Michael K. McMillian

Endogenous apolipoprotein E suppresses LPS-stimulated microglial nitric oxide production

THE human apolipoprotein (apo) E4 isoform is associated with an increased risk for Alzheimers disease (AD) and poor prognosis after acute CNS injury. Addition of human apoE inhibits murine microglial activation in culture, suggesting that microglia might be an important physiological target of apoE. In the present study, we examined the role of endogenous murine apoE in modulating microglial nitric oxide (NO) production following lipopolysaccharide (LPS) stimulation. Brain cultures from apoE-deficient mouse pups showed enhanced NO production relative to cultures from wild-type mice and from transgenic mice expressing the human apoE3 isoform, demonstrating that endogenous apoE produced by glial cultures is capable of inhibiting microglial function. ApoE produced within the brain may suppress microglial reactivity and thus alter the CNS response to acute and chronic injury.

Apolipoprotein E and mimetic peptide initiate a calcium-dependent signaling response in macrophages

Apolipoprotein E (ApoE) is a 34‐kDa cholesterol transport protein that also possesses immunomodulatory properties. In this study, we demonstrate that ApoE initiates a signaling cascade in murine peritoneal macrophages that leads to increased production of inositol triphosphate with mobilization of intracellular Ca2+ stores. This cascade is inhibited by pretreatment with receptor‐associated protein and Ni2+, and it is mediated by a pertussis toxin‐sensitive G protein. These properties are characteristic of signal transduction induced via ligand binding to the cellular receptor, lipoprotein receptor‐related protein. A peptide derived from the receptor‐binding region of ApoE also initiates signal transduction in a manner similar to that of the intact protein, suggesting that this isolated region is sufficient for signal transduction. The ApoE‐mimetic peptide competed for binding with the intact protein, confirming that they both interact with the same site. ApoE‐dependent signal transduction might play a role in mediating the functional properties of this lipoprotein.

Two distinct cytosolic calcium responses to extracellular ATP in rat parotid acinar cells.

1 Increasing concentrations of ATP (0.5 μm‐300 μm) produced a biphasic increase in intracellular calcium concentration [Ca]i in rat parotid acinar cells, reflecting two distinct Cai responses to extracellular ATP. 2 In the absence of Mg2+ (with 3 mm CaCl2 in the buffer solution), the more sensitive response was maximal at 3–5 μm and was not further increased by 30 μm ATP. This response to ATP was not well maintained and was blocked by ADP (0.5 mm). A second, much larger increase in Cai was observed on addition of 300 μm ATP. This larger effect, which we have described previously, appears to be mediated by ATP4–, and was selectively reversed by 4,4′‐di‐isothiocyanato‐dihydrostilbene‐2,2′‐disulphonate as well as by high concentrations of α,β‐methylene ATP. 3 Among ATP analogues, only the putative P2Z agonist, 3′‐0‐(4‐benzoyl)benzoyl‐ATP distinguished between the two responses. This analogue was at least 10 fold more potent than ATP in stimulating the ATP4–‐response, but did not evoke the more sensitive response. The agonist potency series for both responses to ATP was identical for other analogues examined (ATP>ATP‐γS = 2‐methylthio ATP (a P2y‐selective agonist) 72>ADP, ITP and α,β‐methylene ATP (a P2X‐selective agonist)). 4 Although the effect of ATP4– could best be characterized as a P2Z‐type purinoceptor response, this effect was strongly and selectively blocked by reactive blue 2, a putative P2y‐purinoceptor antagonist. Reactive blue 2 may bind to and block P2Z purinoceptors since [γ32P]‐ATP binding to parotid cells was inhibited by this compound. 5 In contrast to the response to ATP4–, the more sensitive response to ATP was potentiated by reactive blue 2 and was less affected by increases in external Mg2+ and Ca2+. 6 Parasympathetic denervation selectively increased the more sensitive response, suggesting that it may be physiologically regulated.

