Arachidonic acid induces ERK activation via Src SH2 domain association with the epidermal growth factor receptor.

Within the kidney, angiotensin II type 2 (AT(2)) receptor mediates phospholipase A(2) (PLA(2)) activation, arachidonic acid release, epidermal growth factor (EGF) receptor transactivation, and mitogen-activated protein kinase activation. Arachidonic acid mimics this transactivation by an undetermined mechanism. The role of c-Src in mediating angiotensin II and arachidonic acid signaling was determined by employing immunocomplex kinase assay, Western blotting analysis, and protein immunoblotting on co-precipitated EGF receptor (EGFR) proteins and agarose conjugates of glutathione S-transferase fusion proteins containing the c-Src homology 2 (SH2) and SH3 domains. Angiotensin II induced extracellular signal-regulated kinase (ERK) activation in primary cultures of rabbit proximal tubule cells via the activation of c-Src and association of the EGFR with the c-Src SH2 domain, effects that were mimicked by arachidonic acid and its inactive analogue eicosatetraynoic acid. Inhibition of PLA(2) by mepacrine and methyl arachidonyl fluorophosphate, AT(2) receptor by PD123319, Src family kinases by, 1-(tert-butyl)-3-(4-chlorophenyl)-4-aminopyrazolo[3,4-d] pyrimidine (PP2) and c-Src by overexpression of a dominant-negative mutant of c-Src abrogated these effects. However, inhibitors of arachidonic acid metabolic pathways did not block these effects. The present work provides a new and novel paradigm for transactivation of a kinase receptor linked to a fatty acid, which may apply to activation of a variety of phospholipases and accompanying arachidonic acid release.

Arachidonic acid directly activates members of the mitogen-activated protein kinase superfamily in rabbit proximal tubule cells.

BACKGROUND: To explore the roles of eicosanoids in arachidonic acid-induced mitogen-activated protein kinase (MAPK) signal transduction, we have shown that exposure of proximal tubular cells to arachidonic acid induces phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), two members of the MAPK superfamily. We observed that ketoconazole, an inhibitor of the cytochrome P450 pathway, blocked ERK but not JNK activation. METHODS: Direct regulation of arachidonic acid on mitogen-activated protein kinase (MAPK) signaling pathways was evaluated more directly by utilizing specific enzyme inhibitors of the cytochrome P450 metabolic pathway and by comparing the relative efficacy of arachidonic acid versus its cytochrome P450 metabolites (exogenous and endogenous), eicosatetraynoic acid (ETYA), and other fatty acids on the phosphorylation of members of the MAPK superfamily (ERKs, JNK, and p38(MAPK)), by utilizing early passage rabbit proximal tubular epithelial cells. RESULTS: Arachidonic acid activated p38(MAPK), a third member of the MAPK superfamily, in a time- and concentration-dependent manner. Studies designed to evaluate the ability of arachidonic acid and its cytochrome P450 metabolites (endogenously and exogenously) to stimulate ERKs, JNK, and p38(MAPK) found four conclusions. First, the metabolites of arachidonic acid generated endogenously by cytochrome P450 2C1 significantly augmented basal ERK activity, whereas the metabolites generated by the 2C2 isozyme significantly augmented basal p38(MAPK) activity. However, their effects were less profound than arachidonic acid itself. In contrast, there were no significant effects with transfection of either isozyme on basal JNK activity. Second, a variety of exogenous cytochrome P450 products were less potent than arachidonic acid on a molar basis in stimulating the activity of all three MAPKs. Third, ketoconazole and 17-octadecynoic acid, inhibitors of the cytochrome P450 pathway, as well as PPOH and DDMS, inhibitors of the epoxygenase and omega-hydroxylase pathways, respectively, failed to significantly reduce the effects of arachidonic acid to activate ERK and p38(MAPK) (JNK was not evaluated). Finally, arachidonic acid, its inactive analog ETYA, and other fatty acids with differing chain lengths and degrees of saturation stimulated the activity of all three MAPKs. CONCLUSIONS: These observations substantiate a role for arachidonic acid and other fatty acids in signaling linked to the MAPK superfamily in rabbit proximal tubular epithelium without the necessity of conversion to cytochrome P450 metabolites.

Ca(2+)-dependent activation of c-jun NH(2)-terminal kinase in primary rabbit proximal tubule epithelial cells.

