Regulation of H1-receptor coupling and H1-receptor mRNA by histamine in bovine tracheal smooth muscle

1. Pretreatment of bovine tracheal smooth muscle (BTSM) with histamine (1-100 microM, 1 h) induced a concentration-dependent desensitization of the contractile response to subsequently administered histamine, with a reduction of the maximum response of 72 +/- 8% (n = 5) following pre-exposure to 100 microM histamine. In contrast, concentration-response curves to the muscarinic agonist, methacholine were not affected following histamine pretreatment, indicating a homologous desensitization. Furthermore, concentration-response curves to NaF, a G-protein activator, were not altered following histamine pre-incubation. 2. The histamine H1-receptor (H1R) desensitization could be antagonized by mepyramine (an H1-receptor antagonist, 1 microM) but not by cimetidine (an H2-receptor antagonist, 10 microM), indicating that the desensitization occurred via stimulation of histamine H1-receptors, without evidence for the involvement of histamine H2-receptors. 3. Indomethacin (10 microM) did not block the H1R desensitization, suggesting no involvement of prostaglandins. Furthermore, histamine pre-incubation in calcium free medium still induced a functional uncoupling of H1R. 4. GF 109203X, a protein kinase C (PKC) inhibitor, and H-7, a non-selective kinase inhibitor, did not antagonize the homologous H1R desensitization. 5. The steady-state level of H1R mRNA, assessed by Northern blot analysis, was not affected by prolonged histamine exposure (100 microM, 0.5, 1, 2, 4, 16 and 24 h). 6. These results suggest that histamine induces desensitization of the H1R at the level of the receptor protein, which involves a mechanism independent of PKC, PKA, PKG and calcium influx, suggesting the involvement of a receptor-specific kinase.     

Receptor-mediated desensitisation of histamine H1 receptor-stimulated inositol phosphate production and calcium mobilisation in GT1-7 neuronal cells is independent of protein kinase C

GT1-7 cells, a clonal line derived from specific tumours of gonadotropin-releasing hormone-secreting neurons from mouse hypothalamus, were used as a model system to investigate the cellular mechanisms underlying the histamine H1 receptor-mediated desensitisation. GT1-7 cells contain H1 receptors, acute stimulation of which leads to the desensitisation of histamine-mediated calcium mobilisation and is manifest as a concurrent reduction in both the magnitude of the calcium transient and of the sustained phase. Acute pretreatment of the cells with the phorbol ester, phorbol 12-myristate 13-acetate, can also ablate the histamine-stimulated calcium mobilisation. In addition, acute H1-receptor stimulation and acute phorbol ester treatment result in the attenuation of histamine-mediated inositol phosphate production. Receptor desensitisation resulting from acute stimulation with histamine is not affected by inhibiting protein kinase C (PKC) activity with Ro 31-7549 or staurosporine. In contrast, the desensitisation of H1-receptor responses induced by direct activation of protein kinase C is preventable by PKC inhibitors. Thus, these results imply that a PKC-dependent mechanism and PKC-independent mechanism are involved in the H1-receptor desensitisation cascade in GT1-7 cells and do not support the involvement of PKC in the receptor-mediated desensitisation of H1 receptor-stimulated calcium and inositol phosphate responses.     

Neurochemical and behavioral effects of ciproxifan, a potent histamine H3-receptor antagonist

