Two-dimensional imaging of soot volume fraction in laminar diffusion flames.

A technique for acquiring two-dimensional soot-volume-fraction measurements in laminar flames has been demonstrated. The technique provides a map of very low noise concentration over a range of wavelengths (250-1100 nm). A noise level of 0.0007 in extinction and a spatial resolution of 30-40 microm for soot concentration were achieved with an arc lamp source that was filtered to provide greater spatial coherence and a CCD detector. The broadband arc lamp source also allowed us to avoid the added noise resulting from speckle with coherent laser sources. Beam steering, due to refractive-index gradients in the flame, was measured and compared with theoretical predictions. The optical arrangement to minimize the effect of beam steering is described. As a result the beam steering had no effect on the soot measurements in the flames examined. Flame-transmission maps obtained with this system in an ethylene/air laminar diffusion flame are presented. Tomographic analysis from use of an Abel inversion of the line-of-sight data to obtain radial profiles of soot concentration is described.

Activation of tyrosine hydroxylase by pituitary adenylate cyclase-activating polypeptide (PACAP-27) in bovine adrenal chromaffin cells.

The effect of pituitary adenylate cyclase-activating polypeptide (PACAP-27) on tyrosine hydroxylase activity has been studied in intact, cultured, bovine adrenal chromaffin cells. Tyrosine hydroxylase activity was determined in situ by measuring the production of 14CO2 following the hydroxylation and rapid decarboxylation of [14C]tyr offered to the cells. PACAP-27 increased tyrosine hydroxylase activity 3-fold over 10 min. With an EC50 of 10-20 nM. PACAP-38 was approximately 2-fold less potent. Removing extracellular Ca2+ reduced basal tyrosine hydroxylase activity and the activation produced by both PACAP-27 and forskolin by about 20%. In the absence of extracellular Ca2+, chelation of intracellular Ca2+ by treating cells with BAPTA-AM (50 microM) caused a consistent 40-50% reduction in basal tyrosine hydroxylase activity and in the responses to forskolin and PACAP-27. The tyrosine hydroxylase activation produced by PACAP-27 was unaffected by the protein kinase C inhibitor Ro 3l-8220 (3 microM), but was reduced by 85% by the protein kinase A inhibitor H89 (10 microM). PACAP-27 increased cellular cyclic AMP levels 3-fold at 100 nM. The results suggest that PACAP-27 activates tyrosine hydroxylase in bovine chromaffin cells through cyclic AMP formation and protein kinase A activation, and that both extracellular and intracellular Ca2+ modulate the effect of the adenylate cyclase/cyclic AMP/protein kinase A signalling pathway on tyrosine hydroxylase activity.

Inhibition of nicotinic responses of bovine adrenal chromaffin cells by the protein kinase C inhibitor, Ro 31-8220.

1. The effects of the protein kinase C inhibitor, Ro 31-8220, on the responses of cultured bovine adrenal chromaffin cells to nicotine, phorbol 12, 13-dibutyrate (PDBu) and K+ have been investigated. 2. Tyrosine hydroxylase activity was measured in situ in intact cells by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. Secretion of endogenous adrenaline and noradrenaline was measured by use of h.p.l.c. with electrochemical detection. Cyclic AMP levels were measured in cell extracts by RIA. 3. Ro 31-8220 produced a concentration-dependent inhibition of 300 nM PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 2 microM and complete inhibition at 10 microM. It had no effect on the responses to forskolin. 4. Ro 31-8220 produced a concentration-dependent inhibition of 5 microM nicotine-stimulated tyrosine hydroxylase activity, adrenaline and noradrenaline secretion and cellular cyclic AMP levels, with an IC50 of about 3 microM and complete inhibition by 10 microM. At concentrations up to 10 microM, Ro 31-8220 had little or no effect on the corresponding responses to 50 mm K+. 5. A structural analogue of Ro 31-8220, bisindolylmaleimide V, that lacks activity as a protein kinase C inhibitor, had no effect up to 10 microM on PDBu-stimulated tyrosine hydroxylase activity or on nicotine-stimulated cyclic AMP levels or noradrenaline secretion and only marginal inhibitory effects on nicotine-stimulated tyrosine hydroxylase activity and adrenaline secretion. 6. A structurally related protein kinase C inhibitor, bisindolylmaleimide I, inhibited PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 1 microM and complete inhibition by 3 microM, but had essentially no effect on nicotine stimulated tyrosine hydroxylase activity or catecholamine secretion. 7. The results suggest that Ro 31-8220 is not only a protein kinase C inhibitor but is also a potent inhibitor of nicotinic receptor responses in adrenal chromaffin cells by a mechanism unrelated to protein kinase C inhibition. The results are consistent with Ro 31-8220 being a nicotinic receptor antagonist.

