targeting and avoidance of these side effects are of utmost importance. Optimizing the balance between drug release, interfollicular permeation, and follicular uptake may allow minimizing these adverse events and simultaneously improve drug delivery, given that one succeeds in targeting a sustained release formulation to the hair follicle. To test this hypothesis, three types of polymeric nanocarriers (nanospheres, nanocapsules, lipid-core nanocapsules) for the potent glucocorticoid clobetasol propionate (CP) were prepared. They all exhibited a sustained release of drug, as was desired. The particles were formulated as a dispersion and hydrogel and (partially) labeled with Rhodamin B for quantification purposes. Follicular uptake was investigated using the Differential Stripping method and was found highest for nanocapsules in dispersion after application of massage. Moreover, the active ingredient (CP) as well as the nanocarrier (Rhodamin B labeled polymer) recovered in the hair follicle were measured simultaneously, revealing an equivalent uptake of both. In contrast, only negligible amounts of CP could be detected in the hair follicle when applied as free drug in solution or hydrogel, regardless of any massage. Skin permeation experiments using heat-separated human epidermis mounted in Franz Diffusion cells revealed equivalent reduced transdermal permeability for all nanocarriers in comparison to application of the free drug. Combining these results, nanocapsules formulated as an aqueous dispersion and applied by massage appeare to be a good candidate to maximize follicular targeting and minimize drug penetration into the interfollicular epidermis. We conclude that such nanotechnology-based formulations provide a viable strategy for more efficient drug delivery to the hair follicle. Moreover, they present a way to minimize adverse effects of potent glucocorticoids by releasing the drug in a controlled manner and simultaneously decreasing interfollicular permeation, offering an advantage over conventional formulations for inflammatory-based skin/scalp diseases.

Effects of high-fat diet and gastric bypass on neurons in the caudal solitary nucleus.

Bariatric surgery is an effective treatment for obesity that involves both peripheral and central mechanisms. To elucidate central pathways by which oral and visceral signals are influenced by high-fat diet (HFD) and Roux-en-Y gastric bypass (RYGB) surgery, we recorded from neurons in the caudal visceral nucleus of the solitary tract (cNST, N=287) and rostral gustatory NST (rNST,N=106) in rats maintained on a HFD and lab chow (CHOW) or CHOW alone, and subjected to either RYGB or sham surgery. Animals on the HFD weighed significantly more than CHOW rats and RYGB reversed and then blunted weight gain regardless of diet. Using whole-cell patch clamp recording in a brainstem slice, we determined the membrane properties of cNST and rNST neurons associated with diet and surgery. We could not detect differences in rNST neurons associated with these manipulations. In cNST neurons, neither the threshold for solitary tract stimulation nor the amplitude of evoked EPSCs at threshold varied by condition; however suprathreshold EPSCs were larger in HFD compared to chow-fed animals. In addition, a transient outward current, most likely an IA current, was increased with HFD and RYGB reduced this current as well as a sustained outward current. Interestingly, hypothalamic projecting cNST neurons preferentially express IA and modulate transmission of afferent signals (Bailey, '07). Thus, diet and RYGB have multiple effects on the cellular properties of neurons in the visceral regions of NST, with potential to influence inputs to forebrain feeding circuits.

Roux-en-Y gastric bypass in rats increases sucrose taste-related motivated behavior independent of pharmacological GLP-1-receptor modulation.

