Human immunodeficiency virus type-1 Tat protein induces nuclear factor (NF)-κB activation and oxidative stress in microglial cultures by independent mechanisms

We have extended our previous findings and shown that human immunodeficiency virus Tat protein, in addition to nitric oxide (NO), stimulated rat microglial cultures to release pro-inflammatory cytokine interleukin-1beta and tumour necrosis factor-alpha in a nuclear factor (NF)-kappaB-dependent manner. At the same time, Tat stimulated the accumulation of free radicals, as indicated by the increased levels of isoprostane 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)), a reliable marker of lipid peroxidation and oxidative stress, by a mechanism unrelated to NF-kappaB activation. The presence of free radical scavengers abrogated Tat-induced 8-epi-PGF(2alpha) accumulation without affecting NO and cytokine production. Consistently, Tat-induced IkappaBalpha degradation - an index of NF-kappaB activation - was not affected by free radical scavengers, but was prevented by an NF-kappaB-specific inhibitor. Our observations indicate that NF-kappaB plays a key role in Tat-dependent microglial activation, and that oxidative stress and NF-kappaB activation induced by Tat occur by independent mechanisms.     

Opposite Regulation of Adenylyl Cyclase by Protein Kinase C in Astrocyte and Microglia Cultures

Abstract: We studied the regulation of cyclic AMP responses by protein kinase C (PKC) in purified astrocyte and microglia cultures obtained from the neonatal rat brain. In astrocytes, a 10-min treatment with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and 4β-phorbol 12,13-didecanoate (4β-PDD) (but not with 4α-PDD) or with diacylglycerol, which activate PKC, dose-dependently enhanced cyclic AMP accumulation induced by the β-adrenergic agonist isoproterenol and the adenylyl cyclase activator forskolin. Such enhancement was prevented by the PKC inhibitors staurosporine and calphostin-C and by down-regulation of PKC and was not related to activation of membrane receptors or G_s proteins or to inhibition of G_i proteins or phosphodiesterases. Instead, the activity of adenylyl cyclase doubled in PMA-treated astrocytes. In microglia, a 10-min treatment with PMA or PKC inhibitors did not affect cyclic AMP accumulation, whereas longer treatments with PMA or 4β-PDD (but not 4α-PDD) inhibited the cyclic AMP response in a time- and dose-dependent manner. Such inhibition was mimicked by staurosporine and calphostin-C. Also, in the case of microglia, the modulation of cyclic AMP responses appeared to occur at the level of adenylyl cyclase, and not elsewhere in the cyclic AMP cascade. The inhibition of microglial adenylyl cyclase was apparently not due to aspecific cytotoxicity. A differential regulation of adenylyl cyclase by PKC in astrocytes and microglia may help to explain qualitative and quantitative differences in the response of these cells to various physiological and pathological stimuli.     

Protein kinase C activation reduces microglial cyclic AMP response to prostaglandin E2 by interfering with EP2 receptors

We studied the modulation by protein kinase C (PKC) of the cyclic AMP (cAMP) accumulation induced by prostaglandin (PG) E2 in rat neonatal microglial cultures. Short pretreatment of microglia with phorbol 12-myristate 13-acetate (PMA) or 4beta-phorbol 12,13-didecanoate, which activate PKC, but not with the inactive 4alpha-phorbol 12,13-didecanoate, substantially reduced cAMP accumulation induced by 1 microM PGE2. The action of PMA was dose and time dependent, and the maximal inhibition (approximately 85%) was obtained after 10-min preincubation with 100 nM PMA. The inhibitory effect of PMA was mimicked by diacylglycerol and was prevented by the PKC inhibitor calphostin C. As PMA did not affect isoproterenol- or forskolin-stimulated cAMP accumulation, we investigated whether activation of PKC decreased cAMP production by acting directly at PGE2 EP receptors. Neither sulprostone (10(-9)-10(-5) M), a potent agonist at EP3 receptors (coupled to adenylyl cyclase inhibition), nor 17-phenyl-PGE2 (10(-6)-10(-5) M), an agonist of EP1 receptors, modified cAMP accumulation induced by forskolin. On the contrary, 11-deoxy-16,16-dimethyl PGE2, which does not discriminate between EP2 and EP4 receptors, both coupled to the activation of adenylyl cyclase, and butaprost, a selective EP2 agonist, induced a dose-dependent elevation of cAMP that was largely reduced by PMA pretreatment, as in the case of PGE2. These results indicated EP2 receptors as a possible target of PKC and suggest that PKC-activating agents present in the pathological brain may prevent the cAMP-mediated microglia-deactivating function of PGE2.     