Previous work from this laboratory demonstrated that arachidonic acid activates c-jun NH(2)-terminal kinase (JNK) through oxidative intermediates in a Ca(2+)-independent manner (Cui X and Douglas JG. Arachidonic acid activates c-jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells. Proc Natl Acad Sci USA 94: 3771-3776, 1997.). We now report that JNK can also be activated via a Ca(2+)-dependent mechanism by agents that increase the cytosolic Ca(2+) concentration (Ca(2+) ionophore A(23187), Ca(2+)-ATPase inhibitor thapsigargin) or deplete intracellular Ca(2+) stores [intracellular Ca(2+) chelator 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM]. The activation of JNK by BAPTA-AM occurs despite a decrease in cytosolic Ca(2+) concentration as detected by the indicator dye fura 2, but appears to be related to Ca(2+) metabolism, because modification of BAPTA with two methyl groups increases not only the chelation affinity for Ca(2+), but also the potency for JNK activation. BAPTA-AM stimulates Ca(2+) influx across the plasma membrane, and the resulting local Ca(2+) increases are probably involved in activation of JNK because Ca(2+) influx inhibitors (SKF-96365, nifedipine) and lowering of the free extracellular Ca(2+) concentration with EGTA reduce the BAPTA-induced JNK activation.

Phospholipase A2-mediated activation of mitogen-activated protein kinase by angiotensin II.

In renal proximal tubule epithelial cells, a membrane-associated phospholipase A2 (PLA2) is a major signaling pathway linked to angiotensin II (Ang II) type 2 receptor (AT2). The current studies were designed to test the hypothesis that membrane-associated PLA2-induced release of arachidonic acid (AA) and/or its metabolites may serve as an upstream mediator of Ang II-induced mitogen-activated protein kinase (MAPK) activation. Ang II stimulated transient dose-dependent phosphorylation of MAPK with a maximum at 1 microM (10 min). Inhibition of PLA2 by mepacrine diminished both AA release and MAPK phosphorylation, induced by Ang II. Furthermore, AA itself induced time- and dose-dependent phosphorylation of MAPK, supporting the importance of PLA2 as a mediator of Ang II signaling. The effects of both Ang II and AA on MAPK phosphorylation were protein kinase C independent and abolished by the inhibitor of cytochrome P450 isoenzyme, ketoconazole. Moreover, 5,6-epoxyeicosatrienoic acid and 14,15-epoxyeicosatrienoic acid, the cytochrome P450-dependent metabolites of AA, significantly stimulated MAPK activity in renal proximal tubule epithelial cells. These observations document a mechanism of Ang II-induced MAPK phosphorylation, mediated by PLA2-dependent release of AA and cytochrome P450-dependent production of epoxy derivatives of AA.

Arachidonic acid mediates angiotensin II effects on p21ras in renal proximal tubular cells via the tyrosine kinase-Shc-Grb2-Sos pathway.

In kidney epithelial cells, an angiotensin II (Ang II) type 2 receptor subtype (AT2) is linked to a membrane-associated phospholipase A2 (PLA2) and the mitogen-activated protein kinase (MAPK) superfamily. However, the intervening steps in this linkage have not been determined. The aim of this study was to determine whether arachidonic acid mediates Ang II's effect on p21ras and if so, to ascertain the signaling mechanism(s). We observed that Ang II activated p21ras and that mepacrine, a phospholipase A2 inhibitor, blocked this effect. This activation was also inhibited by PD123319, an AT2 receptor antagonist but not by losartan, an AT1 receptor antagonist. Furthermore, Ang II caused rapid tyrosine phosphorylation of Shc and its association with Grb2. Arachidonic acid and linoleic acid mimicked Ang II-induced tyrosine phosphorylation of Shc and activation of p21ras. Moreover, Ang II and arachidonic acid induced an association between p21ras and Shc. We demonstrate that arachidonic acid mediates linkage of a G protein-coupled receptor to p21ras via Shc tyrosine phosphorylation and association with Grb2/Sos. These observations have important implications for other G protein-coupled receptors linked to a variety of phospholipases.

VIP antagonist demonstrates differences in VIP- and PHI-mediated stimulation and inhibition of ACTH and corticosterone secretion in rats.