Roux-en-Y gastric bypass (RYGB) surgery has been shown to decrease consummatory responsiveness of rats to high sucrose concentrations, and genetic deletion of glucagon-like peptide-1 receptors (GLP-1R) has been shown to decrease consummatory responsiveness of mice to low-sucrose concentrations. Here we assessed the effects of RYGB and pharmacological GLP-1R modulation on sucrose licking by chow-fed rats in a brief-access test that assessed consummatory and appetitive behaviors. Rats were tested while fasted presurgically and postsurgically and while nondeprived postsurgically and 5 h after intraperitoneal injections with the GLP-1R antagonist exendin-3(9-39) (30 µg/kg), agonist exendin-4 (1 µg/kg), and vehicle in 30-min sessions during which a sucrose concentration series (0.01-1.0 M) was presented in 10-s trials. Other rats were tested postsurgically or 15 min after peptide or vehicle injection while fasted and while nondeprived. Independent of food-deprivation state, sucrose experience, or GLP-1R modulation, RYGB rats took 1.5-3× as many trials as sham-operated rats, indicating increased appetitive behavior. Under nondeprived conditions, RYGB rats with presurgical sucrose experience licked more to sucrose relative to water compared with sham-operated rats. Exendin-4 and exendin-3(9-39) impacted 0.3 M sucrose intake in a one-bottle test, but never interacted with surgical group to affect brief-access responding. Unlike prior reports in both clearly obese and relatively leaner rats given RYGB and in GLP-1R knockout mice, we found that neither RYGB nor GLP-1R blockade decreased consummatory responsiveness to sucrose in our less obese chow-fed rats. Collectively, these results highlight the fact that changes in taste-driven motivated behavior to sucrose after RYGB and/or GLP-1R modulation are very model and measure dependent.

Characterization of compounds on nicotinic acetylcholine receptor alpha7 channels using higher throughput electrophysiology.

Alpha7 nicotinic acetylcholine receptor channels are important ligand-gated ion channels that are fast desensitizing, cation selective and have been implicated in the pathophysiology of schizophrenia and Alzheimer's disease. We report here high quality alpha7 parallel patch clamp recordings using the QPatch automated patch clamp system. The QPatch patch clamps up to 48 cells in parallel with the same high fidelity as conventional patch clamp. EC(50) and IC(50) values were comparable to values obtained with conventional patch clamp. The EC(50) value for acetylcholine (ACh) on the QPatch with area under the curve (AUC) analysis was 26microM compared to a value of 29microM determined from conventional patch clamp experiments. Sequential additions of ACh can be made with minimal decay of the peak amplitude. The competitive alpha7 antagonist methyllycaconitine (MLA) blocked currents with an IC(50) value of 0.25nM which is similar to published IC(50) values for MLA. Finally, two different classes of positive allosteric modulators represented by PNU-120596 and NS-1738 elicited characteristic responses, thus allowing accurate characterization of modulation and measurements of potency. These results demonstrate that alpha7 nicotinic acetylcholine receptor channels can be studied reliably in a higher throughput, parallel manner with the QPatch automated patch clamp system.

Selective open-channel block of Shaker (Kv1) potassium channels by s-nitrosodithiothreitol (SNDTT).

Large quaternary ammonium (QA) ions block voltage-gated K(+) (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to the cytoplasm only when channels are open. S-nitrosodithiothreitol (SNDTT; ONSCH(2)CH(OH)CH(OH)CH(2)SNO) produces qualitatively similar "open-channel block" in Kv channels despite a radically different structure. SNDTT is small, electrically neutral, and not very hydrophobic. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of Kv channels, but not Na(+) or Ca(2)+ channels. Inactivation-removed ShakerB (ShBDelta) Kv1 channels expressed in HEK 293 cells are similarly blocked and were used to study further the action of SNDTT. Dose-response data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficient to produce block. The dissociation constant for the binding of the second SNDTT molecule (K(d2) = 0.14 mM) is lower than that of the first molecule (K(d1) = 0.67 mM), indicating cooperativity. The half-blocking concentration (K(1/2)) is approximately 0.2 mM. Steady-state block by this electrically neutral compound has a voltage dependence (about -0.3 e(0)) similar in magnitude but opposite in directionality to that reported for QA ions. Both nitrosyl groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction (tau approximately 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site, most likely within the channel cavity. Finally, SNDTT is remarkably selective for Kv1 channels. When individually expressed in HEK 293 cells, rat Kv1.1-1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2.