Ciproxifan, i.e., cyclopropyl-(4-(3-1H-imidazol-4-yl)propyloxy) phenyl) ketone, belongs to a novel chemical series of histamine H3-receptor antagonists. In vitro, it behaved as a competitive antagonist at the H3 autoreceptor controlling [3H]histamine release from synaptosomes and displayed similar Ki values (0.5-1.9 nM) at the H3 receptor controlling the electrically-induced contraction of guinea pig ileum or at the brain H3 receptor labeled with [125I]iodoproxyfan. Ciproxifan displayed at least 3-orders of magnitude lower potency at various aminergic receptors studied in functional or binding tests. In vivo, measurement of drug plasma levels, using a novel radioreceptor assay in mice receiving ciproxifan p.o. or i.v., led to an oral bioavailability ratio of 62%. Oral administration of ciproxifan to mice enhanced by approximately 100% histamine turnover rate and steady state level of tele-methylhistamine with an ED50 of 0.14 mg/kg. Ciproxifan reversed the H3-receptor agonist induced enhancement of water consumption in rats with and ID50 of 0.09 +/- 0.04 mg/kg, i.p. In cats, ciproxifan (0.15-2 mg/kg, p.o.) induced marked signs of neocortical electroencephalogram activation manifested by enhanced fast-rhythms density and an almost total waking state. In rats, ciproxifan enhanced attention as evaluated in the five-choice task performed using a short stimulus duration. Ciproxifan appears to be an orally bioavailable, extremely potent and selective H3-receptor antagonist whose vigilance- and attention-promoting effects are promising for therapeutic applications in aging disorders.     

The protein kinase C inhibitors Ro 318220 and GF 109203X are equally potent inhibitors of MAPKAP kinase-1beta (Rsk-2) and p70 S6 kinase

The protein kinase C (PKC) inhibitors Ro 318220 and GF 109203X have been used in over 350 published studies to investigate the physiological roles of PKC. Here we demonstrate that these inhibitors are not selective for PKC isoforms as was previously assumed. Ro 318220 inhibited MAPKAP kinase-1beta (also known as Rsk-2) in vitro (IC50 3nM) more potently than it inhibited mixed PKC isoforms (IC50 5 nM), and it also inhibited p70 S6 kinase (IC50 15 nM). GF 109203X also potently inhibited MAPKAP kinase-1beta (IC50 50 nM) and p70 S6 kinase (IC50 100 nM) with similar potency to PKC isoforms (IC50 30 nM). The inhibition of MAPKAP kinase-1beta, p70 S6 kinase, and probably other protein kinases, may explain many of the effects previously attributed to PKC.     

N alpha-alkylated derivatives of 2-phenylhistamines: synthesis and in vitro activity of potent histamine H1-receptor agonists

New potent N alpha-alkylated histamine H1-receptor agonists have been prepared and functionally evaluated for partial agonist potency and selectivity. N alpha-Methyl-2-(3-trifluoromethylphenyl)histamine contracts ileal segments and aortic rings of guinea-pig with a relative potency of 174% (95% confid. lim. 161-188%) and 217% (164-287%), respectively (histamine: 100%) and is the most potent H1 receptor agonist described so far.     

Modulation of pentagastrin-induced histamine release by histamine H3 receptors in the dog

BACKGROUND: The histamine H3 receptor has been shown to inhibit pentagastrin-induced gastric acid secretion in dogs. Since pentagastrin releases histamine in dogs, we have now assessed whether the effects of H3-receptor ligands may be indirectly mediated by changes in gastric histamine release. METHODS: Pentagastrin infusions (1 or 6 micrograms/kg/h), alone or together with the H3-receptor agonist (R) alpha-methylhistamine (1.2 mumol/kg/h) or the antagonist thioperamide (0.1 mumol/kg/h), were performed in dogs. One group (anaesthetized) was used for enzyme immunoassays of plasma histamine and, when required. (R) alpha-methylhistamine in the gastrosplenic vein, and another group (non-anaesthetized) for measurement of gastric acid secretion. RESULTS: Histamine levels were increased five- and eight-fold after 1 and 6 micrograms/kg/h pentagastrin, respectively, whereas acid output was nearly maximal at the lower dosage. (R) alpha-methylhistamine, at a plasma concentration of 0.15 microM, inhibited histamine release by 78% (P < 0.007) and 37% (not significant) and the total acid output by 44% (P < 0.05) and 19% (not significant) after infusion of 1 and 6 micrograms/kg/h pentagastrin, respectively. Thioperamide, together with pentagastrin in low dose, significantly increased histamine release by 212% (P < 0.05), whereas acid output increased by 34% (not significant). CONCLUSIONS: The histamine H3 receptor mediates a negative feedback control of pentagastrin-induced release of gastric histamine. It is tonically activated by endogenous histamine after pentagastrin in low dosage. The control of acid secretion by the H3 receptor seems to involve modulation of endogenous histamine release, possibly by means of enterochromaffin-like cells.     