Multiple Actions of the Human Immunodeficiency Virus Type-1 Tat Protein on Microglial Cell Functions

The human immunodeficiency virus type-1 (HIV-1) regulatory protein Tat is produced in the early phase of infection and is essential for virus replication. Together with other viral products, Tat has been implicated in the pathogenesis of HIV-1-associated dementia (HAD). As HIV-1 infection in the brain is very limited and macrophage/microglial cells are the only cellular type productively infected by the virus, it has been proposed that many of the viral neurotoxic effects are mediated by microglial products. We and others have shown that Tat affects the functional state of microglial cells, supporting the hypothesis that activated microglia play a role in the neuropathology associated with HIV-1 infection. This review describes the experimental evidence indicating that Tat stimulates microglia to synthesize potentially neurotoxic molecules, including proinflammatory cytokines and free radicals, and interferes with molecular mechanisms controlling cAMP levels, intracellular [Ca2+], and ion channel expression.     

Pro-inflammatory and pro-oxidant properties of the HIV protein Tat in a microglial cell line: attenuation by 17 beta-estradiol

Microglia are activated in humans following infection with human immunodeficiency virus (HIV), and brain inflammation is thought to be involved in neuronal injury and dysfunction during HIV infection. Numerous studies indicate a role for the HIV regulatory protein Tat in HIV-related inflammatory and neurodegenerative processes, although the specific effects of Tat on microglial activation, and the signal transduction mechanisms thereof, have not been elucidated. In the present study, we document the effects of Tat on microglial activation and characterize the signal transduction pathways responsible for Tat's pro-inflammatory effects. Application of Tat to N9 microglial cells increased multiple parameters of microglial activation, including superoxide production, phagocytosis, nitric oxide release and TNF alpha release. Tat also caused activation of both p42/p44 mitogen activated protein kinase (MAPK) and NF kappa B pathways. Inhibitor studies revealed that Tat-induced NF kappa B activation was responsible for increased nitrite release, while MAPK activation mediated superoxide release, TNF alpha release, and phagocytosis. Lastly, pre-treatment of microglial cells with physiological concentrations of 17 beta-estradiol suppressed Tat-mediated microglial activation by interfering with Tat-induced MAPK activation. Together, these data elucidate specific components of the microglial response to Tat and suggest that Tat could contribute to the neuropathology associated with HIV infection through microglial promulgation of oxidative stress.     

Vasoconstrictive effect of hydrogen sulfide involves downregulation of cAMP in vascular smooth muscle cells

Hydrogen sulfide (H(2)S), a new endogenous mediator, produces both vasorelaxation and vasoconstriction. This study was designed to examine whether cAMP mediates the vasoconstrictive effect of H(2)S. We found that NaHS at a concentration range of 10-100 microM (yields approximately 3-30 microM H(2)S) concentration-dependently reversed the vasodilation caused by isoprenaline and salbutamol, two beta-adrenoceptor agonists, and forskolin, a selective adenylyl cyclase activator, in phenylephrine-precontracted rat aortic rings. Pretreatment with NaHS (10-100 microM) for 5 min also significantly attenuated the vasorelaxant effect of salbutamol and forskolin. More importantly, NaHS (5-100 microM) significantly reversed forskolin-induced cAMP accumulation in vascular smooth muscle cells. However, NaHS produced significant, but weaker, vasoconstriction in the presence of N(G)-nitro-l-arginine methyl ester (100 microM), a nitric oxide synthase inhibitor, or in endothelium-denuded aortic rings. Blockade of ATP-sensitive potassium channels with glibenclamide (10 microM) failed to attenuate the vasoconstriction induced by H(2)S. Taken together, we demonstrated for the first time that the vasoconstrictive effect of H(2)S involves the adenyly cyclase/cAMP pathway.     