Alkyne cross metathesis reactions of extended scope.

[figure: see text] A catalyst formed in situ from Mo[N(t-Bu)(Ar)]3 1 (Ar = 3,5-dimethylphenyl) and CH2Cl2 in toluene effects cross metathesis reactions of functionalized alkynes that are beyond reach of more traditional promotors. An application to the synthesis of prostaglandin E2 (PGE2) 19 and the acetylated PGE derivative 18b shows the compatibility of this method with sensitive substrates.

Alkyne metathesis: development of a novel molybdenum-based catalyst system and its application to the total synthesis of epothilone A and C.

Sterically hindered molybdenum(III) amido complexes of the general type [Mo[(tBu)(Ar)N]3] (1), upon treatment with CH2Cl2 or other halogen donors, have been converted into highly effective catalysts for all kinds of alkyne metathesis reactions. Although the actual nature of the propagating species formed in situ is still elusive, halogen transfer to the Mo center of 1 plays a decisive role in the activation of such precatalysts. It was possible to isolate and characterize by X-ray crystallography some of the resulting molybdenum halide derivatives such as 15, 16 and 20 which themselves were shown to be catalytically active. Numerous applications illustrate the performance of the catalytic system 1/CH2Cl2 which operates under mild conditions and tolerates an array of polar functional groups. The wide scope allows the method to be implemented into the total synthesis of sensitive and polyfunctional natural products. Most notable among them is a concise entry into the potent anticancer agents epothilone A (86) and C (88). The macrolide core of these targets is forged by ring closing alkyne metathesis (RCAM) of diyne 113, followed by Lindlar hydrogenation of cycloalkyne 114 thus formed. Since this strategy opens a stereoselective entry into (Z)-alkene 115, the approach is inherently more efficient than previous syntheses based on conventional RCM.

A simultaneous study of the metabolism of apolipoprotein B and albumin in nephrotic patients.

BACKGROUND: The nephrotic syndrome is characterized by proteinuria, hypoalbuminemia and hyperlipidemia. Despite intensive research it is not clear at present what the causal links are between these pathological findings. METHODS: Stable isotope labeled amino acid tracer kinetic analysis was used to simultaneously investigate the metabolism of four apolipoprotein B-containing lipoproteins (VLDL1, VLDL2, IDL and LDL) and albumin in seven patients with nephrotic syndrome and marked hypercholesterolemia, in two additional nephrotic patients with concomitant renal failure and mixed hyperlipidemia, and in a matched group of normolipidemic controls. RESULTS: Increased concentrations of VLDL2, IDL and LDL were due to (a) impaired VLDL2 and IDL delipidation, (b) reduced LDL catabolism, and (c) a trend towards an increased rate of total apolipoprotein B production. The rate of fractional albumin elimination was three times higher in patients than in controls and the rate of albumin synthesis was increased by 45%. No correlations were detectable between rates of apolipoprotein B production and the rate of albumin synthesis. CONCLUSIONS: The results of this study suggest that hyperlipidemia in nephrotic syndrome is predominantly the result of delayed lipoprotein delipidation and catabolism. There is no evidence that it is driven by a general increase of the rate of hepatic protein synthesis.

Calcium release-activated calcium current (ICRAC) is a direct target for sphingosine.

