The presence of 2'', 3''-cyclic AMP 3''-phosphohydrolase in glial cells in tissue culture

Abstract— T he E nzyme 2', 3'-cyclic AMP 3'-phosphohydrolase (CNP) is regarded as a marker for myelin (KURI- HARA and MANDEL, 1970) on the basis of its regional and subcellular distribution (K urihara and T sukada , 1967), its ontogenetic characteristics (KURIHARA and TSUKADA, 1968), and its behaviour in two strains of myelin-deficient mutant mice (K urihara , N ussbaum and M andel , 1969). However we have isolated highly-purified preparations of neuronal plasma membrane from rat brain synaptosomes which contain this enzyme activity (M organ , W olfe , M andel and G ombos , 1971). Two explanations of this finding are possible. The activity could be due to the presence of myelin, but this explanation is ruled out by electron microscopy and by the low level of cerebrosides in the synaptosomal plasma membrane preparations. Myelin is extremely rich in cerebrosides ( norton and A utilio , 1966). The second possibility is that the enzyme, 2', 3'-cyclic AMP 3'-phosphohyrolase, may also be found in the glial cells from which myelin is derived (B unge , B unge and P appas , 1962). To test our hypothesis that 2', 3'-cyclic AMP 3'-phosphohydrolase is not a specific marker for myelin, but is also found, in glial cells, we have examined a tumoral glial cell line maintained in myelin-free tissue culture.     

An antagonistic 5-HT receptor system in the auricles of the systemic heart complex of Sepia officinalis L. (Cephalopoda) shows 5-HT1 and 5-HT4 subtype properties

In pharmacological bioassays on isolated ring-shaped auricle preparations of Sepia officinalis, the action of the specific 5-hydroxytryptamine (5-HT) agonists 8-OH-DPAT (5-HT1a), CP-93129 (5-HT1b), TFMPP (5-HT1b) and RS-67333 (5-HT4) on these autonomously contractile compartments was studied. 8-OH-DPAT and CP-93129 induced mainly positive effects on frequency and tone on the isotonically suspended auricles. The positive effect of 8-OH-DPAT on frequency was blocked by the specific 5-HT1a antagonist NAN-190. The 5-HT1b agonist TFMPP caused similar effects on tone and a positive impact on the auricular amplitude. The highly specific 5-HT4 agonist RS-67333 induced an effect opposite to the action of 5-HT1 agonists inducing mainly negative effects on frequency, amplitude and tone, causing a diastolic standstill at a concentration of 10(-6) M. These negative effects were blocked by the adenylyl cyclase inhibitor SQ-22,536 in the absence of a diastolic standstill. The opposing action of 5-HT1 and 5-HT4 agonists on auricular contractile activity suggests that an antagonistic 5-HT-receptor system exists within the auricular myocardial cells of S. officinalis, probably consisting of 5-HT1- and 5-HT4-like subtypes. The results also suggest that adenylyl cyclase acts as the intracellular target enzyme of both signal transduction mechanisms.     

Effects of Increased Extracellular K on the Elemental Composition and Water Content of Neuron and Glial Cells in Leech CNS

Elemental (Na, Cl, K) and water contents of leech (Macrobdella decora) neurons and glial cells were determined under steady-state exposure to 4, 10, and 20 mM KCl concentrations (bathing media) using x-ray microanalysis for quantitative digital imaging of frozen hydrated and dried cryosections. Effects of furosemide, 5-hydroxytryptamine (5-HT), and ouabain on elemental distribution changes, induced by exposure to 20 mM K, were also determined. Results demonstrated that packet glial cells and neurons accumulated substantial amounts of K that appeared evenly distributed throughout the cytoplasm. Cell water content also increased as a function of increased cytoplasmic K so that the net effect was an unchanged wet-weight K concentration (expressed as millimoles per kilogram wet weight). Dry-weight Na and Cl concentration (expressed as millimoles per kilogram dry weight) increased slightly in glial cells; however, because cell water increased, both Na and Cl (wet-weight) concentrations decreased. Neurons, in contrast, had no significant change in either Na or K on a wet-weight basis, so a relatively constant Na/K ratio was maintained despite a small, but significant, increase in K (dry weight) and cell water. These increases, like those in packet glia, were a function of exposure to different concentrations of extracellular space K. These changes were completely abolished by 10(-4) M ouabain. Neither furosemide nor 5-HT appeared to affect neuronal or glial K wet-weight concentrations. These data show that both glial cells and neurons can act as substantial reservoirs for K while maintaining stable K concentrations (by altering cell water content and elemental composition). This process appears to depend on a functioning Na+, K+-ATPase system.     

