Binding of [3H]saxitoxin to the voltage-dependent Na channels and inhibition of 22Na influx in bovine adrenal medullary cells

The binding characteristics of [3H]saxitoxin and its binding site were examined in bovine adrenal medullary cells. These cells showed a specific binding of [3H]saxitoxin which was saturable and reversible. Scatchard analysis showed a single class of high-affinity binding sites with an equilibrium dissociation constant of 5.8 nM and a maximum binding capacity of 427.2 fmoles/10(7) cells (124.2 fmoles/mg of cell protein). A Hill plot revealed that there were no co-operative interactions among the binding sites. Unlabeled saxitoxin inhibited the specific binding of [3H]saxitoxin as well as veratridine-induced 22Na influx with a similar potency as did tetrodotoxin. However, veratridine, aconitine and scorpion venom, at concentrations that increased 22Na influx, did not inhibit [3H]saxitoxin binding. These results indicate that saxitoxin binds to a specific site on voltage-dependent Na channels and inhibits the influx of 22Na. [3H]Saxitoxin would be useful for the detailed analysis of voltage-dependent Na channels in adrenal medullary cells.     

Potassium channels in cultured bovine adrenal medullary cells: effects of high K, veratridine and carbachol on 86rubidium efflux

To investigate the K permeability mechanism(s) in cultured bovine adrenal medullary cells, we measured the effects of high K, veratridine and carbachol on 86Rb efflux from the 86Rb preloaded cells. In non-stimulated cells, the basal efflux of 86Rb into Krebs-Ringer phosphate buffer containing 5.6 mM K proceeded gradually at the rate of 0.7% of cell 86Rb per min. High K caused a rapid 86Rb efflux; it was considerably reduced in Ca free medium. Mn, Co and Mg strongly inhibited high K-induced 45Ca influx and 86Rb efflux. Veratridine induced a sustained 86Rb efflux; it was inhibited by tetrodotoxin and abolished in Na free sucrose medium, but little affected in Ca free medium. Carbachol evoked a rapid and transient efflux of 86Rb; it amounted to 16.9% of cell 86Rb during 1 min. Carbachol-induced 86Rb efflux was inhibited by hexamethonium and d-tubocurarine. Nicotine caused 86Rb efflux, but muscarine had no effect. Carbachol-induced 86Rb efflux was substantially reduced in Na free sucrose medium, but little affected in Ca free medium. Mn, Co and Mg strongly reduced carbachol-induced 45Ca influx, but they did not appreciably alter carbachol-induced 22Na influx and 86Rb efflux. These results suggest that adrenal medullary cells have, at least, three distinct types of K permeability mechanisms: (1) basal K efflux, (2) Ca dependent K efflux, and (3) Na dependent K efflux. It seems that nicotine receptors mediate K efflux by increasing Na influx via nicotinic receptor-associated ionic channels rather than Ca influx via voltage dependent Ca channels.     

Influx of 22Na through acetylcholine receptor-associated Na channels: relationship between 22Na influx, 45Ca influx and secretion of catecholamines in cultured bovine adrenal medulla cells

The effects of carbachol, veratridine and high K on the influx of 22Na were investigated in relation to the influx of 45Ca and the secretion of catecholamines in cultured bovine adrenal medulla cells, in which stimulation of nicotinic but not muscarinic acetylcholine receptor causes the secretory response. (1) Carbachol caused a rapid influx of 22Na, influx of 45Ca and secretion of catecholamines, all of which occurred within 1 min and leveled off thereafter. Influx of 45Ca and secretion of catecholamines caused by carbachol were not inhibited by tetrodotoxin, but were greatly reduced in Na-free medium. Nicotine evoked an influx of 22Na and it was antagonized by hexamethonium and d-tubocurarine but not by tetrodotoxin. Muscarine had no effect on 22Na influx. The concentration-response curve of carbachol for 22Na influx was quite similar to that for 45Ca influx. (2) Veratridine induced a sustained influx of 22Na, influx of 45Ca and secretion of catecholamines, all of which were antagonized by tetrodotoxin. Influx of 45Ca and secretion of catecholamines due to veratridine were not observed in Na-free medium. (3) High K caused an influx of 45Ca and secretion of catecholamines but did not cause an influx of 22Na. High K-induced influx of 45Ca and secretion of catecholamines were not inhibited by tetrodotoxin nor by Na removal. (4) Magnesium, an inhibitor of voltage-dependent Ca channels, inhibited the influx of 45Ca and secretion of catecholamines caused by carbachol, veratridine and high K. These results indicate that cultured bovine adrenal medulla cells have at least three distinct ion channels: (1) nicotinic acetylcholine receptor-associated Na channels which are not inhibited by tetrodotoxin, (2) voltage-dependent Na channels which are kept activated by veratridine and inhibited by tetrodotoxin and (3) voltage-dependent Ca channels. Influx of Ca through voltage-dependent Ca channels is the common ionic event for the secretion of catecholamines caused by either carbachol, veratridine or high K. It seems that the influx of Na through acetylcholine receptor-associated Na channels as well as voltage-dependent Na channels, activates voltage-dependent Ca channels which triggers the secretion of catecholamines.     