Whole cell patch-clamp recordings were made to study the regulation of the store-operated calcium release-activated calcium current (ICRAC) by metabolites involved in the sphingomyelin pathway in RBL-2H3 cells. Sphingosine, a regulator of cell growth, inhibits ICRAC completely within 200 s and independently from conversion to either sphingosine 1-phosphate or ceramide. Structural analogs of sphingosine, including N,N-dimethylsphingosine, DL-threo-dihydrosphingosine, and N-acetylsphingosine (C2-ceramide) also block ICRAC. This effect is always accompanied by an elevation of whole cell membrane capacitance. These sphingolipids appear, therefore, to accumulate in the plasma membrane and directly block ICRAC channels. Sphingosylphosphorylcholine also increases capacitance but does not inhibit ICRAC, demonstrating structural specificity and that the elevation of capacitance is necessary but not sufficient for block. Nerve growth factor, which is known to break down sphingomyelin, inhibits ICRAC, and this inhibition can be antagonized by reducing sphingosine production with L-cycloserine, suggesting that ICRAC is a physiologically relevant and direct target of sphingosine. We propose that sphingosine directly blocks ICRAC, suggesting that the sphingomyelin pathway is involved in ICRAC regulation.

Effects of anti-C5a monoclonal antibodies on oxygen use in a porcine model of severe sepsis.

METHODS: We analysed the effects of complement depletion and of C5a inhibition on haemodynamic parameters, oxygen delivery (DO2), oxygen consumption (VO2), oxygen extraction ratio (OER) and blood lactate levels after live bacteria infusion in pigs. RESULTS: In the first series of experiments, animals were decomplemented by cobra venom factor (CVF, 125 micrograms kg-1) and challenged with 1.3 x 10(9) Escherichia coli kg-1. In a second series, animals were treated with neutralizing anti-C5a monoclonal antibodies (mAb) T13/9 before infusion of an increased E. coli dosage (1 x 10(10) E. coli kg-1). Administration of Gram-negative bacteria resulted in hypotension, tachycardia, pulmonary hypertension and decreased cardiac output typical for severe sepsis. These alterations were more pronounced in animals challenged with a higher bacteria concentration (1 x 10(10) E. coli kg-1, n = 5) than with a lower dosage (1.3 x 10(9) E. coli kg-1, n = 4). Complement depletion by CVF injection 24 h before E. coli infusion (n = 4), or anti-C5a mAb T13/9 administration (n = 4) had no effect on the changes in haemodynamic parameters and in DO2 associated with E. coli challenge. Application of either 1.3 x 10(9) or 1 x 10(10) E. coli kg-1 resulted in a marked decrease in VO2 and an increase in blood lactate levels, whereas the OER did not change throughout the experiment. In contrast, pretreatment with CVF 24 h before low-dose E. coli (1.3 x 10(9) kg-1) administration resulted in a significant increase in VO2 (P < 0.05) and in OER (P < 0.05) compared with untreated septic animals (n = 4). No hyperlactaemia occurred in complement-depleted septic animals compared with complement-sufficient animals (P < 0.05). Animals challenged with a high E. coli dose (1 x 10(1) kg-1) and treated with anti-C5a mAbs showed a pronounced increase in VO2 and OER (P < 0.05) accompanied by an attenuated increase in lactate levels (P < 0.05) compared with untreated septic animals. CONCLUSION: The results demonstrate an improved oxygen use after complement depletion in this model of severe Gram-negative sepsis. Furthermore, a similar effect was seen after specifically neutralizing C5a by mAbs, indicating a role of C5a in the underlying mechanism.

Fast inactivation of delayed rectifier K conductance in squid giant axon and its cell bodies.

Inactivation of delayed rectifier K conductance (gk) was studied in squid giant axons and in the somata of giant fiber lobe (GFL) neurons. Axon measurements were made with an axial wire voltage clamp by pulsing to VK (approximately -10 mV in 50-70 mM external K) for a variable time and then assaying available gK with a strong, brief test pulse. GFL cells were studied with whole-cell patch clamp using the same prepulse procedure as well as with long depolarizations. Under our experimental conditions (12-18 degrees C, 4 mM internal MgATP) a large fraction of gK inactivates within 250 ms at -10 mV in both cell bodies and axons, although inactivation tends to be more complete in cell bodies. Inactivation in both preparations shows two kinetic components. The faster component is more temperature-sensitive and becomes very prominent above 12 degrees C. Contribution of the fast component to inactivation shows a similar voltage dependence to that of gK, suggesting a strong coupling of this inactivation path to the open state. Omission of internal MgATP or application of internal protease reduces the amount of fast inactivation. High external K decreases the amount of rapidly inactivating IK but does not greatly alter inactivation kinetics. Neither external nor internal tetraethylammonium has a marked effect on inactivation kinetics. Squid delayed rectifier K channels in GFL cell bodies and giant axons thus share complex fast inactivation properties that do not closely resemble those associated with either C-type or N-type inactivation of cloned Kvl channels studied in heterologous expression systems.

