David J. Fogarty

Activation by P2X7 agonists of two phospholipases A2 (PLA2) in ductal cells of rat submandibular gland. Coupling of the calcium-independent PLA2 with kallikrein secretion.

Isolated ductal cells of rat submandibular gland phospholipid pools were labeled with [3H]arachidonic acid (AA). The tracer was incorporated preferentially to phosphatidylcholine (46% of the lipidic fraction). Extracellular ATP induced the release of [3H]AA to the extracellular medium in a time- and dose-dependent manner (EC50 = 220 μm). Among other agents tested, only 2′,3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (Bz-ATP) was able to mimic the effect of ATP (EC50 = 15 μm), without activation of phospholipase C. The purinergic antagonists oxidized ATP, suramin, and Coomassie Blue partly inhibited the response to 1 mm ATP and 100 μmBz-ATP; the response was also blocked by the addition of Mg2+ or Ni2+. Expression of P2X7receptor mRNA in these cells was confirmed by reverse transcription-polymerase chain reaction. In the presence of extracellular calcium, the phospholipase A2 inhibitor 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (a nonspecific inhibitor), arachidonyl trifluoromethylketone (AACOCF3, an inhibitor of the calcium-dependent cytosolic PLA2(cPLA2)), and bromoenol lactone (an inhibitor of the calcium-independent PLA2 (iPLA2)) inhibited the release of [3H]AA induced by ATP and Bz-ATP. In the absence of extracellular calcium, the release of [3H]AA in response to the purinergic agonists was still observed; this response was not affected by AACOCF3 and completely blocked by bromoenol lactone. ATP and Bz-ATP stimulated a calcium-independent secretion of kallikrein, which could be blocked by BEL but which was enhanced by AACOCF3. It is concluded that the P2X7 receptor in ductal cells is coupled to kallikrein secretion through a calcium-dependent cPLA2 and a calcium-independent iPLA2.

Multiple angiotensin receptor subtypes in normal and tumor astrocytes in vitro

A role for neuropeptide receptors in glial tumorigenesis has recently been proposed. Although angiotensin receptors are known to mediate proliferative effects in many cell types, including brain astrocytes, the possible participation of these receptors in glial tumorigenesis remains unknown. In the present study, we have examined the expression of the molecularly defined angiotensin receptor subtypes AT1a, AT1b, and AT2 in normal perinatal rat astrocytes and in a panel of tumor adult astrocytoma cells, using the reverse transcriptase‐polymerase chain reaction (RT‐PCR). Subsequently, we compared the mitogenic effect of the angiotensins A(1–8), A(2–8), A(3–8) and the heptapeptide “metabolite” A(1–7), on both normal and tumor astrocytes, measured in terms of the incorporation of tritiated thymidine. Our results indicate that AT1a, AT1b, and AT2 angiotensin receptor mRNA is commonly expressed by many of these cells. Of notable exception is the astrocytoma U373 which was not found to express AT1 or AT2 mRNA. Chronic (24‐h) incubation of cells with A(1–8) and A(1–7) lead to the induction of mitogenesis, even in the AT1 and AT2 mRNA negative astrocytoma cell line U373. Moreover, pharmacological analysis indicated that the observed mitogenic effects are not mediated by the AT1 or AT2 type receptors, but rather by a novel, specific A(1–7) angiotensin receptor, since mitogenesis was shown to be partially blocked by the A(1–7) analogue D‐Ala7A(1–7) and by the protease inhibitor orthophenanthroline (100 μM). Using Fura‐2 spectrophotometry, we found that activation of this receptor does not alter intracellular calcium levels; however, preincubation with the protein kinase kinase inhibitor U0126 (10 μM) was found to inhibit these mitogenic effects partially. Overall, these results which demonstrate that normal and tumor astrocytes express a greater variety of angiotensin receptor subtypes than previously thought, support the idea that A(1–7) and its receptor signaling system may play an important role in shaping the astrocyte population during development. Moreover, the untimely expression of this A(1–7) receptor may represent an important etiological component in the development of brain astrocytomas. GLIA 39:304–313, 2002.

Angiotensin receptor-like immunoreactivity in adult brain white matter astrocytes and oligodendrocytes.

Most of the physiological effects of brain angiotensins are currently believed to be mediated by angiotensin receptors located principally on neurons. However, numerous studies in vitro have demonstrated the presence of functional angiotensin receptors on brain astrocytes, raising the possibility that glial cells may also participate in mediating the effects of the central renin–angiotensin system. Nevertheless, it is uncertain whether these cells in situ express angiotensin receptors, raising questions about the physiological significance of results observed in cell cultures. We have examined the distribution of angiotensin receptor‐like immunoreactivity in glial cells in white matter tracts in the adult CNS, using a panel of antisera to the AT1 and AT2 angiotensin receptors. Antiserum preadsorption and/or Western blot demonstrated the specificity of the antisera in brain tissue. In immunohistochemical experiments, the AT1 antisera selectively labeled AT1‐expressing neurons in the piriform cortex, whereas the AT2 antiserum stained cells in the trigeminal motor nucleus, these being nuclei known to express AT1 and AT2 receptors, respectively. Using double‐label immunohistochemistry, we observed AT1‐ and AT2‐immunoreactive astrocytes and oligodendrocytes in white matter tracts, which include the rat cerebellar white matter, periventricular white matter, and optic nerve, in addition to the bovine corpus callosum and human subcortical white matter. In contrast, astrocytes in the gray matter region of the cerebral cortex were not found to be angiotensin receptor‐like immunoreactive. These results demonstrate the presence of AT1 and/or AT2 angiotensin receptor‐like immunoreactivity in brain white matter macroglial cells in situ and support the idea that glial cells may play a more important role in the central renin‐angiotensin system than previously thought. GLIA 35:131–146, 2001.

