Parathyrin and calcitonin stimulate cyclic AMP accumulation in cultured murine brain cells.

Despite the key role Ca2+ plays in the nervous system, biochemical actions on neural tissue of the Ca2+-regulating peptide hormones parathyrin and calcitonin were unknown. Until a few years ago only neurons, but not glial cells, were considered as targets for peptide hormones. Our recent observation that peptide hormones do indeed act on glial cells is extended by the present report that these cells respond to the calcaemic peptide hormones parathyrin and calcitonin. In cultured murine brain cells mainly consisting of glioblasts, parathyrin stimulates the accumulation of cyclic AMP. The half-maximal effect is elicited at 30 nM parathyrin. With rat brain cells the effects are three times those observed with mouse brain cells. Calcitonin, which is less potent than parathyrin, elevates the concentration of cyclic AMP only in rat brain cells. If properly occupied, the inhibitory receptors present on the cells lower the increase in the level of cyclic AMP evoked by parathyrin and, to some extent, that elicited by calcitonin. The results suggest that: (i) these or closely related hormones might exert regulatory functions in brain; and (ii) glial cells must be considered in discussions of the targets of the calcaemic and other peptide hormones.     

ADENOSINE REGULATES VIA TWO DIFFERENT TYPES OF RECEPTORS, THE ACCUMULATION OF CYCLIC AMP IN CULTURED BRAIN CELLS

Abstract— In cell cultures of glial character derived from perinatal mouse brain adenosine elicits two effects. (a) At submicromolar concentrations It inhibits the increase in the intracellular level of cyclic AMP caused by β-adrenoceptor agonists. (b) At concentrations above micromolar it increases the level of cyclic AMP in the cultures. These two effects are mediated by two different adenosine receptors present on the outer surface of the cells. This is concluded from the following evidence. (a) Both effects are antagonized by methylxanthines but not by blockage of adenosine uptake or inhibition of phosphodiesterase activity. (b) In both cases activity depends on the integrity of the ribose moiety of the nucleotide. Substituents of the purine system are tolerated comparatively well. (c) The order of potency of adenosine analogues is different for the two effects. We suggest the name A1 receptors for those that mediate the inhibition and A2 for those that mediate the stimulation of cyclic AMP accumulation.     

Expression and signaling of group I metabotropic glutamate receptors in astrocytes and microglia

Stimulation of astrocytes with the excitatory neurotransmitter glutamate leads to the formation of inositol 1,4,5-trisphosphate and the subsequent increase of intracellular calcium content. Astrocytes express both ionotropic receptors and metabotropic glutamate (mGlu) receptors, of which mGlu5 receptors are probably involved in glutamate-induced calcium signaling. The mGlu5 receptor occurs as two splice variants, mGlu5a and mGlu5b, but it was hitherto unknown which splice variant is responsible for the glutamate-induced effects in astrocytes. We report here that both mRNAs encoding mGlu5 receptor splice variants are expressed by cultured astrocytes. The expression of mGlu5a receptor mRNA is much stronger than that of mGlu5b receptor mRNA in these cells. In situ hybridization experiments reveal neuronal expression of mGlu5b receptor mRNA in adult rat forebrain but a strong neuronal expression of mGlu5a mRNA only in olfactory bulb. Signals for mGlu5a receptor mRNA in the rest of the brain were diffuse and weak but consistently above background. Activation of mGlu5 receptors in astrocytes yields increases in inositol phosphate production and transient calcium responses. It is surprising that the rank order of agonist potency [quisqualate > (2S,1 'S,2'S)-2-(carboxycyclopropyl)glycine = trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (1S,3R-ACPD) > glutamate] differs from that reported for recombinantly expressed mGlu5a receptors. The expression of mGlu5a receptor mRNA and the occurrence of 1S,3R-ACPD-induced calcium signaling were found also in cultured microglia, indicating for the first time expression of mGlu5a receptors in these macrophage-like cells.     

IL-6 expression induced by adenosine A2b receptor stimulation in U373 MG cells depends on p38 mitogen activated kinase and protein kinase C

Adenosine binds to a class of G-protein coupled receptors, which are further distinguished as A(1), A(2a), A(2b) and A(3) adenosine receptors. As we have shown earlier, the stable adenosine analogue NECA (N6-(R)-phenylisopropyladenosine) stimulates IL-6 expression in the human astrocytoma cell line U373 MG via the A(2b) receptor. The mechanism by which NECA promotes astrocytic IL-6 expression has not been identified. By using various inhibitors of signal transduction, we found that p38 mitogen-activated protein kinases (MAPK) activation (inhibitor SB202190), but not extracellular signal-regulated kinase (ERK) (PD98059) and c-jun N-terminal kinase (JNK)(SP600125), is essential in the NECA-induced signalling cascade that leads to the increase in IL-6 synthesis in U373 MG cells. Results obtained with protein kinase C (PKC) inhibitors that have different substrate specificities, indicated that the PKC delta and epsilon isoforms are also involved in adenosine receptor A(2b) dependent upregulation of IL-6 expression. This is supported by the fact that NECA induced the activation of PKC delta and epsilon in U373 MG cells.     