The Ca++/calmodulin-dependent protein kinase II inhibitors KN62 and KN93, and their inactive analogues KN04 and KN92, inhibit nicotinic activation of tyrosine hydroxylase in bovine chromaffin cells.

The possible role of Ca++/calmodulin-dependent protein kinase II (CAM-K-II) in the nicotinic activation of tyrosine hydroxylase in intact cultured bovine adrenal chromaffin cells has been investigated. Over the concentration range 3-30 microM, KN62, a specific CAM-K-II inhibitor, inhibited basal tyrosine hydroxylase activity and the activity stimulated by nicotine or K+ depolarisation. KN04, a structural analogue of KN62 which does not inhibit CAM-K-II, produced an identical concentration-dependent inhibition of basal and nicotine-stimulated tyrosine hydroxylase activity. Another CAM-K-II inhibitor, KN93, also inhibited nicotine and K+ stimulation of tyrosine hydroxylase activity; however, an inactive analogue of KN93, KN92, mimicked these effect. The results suggest that the inhibition of nicotine- and K+-stimulated tyrosine hydroxylase activity by KN62 and KN93 is not due to their ability to inhibit CAM-K-II.

Nerves containing nitric oxide synthase and their possible function in the control of catecholamine secretion in the bovine adrenal medulla.

NADPH-diaphorase reactivity and neuronal nitric oxide synthase (nNOS) immunostaining have been localised in sections of bovine adrenal glands. Both were present in nerve fibres and terminals in the subcapsular region and running between zona glomerulosa cells, amongst the medullary chromaffin cells, between large ganglion cells in rare encapsulated medullary ganglia and in large nerve bundles running through the cortex. Occasional isolated fibres were stained in deeper cortical layers. Both NADPH-diaphorase reactivity and nNOS immunoreactivity were present in a population of ganglion cells located individually or in small groups at the medullary-cortical boundary. NADPH-diaphorase reactivity was also found in all cortical cells (zona glomerulosa cells being more densely stained than other cortical cells) and in large fibrous structures in large nerve bundles (tentatively identified as glial cells): these structures were not stained with antisera to nNOS. Chromaffin cells were not stained with either technique. The possible role of neurally-released nitric oxide in the regulation of nerve-induced catecholamine secretion from chromaffin cells was investigated in isolated, perfused, bovine adrenal glands. The secretion of both adrenaline and noradrenaline in response to field stimulation of adrenal nerves at either 2 Hz or 10 Hz was unaffected by the presence of N omega-nitro-L-arginine (30 microM), sodium nitroprusside (10 microM) or L-arginine (100 microM) in the perfusing solution. It is concluded that, although nitric oxide may be generated and released from adrenal medullary nerves innervating chromaffin cells, it does not play a direct role in the acute regulation of adrenal catecholamine secretion.

Protein kinase A and nicotinic activation of bovine adrenal tyrosine hydroxylase.

1. Stimulation of nicotinic cholinoceptors on bovine chromaffin cells increases phosphorylation of three serine residues in tyrosine hydroxylase (TOH) and activates TOH. One of the serines is a target for protein kinase A phosphorylation, and phosphorylation of this serine is adequate alone to cause TOH activation. The role of protein kinase A in nicotinic activation of TOH was therefore investigated. 2. TOH activity was studied in situ in intact, cultured, bovine adrenal chromaffin cells, by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. 3. Nicotine (5 microM), forskolin (1 microM) and 8-bromo-cyclic AMP (8-Br-cyclic AMP, 1 mM) each increased TOH activity by up to 200% over 10 min. The effect of nicotine was completely abolished by removal of extracellular Ca2+. 4. TOH activation by all three drugs was blocked by H89 (3-20 microM), which inhibits protein kinase A by competing for the ATP binding site on the kinase. Adenosine 3':5'-cyclic monophosphorothioate Rp-diastereomer (Rp-cAMPS) (1 mM), an inhibitor of protein kinase A that competes with cyclic AMP for the regulatory subunit of the kinase, abolished the activation of TOH by nicotine, and reduced that by forskolin and 8-Br-cyclic AMP. Both H89 and Rp-cAMPS inhibited basal TOH activity by 50-80%. 5. A structural analogue of H89, H85 (3-20 microM), which lacks activity as a protein kinase A inhibitor, did not inhibit either the activation of TOH by nicotine (5 microM) or basal TOH activity. Neither sodium nitroprusside (0.3-1O microM) nor 8-Br-cyclic GMP (1 mM) increased TOH activity.6. In digitonin-permeabilized chromaffin cells, forskolin (3 microM), cyclic AMP (10 microM) and Ca2+ (approx.2 micro M free Ca2+) each increased TOH activity. The response to all three drugs was blocked by H89(10 microM), which also reduced basal TOH activity in the permeabilized cells.7. Maximal activation of TOH by forskolin was achieved with 10 micro M forskolin. This concentration was less than the EC50 for forskolin-induced cyclic AMP accumulation in these cells. The activations of TOH by forskolin (1O microM) and nicotine (5 microM) were additive.8. The results indicate that both basal TOH activity and nicotinic activation of TOH in bovine chromaffin cells require protein kinase A activity. However, it is unlikely that nicotinic activation of TOH is directly mediated by an activation of protein kinase A in response to elevated cyclic AMP levels.It is possible that protein kinase A plays a permissive role in allowing nicotinic cholinoceptors to activate TOH by another signalling pathway.