The nitric oxide/cGMP pathway couples muscarinic receptors to the activation of Ca2+ influx.

Inward currents activated by 8-bromc-cGMP and by muscarinic agonist were compared in N1E-115 mouse neuroblastoma cells using perforated-patch voltage clamp and Fura-2 imaging. The cGMP analog activates a voltage-independent inward current that is carried at least in part by Ca2+ because it persists in Na(+)-free saline when Ca2+ is present and is blocked by external Mn2+ and Ba2+. The current is similar to the inward current that develops during stimulation of M1 muscarinic receptors, and the currents activated by agonist and by 8-bromo-cGMP are not additive, indicating that the same pathway is involved. Inhibition of cGMP production with NG-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide (NO)-synthase, prevents activation of Ca2+ current by agonist without affecting the content of intracellular Ca2+ stores or the ability of agonist to mobilize Ca2+. The inhibition is overcome by 8-bromo-cGMP. LY83583, a competitive inhibitor of guanylyl cyclase, reversibly blocks activation of Ca2+ current by agonist, again without affecting the content of Ca2+ stores or Ca2+ release. Rp-8-pCPT-cGMPS, an inhibitory analog of cGMP, also reduces the Ca2+ current and reduces Ca2+ influx during muscarinic activation. It is concluded that cGMP is the necessary and sufficient intermediate in the pathway linking muscarinic receptor occupancy to the activation of voltage-independent Ca2+ current. The pathway involves positive feedback. Calcium entering via voltage-independent channels preferentially stimulates NO-synthase, which leads to enhanced cGMP production and greater Ca2+ influx. Positive feedback may explain the rapid increase in cGMP that occurs during muscarinic receptor activation.

The relationship between depletion of intracellular Ca2+ stores and activation of Ca2+ current by muscarinic receptors in neuroblastoma cells.

The relationship between the depletion of IP3-releasable intracellular Ca2+ stores and the activation of Ca(2+)-selective membrane current was determined during the stimulation of M1 muscarinic receptors in N1E-115 neuroblastoma cells. External Ca2+ is required for refilling Ca2+ stores and the voltage-independent, receptor-regulated Ca2+ current represents a significant Ca2+ source for refilling. The time course of Ca2+ store depletion was measured with fura-2 fluorescence imaging, and it was compared with the time course of Ca2+ current activation measured with nystatin patch voltage clamp. At the time of maximum current density (0.18 + .03 pA/pF; n = 48), the Ca2+ content of the IP3-releasable Ca2+ pool is reduced to 39 + 3% (n = 10) of its resting value. Calcium stores deplete rapidly, reaching a minimum Ca2+ content in 15-30 s. The activation of Ca2+ current is delayed by 10-15 s after the beginning of Ca2+ release and continues to gradually increase for nearly 60 s, long after Ca2+ release has peaked and subsided. The delay in the appearance of the current is consistent with the idea that the production and accumulation of a second messenger is the rate-limiting step in current activation. The time course of Ca2+ store depletion was also measured after adding thapsigargin to block intracellular Ca2+ ATPase. After 15 min in thapsigargin, IP3-releasable Ca2+ stores are depleted by > 90% and the Ca2+ current is maximal (0.19 + 0.05 pA/pF; n = 6). Intracellular loading with the Ca2+ buffer EGTA/AM (10 microM; 30 min) depletes IP3-releasable Ca2+ stores by between 25 and 50%, and it activates a voltage-independent inward current with properties similar to the current activated by agonist or thapsigargin. The current density after EGTA/AM loading (0.61 + 0.32 pA/pF; n = 4) is three times greater than the current density in response to agonist or thapsigargin. This could result from partial removal of Ca(2+)-dependent inactivation.