Potentiation by Bay-K-8644 of the adrenal catecholamine secretory response to Ca re-introduction and ouabain: possible activation of Ca influx linked with Na efflux

Catecholamine (CA) secretion induced by Ca re-introduction or ouabain in the presence of Ca is markedly potentiated by Bay-K-8644 in the perfused cat adrenal. The mechanism of potentiation by this Ca agonist was investigated using perfused cat adrenal and isolated bovine chromaffin cells. The stimulatory effect of Bay-K-8644 on the response to Ca was very slight when cat adrenal was perfused with a Ca-deficient medium, in which 1 mM Mg was added or the concentration of Na was lowered. The inhibitory effect of Mg was reversed by inhibition of the Na pump with K deprivation, ouabain, or KCN. The secretion induced by ouabain during maintained depolarization at a lowered concentration (0.25 mM) of Ca, which is supposed to be due to Ca influx in exchange for Na efflux, was larger in the Bay-K-8644 treated adrenal than that in the untreated adrenal. The increase in secretion by the delayed addition of Bay-K-8644 during perfusion with a high K medium containing ouabain was larger when the concentration of Na in a high K medium was higher. When isolated chromaffin cells were stimulated with a Na-free (Tris) medium containing 0.5 mM Ca, CA secretion from and 45Ca uptake into the cells preincubated with a divalent cation-free medium were potentiated by Bay-K-8644. The stimulatory effect of Bay-K-8644 was not seen when a Ca-free treatment medium contained Mg or lacked Na, but the inhibitory effect of Mg was reversed by the addition of ouabain or KCN to the pretreatment medium. When isolated cells preloaded with 45Ca were superfused with a Ca-free medium, Ca re-introduction increased the rate of Ca efflux only under conditions in which significant increases in CA secretion and 45Ca uptake were previously observed under the static incubation system. Bay-K-8644 further increased this Ca efflux rate under these conditions. These results support the view that Ca influx linked with Na efflux is through a pathway with properties similar to those of voltage-dependent Ca channels, and suggest that this Ca pathway is activated by Bay-K-8644, which is an activator of voltage-dependent Ca channels.     

High-affinity and selectivity of neosurugatoxin for the inhibition of 22Na influx via nicotinic receptor-ion channel in cultured bovine adrenal medullary cells: comparative study with histrionicotoxin

In cultured bovine adrenal medullary cells, neosurugatoxin and histrionicotoxin inhibited carbachol-induced influx of 22Na, 45Ca and secretion of catecholamines with IC50 of 27 nM and 3 microM, respectively. The inhibitory effects of neosurugatoxin were reversed by the increased concentrations of carbachol, whereas those of histrionicotoxin were not. Histrionicotoxin at concentrations higher than 10 microM also reduced veratridine-induced influx of 22Na, 45Ca and secretion of catecholamines, while neosurugatoxin had no effects. High K-induced 45Ca influx and catecholamine secretion were not altered by either neosurugatoxin or histrionicotoxin. The present findings suggest (1) neosurugatoxin competitively inhibits nicotinic receptor-ion channel complex at nanomolar concentrations, but has no effects on voltage-dependent Na channel and voltage-dependent Ca channel; (2) histrionicotoxin at micromolar concentrations non-competitively suppresses nicotinic receptor-ion channel complex. Higher concentrations of histrionicotoxin also interferes with voltage-dependent Na channel, but has no effect on voltage-dependent Ca channel; (3) neosurugatoxin, due to its high-affinity and selectivity, may be a useful probe for studying nicotinic receptors in nervous tissues.     

cis-unsaturated fatty acids stimulate catecholamine secretion, tyrosine hydroxylase and protein kinase C in adrenal medullary cells