Methylene blue blocks cGMP production and disrupts directed migration of microglia to nerve lesions in the leech CNS

Migration and accumulation of microglial cells at sites of injury are important for nerve repair. Recent studies on the leech central nervous system (CNS), in which synapse regeneration is successful, have shown that nitric oxide (NO) generated immediately after injury by endothelial nitric oxide synthase (eNOS) stops migrating microglia at the lesion. The present study obtained results indicating that NO may act earlier, on microglia migration, and aimed to determine mechanisms underlying NO's effects. Injury induced cGMP immunoreactivity at the lesion in a pattern similar to that of eNOS activity, immunoreactivity, and microglial cell accumulation, which were all focused there. The soluble guanylate cyclase (sGC) inhibitor methylene blue (MB) at 60 microM abolished cGMP immunoreactivity at lesions and blocked microglial cell migration and accumulation without interfering with axon conduction. Time-lapse video microscopy of microglia in living nerve cords showed MB did not reduce cell movement but reduced directed movement, with significantly more cells moving away from the lesion or reversing direction and fewer cells moving toward the lesion. The results indicate a new role for NO, directing the microglial cell migration as well as stopping it, and show that NO's action may be mediated by cGMP.     

Calcium-independent and cAMP-dependent modulation of soluble guanylyl cyclase activity by G protein-coupled receptors in pituitary cells

It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca(2+) and activating Ca(2+)-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca(2+) signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that G(s)-coupled receptors stimulated de novo cGMP production. Furthermore, cholera toxin (an activator of G(s)), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of G(s)-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells.     

Epidermal growth factor stimulates cAMP accumulation in cultured rat cardiac myocytes.

We have previously shown that epidermal growth factor (EGF) augments cAMP accumulation in the heart and stimulates cardiac adenylyl cyclase via a G protein mediated mechanism (Nair et al., 1989). More recently, employing an antibody against the carboxy-terminus decapeptide of Gs alpha, we have demonstrated that Gs alpha mediates the effects of EGF on cardiac adenylyl cyclase (Nair et al., 1990). Since the heart comprises of a variety of cell types, the purpose of the studies presented here was to determine whether or not the effects of EGF on adenylyl cyclase were mediated in cardiac myocytes or noncardiomyocytes. Therefore, cultures of ventricular cardiomyocytes and noncardiomyocytes from neonatal rat hearts were established and characterized. Apart from the differences in cellular morphology, cardiomyocytes but not the noncardiomyocytes employed in our studies expressed the alpha- and beta-myosin heavy chain (MHC) mRNA and the beta-MHC protein. Additionally, as described previously, treatment of cardiomyocytes with thyroid hormone increased alpha-MHC mRNA and decreased the expression of beta-MHC mRNA, indicating that the cardiomyocytes employed in our studies were responding in a physiologically relevant manner. EGF in a time-dependent manner increased cAMP accumulation in the cardiomyocytes but not in noncardiomyocytes. Maximum and half-maximum effects were observed at 100 nM and 2 nM concentrations of EGF, respectively. As determined by the presence of immunoreactive EGF receptors and tyrosine phosphorylation of the 170 kDa protein in membranes of cardiomyocytes and noncardiomyocytes, both the cell populations contained functional EGF receptors. Therefore, the differential effects of EGF on cAMP accumulation in the two cell populations appear to be due to differential coupling of the EGF receptors to the adenylyl cyclase system rather than the absence of EGF receptors in noncardiomyocytes. Consistent with our previous findings in isolated membranes and perfused rat hearts, EGF-elicited increase in cAMP accumulation in cardiomyocytes did not involve activation of beta-adrenoreceptors and was abolished by prior treatment of cells with cholera toxin. Overall, our findings demonstrate that EGF-elicited increase in cAMP accumulation in the heart is the reflection of changes in cAMP content of cardiomyocytes and not noncardiomyocytes.     