Stimulation by Bradykinin, Angiotensin II, and Carbachol of the Accumulation of Inositol Phosphates in PC-12 Pheochromocytoma Cells: Differential Effects of Lithium Ions on Inositol Mono-and Polyphosphates

Rat PC-12 pheochromocytoma cells respond to stimulation with bradykinin, angiotensin II, and carbachol with an increased formation of labeled inositol phosphates after preincubation of the cells with [3H]inositol. Li+ potentiates greatly the agonist-induced increase in amount of inositol mono-, bis-, and trisphosphate but not the increase in amount of inositol tetrakisphosphate. Separation of the isomers of inositol trisphosphate shows that the lithium-induced increase in amount of inositol trisphosphate is due to potentiation evoked by lithium of the accumulation of inositol-1,3,4-trisphosphate.     

Polyploid rat glioma cells. Production, oscillations of membrane potential and response to neurohormones

Due to their small size, electrophysiological investigation of C6 rat glioma cells by intracellular recording with microelectrodes is very difficult. In order to facilitate such electrophysiological studies, the degree of ploidy of C6 cells was increased in three successive steps. At each step complementary drug-resistant mutants of C6 were fused and hybrid cell clones isolated in selective culture medium. Cell volume was measured by electronic cell sizing and DNA content per cell assessed by impulse cytofluorimetry. In the final polyploid line, C6-4-2, both parameters were found to be increased 6-fold in comparison with the original cell line C6. In response to β-adrenergic agonists and prostaglandin E₁ the intracellular concentration of cAMP was raised in C6-4-2 cells. Stable membrane potentials could be recorded from the polyploid glioma cells much more easily than from the original C6 cells. The plasma membrane of the C6-4-2 cells could hyperpolarize spontaneously with a frequency of about 1 cycle/min. Elevation of the concentration of Ca²⁺ ions (but not other earth alkali ions) in the medium induced the onset of the spontaneous hyperpolarizations. The membranes of the C6-4-2 cells also hyperpolarized in response to acetylcholine, γ-aminobutyric acid (GABA) and glutamate.     

Local stimulation of the adenosine A2B receptors induces an increased release of IL-6 in mouse striatum: an in vivo microdialysis study

Both adenosine and interleukin-6 (IL-6) have been implicated in the pathophysiology of, e.g., epileptic seizures, traumatic brain injury, and affective disorders. Stimulation of adenosine A_2B receptors on astrocytes in vitro leads to the increased synthesis and secretion of IL-6. We investigated whether or not activation of adenosine receptors evokes an increase of IL-6 release also in vivo . 5'- N -ethylcarboxamidoadenosine, a non-specific adenosine-agonist or vehicle was administered into the striatum of freely moving mice by reverse microdialysis. A statistical significant increase of the IL-6 concentration in the perfusate was detected already 60 min after 5'- N -ethylcarboxamidoadenosine administration. IL-6 increased progressively and reached a maximum after 240 min. This effect appears to be mediated through adenosine A_2B receptors since it was counteracted by the specific A_2B receptor antagonist MRS1706 but not by the specific A₁ receptor antagonist DPCPX. We conclude that adenosine via activation of A_2B receptors evokes IL-6 release also in vivo .     

Corticotropin Peptides and Melanotropins Elevate the Level of Adenosine 3'': 5''-Cyclic Monophosphate in Cultured Murine Brain Cells

Cell cultures derived from mouse and rat brain and consisting mainly of astroblasts are known to respond to several hormones by increasing or decreasing their intracellular concentration of cyclic AMP. In the present study these cultures were analyzed for their susceptibility to various additional hormonal and other neuroactive compounds. Only the peptides of the corticotropin (ACTH)/melanotropin (MSH) family were found active. Their potency for elevating the intracellular level of cyclic AMP decreases in the sequence (values for the half-maximally stimulating concentrations, EC50, in parentheses) ACTH-(1-24) (10 m) greater than alpha-,beta-MSH (30 nm) greater than ACTH (greater than or equal to 100 nm) gamma-MSH, ACTH-(1-10), -(4-10), -(4-11) (greater than or equal to 0.5 microM). The lack of additivity of the maximal effects of the peptides suggests that they all act at the same receptor. The stimulation exerted by these peptides is partially suppressed by hormones known to inhibit cyclic AMP formation in that culture, i.e., noradrenaline (acting via an alpha-adrenergic receptor), adenosine (acting via an A1 receptor), and somatostatin. It is concluded that the receptors for the ACTH/MSH peptides and the inhibitory hormones are located on the same cells, presumably the astroblasts. The maximal response to ACTH and alpha- and beta-MSH depends strongly on the age of culture. The results are discussed in view of the facts that (1) peptides of the ACTH/MSH family affect behavior and learning in animals, and (2) ACTH and alpha-MSH occur in brain.