Serotonin sensitive adenylate cyclase in horse brain synaptosomal membranes.

In different membranal preparations isolated from horse brain stritum we have shown the existence of an adenylate cyclase system sensitive to serotonin (5-HT). Activation of the adenylate cyclase was determined by measuring cAMP using a radioimmunoassay. This serotoninergic sensitive enzyme is characterized by a high apparent affinity constant (in the nanomolar range), located on synaptosomal membranes. It is inhibited by antiserotoninergic drugs (cyproheptadine, cinanserin, methysergide, LSD), and synergistically activated by GTP. This serotoninergic activation is clearly additive to the activation induced by dopamine. It appears different from the adenylate cyclase system previously described in the literature which is also activated by 5-HT, but which has a low apparent affinity constant (in the micromolar range); the latter is apparently located in non-synaptosomal membranes, and its activation by 5-HT is non-additive to the activation induced by dopamine.The serotoninergic sensitive adenylate cyclase reported in this study, might be related to the serotoninergic binding system which we have previously described which has similar affinity constant, a similar subcellular distribution and which is inhibited in the same concentration ranges by antiserotoninergic drugs. These two systems might represent a synaptosomal serotoninergic receptor complex.     

On the presence of serotonin in mammalian cardiomyocytes

Pleiotropic effects of serotonin (5-HT) in the cardiovascular system are well documented. However, it remains to be elucidated, whether 5-HT is present in adult mammalian cardiomyocytes. To address this issue, we investigated the levels of 5-HT in blood, plasma, platelets, cardiac tissue, and cardiomyocytes from adult mice and for comparison in human right atrial tissue. Immunohistochemically, 5-HT was hardly found in mouse cardiac tissue, but small amounts could be detected in renal preparations, whereas adrenal preparations revealed a strong positive immunoreaction for 5-HT. Using a sensitive HPLC detection system, 5-HT was also detectable in the mouse heart and human atrium. Furthermore, we could identify 5-HT in isolated cardiomyocytes from adult mice. These findings were supported by detection of the activity of 5-HT-forming enzymes-tryptophan hydroxylase and aromatic L-amino acid decarboxylase-in isolated cardiomyocytes from adult mice and by inhibition of these enzymes with p-chlorophenylalanine and 3-hydroxybenzyl hydrazine. Addition of the first intermediate of 5-HT generation, that is 5-hydroxytryptophan, enhanced the 5-HT level and inhibition of monoamine oxidase by tranylcypromine further increased the level of 5-HT. Our findings reveal the presence and synthesis of 5-HT in cardiomyocytes of the mammalian heart implying that 5-HT may play an autocrine and/or paracrine role in the heart.     

The regulation of 5-hydroxytryptamine release from superfused synaptosomes by 5-hydroxytryptamine and its immediate precursors

The effect of L-tryptophan, 5-hydroxy-L-tryptophan (5-HTP), and 5-hydroxytryptamine (5-HT) on the K+-evoked release of [3H]5-HT from superfused rat brain synaptosomes was studied. 5-HT at concentrations above 10 nM significantly inhibited the K+-evoked release of [3H]5-HT. A slight enhancement of [3H]5-HT release was observed at a concentration of 5nM. In contrast tryptophan at a concentration of 10 nM significantly enhanced [3H]5-HT release with little effect at higher concentrations. 5-HTP did not significantly effect [3H]5-HT release. The results confirm previous findings that 5-HT inhibits its own release from nerve endings, and demonstrate that low concentrations of tryptophan in the synaptic region may act as a positive feedback regulator of 5-HT release.     