Morphine and HIV-Tat increase microglial-free radical production and oxidative stress: possible role in cytokine regulation

Opiate abuse alters the progression of human immunodeficiency virus and may increase the risk of neuroAIDS. As neuroAIDS is associated with altered microglial reactivity, the combined effects of human immunodeficiency virus-Tat and morphine were determined in cultured microglia. Specifically, experiments determined the effects of Tat and morphine on microglial-free radical production and oxidative stress, and on cytokine release. Data show that combined Tat and morphine cause early and synergistic increases in reactive oxygen species, with concomitant increases in protein oxidation. Furthermore, combined Tat and morphine, but not Tat or morphine alone, cause reversible decreases in proteasome activity. The effects of morphine on free radical production and oxidative stress are prevented by pre-treatment with naloxone, illustrating the important role of opioid receptor activation in these phenomena. While Tat is well known to induce cytokine release from cultured microglia, morphine decreases Tat-induced release of the cytokines tumor necrosis factor-α and interleukin-6, as well as the chemokine monocyte chemoattractant protein-1 (MCP-1). Finally, experiments using the reversible proteasome inhibitor MG115 show that temporary, non-cytotoxic decreases in proteasome activity increase protein oxidation and decrease tumor necrosis factor-α, interleukin-6, and MCP-1 release from microglia. Taken together, these data suggest that oxidative stress and proteasome inhibition may be involved in the immunomodulatory properties of opioid receptor activation in microglia.     

Identification of RGS2 and type V adenylyl cyclase interaction sites

The production of cAMP is controlled on many levels, notably at the level of cAMP synthesis by the enzyme adenylyl cyclase. We have recently identified a new regulator of adenylyl cyclase activity, RGS2, which decreases cAMP accumulation when overexpressed in HEK293 cells and inhibits the in vitro activity of types III, V, and VI adenylyl cyclase. In addition, RGS2 blocking antibodies lead to elevated cAMP levels in olfactory neurons. Here we examine the nature of the interaction between RGS2 and type V adenylyl cyclase. In HEK293 cells expressing type V adenylyl cyclase, RGS2 inhibited Galpha(s)-Q227L- or beta(2)-adrenergic receptor-stimulated cAMP accumulation. Deletion of the N-terminal 19 amino acids of RGS2 abolished its ability to inhibit cAMP accumulation and to bind adenylyl cyclase. Further mutational analysis indicated that neither the C terminus, RGS GAP activity, nor the RGS box domain is required for inhibition of adenylyl cyclase. Alanine scanning of the N-terminal amino acids of RGS2 identified three residues responsible for the inhibitory function of RGS2. Furthermore, we show that RGS2 interacts directly with the C(1) but not the C(2) domain of type V adenylyl cyclase and that the inhibition by RGS2 is independent of inhibition by Galpha(i). These results provide clear evidence for functional effects of RGS2 on adenylyl cyclase activity that adds a new dimension to an intricate signaling network.     

Release of calcium from inositol 1,4,5-trisphosphate receptor-regulated stores by HIV-1 Tat regulates TNF-alpha production in human macrophages

HIV-1 protein Tat is neurotoxic and increases macrophage and microglia production of TNF-alpha, a cytopathic cytokine linked to the neuropathogenesis of HIV dementia. Others have shown that intracellular calcium regulates TNF-alpha production in macrophages, and we have shown that Tat releases calcium from inositol 1,4, 5-trisphosphate (IP3) receptor-regulated stores in neurons and astrocytes. Accordingly, we tested the hypothesis that Tat-induced TNF-alpha production was dependent on the release of intracellular calcium from IP3-regulated calcium stores in primary macrophages. We found that Tat transiently and dose-dependently increased levels of intracellular calcium and that this increase was blocked by xestospongin C, pertussis toxin, and by phospholipase C and type 1 protein kinase C inhibitors but not by protein kinase A or phospholipase A2 inhibitors. Xestospongin C, BAPTA-AM, U73122, and bisindolylmalemide significantly inhibited Tat-induced TNF-alpha production. These results demonstrate that in macrophages, Tat-induced release of calcium from IP3-sensitive intracellular stores and activation of nonconventional PKC isoforms play an important role in Tat-induced TNF-alpha production.     