Histamine H1- and H2-receptor vasodilation of canine intestinal circulation

Studies were conducted in anesthetized dogs to determine whether the mesenteric vasodilator response to histamine is mediated by H1 receptors alone or whether H2 receptors are also involved in the response. Evidence favoring a role for both receptors included: 1) the vasodilator response to histamine was inhibited by either the H1-receptor antagonist, tripelennamine, or the H2-receptor antagonist, metiamide; 2) both the H1 agonist, 2-methylhistamine, and the H2 agonist, 4-methylhistamine, induced dilator responses in the mesenteric circulation; and 3) two temporal patterns of vasodilation could be distinguished, namely a transient spike and subsequent fade of blood flow (seen with either the H1 agonist or with histamine after H2-receptor blockade) and a sustained and stable increase in flow (seen with either the H2 agonist or with histamine after H1 blockade). Metiamide appeared to be a potent inhibitor of the mesenteric vasodilator response to histamine at least equal to tripelennamine.     

Theophylline derivatives as potential histamine H3-receptor antagonists

Previous results of histamine H3-receptors investigations allowed to formulate a general structure of H3-receptor antagonists. According to this model a series of compounds were obtained. As heterocycles they contained a theophylline moiety connected with a polar group (amine, ester, amide, and thiourea function) via an alkyl chain linked by a spacer to a lipophilic residue. The common distance between xanthine moiety and lipophilic rest was a six-link-chain. Selected compounds did not show significant H3-receptor antagonist activity and were weak antagonists at histamine H1-receptors.     

Role of PKC in the effects of alpha1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

The effects of alpha1-adrenoceptor stimulation on intracellular Ca2+ transients, contractility and L-type Ca2+ current (ICa,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanisms of the positive inotropic effect of alpha1-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an alpha1-adrenergic agonist, at concentrations of 50-100 microM elicited a biphasic inotropic response: a transient negative inotropic response (22.9+/-6.0% of control) followed by a sustained positive inotropic response (61.0+/-8.4%, mean+/-SE, n=12). The Ca2+ transient decreased by 10.2+/-3.9% during the negative inotropic phase, while it increased by 67.7+/-10% (n=12) during the positive inotropic phase. These effects were inhibited by prazosin (1 microM), a alpha1-adrenergic antagonist. Phenylephrine increased the ICa,L by 60.8+/-21% (n=5) during the positive inotropic phase. To determine whether activation of PKC is responsible for the increases in Ca2+ transients, contractile amplitude and ICa,L during alpha1-adrenoceptor stimulation, we tested the effects of 4beta-phorbol 12-myristate 13-acetate (PMA), a PKC activator, and of bisindolylmaleimide I (GF109203X) and staurosporine, both of which are PKC inhibitors. PMA mimicked phenylephrine's effects on Ca2+ transients, contractile amplitude and ICa,L. PMA (100 nM) increased the Ca2+ transient, contractile amplitude and ICa,L by 131+/-17%, 137+/-25% (n=8), and 81.1+/-26% (n=5), respectively. Prior exposure to GF109203X (1 microM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca2+ transients, contractile amplitude and ICa,L. Our study suggests that during alpha1-adrenoceptor stimulation increase in ICa,L via PKC causes an increase in Ca2+ transients and thereby in the contractile force of the ventricular myocytes.     