In digitonin-permeabilized bovine adrenal medullary cells, arachidonic acid and oleic acid, the cis-unsaturated fatty acids, enhanced Ca2+-induced secretion of catecholamines, whereas elaidic acid, a trans-unsaturated fatty acid and stearic acid, a saturated fatty acid, had no effect. Indomethacin, an inhibitor of cyclooxygenase and nordihydroguaiaretic acid, an inhibitor of lipoxygenase, failed to inhibit the stimulatory effect of arachidonic acid. Stimulation of catecholamine secretion by arachidonic acid was abolished by the removal of adenosine 5'-triphosphate and Mg2+ from the incubation medium. Pretreatment of the cells with phorbol 12-myristate 13-acetate, an activator of protein kinase C, enhanced Ca2+-induced catecholamine secretion. In cells pretreated with phorbol 12-myristate 13-acetate, the stimulatory effect of arachidonic acid on Ca2+-induced catecholamine secretion was greatly reduced. In digitonin-permeabilized cells, arachidonic acid and oleic acid enhanced Ca2+-induced activation of tyrosine hydroxylase in the presence of adenosine 5'-triphosphate and Mg2+, whereas elaidic acid and stearic acid did not activate the enzyme. In a soluble fraction of adrenal medullary cells, 32P incorporation to histone by protein kinase C was increased by arachidonic acid and oleic acid, but not by elaidic acid and stearic acid. These results suggest that cis-unsaturated fatty acids modulate Ca2+-induced catecholamine secretion and tyrosine hydroxylase activity by activation of protein kinase C in adrenal medullary cells.     

No correlation between membrane potential and increased cytosolic free Ca2+ concentration, 86Rb+ influx or subsequent [3H]-thymidine incorporation in neoplastic human B cells stimulated with antibodies to surface immunoglobulin

We have followed the changes in the membrane potential of neoplastic B cells after addition of antibodies to cell specific surface immunoglobulin heavy chains (anti-Ig). Two methods were used, both based on the distribution of lipophilic fluorescent indicators: the anionic bis(1,3-diethylthiobarbiturate)-trimethine oxonol (fluorescence measurement made on whole cell suspensions) and the cationic 3,3-dihexyloxacarbocyanine iodide (diOC6-(3] (using flow cytofluorimetric analysis). By the latter method, the resting membrane potential was calculated to range from -40 to -63 mV. The stimulatory effect of anti-Ig was investigated on two B cell populations. In one, which may be stimulated in this way to [3H]-thymidine incorporation, the membrane potential was apparently unchanged, at least during the first hour of stimulation. In the other population, anti-Ig induced a rapid depolarization (oxonol method), but these cells did not, on the other hand, respond with [3H]-thymidine incorporation. The nature of this depolarization was further investigated. Both Na+ and K+ permeabilities appeared increased, while Ca2+ influx did not contribute to the change in membrane potential. Nor did depolarization itself change free cytosolic Ca+ concentration, [Ca2+]i, as estimated by the quin-2 method. Furthermore, anti-Ig stimulation increased [Ca2+]i in these cells, even when depolarization was abolished by lowering the extracellular Na+ concentration. Thus, in cells where depolarization could be demonstrated, it was linked neither to early changes in [Ca2+]i nor to late [3H]-thymidine incorporation. In contrast, such effects of anti-Ig stimulation were observed in other cells without any significant early change in the membrane potential.     

The efflux of 86Rb and [3H]5-HT from human platelets during continuous perfusion: effects of potassium-induced membrane depolarization and thrombin stimulation

In order to study the ionic efflux or granule release from human platelets following pulse exposure to various stimuli, a method for continuous perfusion of platelets was developed. The method was applied to compare the effects of membrane depolarization and thrombin stimulation on the release of 86Rb and [3H]5-HT. Washed and preloaded human platelets were placed on a membrane filter in a temperature controlled polypropylene chamber, and subsequently perfused with buffer. After an initial washout period the efflux of 86Rb or [3H]5-HT reached steady, low levels. K+ induced concentration dependent increases in 86Rb efflux, corresponding to a depolarization of the membrane potential, whereas the efflux of [3H]5-HT was unaltered. Thrombin induced concentration dependent increases in the efflux of both 86Rb and [3H]5-HT. Pretreatment with K+ 12 or 30 mM did not alter the [3H]5-HT efflux induced by thrombin 0.1 U ml-1. Scanning electron micrographs of platelets on the filter showed that the unstimulated platelets had regular shape, whereas after addition of thrombin there was formation of pseudopods and minor aggregates. The effect of potassium-induced membrane depolarization on platelet aggregation was also studied. High concentration of K+ did not induce aggregation or shape change during 2 or 10 minutes of incubation. K+ had little or no effect on aggregation induced by ADP 2 microM or thrombin 0.4 U ml-1. The results from release experiments and aggregation tests argue against an immediate coupling between membrane potential and platelet reactivity.     