Calcium requirement for cGMP production during muscarinic activation of N1E-115 neuroblastoma cells.

Muscarinic agonists elicit large increases in intracellular Ca2+ and guanosine 3',5'-cyclic monophosphate (cGMP) in N1E-115 neuroblastoma cells. Both signals are blocked in cells loaded with the Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid showing that the increase in intracellular Ca2+ concentration ([Ca2+]i) is necessary to stimulate cGMP accumulation. Inhibition of nitric oxide synthase (NOS) blocks the cGMP response without affecting the peak amplitude of the intracellular Ca2+ signal, and it is concluded that Ca(2+)-dependent activation of NOS is required for cGMP production. cGMP accumulation is reduced by 60% when cells are bathed in Ca(2+)-free saline, but the peak change in [Ca2+]i is not affected. This suggests that Ca2+ influx is strongly coupled to the activation of cGMP production, even though it makes a smaller contribution to the intracellular Ca2+ signal than does Ca2+ release. Thapsigargin, which releases Ca2+ from intracellular stores, activates Ca2+ influx and increases cGMP. The cGMP increase is transient and follows approximately the same time course as Ca2+ store depletion. Ca2+ influx remains activated after store depletion, however, which indicates that influx alone cannot sustain cGMP production. It is concluded that summation of Ca2+ influx and Ca2+ release is necessary to reach a threshold Ca2+ level needed to stimulate cGMP accumulation. Because of the large contribution from Ca2+ influx, we suggest that NOS or a cofactor necessary for its activation may be located close to Ca2+ channels in the membrane.

Calcium current activated by muscarinic receptors and thapsigargin in neuronal cells.

The activation of muscarinic receptors in N1E-115 neuroblastoma cells elicits a voltage-independent calcium current. The current turns on slowly, reaches its maximum value approximately 45 s after applying the agonist, is sustained as long as agonist is present, and recovers by one half in approximately 10 s after washing the agonist away. The current density is 0.11 +/- 0.08 pA/pF (mean +/- SD; n = 12). It is absent in zero-Ca++ saline and reduced by Mn++ and Ba++. The I(V) curve characterizing the current has an extrapolated reversal potential > +40 mV. The calcium current is observed in cells heavily loaded with BAPTA indicating that the calcium entry pathway is not directly gated by calcium. In fura-2 experiments, we find that muscarinic activation causes an elevation of intracellular Ca++ that is due to both intracellular calcium release and calcium influx. The component of the signal that requires external Ca++ has the same time course as the receptor operated calcium current. Calcium influx measured in this way elevates (Ca++)i by 89 +/- 41 nM (n = 7). Thapsigargin, an inhibitor of Ca++/ATPase associated with the endoplasmic reticulum (ER), activates a calcium current with similar properties. The current density is 0.22 +/- 0.20 pA/pF (n = 6). Thapsigargin activated current is reduced by Mn++ and Ba++ and increased by elevated external Ca++. Calcium influx activated by thapsigargin elevates (Ca++)i by 82 +/- 35 nM. The Ca++ currents due to agonist and due to thapsigargin do not sum, indicating that these procedures activate the same process. Carbachol and thapsigargin both cause calcium release from internal stores and the calcium current bears strong similarity to calcium-release-activated calcium currents in nonexcitable cells (Hoth, M., and R. Penner. 1993. Journal of Physiology. 465:359-386; Zweifach, A., and R. S. Lewis, 1993. Proceedings of the National Academy of Sciences, USA. 90:6295-6299).