Previous studies in our laboratory have demonstrated that PVN administration of equimolar doses of VIP and PHI induce similar increases in plasma ACTH and CORT concentrations via the release of CRF and vasopressin in fasted, freely moving rats studied during the early light cycle. The purpose of these investigations was to determine whether VIP and PHI act via the same receptor and/or mechanism. Individual studies involving the PVN administration of either VIP or PHI in doses ranging from 0.3 to 30.0 nmol/rat demonstrated that VIP increases both ACTH and CORT secretion throughout the administered range. In contrast, PHI was an effective stimulant in doses up to 15 nmol/rat but had no effect on either ACTH or CORT at a dose of 30 nmol/rat thus yielding a bell-shaped dose-response curve. When increasing doses of PHI (0.15-3.0 nmol/rat) were administered against a background of VIP (3.0 nmol/rat) predictably additive responses were observed; however, when increasing doses of VIP (0.15-3.0 nmol/rat) were administered with PHI (3.0 nmol/rat) only the higher doses of VIP facilitated the PHI-induced secretion while the lower doses of VIP actually reduced the PHI-induced ACTH secretion. Finally, pretreatment with [Lys1, Pro2,5, Arg3,4, Tyr6]-VIP, anVIP (1.5 nmol/rat) totally suppressed VIP-induced ACTH secretion but had no effect on PHI-induced secretion. These studies collectively suggest that VIP and PHI utilize different receptors/mechanisms to regulate HPA secretion. Furthermore, when a range of doses of anVIP (1.5-30.0 nmol/rat) was tested against VIP (3.0 nmol/rat), ACTH secretion was totally suppressed at all doses of the antagonist. However, the maximal reduction of CORT secretion occurred at the lowest dose of anVIP and increasing doses were less and less effective, suggesting that not only PHI but VIP may also stimulate and inhibit HPA secretion. While both the stimulatory and the inhibitory actions of PHI appear to involve ACTH, only the stimulatory action of VIP is ACTH-dependent.

A possible involvement of VIP in feeding-induced secretion of ACTH and corticosterone in the rat.

For decades it has been known that brain/gut peptides are released during the ingestion of a meal. Although a multitude of actions have been attributed to these peptides acting in the brain, including the release or inhibition of a variety of pituitary hormones, the actual physiological roles of these substances in the brain have not been confirmed. For the first time, we have demonstrated that feeding-induced hypothalamic-pituitary-adrenal (HPA) secretion may involve the brain/gut peptide, vasoactive intestinal peptide (VIP), acting in the paraventricular nucleus (PVN) of the hypothalamus. The effects of 24 h fasting and refeeding on the release of plasma ACTH and CORT secretion in male rats were investigated. Blood samples were collected 5 min prior to PVN administration of saline or VIP antagonist, [Lsy, Pro, Arg, Tyr]-VIP and 30 min after refeeding. Plasma ACTH and CORT concentrations were significantly increased by 43 and 485%, respectively, by 30 min ingestion of food. Pretreatment with the VIP antagonist (0.75 and 1.5 nmol/rat) significantly reduced the food-induced ACTH response by 69 and 76% and the CORT response by 58 and 65%, respectively. There were no significant differences in food-intake among groups. These results suggest that one potential role of hypothalamic VIP may involve activation of hypothalamic-releasing factors to regulate ACTH and CORT levels during or after a meal.

Involvement of vasopressin and corticotropin-releasing hormone in VIP- and PHI-induced secretion of ACTH and corticosterone.

The relative roles of hypothalamic corticotropin-releasing-hormone (CRH) and vasopressin (AVP) as mediators of the stimulant effect of vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) on ACTH and corticosterone (CORT) secretion, were examined using receptor blockade of endogenous CRH and AVP. ACTH and CORT secretion were stimulated 6- and 7-fold, respectively, by PVN infusion of VIP (3.0 nmol) and 6- and 9-fold, respectively, by PHI (3.0 nmol). ACTH and CORT stimulation by VIP were inhibited 78 and 72%, respectively, by pretreatment with the CRF antagonist, 59 and 57%, respectively, by pretreatment with the AVP antagonist and about 78% by combined pretreatment with the CRF and AVP antagonists. PHI-induced stimulation of ACTH and CORT was inhibited 89 and 81%, 73 and 59% and 93% by pretreatment with the CRF- or AVP-antagonist or combined administration, respectively. These results support the hypothesis that the activation of the hypothalamic-pituitary-adrenal (HPA) axis by VIP and PHI is mediated through the release of endogenous CRH. AVP also plays a role in this response, possibly by enhancing the activity of CRH in a synergistic manner.

Vasoactive intestinal peptide stimulates ACTH and corticosterone release after injection into the PVN.

Vasoactive intestinal peptide (VIP), a neuropeptide originally isolated from the intestine, is widely distributed in the central and peripheral nervous systems and exhibits a broad range of biological actions. In the present study the effects of VIP on plasma ACTH and corticosterone (CORT) secretion were investigated in fasted, freely-moving male rats. Male rats, chronically implanted with a hypothalamic paraventricular nucleus (PVN) cannula, were injected with VIP doses (0.15-3.0 nmol/rat) of VIP or saline (control). Blood samples were collected (0.6 ml) from an intravenous catheter immediately preceding and at 15, 30, 60, 90 and 120 min following peptide administration. PVN administration of VIP increased the plasma ACTH and CORT levels in a dose-dependent manner and the maximal effect was obtained at 15 min after administration. At the highest dose tested, VIP increased ACTH and CORT to 167% and 342% of time-matched controls, respectively. These results demonstrate that the PVN is a sensitive site for VIP-induced elevation of plasma ACTH and CORT and imply that the VIP binding sites and immunoreactive terminals previously identified in this region may be involved in the central regulation of the pituitary-adrenal axis.