Tricyclic antidepressant amitriptyline activates fibroblast growth factor receptor signaling in glial cells: involvement in glial cell line-derived neurotrophic factor production

Recently, both clinical and animal studies demonstrated neuronal and glial plasticity to be important for the therapeutic action of antidepressants. Antidepressants increase glial cell line-derived neurotrophic factor (GDNF) production through monoamine-independent protein-tyrosine kinase, extracellular signal-regulated kinase (ERK), and cAMP responsive element-binding protein (CREB) activation in glial cells (Hisaoka, K., Takebayashi, M., Tsuchioka, M., Maeda, N., Nakata, Y., and Yamawaki, S. (2007) J. Pharmacol. Exp. Ther. 321, 148-157; Hisaoka, K., Maeda, N., Tsuchioka, M., and Takebayashi, M. (2008) Brain Res. 1196, 53-58). This study clarifies the type of tyrosine kinase and mechanism of antidepressant-induced GDNF production in C6 glioma cells and normal human astrocytes. The amitriptyline (a tricyclic antidepressant)-induced ERK activation was specifically and completely inhibited by fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors and siRNA for FGFR1 and -2. Treatment with amitriptyline or several different classes of antidepressants, but not non-antidepressants, acutely increased the phosphorylation of FGFRs and FGFR substrate 2α (FRS2α). Amitriptyline-induced CREB phosphorylation and GDNF production were blocked by FGFR-tyrosine kinase inhibitors. Therefore, antidepressants activate the FGFR/FRS2α/ERK/CREB signaling cascade, thus resulting in GDNF production. Furthermore, we attempted to elucidate how antidepressants activate FGFR signaling. The effect of amitriptyline was inhibited by heparin, non-permeant FGF-2 neutralizing antibodies, and matrix metalloproteinase (MMP) inhibitors. Serotonin (5-HT) also increased GDNF production through FGFR2 (Tsuchioka, M., Takebayashi, M., Hisaoka, K., Maeda, N., and Nakata, Y. (2008) J. Neurochem. 106, 244-257); however, the effect of 5-HT was not inhibited by heparin and MMP inhibitors. These results suggest that amitriptyline-induced FGFR activation might occur through an extracellular pathway, in contrast to that of 5-HT. The current data show that amitriptyline-induced FGFR activation might occur by the MMP-dependent shedding of FGFR ligands, such as FGF-2, thus resulting in GDNF production.     

Increasing external K+ blocks phase shifts in a circadian rhythm produced by serotonin or 8-benzylthio-cAMP

Serotonin (5-HT) phase shifts the circadian rhythm from the isolated eye of Aplysia. The discovery of the mechanisms involved in phase shifting by 5-HT may help elucidate the nature of the circadian oscillator. We have found that 5-HT appears to phase shift by causing a change in membrane K+ conductance. Solutions containing zero K+(0-K+) phase shift the rhythm and the phase response curve (PRC) for 0-K+ is similar to one previously obtained for 5-HT. The similarity in PRCs for 0-K+ and 5-HT suggested that these treatments may be phase shifting the rhythm through a common mechanism. The nonadditivity of phase shifting by 0-K+ and 5-HT supports this suggestion. A common mechanism of action of 5-HT and 0-K+ might be effects on membrane potentials. The possible involvement of a membrane potential change in mediating the effect of 5-HT and the lack of an effect of large reductions in Na+, Cl-, and Ca2+ ions on phase shifting by 5-HT led us to examine the role of K+ ions in phase shifting by 5-HT. A change in K+ conductance may mediate the effects of 5-HT on the rhythm because HiK (30mM) solutions blocked the phase shift normally produced by 5-HT. The conductance change produced by 5-HT may be an increase in K+ conductance which would produce a hyperpolarization and not a decrease in K+ conductance which would produce a depolarization since depolarizing treatments, HiK (30-110mM), had no effect on the rhythm at the phase where 5-HT produces its largest phase shifts. Since we previously found that the effects of 5-HT appear to be mediated by cAMP, we examined whether HiK solutions could block the effects of 8-benzylthio-cAMP on the rhythm. HiK (40mM) blocked the phase shifts normally produced by 8-benylthio-cAMP. Our working hypothesis for the 5-HT phase-shifting pathway based on these results is 5-HT leads to increased cAMP leads to elevates K+ conductance leads to membrane hyperpolarization leads to phase shifts the rhythm.     