Inhibition of [3H]catecholamine release and Ca2+ currents by prostaglandin E2 in rabbit carotid body chemoreceptor cells.

Basal release of [3H]catecholamine ([3H]CA) from rabbit carotid bodies (CBs), previously incubated in the presence of [3H]tyrosine, was not significantly modified by prostaglandin E2 (PGE2). On the contrary, PGE2 (3-300 nM) produced a dose-dependent inhibition of the low PO2-evoked release of [3H]CA. The inhibition was greatest (55%) at a low intensity of hypoxic stimulation (incubating solution PO2 approximately 66 mmHg) and decreased with increasing intensities of hypoxia. Chronic denervation of the CB did not modify the response to PGE2. The release of [3H]CA induced by incubating the CBs in a hypercapnic-acidic solution (PCO2 approximately 132 mmHg; pH = 6.60) and by dinitrophenol (100 microM) was not significantly modified by 300 nM PGE2. PGE2 (300 nM) inhibited the release of [3H]CA elicited by incubating the CBs in a high K+ (35 mM)-containing solution. The release response elicited by high K+ (25 mM) was strongly augmented by a dihydropyridine agonist of Ca2+ channels, Bay K 8644, at a concentration of 1 microM. The Bay K 8644 effect was partly inhibited by PGE2 (300 nM). Using whole-cell recordings in freshly dispersed or short-term cultured chemoreceptor cells from adult rabbits it was found that Ca2+ currents (ICa) were reversibly inhibited by bath application of PGE2. A good parallelism exits between the dose-response curves for PGE2 inhibition of ICa in isolated chemoreceptor cells and high extracellular [K+]- or hypoxia-evoked release of [3H]CA from the whole CB.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous secretion of catecholamines from the adrenal medulla and of [3H]norepinephrine from sympathetic nerves from a single test preparation: different effects of agents on the secretion

The uptake and release of catecholamines was investigated in the isolated perfused adrenal gland of the rat after preloading the preparation with [3H]norepinephrine, and the effects of various agents were examined on the stimulation-evoked secretion of catecholamines and total tritium. Large quantities of tritium were found in the adrenal medulla after either intravenous injection of [3H]norepinephrine to the rat, or perfusion of the isolated adrenal gland with Krebs-bicarbonate solution containing [3H]norepinephrine. The retention of the tritium was inhibited 90% by desipramine. Acute treatment with guanethidine and chronic treatment with 6-hydroxydopamine abolished the secretion of tritium without affecting the secretion of catecholamines evoked at 1 Hz. Nicotine, muscarine and acetylcholine enhanced the secretion of catecholamines but not tritium, whereas tyramine and ephedrine enhanced the secretion of tritium but not catecholamines. It is concluded that chromaffin cells do not possess the norepinephrine uptake mechanism and that the uptake of [3H]norepinephrine occurs mainly in sympathetic nerve terminals present in the adrenal gland and the surrounding blood vessels (adrenal and renal veins). The differential localization of [3H]norepinephrine and catecholamines allowed us to test the effects of a variety of pharmacological agents that alter neurotransmitter release by acting on receptors on the neuronal membrane, acting on sodium and potassium channels, or acting to alter the intracellular concentrations of adenosine 3',5'-cyclic monophosphate and protein kinase C. Transmural stimulation (1 Hz for a total of 300 pulses) markedly enhanced the release of catecholamines and tritium which was blocked by tetrodotoxin (sodium channel-blocker) and potentiated by tetraethylammonium and gallamine (potassium channel-blockers). Phentolamine, an alpha adrenergic blocking agent which acts on both alpha-1 and alpha-2 receptors, caused a 3- to 4-fold facilitation of the tritium secretion while inhibiting catecholamine secretion by 45%. [Met]enkephalin almost completely inhibited the evoked-secretion of tritium but had very little effect on the secretion of catecholamines. Forskolin inhibited the tritium secretion by 80% but produced more than a 2-fold facilitation of catecholamine secretion. Phorbol 12,13-dibutyrate caused facilitation of evoked secretion of both catecholamines and tritium. A combination of phorbol ester and forskolin had a synergistic effect on stimulation-evoked secretion of catecholamines, whereas phorbol ester partially reversed the inhibitory effects of forskolin on the tritium secretion.(ABSTRACT TRUNCATED AT 400 WORDS)     

A comparative study on the uptake in vitro of [3H]thymidine, [131I]5-iodo-2'-deoxyuridine and [3H]deoxycytidine in mouse spleen cells using double isotope autoradiography.