Membrane toxicity of the protein kinase C inhibitor calphostin A by a free-radical mechanism.

The effects of calphostin A on cytoplasmic calcium levels, receptor-mediated calcium release, and membrane input resistance were measured in neuroblastoma cells. Calphostin A is a lipophilic, light-sensitive perylenequinone that generates singlet oxygen when illuminated. It inhibits the activity of protein kinase C (IC50 = 250 nM), but only in the presence of light. Phorbol esters normally attenuate carbachol-evoked calcium release. This effect was blocked by simultaneous exposure to light and calphostin A (40 nM) for 30 min. At higher doses (0.5-1 microM) calphostin A also approximately doubled the resting calcium level and decreased cell input resistance by 51%. These toxic effects did not occur in the dark or after preincubation with the antioxidant alpha-tocopherol. These data support the hypothesis that the calphostins act by partitioning into the membrane and producing singlet oxygen and endoperoxides which then irreversibly modify protein kinase C and other membrane proteins and lipids.

Membrane toxicity of the protein kinase C inhibitor calphostin A by a free-radical mechanism.

The effects of calphostin A on cytoplasmic calcium levels, receptor-mediated calcium release, and membrane input resistance were measured in neuroblastoma cells. Calphostin A is a lipophilic, light-sensitive perylenequinone that generates singlet oxygen when illuminated. It inhibits the activity of protein kinase C (IC50 = 250 nM), but only in the presence of light. Phorbol esters normally attenuate carbachol-evoked calcium release. This effect was blocked by simultaneous exposure to light and calphostin A (40 nM) for 30 min. At higher doses (0.5-1 microM) calphostin A also approximately doubled the resting calcium level and decreased cell input resistance by 51%. These toxic effects did not occur in the dark or after preincubation with the antioxidant alpha-tocopherol. These data support the hypothesis that the calphostins act by partitioning into the membrane and producing singlet oxygen and endoperoxides which then irreversibly modify protein kinase C and other membrane proteins and lipids.

Differential visualization of cholinesterasic neuronal somata and fibers by use of modifications of acetylcholinesterase pharmacohistochemistry.

We modified existing acetylcholinesterase (AChE) histochemical techniques to visualize cholinergic neuronal somata and processes in the same rat brain. The AChE staining procedure of Karnovsky and Roots, combined with diisopropylfluorophosphate (DFP) pre-treatment, have previously allowed visualization of neuronal somata. Fibers have been visualized by nickel-diaminobenzidine (DAB)-hydrogen peroxide histochemical intensification. We combined novel modifications of these techniques to visualize cell bodies and fibers simultaneously. Maximal staining of neuronal somata occurs with a 1:1 dilution of both the Karnovsky-Roots medium and the nickel-DAB solution; diluting the Karnovsky-Roots medium 1:10 and increasing the concentration of the nickel-DAB solution twofold allows visualization of fibers previously unstainable with DFP pre-treatment and the classical AChE protocol. It is now possible to visualize both neuronal somata and fibers in adjacent sections of the same brain, in a quick and inexpensive manner.

Intracellular calcium release in N1E-115 neuroblastoma cells is mediated by the M1 muscarinic receptor subtype and is antagonized by McN-A-343.

Experiments using muscarinic receptor antagonists were done to determine which muscarinic receptor subtypes(s) mediate carbachol-evoked calcium release in N1E-115 cells. McN-A-343 and a new analog, (+/-)BN228, were weak antagonists and neither compound caused release on its own. The rank order of potency was 4-DAMP greater than pirenzepine greater than AFDX116 greater than (+/-)BN228 and McN-A-343. This profile, pirenzepine's high potency (19-fold greater than AFDX116) and its IC50 of 31 nM suggest that calcium release in this neuronal cell line is mediated by the M1 muscarinic receptor subtype.