A rare inherited euchromatic heteromorphism on chromosome 1.

Extra genetic material that is euchromatic is generally regarded to be associated with phenotypic abnormalities. However, recent studies suggest that this is not always the case. Chromosome analysis was performed on amniotic fluid cells from a 37-year-old phenotypically normal patient referred for advanced maternal age. All the cells analysed showed a karyotype of 46,XY,1p+. The 1p+ chromosome had extra genetic material of uncertain origin in chromosome band region 1p21-->31. Chromosome analysis on the father revealed a normal 46,XY male karyotype. The mother's karyotype showed the same 1p+ chromosome. C and Q banding, as well as silver staining studies, in both the mother and the fetus support the interpretation that the extra chromosomal material was euchromatic in nature. This 1p+ chromosome may be characterized as a euchromatic heteromorphism. Euchromatic heteromorphisms not associated with phenotypic abnormalities have been reported for chromosomes 9 and 16. To the best of our knowledge, this is the first report involving this type of cytogenetic anomaly on chromosome number 1 in a phenotypically normal mother and infant.

Non-cholinergic nervous control of catecholamine secretion from perfused bovine adrenal glands.

1. Field stimulation of adrenal nerves was used to study nervous control of adrenal catecholamine secretion in isolated, retrogradely perfused, bovine adrenal glands. 2. Secretion of both adrenaline and noradrenaline was maximal at 10 Hz. Secretion at 2 Hz was < 10% of maximum. Stimulating with trains of pulses at ten times the average frequency for 1 s out of every 10 s gave 2-fold greater secretion at 2 Hz average frequency, similar release at 5 Hz, and only half the secretion at 10 Hz, compared to continuous stimulation at the average frequency. 3. At 10 Hz, adrenaline and noradrenaline secretion was virtually abolished by tetrodotoxin (1 microM), but was only reduced by 75% by prolonged perfusion with a combination of mecamylamine (5 microM) and atropine (1 microM). Mecamylamine and atropine completely abolished the secretory response to 2 Hz stimulation. Tetrodotoxin had no significant effect on secretion induced by perfusing glands with nicotine (5 microM), while mecamylamine abolished this response. Mecamylamine and atropine had no effect on secretion induced by K+ depolarization. 4. The secretion of adrenaline and noradrenaline induced by 10 Hz stimulation was not inhibited by naloxone at either 1 or 30 microM. 5. The results suggest that bovine adrenal chromaffin cells, like those in the rat, receive a significant non-cholinergic secretomotor innervation. In contrast to the rat, however, the non-cholinergic component in the bovine adrenal is negligible at low-frequency nerve stimulation and substantial at higher frequencies, and is not antagonized by naloxone. The identity of the non-cholinergic transmitter remains to be determined.

Regulation of cyclic AMP levels by phorbol esters in bovine adrenal medullary cells.

Cyclic AMP responses to phorbol esters were studied in cultured bovine adrenal medullary cells. Phorbol esters that activate protein kinase C (PKC: phorbol 12,13-dibutyrate, phorbol 12,13-didecanoate) increased cellular cyclic AMP levels by up to 100% over 5 min, and this was maintained for up to 3 h. The effect was mimicked by 1,2-dioctanoyl-sn-glycerol but not by inactive phorbol esters. The effect of active phorbol esters was concentration dependent over the range 50-500 nM, and was abolished by the PKC inhibitor, Ro 31-8220 (10 microM). The response was enhanced by 3-isobutyl-1-methylxanthine (1 mM) and by forskolin (0.3 microM), was enhanced following pertussis toxin pretreatment (100 ng/ml, 7.5 h) and was unaffected by removing extracellular Ca2+. The phorbol ester cyclic AMP response was additive with that to K+ depolarisation, and synergised with those to prostaglandin E1 and dimaprit. The results indicate PKC activation increases cyclic AMP formation in bovine adrenal medullary cells, probably by a direct action on adenylate cyclase or Gs.

Regulation of cyclic AMP metabolism in bovine adrenal medullary cells.