The uptake of 3H-labeled thymidine (3H-TdR), 131I-labelled 5-iodo-2'-deoxyuridine (131IUdR) and 3H-labelled deoxycytidine (3H-CdR) by mouse spleen cells in vitro was studied using an autoradiographic method for the separate detection of 3H and 131I in cell smears. The experiment was performed in two steps. In one half of a pooled spleen cell suspension uptake of 3H-TdR and 131IUdR was compared; in the other half of the suspension uptake of 3H-CdR and 131IUdR was compared. Only two nucleated spleen cells were found which had taken up only 3H-CdR. All other scanned cells were double labelled. Generally the nucleotides were taken up according to a rather fixed relation. It was further found that 3H-CdR and 3H-TdR were incorporated to about the same extent. However, cells which were incubated with 131IUdR and 3H-TdR took up the 131IUdR to a lesser extent than cells which were incubated with 131IUdR and 3H-CdR.     

Release of [3H]norepinephrine from synaptic vesicles isolated from rat brain after the intracisternal administration of [3H]norepinephrine: influence of nucleotides, ions and drugs, and destabilization of transmitter storage caused by acute or chronic lithium administration

Following the intracisternal administration of [³H]norepinephrine to rats pretreated with a monoamine oxidase inhibitor, synaptic vesicles containing the radio label could be isolated from isotonically prepared microsomal fractions of rat brain. Incorporation of [³H]norepinephrine into the vesicles was reduced by pretreatment of the rats with desmethylimipramine and was also reduced if the rats had not been pretreated with a monoamine oxidase inhibitor. Incorporation of the label was totally eliminated by pretreatment with reserpine. Release in vitro of [³H]norepinephrine from the labeled vesicles was monophasic with a half-time of about 12 min at 30°C. The release was slowed by addition of adenosine 5′-triphosphate plus Mg²⁺ by a mechanism different from that of the vesicular amine uptake system; this was shown by the failure of inhibitors of adenosine triphosphate-Mg²⁺-stimulated uptake (reserpine,N-ethylmaleimide, lithium) to block the effect of adenosine triphosphate plus Mg²⁺ on release. Several other nucleotides also were able to slow the release of [³H]norepinephrine. Unlike adrenomedullary vesicles, rat brain synaptic vesicles did not show enhancement of amine release by chloride in the presence or absence of adenosine triphosphate plus Mg²⁺. The yield of labeled vesicles was substantially reduced if vesicles were prepared by hypotonic lysis of synaptosomes instead of isotonically from the microsomal fraction; the isotonic preparation appears to be superior for studies of vesicle uptake and storage properties.This preparation is readily utilizable for studies of the effects of in vivo administration of drugs thought to act on vesicular storage of catecholamines, a point illustrated by the destabilization of norepinephrine storage caused by acute or chronic lithium administration.     

Alpha 2-adrenoceptor agonist-mediated inhibition of [3H]noradrenaline release from rat hippocampus is reduced by 4-aminopyridine, but that caused by an adenosine analogue or omega-conotoxin is not

The inhibitory effect of an adenosine analogue, R-PIA, and an alpha 2-adrenoceptor agonist, UK 14,304, on [3H]NA efflux from field-stimulated rat hippocampal slices was examined. The effect of 0.1 microM UK 14,304 was mimicked by 30 nM omega-conotoxin and by 10 microM cadmium chloride, inhibitors of N- and L-type Ca2+ channels. R-PIA (1 microM) had no effect per se, but caused a clear-cut inhibition after blockade of the pre-synaptic alpha 2-receptor by yohimbine. 4-Aminopyridine (4-AP) caused a dose-dependent increase in evoked transmitter release. At 30 microM 4-AP did not affect the actions of omega-conotoxin or cadmium chloride. The pre-synaptic effect of R-PIA was similarly unaffected by 30 microM 4-AP. The pre-synaptic effect of UK 14,304 was virtually abolished by 4-AP (30 microM). The effect of UK 14,304 (0.1 microM) could be partly restored by reducing the Ca2+ concentration during treatment with 4-AP (22% inhibition compared to 42% with normal Ca2+). The magnitude of increase in evoked [3H]NA efflux by yohimbine (1 microM) was decreased by 4-AP in a concentration-dependent manner from 142% increase in controls to 21% at 100 microM 4-AP. The present results indicate that NA release is reduced by somewhat different mechanisms by pre-synaptic alpha 2- and adenosine A1-receptors. Furthermore, the results indicate that pre-synaptic A1-receptors on hippocampal NA neurons do not primarily regulate 4-AP-dependent potassium channels, but they might act directly on a Ca2+ conductance.     