Coomassie Brilliant Blue G is a more potent antagonist of P2 purinergic responses than Reactive Blue 2 (Cibacron Blue 3GA) in rat parotid acinar cells

The ability of Brilliant Blue G (Coomassie Brilliant Blue G) and Reactive Blue 2 (Cibacron Blue 3GA) to block the effects of extracellular ATP on rat parotid acinar cells was examined by evaluating their effects on ATP-stimulated 45Ca2+ entry and the elevation of [Ca2+]i (Fura 2 fluorescence). ATP (300 microM) increased the rate of Ca2+ entry to more than 25-times the basal rate and elevated [Ca2+]i to levels more than three times the basal value. Brilliant Blue G and Reactive Blue 2 greatly reduced the entry of 45Ca2+ into parotid cells, but the potency of Brilliant Blue G (IC50 approximately 0.4 microM) was about 100-times that of Reactive Blue 2. Fura 2 studies demonstrated that inhibitory concentrations of these compounds did not block the cholinergic response of these cells, thus demonstrating the selectivity of the dye compounds for purinergic receptors. Unlike Reactive Blue 2, effective concentrations of Brilliant Blue G did not substantially quench Fura 2 fluorescence. The greater potency of Brilliant Blue G suggests that it may be very useful in identifying P2-type purinergic receptors, especially in studies which utilize fluorescent probes.

Elevation of [Ca2+]i and the Activation of Ion Channels and Fluxes by Extracellular ATP and Phospholipase C-Linked Agonists in Rat Parotid Acinar Cells

Extracellular ATP initiates a variety of changes in the parotid acinar cell. The initial effect appears to be the entry of Ca2+ (and perhaps Na+), and a series of ion transport events result from the subsequent elevation of [Ca2+]i. Agonists of phospholipase C-linked receptors elevate [Ca2+]i by a different pathway, involving the generation of inositol polyphosphate compounds, but share in the subsequent initiation of the ion transport events. Although the maintenance of the physiological changes may depend on specific inositol polyphosphate intermediates, the critical initiating factor is the elevation of [Ca2+]i. Fluid secretion by the parotid gland is triggered by the action of neurotransmitters, which alter the membrane permeability of the acinar cell. The similarities between the two receptor-mediated activation pathways suggests that ATP may act as a neurotransmitter and play a role in the control of fluid secretion. Basing our analysis on the purinoceptor characteristics outlined by Gordon, we suggest that the parotid receptor belongs to the P2Z class, which is highly sensitive to ATP4-. Basing his analysis on the earlier report by Gallacher of the effects of ATP on mouse parotid cells, Gordon placed the parotid purinoceptor in a different P2 subclass (P2Y). However, our findings of an increased potency of ATP in the absence of Mg2+, as well as the potency order of different nucleotides, indicate that the P2Z class is a more appropriate category.

Rapid desensitization of substance P- but not carbachol-induced increases in inositol trisphosphate and intracellular Ca++ in rat parotid acinar cells

The effects of supramaximal concentrations of substance P and the cholinergic agonist carbachol on the accumulation of inositol trisphosphate and the elevation of the intracellular free calcium concentration were compared in rat parotid acinar cells. Substance P was fully as effective as carbachol at initial times, but there was a rapid loss of the substance P responses while the effects of carbachol were well maintained. The loss of the substance P responses represented desensitization rather than degradation of the peptide since further additions of substance P were without effect. Desensitization to substance P did not involve long-term loss of substance P receptors as it was fully reversible in less than twenty minutes, the minimum time to extensively wash previously desensitized cells.

Evidence for calcium mediation of gonadotropin releasing hormone action in the pituitary

Abstract Despite the relatively long time since the isolation, characterization, and complete chemical synthesis of the gonadotropin releasing hormone (GnRH), very little information has become available which has elucidated the manner by which this hormone evokes gonadotropin release from the pituitary. Recently, a line of evidence has developed which suggests that calcium (Ca 2+ ) may play a central role in GnRH stimulation of gonadotropin release from cultured rat pituitary cells.