The flavonoid rutin induces astrocyte and microglia activation and regulates TNF-alpha and NO release in primary glial cell cultures

Astrocyte and microglia cells play an important role in the central nervous system (CNS). They react to various external aggressions by becoming reactive and releasing neurotrophic and/or neurotoxic factors. Rutin is a flavonoid found in many plants and has been shown to have some biological activities, but its direct effects on cells of the CNS have not been well studied. To investigate its potential effects on CNS glial cells, we used both astrocyte primary cultures and astrocyte/microglia mixed primary cell cultures derived from newborn rat cortical brain. The cultures were treated for 24 h with rutin (50 or 100 μmol/L) or vehicle (0.5% dimethyl sulfoxide). Mitochondrial function on glial cells was not evidenced by the MTT test. However, an increased lactate dehydrogenase activity was detected in the culture medium of both culture systems when treated with 100 μmol/L rutin, suggesting loss of cell membrane integrity. Astrocytes exposed to 50 μmol/L rutin became reactive as revealed by glial fibrillary acidic protein (GFAP) overexpression and showed a star-like phenotype revealed by Rosenfeld’s staining. The number of activated microglia expressing OX-42 increased in the presence of rutin. A significant increase of nitric oxide (NO) was observed only in mixed cultures exposed to 100 μmol/L rutin. Enhanced TNFα release was observed in astrocyte primary cultures treated with 100 μmol/L rutin and in mixed primary cultures treated with 50 and 100 μmol/L, suggesting different sensitivity of both activated cell types. These results demonstrated that rutin affects astrocytes and microglial cells in culture and has the capacity to induce NO and TNFα production in these cells. Hence, the impact of these effects on neurons in vitro and in vivo needs to be studied.     

Nerve Growth Factor Amplifies Cyclic AMP Production in the HT4 Neuronal Cell Line

Abstract: There has been considerable interest and controversy in the relationship between nerve growth factor (NGF) and the cyclic AMP (cAMP) second messenger system. We have used a novel, neuronal cell line (HT4) to investigate the effect of NGF on the adenylyl cyclase signaling system. Treatment of cells with NGF (100 ng/ml 15 min) amplified cAMP accumulation (≈75%) in response to activation of adenosine A₂ receptors (5 min) with 5′-N-ethylcarboxamidoadenosine or activation of adenylyl cyclase directly with forskolin. Basal cAMP accumulation was not altered by NGF. This amplification appears to be mediated by activation of protein kinase C (PKC) because (1) it was mimicked by activators (phorbol esters and a diacylglycerol analogue) of PKC, (2) the effects of NGF and phorbol ester on cAMP accumulation were not additive, (3) NGF amplification of cAMP accumulation was abolished by down-regulation of PKC, (4) NGF increased cytosolic PKC activity, and (5) inhibitors of PKC blocked the NGF-induced amplification of cAMP accumulation. Although NGF-induced amplification of cAMP accumulation was dependent upon PKC, mechanisms other than the classic activation pathway (i.e., hydrolysis of inositol phospholipids or the production of diacylglycerol) appeared to mediate PKC activation by NGF. The tyrosine kinase inhibitor, lavendustin A, blocked NGF-mediated amplification of cAMP accumulation, suggesting a novel interaction between a tyrosine kinase and protein kinase C.     

Regulation of adenylyl cyclase, ERK1/2, and CREB by Gz following acute and chronic activation of the delta-opioid receptor