Peptide histidine isoleucine-induced elevations in ACTH and corticosterone in the rat.

Peptide histidine isoleucine (PHI) is one of many neuropeptides found in both the periphery and the central nervous system (CNS). In the present work, we compared the effects of intravenous (IV) and hypothalamic (PVN) administration of PHI on plasma ACTH and corticosterone (CORT) secretion 2 h after the onset of light in fasted, freely moving male rats. The PVN administration of PHI (0.15, 0.30, 1.50, and 3.0 nmol/rat) elicited significant, dose-dependent increases in plasma ACTH and CORT concentrations that were maximal at 15 min postadministration and fell gradually over 60-120 min. The highest dose of PHI increased ACTH and CORT to 821% and 340% of time-matched control levels, respectively. The IV injection of PHI (3.0 nmol/rat) also raised plasma ACTH and CORT levels in these animals. However, the peak response (at 15 min postinjection) to the same dose of PHI was much lower following IV administration (ACTH 188% of control; CORT 210% of control) than after PVN administration. These results suggest that endogenous PHI may be a physiological regulator of ACTH and CORT secretion in rats and that the PVN is a major site for its action.

Activation of ATP-sensitive K+ channels by cromakalim. Effects on cellular K+ loss and cardiac function in ischemic and reperfused mammalian ventricle.

Pharmacological modulation of [K+]o accumulation and action potential changes during acute myocardial ischemia is under evaluation as a promising new antiarrhythmic and cardioprotective strategy during myocardial ischemia and reperfusion. We studied the effects of cromakalim, a K+ channel opener that activates ATP-sensitive K+ channels, in isolated arterially perfused rabbit interventricular septa subjected to ischemia and reperfusion and, through use of the patch clamp technique, in inside-out membrane patches excised from guinea pig ventricular myocytes. During aerobic perfusion, 5 microM cromakalim shortened action potential duration (APD) from 217 +/- 7 to 201 +/- 10 msec, had no effect on [K+]o, and reduced tension by 17 +/- 3% (n = 11). During ischemia, pretreatment with 5 microM cromakalim resulted in 1) more rapid APD shortening (71 +/- 9 versus 166 +/- 7 msec at 10 minutes and 63 +/- 12 versus 122 +/- 8 msec at 30 minutes), 2) similar [K+]o accumulation after 10 minutes (8.9 +/- 0.3 versus 9.6 +/- 0.5 mM) but a trend toward increased [K+]o accumulation after 30 minutes (11.0 +/- 1.7 versus 9.6 +/- 1.0 mM), and 3) similar times for tension to decline to 50% of control (2.14 +/- 0.16 versus 2.14 +/- 0.19 minutes) but shorter time to fall to 20% of control (4.34 +/- 0.33 versus 4.90 +/- 0.22 minutes; p = 0.003). After 60 minutes of reperfusion following 30 minutes of ischemia, recovery of function was similar, with a trend toward better recovery of developed tension (to 58 +/- 9% versus 39 +/- 10% of control; p = 0.18) and tissue ATP levels in cromakalim-treated hearts but no differences in APD or rest tension. Thus, 5 microM cromakalim had mild effects in normal heart but greatly accelerated APD shortening during ischemia without markedly increasing [K+]o accumulation, possibly because the more rapid APD shortening reduced the time-averaged driving force for K+ efflux through ATP-sensitive K+ channels. A significant cardioprotective effect during 30 minutes of ischemia plus 60 minutes of reperfusion could not be demonstrated in this model. In excised membrane patches studied at room temperature, the ability of cromakalim to activate ATP-sensitive K+ channels was significantly potentiated by 100 microM but not 15 microM cytosolic ADP, suggesting that in addition to the modest fall in cytosolic ATP during early ischemia, the rapid increases in cytosolic ADP may further sensitize cardiac ATP-sensitive K+ channels to activation by cromakalim.(ABSTRACT TRUNCATED AT 400 WORDS)

Correspondence between subjective report of temporomandibular disorder symptoms and clinical findings.

Correspondence between subjective reports of temporomandibular disorder symptoms and clinical findings was examined in 65 volunteers. Significant relationships showed that the greater the number of reported symptoms, the greater the likelihood that positive signs would be found during examination. The symptom survey questionnaires served as a valid method for screening patients for temporomandibular disorder-related signs.

Antecedents of dental fear.

Early dental experiences were described in essay form by 225 college students reporting either "high" or "low" fear of dentistry. Antecedent factors which appeared to differentiate the two groups most clearly included pain experiences and dentist behaviors. Results were interpreted as supportive of a social-learning model of dental fear acquisition and their implications for dental training and practice were discussed.