Regulation of Glial Na+/K+-ATPase by Serotonin: Identification of Participating Receptors

The purpose of the present study was the characterization of the receptors participating in the regulatory mechanism of glial Na⁺/K⁺-ATPase by serotonin (5-HT) in rat brain. The activity of the Na⁺ pump was measured in four brain regions after incubation with various concentrations of serotoninergic agonists or antagonists. A concentration-dependent increase in enzyme activity was observed with the 5-HT_1A agonist R (+)-2-dipropylamino-8-hydroxy-1,2,3, 4-tetrahydronaphthalene hydrobromide (8-OH-DPAT) in homogenates or in glial membrane enriched fractions from cerebral cortex and in hippocampus. Spiperone, a 5-HT_1A antagonist, completely inhibited the response to 8-OH-DPAT but had no effect on Na⁺/K⁺-ATPase activity in cerebellum where LSD, a 5-HT₆ agonist, elicited a dose-dependent response similar to that of 5-HT. In brainstem, a lack of reponse to 5-HT and other agonists was confirmed. Altogether, these results show that serotonin modulates glial Na⁺/K⁺-ATPase activity in the brain, apparently not through only one type of 5-HT receptor. It seems that the receptor system involved is different according to the brain region. In cerebral cortex, the response seems to be mediated by 5-HT_1A as well as in hippocampus but not in cerebellum where 5-HT₆ appears as the receptor system involved.     

Cytochemical localization of Na+/K+-ATPase activity in cochlear strial marginal cells after various catecholamine administrations

Sodium/potassium-activated adenosine triphosphatase (Na+/K+-ATPase) activity in the kidney and brain is high, and is regulated by catecholamines. Na+/K+-ATPase activity is also high in the basolateral infoldings of the strial marginal cells, where it aids in maintaining the characteristic electrolyte composition of the endolymph. To clarify the involvement of humoral control in strial function, particularly the role of catecholamines, the K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity of strial marginal cells was investigated in guinea pigs using a cerium-based cytochemical method. The effects of reserpine, serotonin (5-HT), norepinephrine (NE), epinephrine (EP), both alone and in combination, were studied. High doses of reserpine cause depletion of sympathetic substances. Strial K+-NPPase activity was decreased after reserpine or dopamine treatment, and was increased after 5-HT, NE, and EP treatment. After reserpinization, repeated treatment with 5-HT, NE, or EP led to detectable strial enzyme activity. Thus, exogenous 5-HT, NE, and EP were able to restore strial K+-NPPase activity in the reserpine-treated animals. These results suggested that biogenic amines regulate strial K+-NPPase activity. Thus, the function of the stria vascularis may be regulated by the opposing actions of these catecholamines, and 5-HT.     

Effect of Chronic Lithium Treatment on 5-Hydroxytryptamine Autoreceptors and Release of 5-[3H]Hydroxytryptamine from Rat Brain Cortical, Hippocampal, and Hypothalamic Slices

The effect of acute and chronic lithium treatments on 5-hydroxytryptamine (5-HT, serotonin) release and on its regulation by presynaptic 5-HT autoreceptors was studied in [3H]5-HT preloaded superfused rat brain slices. The [3H]5-HT overflow evoked by a 30-s exposure to 65 mM K+ was increased after 3 weeks of ingestion of lithium-containing diet in the three brain areas examined. Acute injection of 4 mEq/kg lithium chloride did not affect 5-HT release. The K+-induced release observed in both control and chronically lithium-treated animals was Ca2+-dependent. Chronic lithium treatment was also found to be associated with a decrease in basal [3H]5-HT overflow in the cortex and hypothalamus but not in hippocampus [corrected]. The Ca2+-independent overflow induced by fenfluramine was also decreased in cortical slices from lithium-treated animals. The sensitivity of the inhibitory 5-HT autoreceptors was assessed by the response to the 5-HT agonist 5-methoxytryptamine. The results indicate a marked reduction in the maximal inhibition of [3H]5-HT release induced by 5-methoxytryptamine in slices obtained from animals which have been treated with lithium for 3 weeks. These data suggest that the functional down regulation of the prejunctional 5-HT sites may be responsible for the increase in K+-stimulated 5-HT overflow in brain slices of animals treated chronically with lithium.     