Long-term increase of hippocampal excitability by histamine and cyclic AMP

The action of histamine (HA) on rat hippocampal CA1 pyramidal cells in vitro was investigated in slices perfused with solution containing 0.2 mM Ca2+/4.0 mM Mg2+. Extracellular recordings of the spontaneous discharges occurring under these conditions revealed that HA caused a long-lasting increase in cell firing. The HA-effects were dose-dependent, in that low concentrations of HA (0.1-0.5 microM) exhibited an initial transient depression of cell firing and practically no long-lasting action, whereas higher concentrations of HA (1-10 microM) exerted strong, non-declining increases. The H1-receptor antagonist mepyramine (1 microM) blocked the initial depression of firing and attenuated the long-lasting HA-mediated excitation. Pure H1-receptor activation, tested with the H1-receptor agonist 2-(3-fluorphenyl)histamine (1-10 microM) depressed cell firing, similar to the low dose effects of HA. HA-induced excitations were prevented by the H2-receptor antagonist cimetidine (10-50 microM), and mimicked by the very potent H2-receptor agonist impromidine (1 or 3 microM) which was, however, less effective compared to equal concentrations of HA. H3-receptor activation by R-alpha-methylhistamine had no significant effect on cell firing. Thus, histamine H1 and H2 receptors seem to cooperate in producing this long-lasting augmentation of excitability. 8-Bromo-cyclic AMP monophosphate (8-Br-cAMP, 50-100 microM) mimicked the long-term excitation, whereas the adenylyl-cyclase inhibitor 9-tetrahydro-2-furyladenine (THFA, 100-500 microM) or the PKA-inhibitor Rp-adenosine-3'5'-cyclic monophosphate (Rp-cAMPS, 10 microM) blocked it, indicating that the HA-mediated increase of excitability in the hippocampus is dependent on the adenylate cyclase/PKA-signal transduction cascade. DL-2-Amino-5-phosphonopentanoic acid (APV, 50 microM) significantly attenuated the magnitude of the HA-induced enhancement, indicating an NMDA receptor-dependent component. Other biogenic amines, acting through receptors positively coupled to adenylyl cyclase, elicited similar responses as HA, indicating common mechanisms by which these substances modulate excitability in CA1 pyramidal cells.     

Homologous desensitization of bombesin-induced increases in intracellular Ca2+ in quiescent Swiss 3T3 cells involves a protein kinase C-independent mechanism

Addition of bombesin to Swiss 3T3 cells causes a rapid and transient increase in the intracellular concentration of Ca2+ ([Ca2+]i), which is followed by desensitization to a subsequent addition of the peptide. The concentrations of bombesin used to study this acute cellular desensitization (0.1-0.5 nM) did not deplete the intracellular pool of Ca2+ released by inositol(1,4,5)trisphosphate, as shown by addition of vasopressin after consecutive additions of bombesin. Two lines of evidence support the conclusion that activation of protein kinase C (PKC) does not mediate the acute homologous desensitization of Ca2+ responses induced by bombesin. First, long-term treatment (48 h) of Swiss 3T3 cells with phorbol 12,13-dibutyrate (PDB) to deplete PKC did not prevent homologous desensitization. The responses to second additions of bombesin at 0.1, 0.25, and 0.5 nM were 42%, 26% and 11% of the initial responses, respectively. Second, the PKC inhibitor GF 109203X did not alter homologous desensitization at concentrations that completely prevented the inhibition of Ca2+ mobilization induced by PDB and blocked PDB-mediated phosphorylation of the prominent PKC substrate 80K/MARCKS. We conclude that acute homologous desensitization of Ca2+ responses induced by bombesin occurs through a PKC-independent mechanism.     