Extracellular ATP elevates intracellular free calcium in rat parotid acinar cells

The effect of extracellular ATP on intracellular free Ca2+ was characterized in quin2-loaded parotid acinar cells. ATP specifically increased the intracellular Ca2+ concentration six-fold above a basal level of 180 nM. Of other purine nucleotides tested, only adenylylthiodiphosphate (ATP gamma S) had significant activity. ATP and the muscarinic agonist carbachol increased intracellular Ca2+ even in the absence of extracellular Ca2+. Both agonists stimulated K+ release, which was followed by reuptake of K+, even in the continued presence of agonist. In the absence of Mg2+, ATP was much more potent but no more efficacious in elevating intracellular Ca2+, suggesting that ATP4- is the active species. The effect of ATP was reversed by removal with hexokinase, arguing against a role for an active contaminant of ATP and against a non-specific permeabilizing effect of extracellular ATP. Lactate dehydrogenase release was unaffected by a maximally effective concentration of ATP. These observations are consistent with a possible neurotransmitter role for ATP in the rat parotid gland.

Protection against glutamate toxicity through inhibition of the p44/42 mitogen-activated protein kinase pathway in neuronally differentiated P19 cells

Excessive levels of the neurotransmitter glutamate trigger excitotoxic processes in neurons that lead to cell death. N-Methyl-D-aspartate (NMDA) receptor over-activation is a key excitotoxic stimulus that leads to increases in intracellular calcium and activation of downstream signaling pathways, including the p44/42 mitogen-activated protein (MAP) kinase pathway. In the present study, we have demonstrated that 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a potent and selective inhibitor of the p44/42 MAP kinase signaling pathway, prevents glutamate-induced death in neuronally differentiated P19 cells. In addition, we show that differentiated, but not undifferentiated, P19 cells expressed zeta1, epsilon1, and epsilon2 subunits of the NMDA receptor. Differentiated P19 cells exhibited specific NMDA receptor binding and intracellular calcium responses to glutamate that were blocked by the selective NMDA receptor antagonist [5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), but not U0126. Glutamate treatment of differentiated P19 cells triggered a rapid and sustained induction in p42 MAP kinase phosphorylation that was blocked by U0126. Pretreatment of differentiated P19 cells with U0126, but not other classes of protein kinase inhibitors, protected against glutamate-induced cell death. Post-treatment with U0126, even as late as 6 hr after glutamate application, also protected against glutamate toxicity. These results suggest that the p44/42 MAP kinase pathway may be a critical downstream signaling pathway in glutamate receptor-activated toxicity.

Regulation of tyrosine hydroxylase in olfactory bulb cultures: selective inhibition of depolarization-induced increase by endogenous opioids.

Regulation of tyrosine hydroxylase (TH) by second messenger pathway activators was examined in rat olfactory bulb cell cultures. The number of TH-immunoreactive neurons was increased 2-3-fold by 36 h treatments with forskolin (Fsk, 10(-6) M) or phorbol myristate acetate (PMA, 10(-7) M), but was not significantly increased by a depolarizing concentration of KCl (45 mM). In contrast, KCl increased media [Met5]enkephalin (ME) immunoreactivity 2-fold in these cultures, equivalent to stimulation with Fsk or PMA. The possibility was examined that ME or another opioid produced by the cultures selectively inhibited the TH response to KCl. Pretreatment with the opioid receptor antagonist naloxone (10(-6) M) greatly increased the number of TH-immunoreactive neurons observed in response to KCl treatment, but had no effect on basal or Fsk-stimulated TH immunostaining, nor on basal or stimulated ME release. The increase in TH-immunoreactivity observed with combined KCl plus naloxone treatment was prevented by pretreating the cultures with the calcium channel blocker nimodipine (10(-6) M), which had no effect on Fsk stimulation or basal TH immunostaining. These data suggest that endogenous opioids selectively inhibit KCl-stimulated Ca2+ entry and thus TH induction in olfactory bulb cell cultures. These cultures offer a simple model system for further study of TH regulation in dopaminergic neurons.