Calcium influx into dendrites of the leech Retzius neuron evoked by 5-hydroxytryptamine

5-Hydroxytryptamine (5-HT) is a ubiquitous neurotransmitter and neuromodulator that affects neural circuits and behaviours in vertebrates and invertebrates. In the present study, we have investigated 5-HT-induced Ca(2+) transients in subcellular compartments of Retzius neurons in the leech central nervous system using confocal laser scanning microscopy, and studied the effect of 5-HT on the electrical coupling between the Retzius neurons. Bath application of 5-HT (50mM) induced a Ca(2+) transient in axon, dendrites and cell body of the Retzius neuron. This Ca(2+) transient was significantly faster and larger in dendrites than in axon and cell body, and was half-maximal at a 5-HT concentration of 5-12mM. The Ca(2+) transient was suppressed in the absence of extracellular Ca(2+) and by methysergide (100mM), a non-specific antagonist of metabotropic 5-HT receptors, and was strongly reduced by bath application of the Ca(2+) channel blocker Co(2+) (2mM). Injection of the non-hydrolysable GTP analogue GTPgammaS increased and prolonged the dendritic 5-HT-induced Ca(2+) transient. The non-selective protein kinase inhibitor H7 (100mM) and the adenylate cyclase inhibitor SQ22536 (500 mM) did not affect the Ca(2+) transient, and the membrane-permeable cAMP analogue dibutyryl-cAMP (500 mM) did not mimic the effect of 5-HT application. 5-HT reduced the apparent electrical coupling between the two Retzius neurons, whereas suppression of the Ca(2+) influx by removal of external Ca(2+) improved the transmission of action potentials at the electrical synapses which are located between the dendrites of the adjacent Retzius neurons. The results indicate that 5-HT induces a Ca(2+) influx through calcium channels located primarily in the dendrites, and presumably activated by a G protein-coupled 5-HT receptor. The dendritic Ca(2+) increase appears to modulate the excitability of, and the synchronization between, the two Retzius neurons.     

Metabolic origins of 5-hydroxytryptamine in enteric neurons in a teleostean fish (Platycephalus bassensis), a toad (Bufo marinus) and the guinea-pig

1. 5-Hydroxytryptamine (5-HT) content and synthesis in mucosa-free intestine of guinea-pig, the teleost Platycephalus bassensis and the amphibian Bufo marinus was studied by HPLC with electrochemical detection or by TLC. 2. The 5-HT content of small intestine was: guinea-pig 0.58; Bufo: 1.23; Platycephalus: 26.88 nmol/g. 3. Intestine from each species synthesized 5-HT from exogenous 5-HTP. 4. Platycephalus preparations synthesized labelled 5-HT from 14C-tryptophan, but no labelled 5-HT was detected after similar incubation of guinea-pig or Bufo preparations. 5. Incubation of guinea-pig preparations with tryptophan did not increase tissue 5-HT or 5-HIAA content. 6. 5-HT in Platycephalus enteric neurons may be synthesized from tryptophan in situ; 5-HT in Bufo and guinea-pig neurons may be synthesized elsewhere, perhaps in enterochromaffin cells.     

Airway smooth muscle relaxation induced by 5-HT(2A) receptors: role of Na(+)/K(+)-ATPase pump and Ca(2+)-activated K(+) channels