α2-Aclrenoceptor agonist-mediated inhibition of [3H]noradrenaline release from rat hippocampus is reduced by 4-aminopyridine,but that caused by an adenosine analogue or co-conotoxin is not

The inhibitory effect of an adenosine analogue, R-PIA, and an α₂-adrenoceptor agonist, UK 14,304, on [³H]NA efflux from field-stimulated rat hippocampal slices was examined. The effect of 0.1 μm UK 14,304 was mimicked by 30 nM w-conotoxin and by 10 μM cadmium chloride, inhibitors of N- and L-type Ca²⁺ channels. R-PIA (1 μM) had no effect per se, but caused a clear-cut inhibition after blockade of the pre-synaptic α₂-receptor by yohimbine. 4-Aminopyridine (4-AP) caused a dose-dependent increase in evoked transmitter release. At 30 μM 4-AP did not affect the actions of ω-conotoxin or cadmium chloride. The pre-synaptic effect of R-PIA was similarly unaffected by 30 μM 4-AP. The presynaptic effect of UK 14,304 was virtually abolished by 4-AP (30 μM). The effect of UK 14,304 (0.1 μM) could be partly restored by reducing the Ca²⁺ concentration during treatment with 4-AP (22% inhibition compared to 42% with normal Ca²⁺). The magnitude of increase in evoked [³H]NA efflux by yohimbine (1 μM) was decreased by 4-AP in a concentration-dependent manner from 142% increase in controls to 21 % at 100 μM 4-AP. The present results indicate that NA release is reduced by somewhat different mechanisms by pre-synaptic α₂- and adenosine Aj-receptors. Furthermore, the results indicate that pre-synaptic A_rreceptors on hippocampal NA neurons do not primarily regulate 4-AP-dependent potassium channels, but they might act directly on a Ca²⁺ conductance.     

Influence of renal sympathectomy, sodium depletion and prostaglandin synthetase inhibition on prostaglandin production and [3H]PGE2 binding characteristics in rat kidney

The effects of renal sympathectomy (unilateral, renal microsurgical denervation), sodium depletion (hypovolaemia) and prostaglandin synthetase inhibition on the rate of prostaglandin synthesis and [3H]PGE2 binding characteristics were studied in the rat kidney. The intrarenal rate of prostaglandin synthesis was measured by monitoring the urinary excretion of 6-keto-PGF1 alpha, the stable hydration product of prostacyclin. Dietary sodium restriction was associated with a 99% decrease in urinary sodium excretion (P less than 0.001) and a 17% decrease of urine volume (n.s.). Renal denervation or sodium deprivation changed neither the rate of excretion of 6-keto-PGF1 alpha nor the density or affinity of [3H]PGE2 binding sites as compared to control. However, in sodium-depleted rats, prostaglandin synthesis inhibition, induced by naproxen, decreased the urinary excretion of 6-keto-PGF1 alpha by 40% (P = 0.011) and increased the number of [3H]PGE2 binding sites by almost 30% (P = 0.031) with no change in binding affinities as compared with sodium-depleted controls. In contrast, sulindac was not able to suppress the renal synthesis and excretion of 6-keto-PGF1 alpha, and did not modulate the [3H]PGE2 binding characteristics. The lack of effect on the excretion of 6-keto-PGF1 alpha and on the [3H]PGE2 binding characteristics supports the view that sulindac spares renal prostaglandin synthesis.     

Accumulation of [3H]fucose-labelled glycoproteins in the Golgi apparatus of dorsal root ganglion neurons during inhibition of fast axonal transport caused by exposure of the ganglion to Co2+-containing or Ca2+-free medium

Previous in vitro studies have established that Co2+-containing or Ca2+-free media interfere with the initiation of the fast axonal transport of proteins. The present study has used light- and electron-microscope radioautography to compare the distribution of [3H]fucose-labelled glycoproteins in neuronal cell bodies of control dorsal root ganglia and ganglia incubated for 16-17 h in Ca2+-free medium or in medium containing 0.18 mM Co2+. The radioautographic reaction in control cell bodies was diffusely scattered throughout the cytoplasm; grain counts revealed that 22% of the reaction was associated with elements of the Golgi apparatus and 78% was over other organelles and the remainder of the cytoplasm. In most experimental cell bodies, 78% of the silver grains were clustered over elements of the Golgi complex whereas other organelles and the remainder of the cytoplasm were comparatively much less labelled; structural alterations of the Golgi apparatus were also produced by the modified media. In parallel studies where the radioactivity in nerve trunks and ganglia was measured by liquid scintillation counting, it was found that the Ca2+-free medium and the Co2+-containing medium both reduced by approximately 80% the quantity of [3H]fucose-labelled glycoproteins which were carried by the fast axonal transport system; they did so without interfering with the incorporation of [3H]fucose into glycoproteins. The results indicate that in the presence of Co2+ or in the absence of Ca2+ the proteins which are destined for fast axonal transport accumulate at the Golgi apparatus of neuronal cell bodies. These results thus suggest that Ca2+ is required for proteins to leave the Golgi region in transit to the fast axonal transport system.     