The role of histamine H1, H2 and H3 receptors on enteric ascending synaptic transmission in the guinea pig ileum

The role of histamine H1-, H2- and H3-receptors was studied on neural transmission in ascending excitatory pathways of the guinea pig ileum. A two-compartment (oral and anal compartments) bath was used: ascending neural pathways were activated by electrical stimulation in the anal compartment and the resulting contraction of the circular muscle in the oral compartment was recorded. Drugs were applied in the anal compartment and each agonist was evaluated in the presence of the antagonists of the other two receptors. In the presence of cimetidine (10 microM) and thioperamide (1 microM), histamine (0.03-3 microM) depressed the nerve-mediated contractions (5-70% inhibition, P <.05-.01). The inhibitory effect of histamine was antagonized by mepyramine. At the higher concentrations (10 and 30 microM), histamine elicited contractions of the circular muscle in the oral compartment, and these were abolished by mepyramine (1 microM) and tetrodotoxin (0.6 microM). The H2 agonists dimaprit (30 and 100 microM) and amphamine (0.1-300 microM) produced small contractions of the circular muscle in the oral compartment. These contractile responses were abolished by tetrodotoxin (0.6 microM) and cimetidine (10 microM). The H3 agonist R-alpha-methylhistamine (0.001-1 microM) inhibited (2-58%, P <.05) the nerve-mediated contractions. This inhibitory effect was antagonized by the H3 antagonist thioperamide. These results indicate that 1) histamine, acting at H1 receptors, at lower concentrations depresses synaptic transmission, although at higher concentrations activates the enteric excitatory ascending pathway; 2) activation of H2 receptors by H2 agonists stimulates the enteric excitatory ascending pathways and 3) activation of H3 receptors inhibits synaptic transmission.     

The muscarinic M1 agonist xanomeline increases soluble amyloid precursor protein release from Chinese hamster ovary-m1 cells

The functionally selective M1 agonist xanomeline, which is currently undergoing clinical trials as a therapy for Alzheimer's disease, was compared to the muscarinic agonist carbachol for effects on secretion of soluble amyloid precursor protein (APPs) from Chinese hamster ovary cells transfected with the human m1 receptor (CHO-m1). Release of APPs from CHO-m1 cells was increased maximally (4-10 fold) by 100 microM carbachol (EC50 = 11 microM) and by 100 nM xanomeline (EC50 = 10 nM). Stimulation of APPs secretion by xanomeline and carbachol was blocked by preincubation with 1 microM atropine. Carbachol did not stimulate APPs secretion from non-transfected CHO cells. Pilocarpine at 1 mM also increased APPs release. The efficacy of carbachol, xanomeline and pilocarpine for stimulating APPs secretion did not differ significantly. Activation of protein kinase C (PKC) in m1 transfected cell lines by 1 microM phorbol dibutyrate (PDBu) increased APPs release, and this was inhibited 97% by the PKC inhibitor bisindolemalemide. The PKC inhibitor decreased xanomeline and carbachol-stimulated APPs secretion by only 25-30%. These results demonstrate that xanomeline increased APPs release by activation of m1 muscarinic receptors and support the possibility that cholinergic replacement therapy for Alzheimer's Disease may reduce amyloid deposition.     

Histamine, histamine antagonists and regional blood flow

The effects of mepyramine, a selective histamine H1-receptor antagonist, and metiamide, a selective histamine H2-receptor antagonist, have been determined on the cardiovascular response to intravenous infusions of a large dose of histamine, 1 X 10(-7) mol/kg/min. Pretreatment with either mepyramine, 2.5 X 10(-6) mol/kg, or metiamide, 2 X 10(-6) mol/kg/min, had no significant effect on the fall in blood pressure or fall in total peripheral resistance during histamine infusion. Pretreatment with mepyramine, 2.5 X 10(-6) mol/kg, and metiamide, 2 X 10(-6) mol/kg/min, abolished the fall in blood pressure and total peripheral resistance during infusion of histamine. Histamine-induced vasodilatation in the stomach was unaltered by pretreatment with mepyramine but abolished by pretreatment with metiamide. Histamine-induced coronary vasodilation was reduced slightly by pretreatment with mepyramine and substantially by pretreatment with metiamide. Coronary vasodilatation was abolished by treatment with mepyramine and metiamide.     