On how altered glutamate homeostasis may contribute to demyelinating diseases of the CNS.

Glial cells communicate reciprocally with neurons in multiple ways, both in synaptic and non-synaptic regions of the central nervous system. In the latter, neuron to glial and glial to glial signals can be mediated by neurotransmitters. Here, we review the presence and some of the functional properties of glutamate transporters and receptors in oligodendrocytes. In addition, we present data illustrating that alterations in glutamate homeostasis can be excitotoxic to oligodendroglia and that the tissue lesions caused by overactivation of glutamate receptors resemble those observed in demyelinating diseases such as multiple sclerosis. Overall, this information indicates that aberrant glutamate signaling may contribute to the development of some white matter pathologies.

KA1-like kainate receptor subunit immunoreactivity in neurons and glia using a novel anti-peptide antibody

Functional kainate receptors can be formed by various combinations of subunits with low (GluR5, GluR6 and GluR7) or high affinity (KA1 and KA2) for kainate. The precise contribution of each subunit to native receptors, as well as their distribution within the central nervous system (CNS) is still unclear. Here, we describe the presence of KA1-like immunoreactivity in both neurons and glial cells of the CNS, using a newly developed antiserum to a specific carboxy terminus epitope of the KA1 subunit. Intense immunoreactivity was observed in the CA3 area of the rat hippocampus. Electron microscopy revealed that immunostaining was present in dendritic structures postsynaptic to commissural-associational fibers, rather than in those contacted by mossy fiber terminals. We also observed immunostaining of CA1 pyramidal cell apical dendrites. In the cerebral cortex, KA1-like immunostaining was observed in many pyramidal neuron somata, mainly in layer V, and along their apical dendrites. A subset of gamma-amino-butyric acidic cells were also intensely stained. In the cerebellum, the antiserum selectively stained Purkinje cell somata and their dendrites as well as Bergmann glial processes. Other types of macroglia were also labeled by the KA1 antiserum. Thus, optic nerve oligodendrocytes both in vitro and in situ and cultured astrocytes were densely stained. Our results indicate that KA1-type subunits are more widely distributed throughout the CNS than previously thought. This newly developed antiserum may help to clarify the properties of kainate receptors containing KA1 or KA1-type subunits within the normal and pathological brain.

Regulation by P2 agonists of the intracellular calcium concentration in epithelial cells freshly isolated from rat trachea.

Epithelial cells were isolated from rat trachea by incubation of the organ in a calcium-free medium. The intracellular concentration of calcium ([Ca(2+)](i)) was measured with the calcium-sensitive fluorescent dye fura2. In resting conditions, the cells maintained a low [Ca(2+)](i) in spite of the presence of millimolar concentration of calcium in the incubation medium. These cells had retained intracellular stores of calcium which were emptied after exposure of the cells to thapsigargin, an inhibitor of intracellular calcium ATPases. Substance P (125 nM) transiently increased 2.5-fold the [Ca(2+)](i). ATP (1 mM) doubled the [Ca(2+)](i) after a few seconds and further induced a sustained increase of the [Ca(2+)](i). Coomassie blue fully blocked the response to ATP and extracellular magnesium only inhibited the delayed response to ATP. Among purinergic analogs, only benzoyl-ATP (Bz-ATP), an agonist on P2X ionotropic purinergic receptors, reproduced the response to ATP. UTP and 2-methylthioATP (two agonists on P2Y metabotropic purinergic receptors) transiently increased the [Ca(2+)](i). Thapsigargin, ATP and Bz-ATP increased the uptake of extracellular calcium. RT-PCR analysis revealed that two metabotropic receptors (P2Y(1) and P2Y(2)) and two ionotropic receptors (P2X(4) and P2X(7)) were expressed by the cells present in the suspension. It is concluded that purinergic agonists can modulate the response of rat tracheal epithelial cells by several mechanisms. The activation of metabotropic receptors should mobilize intracellular IP(3)-sensitive calcium pools. The activation of the ionotropic receptors should not only open a non-specific cation channel leading to the entry of calcium but should also induce the formation of pores in cells expressing the P2X(7) receptors, which could be deleterious to these cells.

Expression and Function of Neurotransmitter Receptors in Glial Cells of the Central Nervous System

Over the last decade, it has been clearly established that glial cells express a plethora of neurotransmitter and neuropeptide receptors. However, little information is still available about the functions mediated by these glial receptors. Since the mainstream hypothesis about the roles of glia considers these cells as providing structural and trophic support to neurons, it is likely that the receptors expressed by glial cells are key mediators of these phenomena. We summarize in this chapter recent work carried out in our laboratory illustrating the expression of glutamate, angiotensin II and ATP receptors, their molecular nature and some of the putative functions mediated by them. Overall, we have observed that these receptors are expressed in a highly heterogenous manner in glial cells and that they may participate in regulating the size of the glial cell population as well as in the production of trophic support relevant to brain repair.