The capacity of cultured bovine adrenal medullary cells to metabolize and export cyclic AMP has been studied. Basal cellular cyclic AMP levels were increased 50% by 100 microM 3-isobutyl-1-methylxanthine (IBMX) and rolipram, a class IV (cyclic AMP-specific) phosphodiesterase (PDE) inhibitor. They were not affected by inhibition of class I (Ca2+/calmodulin-dependent), class III (cyclic GMP-inhibited) or class V PDE (cyclic GMP-specific) with vinpocetine or 3-isobutyl-8-methoxymethyl-1-methylxanthine (8-methoxymethyl-IBMX), SK&F 94120, or MB 22,948, respectively, all at 100 microM. Furthermore, only IBMX and rolipram enhanced the cyclic AMP response to 0.3 microM forskolin. Rolipram had an EC50 of < or = 1 microM and was equally effective at 100 microM and 1 mM. IBMX enhanced cyclic AMP levels significantly more at 1 mM than at 100 microM. Neither vinpocetine nor 8-methoxymethyl-IBMX (100 microM) enhanced the Ca(2+)-dependent cyclic AMP response to K+ depolarization. Elevation of cyclic GMP levels with sodium nitroprusside (10 or 100 microM), to activate any cyclic GMP-stimulated class II PDE and to inhibit any cyclic GMP-inhibited class III PDE, also had no effect on basal or forskolin-stimulated cyclic AMP levels. In the presence of IBMX (1 mM), forskolin (5 microM) caused a rapid and large increase in cellular cyclic AMP levels which was maximal after about 5 min and declined slightly over 3 hr. Over this period, extracellular cyclic AMP levels rose almost linearly reaching levels 2-3 times those in the cells. The results indicate bovine adrenal medullary cells have a high capacity for sustained cyclic AMP export. Furthermore, two PDE isozymes appear to degrade cyclic AMP in these cells, a rolipram-sensitive, cyclic AMP-specific, class IV isozyme and a rolipram-insensitive isoform.

Histamine-induced increases in cyclic AMP levels in bovine adrenal medullary cells.

1. The effect of histamine on cellular cyclic AMP levels in cultured bovine adrenal medullary cells has been studied. 2. Histamine (0.3-30 microM) increased cyclic AMP levels transiently, with a maximal response after 5 min, a smaller response after 20 min, and no increase seen after 80 or 180 min. The EC50 at 5 min was approximately 2 microM. Histamine had no effect on cyclic AMP release from the cells over 5 min, but increased it after 90 min. 3. The cyclic AMP response to 5 microM histamine was reduced by 45% by 1 microM mepyramine and by almost 30% by 1 microM cimetidine, and was abolished by the combination of both antagonists. Cimetidine at 100 microM did not inhibit the response to histamine more than 1 microM cimetidine. The H3-receptor antagonist, thioperamide (1 microM), had no effect on the response to histamine. 4. The H1-receptor agonist, 2-thiazolyethylamine (5-100 microM) and the H2-receptor agonist, dimaprit (5-100 microM), each induced a cyclic AMP response, and gave more-than-additive responses when combined. The H3 agonist (R) alpha-methylhistamine (100 microM) had no effect either on its own or in combination with either the H1 or the H2 agonist. The response to 100 microM 2-thiazolylethylamine was unaffected by cimetidine (100 microM). 5. The cyclic AMP responses to 5 microM histamine, 100 microM thiazolylethylamine and 100 microM dimaprit were each weakly enhanced in the presence of 1 mM 3-isobutyl-1-methylxanthine. The response to dimaprit was enhanced more than 10 fold in the presence of 0.3 microM forskolin, while the responses to histamine and thiazolylethylamine were weakly enhanced.6. The cyclic AMP response to 5 microM histamine was partially reduced in the absence of extracellular Ca2 and the residual response was fully antagonized by 1 microM cimetidine and was unaffected by 1 microM mepyramine.In the absence of Ca2 , the cyclic AMP response to 100 microM thiazolylethylamine was abolished, while that to 100 microM dimaprit was unaffected.7. Reincubation of 5 microM histamine solutions with a second set of chromaffin cells, following prior incubation with another set of cells, induced a cyclic AMP response in the fresh cells. This response was reduced by a combination of mepyramine and cimetidine to the same degree as the response to fresh 5 microm histamine solutions.8. The results indicate that histamine increases cellular cyclic AMP levels in bovine chromaffin cells by three mechanisms: by acting on H1 receptors, by acting on H2 receptors, and by an interaction between H, and H2 receptors. The H1 response does not require concomitant activation of H2 receptors, is fully dependent on extracellular Ca2 +, does not depend on secreted chromaffin cell products, and is not due to reduced cyclic AMP degradation or export. The H2 cyclic AMP response is the first functional response reported for H2 receptors on chromaffin cells, is independent of Ca2 , is not due to reduced cyclic AMP export or degradation, and is likely to be mediated via a direct action through Gs. The role of these different mechanisms in the regulation of cyclic AMP-dependent processes in chromaffin cells by histamine is under investigation.