Cellular relocalisation of protein kinase C-theta caused by staurosporine and some of its analogues

The microbial product staurosporine is a protein kinase C (PKC) inhibitor with some phorbol ester-agonistic properties. It is known to cause the translocation of the PKC isoenzymes epsilon and delta from the cellular cytosol to the membrane and nucleus. We tested the hypothesis that it also affects the cellular localisation of the novel PKC isoenzyme theta, and that staurosporine analogues, some of which are currently under clinical evaluation as potential anticancer drugs, have a similar effect. Their ability to alter PKC-theta distribution was studied in human-derived A549 lung carcinoma cells. Western blot analysis and confocal microscopy after indirect immunofluorescence staining showed that staurosporine (100 nM), like the phorbol ester 12-O-tetradecanoylphorhol-13-acetate (25 nM) caused the translocation of PKC-theta from the cytosol to the membrane and the nucleus. The bisindolylmaleimide GF 109203X mimicked staurosporine, but had a weaker effect. Ro 31-8220 and UCN-01 decreased cytosolic PKC-theta only at 1 microM. CGP 41251 had no effect on PKC-theta in either experimental design. The results show that some, but not all, staurosporine analogues share the partial phorbol ester-agonistic PKC-translocatory activity of the parent molecule.     

Protein kinase C activation potentiates the rapid secretion of the amyloid precursor protein from rat cortical synaptosomes

In this study we have used the presynaptic-rich rat cerebrocortical synaptosomal preparation to investigate the proteolytic cleavage of the amyloid precursor protein (AbetaPP) by the alpha-secretase pathway within the betaA4 domain to generate a soluble secreted N-terminal fragment (AbetaPPs). AbetaPP was detected in crude cortical synaptosomal membranes, although at a lower density than that observed in whole-tissue homogenates. Protein kinase C (PKC) activation induced a translocation of the conventional PKC isoform beta1 and novel PKCepsilon from cytosol to membrane fractions, but there was no alteration in the proportion of AbetaPP associated with the Triton-soluble and -insoluble fractions. AbetaPPs was constitutively secreted from cortical synaptosomes, with this secretion being enhanced significantly by the direct activation of PKC with phorbol ester. The PKC-induced secretion of AbetaPPs was only partially blocked by the PKC inhibitor GF109203X (2.5 microM), whereas the phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein was significantly inhibited by GF109203X. The differential sensitivities of the MARCKS phosphorylation and AbetaPPs secretion to GF109203X may imply that different PKC isoforms are involved in these two events in the synaptosomal system. These findings strongly suggest that the alpha-secretase activity leading to the secretion of AbetaPPs can occur at the level of the presynaptic terminal.     

Inhibition by dexamethasone of antigen-induced c-Jun N-terminal kinase activation in rat basophilic leukemia cells

Antigen stimulation of IgE-sensitized rat basophilic leukemia RBL-2H3 cells induced activation of c-Jun N-terminal kinase (JNK) within a few minutes with maximum activity attained 40 min later. The increase in JNK activity was accompanied with an increase in phosphorylation of c-Jun in the cells. The Ag-induced JNK activation was inhibited by the phosphatidylinositol 3-kinase inhibitors wortmannin (10-100 nM) and LY 294002 (100 microM) but not by the protein kinase C inhibitors calphostin C (1 and 3 microM) and Ro 31-8425 (1 and 3 microM). Pretreatment with dexamethasone (10 and 100 nM) for 18 h inhibited the Ag-induced increase in JNK activity in a concentration-dependent manner. At least 6 h of preincubation with dexamethasone was necessary to inhibit the Ag-induced JNK activation. The phosphorylation of c-Jun induced by the Ag stimulation was reduced by pretreatment with dexamethasone without reduction of the content of c-Jun protein. The Ag-induced activation of the JNK kinase kinase mitogen-activated protein kinase-extracellular signal-regulated kinase kinase-1 was also inhibited by pretreatment with dexamethasone at 10 and 100 nM. These findings indicate that dexamethasone reduces JNK protein level and inhibits the Ag-induced activation of JNK resulting in the inhibition of c-Jun phosphorylation.     