Effects of N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate, adenosine, and of oxotremorine and 3-isobutyl-1-methylxanthine on the electrically evoked [3H]acetylcholine secretion in the guinea-pig ileum myenteric plexus

The guinea-pig ileum longitudinal muscle-myenteric plexus preparation, pre-incubated with [3H]choline, was mounted in an organ bath and superfused with Tyrode's solution. [3H]Acetylcholine secretion was evoked by 150 electrical shocks at 0.5 Hz. N6,2'-O-Dibutyryladenosine 3',5'-cyclic monophosphate (dibutyryl cyclic AMP) enhanced the [3H]acetylcholine secretion in the presence of eserine and the adenosine receptor antagonist 8-phenyltheophylline (10 mumol l-1). Conversely, in the absence of 8-phenyltheophylline the [3H]acetylcholine secretion was reduced by dibutyryl cyclic AMP. In the absence and presence of 8-phenyltheophylline (apparent KD = 12 mumol l-1), adenosine reduced the [3H]acetylcholine secretion to 33% of control (IC50 = 8 mumol l-1) and to 48% of control (IC50 = 14 mumol l-1) respectively. Neither butyrate, dibutyryl cyclic GMP nor guanosine altered the [3H]acetylcholine secretion. Interaction experiments with 3-isobutyl-1-methylxanthine and oxotremorine were done in the absence of eserine, i.e. when oxotremorine is effective. Oxotremorine depressed the fractional secretion of [3H]acetylcholine with a 'maximal inhibition' of 13% of control (IC50 = 10 nmol l-1). In the presence of 3-isobutyl-1-methylxanthine (5 mmol l-1) oxotremorine depressed the secretion to 2% of control with an apparent IC50 value of 0.9 mumol l-1. 3-Isobutyl-I-methylxanthine (0.01-4 mmol l-1) enhanced the fractional secretion of [3H]acetylcholine with a 'maximal enhancement' value of 232% of control (EC50 = 0.19 mmol l-1). The presence of oxotremorine (30 nmol l-1) counteracted, and higher concentrations reversed, the enhancement caused by 3-isobutyl-1-methylxanthine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential release of coexisting neurotransmitters: frequency dependence of the efflux of substance P, thyrotropin releasing hormone and [3H]serotonin from tissue slices of rat ventral spinal cord

In few systems has the release of coexisting classical and peptide neurotransmitters been studied. Here the release of substance P-like immunoreactivity (SP-LI), thyrotropin releasing hormone-like immunoreactivity (TRH-LI) and [3H]serotonin ([3H]5-HT) from tissue slices of rat ventral spinal cord was investigated in a superfusion system. The slices were stimulated electrically with field stimulation (900 pulses, 2 ms duration, 36 V) at frequencies between 0.25 Hz and 40 Hz. The evoked fractional release of SP-LI increased significantly from 0.46 to 1.24% of the total tissue store when the frequency of stimulation was changed from 3 to 10 Hz, while the evoked fractional release of TRH-LI increased significantly from 0.28 to 0.71% of the total tissue store with increasing frequency of stimulation between 0.5 and 3 Hz. The evoked fractional release of [3H]5-HT did not show any significant change when the frequency of stimulation was changed in the frequency range of 0.25-40 Hz but remained between 5.6 and 7.2% of the total tissue store. It appears that at frequencies lower than 0.5-1 Hz these 5-HT/SP/TRH neurons may function predominantly as serotonergic neurons. At 3 Hz stimulation with 900 pulses the extracellular Ca2+ concentration required for half-maximal release of [3H]5-HT was 1.2 mmol l-1, while for half-maximal release of SP-LI significantly higher concentrations of Ca2+ (4.2 mmol l-1) would be required.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of N6, 2‘-0-dibutyryladenosine 3’,5‘-cyclic monophosphate,adenosine, and of oxotremorine and 3-isobutyl-1-methylxanthine on the electrically evoked [3H] acetylcholine secretion in the guinea-pig ileum myenteric plexus