Opioid tolerance and physical dependence in mammals can be rapidly induced by chronic exposure to opioid agonists. Recently, opioid receptors have been shown to interact with the pertussis toxin (PTX)-insensitive Gz (a member of the Gi subfamily), which inhibits adenylyl cyclase and stimulates mitogen-activated protein kinases (MAPKs). Here, we established stable human embryonic kidney 293 cell lines expressing delta-opioid receptors with or without Gz to examine the role of Gz in opioid receptor-regulated signaling systems. Each cell line was acutely or chronically treated with [D-Pen2,D-Pen5]enkephalin (DPDPE), a delta-selective agonist, in the absence or presence of PTX. Subsequently, the activities of adenylyl cyclase, cyclic AMP (cAMP)-dependent response element-binding proteins (CREBs), and MAPKs were measured by determining cAMP accumulation and phosphorylation of CREBs and the extracellular signal-regulated protein kinases (ERKs) 1 and 2. In cells coexpressing Gz, DPDPE inhibited forskolin-stimulated cAMP accumulation in a PTX-insensitive manner, but Gz could not replace Gi to mediate adenylyl cyclase supersensitization upon chronic opioid treatment. DPDPE-induced adenylyl cyclase supersensitization was not associated with an increase in the phosphorylation of CREBs. Both Gi and Gz mediated DPDPE-induced activation of ERK1/2, but these responses were abolished by chronic opioid treatment. Collectively, our results show that although Gz mediated opioid-induced inhibition of adenylyl cyclase and activation of ERK1/2, Gz alone was insufficient to mediate opioid-induced adenylyl cyclase supersensitization.     

Induction of Nitric Oxide Synthase in Glial Cells

Primary astrocyte cultures, C6 glioma cells, and N18 neuroblastoma cells were assayed for nitric oxide synthase (NOS) activity with a bioassay of cyclic GMP production in RFL-6 fibroblasts. Treatment of astrocyte cultures for 16-18 h with lipopolysaccharide (LPS) induced NOS-like activity that was L-arginine and NADPH dependent, Ca2+ independent, and potentiated by superoxide dismutase. Induction was evident after 4 h, was dependent on the dose of LPS, and required protein synthesis. Treatment of astrocyte cultures with leucine methyl ester reduced microglial cell contamination from 7 to 1%, with a loss of 44% of NOS-like activity. C6 cells treated with LPS also showed Ca(2+)-independent and L-arginine-dependent NOS-like activity. N18 cells demonstrated constitutive Ca(2+)-dependent NOS-like activity that was not enhanced by LPS induction. These data indicate that NOS-like activity can be induced in microglia, astrocytes, and a related glioma cell line as it can in numerous other cell types, but not in neuron-like N18 cells.     

Alkaline pH- and cAMP-induced V-ATPase membrane accumulation is mediated by protein kinase A in epididymal clear cells

In the epididymis, low luminal bicarbonate and acidic pH maintain sperm quiescent during maturation and storage. The vacuolar H(+)-ATPase (V-ATPase) in epididymal clear cells plays a major role in luminal acidification. We have shown previously that cAMP, luminal alkaline pH, and activation of the bicarbonate-regulated soluble adenylyl cyclase (sAC) induce V-ATPase apical accumulation in these cells, thereby stimulating proton secretion into the epididymal lumen. Here we examined whether protein kinase A (PKA) is involved in this response. Confocal immunofluorescence labeling on rat epididymis perfused in vivo showed that at luminal acidic pH (6.5), V-ATPase was distributed between short apical microvilli and subapical endosomes. The specific PKA activator N(6)-monobutyryl-3'-5'-cyclic monophosphate (6-MB-cAMP, 1 mM) induced elongation of apical microvilli and accumulation of V-ATPase in these structures. The PKA inhibitor myristoylated-PKI (mPKI, 10 microM) inhibited the apical accumulation of V-ATPase induced by 6-MB-cAMP. Perfusion at pH 6.5 with 8-(4-chlorophenylthio)-2-O-methyl-cAMP (8CPT-2-O-Me-cAMP; 10 microM), an activator of the exchange protein activated by cAMP (Epac), did not induce V-ATPase apical accumulation. When applied at a higher concentration (100 microM), 8CPT-2-O-Me-cAMP induced V-ATPase apical accumulation, but this effect was completely inhibited by mPKI, suggesting crossover effects on the PKA pathway with this compound at high concentrations. Importantly, the physiologically relevant alkaline pH-induced apical V-ATPase accumulation was completely inhibited by pretreatment with mPKI. We conclude that direct stimulation of PKA activity by cAMP is necessary and sufficient for the alkaline pH-induced accumulation of V-ATPase in clear cell apical microvilli.     

Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding.

In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.