AIMS: Although 5-hydroxytryptamine (5-HT) contracts airway smooth muscle in many mammalian species, in guinea pig and human airways 5-HT causes a contraction followed by relaxation. This study explored potential mechanisms involved in the relaxation induced by 5-HT. MAIN METHODS: Using organ baths, patch clamp, and intracellular Ca(2+) measurement techniques, the effect of 5-HT on guinea pig airway smooth muscle was studied. KEY FINDINGS: A wide range of 5-HT concentrations caused a biphasic response of tracheal rings. Response to 32 muM 5-HT was notably reduced by either tropisetron or methiothepin, and almost abolished by their combination. Incubation with 10 nM ketanserin significantly prevented the relaxing phase. Likewise, incubation with 100 nM charybdotoxin or 320 nM iberiotoxin and at less extent with 10 muM ouabain caused a significant reduction of the relaxing phase induced by 5-HT. Propranolol, L-NAME and 5-HT(1A), 5-HT(1B)/5-HT(1D) and 5-HT(2B) receptors antagonist did not modify this relaxation. Tracheas from sensitized animals displayed reduced relaxation as compared with controls. In tracheas precontracted with histamine, a concentration response curve to 5-HT (32, 100 and 320 muM) induced relaxation and this effect was abolished by charybdotoxin, iberiotoxin or ketanserin. In single myocytes, 5-HT in the presence of 3 mM 4-AP notably increased the K(+) currents (I(K(Ca))), and they were completely abolished by charybdotoxin, iberiotoxin or ketanserin. SIGNIFICANCE: During the relaxation induced by 5-HT two major mechanisms seem to be involved: stimulation of the Na(+)/K(+)-ATPase pump, and increasing activity of the high-conductance Ca(2+)-activated K(+) channels, probably via 5-HT(2A) receptors.     

Mevalonate kinase in green leaves and etiolated cotyledons of the French bean Phaseolus vulgaris

1. Partially purified preparations of mevalonate kinase were obtained from green leaves and etiolated cotyledons of Phaseolus vulgaris. 2. After removal of interfering polyphenols both enzyme preparations behaved identically on gel filtration, ion-exchange chromatography and density-gradient centrifugation. 3. The kinetic parameters of the preparations from the two sources were indistinguishable. The preparation from etiolated cotyledons had a K(m) of 4.26x10(-5)m for RS-mevalonate and 1.54x10(-3)m for ATP. The preparation from green leaves had a K(m) of 4.55x10(-5)m for RS-mevalonate and 1.75x10(-3)m for ATP. The pH optimum of both enzyme preparations was pH7.0. 4. The effect of inhibitors on the two enzyme preparations was similar, both being inhibited by reagents known to react with thiol groups, and the two preparations had similar inhibitor constants for competitive inhibition by prenyl pyrophosphates. 5. The molecular weight of the enzyme in both preparations was estimated to be 100000; the enzymes from the two preparations had similar mobilities on polyacrylamide-gel electrophoresis.     

Fenfluramine-induced pulmonary vasoconstriction: role of serotonin receptors and potassium channels.

The anorexic agent fenfluramine considerably increases the risk of primary pulmonary hypertension. The mechanism of this effect is unknown. The appetite-reducing action of fenfluramine is mediated by its interaction with the metabolism of serotonin [5-hydroxytryptamine (5-HT)] in the brain. We tested the hypothesis that the pulmonary vasoconstrictive action of fenfluramine is at least in part mediated by 5-HT receptor activation. In addition, we sought to determine whether pharmacological reduction of voltage-gated potassium (K(V)) channel activity would potentiate the pulmonary vascular reactivity to fenfluramine. Using isolated rat lungs perfused with Krebs-albumin solution, we compared the inhibitory effect of ritanserin, an antagonist of 5-HT(2) receptors, on fenfluramine- and 5-HT-induced vasoconstriction. Both 5-HT (10(-5) mol/l) and fenfluramine (5 x 10(-4) mol/l) caused significant increases in perfusion pressure. Ritanserin at a dose (10(-7) mol/l) sufficient to inhibit >80% of the response to 5-HT reduced the response to fenfluramine by approximately 50%. A higher ritanserin dose (10(-5) mol/l) completely abolished the responses to 5-HT but had no more inhibitory effect on the responses to fenfluramine. A pharmacological blockade of K(V) channels by 4-aminopyridine (3 x 10(-3) mol/l) markedly potentiated the pulmonary vasoconstrictor response to fenfluramine but was without effect on the reactivity to 5-HT. These data indicate that the pulmonary vasoconstrictor response to fenfluramine is partly mediated by 5-HT receptors. Furthermore, the pulmonary vasoconstrictor potency of fenfluramine is elevated when the K(V)-channel activity is low. This finding suggests that preexisting K(V)-channel insufficiency may predispose some patients to the development of pulmonary hypertension during fenfluramine treatment.     