The guinea-pig ileum longitudinal muscle-myenteric plexus preparation, pre-incubated with [³H]choline, was mounted in an organ bath and superfused with Tyrode's solution. [³H]Acetylcholine secretion was evoked by 150 electrical shocks at 0.5 Hz. >N⁶,2′-0-Dibutyryladenosine 3′,5′-cyclic monophosphate (dibutyryl cyclic AMP) enhanced the [³H]acetylcholine secretion in the presence of eserine and the adenosine receptor antagonist 8-phenyltheophylline (10 μmol 1^-1). Conversely, in the absence of 8-phenyltheophylline the [³H]acetylcholine secretion was reduced by dibutyryl cyclic AMP. In the absence and presence of 8-phenyltheophylline (apparent K_D = 12 μ.mol 1^-1), adenosine reduced the [³H]acetylcholine secretion to 33% of control (IC₅₀ = 8, μmol l^-1) and to 48% of control (IC₅₀ = 14 μmol l^-1) respectively. Neither butyrate, dibutyryl cyclic GMP nor guanosine altered the [³H]acetylcholine secretion. Interaction experiments with 3-isobutyl-1-methylxanthine and oxotremorine were done in the absence of eserine, i.e. when oxotremorine is effective. Oxotremorine depressed the fractional secretion of [³H]acetylcholine with a ‘maximal inhibition’ of 13% of control (IC₅₀ = 10 nmol l^-1). In the presence of 3-isobutyl-1-methylxanthine (5 mmol l^-1) oxotremorine depressed the secretion to 2% of control with an apparent IC₅₀) value of 0.9 μmol l^-1 3-Isobutyl-I-methylxanthine (0.01–4 mmol l^-1) enhanced the fractional secretion of [³H]acetylcholine with a ‘maximal enhancement’ value of 232% of control (EC₅₀ = 0.19 mmol l^-1). The presence of oxotremorine (30 nmol l^-1) counteracted, and higher concentrations reversed, the enhancement caused by 3–isobutyl-I-methylxanthine. The results are consistent with the suggestion that the presynaptic muscarinic acetylcholine receptor-mediated control of the secretion of [³H]acetylcholine in the guinea-pig ileum myenteric plexus may be mediated both by cyclic AMP and calcium ions.     

A novel TRH analog, Glp-Asn-Pro-D-Tyr-D-TrpNH2, binds to [3H][3-Me-His2]TRH-labelled sites in rat hippocampus and cortex but not pituitary or heterologous cells expressing TRHR1 or TRHR2

Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is a novel synthetic peptide that mimics and amplifies central actions of thyrotropin-releasing hormone (TRH) in rat without releasing TSH. The aim of this study was to compare the binding properties of this pentapeptide and its all-L counterpart (Glp-Asn-Pro-Tyr-TrpNH(2)) to TRH receptors in native rat brain tissue and cells expressing the two TRH receptor subtypes identified in rat to date, namely TRHR1 and TRHR2. Radioligand binding studies were carried out using [(3)H][3-Me-His(2)]TRH to label receptors in hippocampal, cortical and pituitary tissue, GH4 pituitary cells, as well as CHO cells expressing TRHR1 and/or TRHR2. In situ hybridization studies suggest that cortex expresses primarily TRHR2 mRNA, hippocampus primarily TRHR1 mRNA and pituitary exclusively TRHR1 mRNA. Competition experiments showed [3-Me-His(2)]TRH potently displaced [(3)H][3-Me-His(2)]TRH binding from all tissues/cells investigated. Glp-Asn-Pro-D-Tyr-D-TrpNH(2) in concentrations up to 10(-5)M did not displace [(3)H][3-Me-His(2)]TRH binding to membranes derived from GH4 cells or CHO-TRHR1 cells, consistent with its lack of binding to pituitary membranes and TSH-releasing activity. Similar results were obtained for the corresponding all-L peptide. In contrast, both pentapeptides displaced binding from rat hippocampal membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.7+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2), analogous to cortical membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.8+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2). Neither peptide, however, displaced [(3)H][3-Me-His(2)]TRH binding to CHO-TRHR2. Thus, this study reveals for the first time significant differences in the binding properties of native and heterologously expressed TRH receptors. Also, the results raise the possibility that Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is not displacing [(3)H][3-Me-His(2)]TRH from a known TRH receptor in rat cortex, but rather a hitherto unidentified TRH receptor.