Postnatal expression of H1-receptor mRNA in the rat brain: correlation to L-histidine decarboxylase expression and local upregulation in limbic seizures

Histamine is implicated in the regulation of brain functions through three distinct receptors. Endogenous histamine in the brain is derived from mast cells and neurons, but the importance of these two pools during early postnatal development is still unknown. The expression of histamine H1-receptor in the rat brain was examined using in situ hybridization during postnatal development and in adults. For comparison, the expression of L-histidine decarboxylase (HDC) in the two pools was revealed. H1-receptor was evenly expressed throughout the brain on the first postnatal days, but resembled the adult, uneven pattern already on postnatal day 5 (P5). HDC was expressed in both mast cells and tuberomammillary neurons from birth until P5, after which the mast cell expression was no more detectable. In adult rat brain, high or moderate levels of H1-receptor expression were found in the hippocampus, zona incerta, medial amygdaloid nucleus and reticular thalamic nucleus. In most areas of the adult brain the expression of H1-receptor mRNA correlates well with binding data and histaminergic innervation. A notable exception is the hypothalamus, with high fibre density but moderate or low H1-receptor expression. Systemic kainic acid administration induced increased expression of H1-receptor mRNA in the caudate-putamen and dentate gyrus, whereas no change was seen in the hippocampal subfields CA1-CA3 or in the entorhinal cortex 6 h after kainic acid injections. This significant increase supports the concept that histaminergic transmission, through H1-receptor, is involved in the regulation of seizure activity in the brain.     

Useful applications and limits of battery powered implants in functional electrical stimulations

Noradrenaline (NA) (1-10 microM), dibutyryl-cAMP (1-5 mM), and forskolin (10-20 microM) increased cytosolic Ca2+ concentration ([Ca2+]i) in isolated arginine-vasopressin (AVP)-containing neurons in the hypothalamic supraoptic nucleus (SON). The NA-induced increase in [Ca2+]i in AVP-containing neurons was abolished by a specific alpha1-antagonist, prazosin (1 microM) and was markedly reduced when treated with a protein kinase A (PKA) blocker, H89 (40 microM). The NA-induced [Ca2+]i was not altered by a protein kinase C (PKC) inhibitor, calphostin C (0.1 microM) and a PKC activator, TPA (100 nM). In general, NA, a known neurotransmitter in the SON, activates AVP-containing neurons via alpha1-receptor which is linked to stimulation of cAMP-PKA-regulated Ca2+ signaling pathway.     

PKC and tyrosine kinase involvement in amyloid beta (25-35)-induced chemotaxis of microglia

Microglia are activated by amyloid beta (Abeta) in vivo and in vitro, and Abeta-activated microglia may be involved in the pathogenesis of Alzheimer's disease (AD). We investigated the mechanism of microglial chemotaxis induced by Abeta (25-35), an active fragment of Abeta. Abeta (25-35) 0.1 and 1 nM stimulated microglial chemotaxis. The protein kinase C (PKC) inhibitors chelerythrine (0.5 and 2 microM), calphostin C (1 microM) and staurospine (10 nM) significantly inhibited the microglial chemotaxis induced by Abeta (25-35) (1 nM). The chemotactic effect of Abeta (25-35) on microglia was desensitized by pretreatment of microglia with 1 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA). Pretreatment of cells with Abeta (25-35) (1 nM) also desensitized the chemotactic effect by Abeta (25-35) (1 nM). The desensitization by TPA or Abeta (25-35) was inhibited when staurosporine was present in the pretreatment media. The tyrosine kinase inhibitor herbimycin A (0.1 and 1 microM) significantly inhibited the microglial chemotaxis induced by Abeta (25-35) (1 nM). Based on these observations, it seems likely that PKC and tyrosine kinase are involved in the Abeta-induced chemotaxis of microglia.