Inhibition of insulin secretion by KN-62, a specific inhibitor of the multifunctional Ca2+/calmodulin-dependent protein kinase II.

The effects of KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CamPKII), on insulin secretion and protein phosphorylation were studied in rat pancreatic islets and RINm5F cells. KN-62 was found to dose-dependently inhibit autophosphorylation of CamPKII in subcellular preparations of RINm5F cells (K0.5 = 3.1 +/- 0.3 microM), but had no effect on protein kinase C or myosin light chain kinase activity. KN-62, but not the inactive analogue KN-04, dose-dependently inhibited glucose-induced insulin release (K0.5 = 1.5 +/- 0.5 microM) in a manner similar to the inhibition of CamPKII autophosphorylation. KN-62 (10 microM) inhibited carbachol (in the presence of 8 mM glucose) and potassium-stimulated insulin secretion from islets by 53% and 59%, respectively. These results support a role of CamPKII in glucose-sensitive insulin secretion.     

Na+ K+ ATPase activity in synaptosomes and myelin of developing control and intra-uterine growth retarded rats: effects of lead and serotonin

An intrauterine growth retarded (IUGR) model based on restriction of blood supply to fetuses at 17 days of pregnancy in rats was studied. We investigated in vitro the effects of lead on Na⁺K⁺ ATPase activity in synaptosomes and myelin of IUGR and control rats from 6 to 60 days after birth. In both groups an age-dependent effects existed in synaptosomes for the lowest doses of lead. The experimental group tended to be more sensitive to the metal than the control group and the Na⁺K⁺ATPase activity was less inhibited in the younger rats as compared to mature rats. Serotonin (5-HT) added to the subcellular preparations produced different changes in Na⁺K⁺ATPase activity. In synaptosomes, 5-HT stimulated the enzyme activity in a dose-related manner and apparently reversed the inhibiton induced by lead up to 22 days after birth in the control group. This action was less marked in the IUGR group. In myelin fractions, the Na⁺K⁺ATPase activity was inhibited by lead in both groups but the “protective effect of monoamines” was never observed. The Na⁺K⁺ ATPase activity was modulated by monoamines in synaptosomes and not in myelin, perhaps through a mechanism involving soluble factor(s).     

The inhibitory actions of acebutolol and propranolol on the contractile response to 5-hydroxytryptamine in various isolated vascular smooth muscles

Pretreatment with acebutolol or propranolol at high concentrations had an inhibitory effect on the contractile response to 5-hydroxytryptamine (5-HT) in most vascular smooth muscles such as rabbit aorta and basilar, mesenteric, renal, femoral arteries and cat coronary artery. The inhibitory actions of both agents were generally greater than on the responses to excess Ca2+ and potassium. In rabbit renal arteries, acebutolol had no effect on the response to 5-HT but inhibited the responses to excess Ca2+ and potassium. Propranolol had a marked inhibitory effect on the response to 5-HT. In all preparations used, the contractions induced by norepinephrine (NE) and histamine showed a much greater resistance to the effect of acebutolol and propranolol than the contractions induced by 5-HT, Ca2+ and potassium. Nifedipine had no inhibitory effect on the response to 5-HT in most of the preparations. Nifedipine inhibited the response to 5-HT only in the basilar arteries. The inhibitory actions of propranolol on the response to 5-HT was greater than that of acebutolol. The inhibitory action of acebutolol and propranolol on the response to 5-HT may be related to mechanisms other than the beta-adrenoceptor blocking action of the drugs. The possible mechanisms of inhibitory action of both beta-adrenoceptor antagonists on 5-HT are discussed.     

DISC ELECTROPHORETIC SEPARATION OF PROTEINS IN NEURONAL, GLIAL AND SUBCELLULAR FRACTIONS FROM CEREBRAL CORTEX

Abstract— Soluble proteins were studied in preparations from rabbit brain cortex enriched in neuronal or glial cells and in subcellular cortical fractions. Analytical polyacrylamide gels were used for acidic (pH 9-5) and basic (pH 4-3) proteins and qualitative and quantitative differences are described. The isozymes of lactic dehydrogenase, brain specific proteins and radioactive labelling patterns were used to characterize some soluble proteins.