Dasatinib (BMS-354825) is active in Philadelphia chromosome-positive chronic myelogenous leukemia after imatinib and nilotinib (AMN107) therapy failure

Developing strategies to counteract imatinib resistance constitutes a challenge in chronic myelogenous leukemia (CML). Therapy with the tyrosine kinase inhibitors nilotinib (AMN107) and dasatinib (BMS-354825) has produced high rates of hematologic and cytogenetic response. Src kinase activation has been linked to Bcr-Abl-mediated leukemogenesis and CML progression. In addition to binding Abl kinase with less stringent conformational requirements than imatinib, dasatinib is a potent Src kinase inhibitor. In the current study, we report on 23 patients with CML (19 of them in accelerated or blastic phases) treated with dasatinib after treatment failure with both imatinib and nilotinib. More than half (13; 57%) of 23 patients responded to dasatinib: 10 (43%) had a complete hematologic response (CHR), including 7 (30%) who had a cytogenetic response (2 complete, 4 partial, and 1 minor). These results suggest that dasatinib may be active in some patients after failure with both imatinib and nilotinib.     

Dasatinib (BMS-354825) inhibits KITD816V, an imatinib-resistant activating mutation that triggers neoplastic growth in most patients with systemic mastocytosis

Mastocytosis is associated with an activating mutation in the KIT oncoprotein (KITD816V) that results in autophosphorylation of the KIT receptor in a ligand-independent manner. This mutation is inherently resistant to imatinib and, to date, there remains no effective curative therapy for systemic mastocytosis associated with KITD816V. Dasatinib (BMS-354825) is a novel orally bioavailable SRC/ABL inhibitor that has activity against multiple imatinib-resistant BCR-ABL isoforms in vitro that is presently showing considerable promise in early-phase clinical trials of chronic myeloid leukemia (CML). Pharmacokinetic analysis suggests that high nanomolar concentrations of dasatinib can be achieved safely in humans. In this study, we demonstrate significant inhibitory activity of dasatinib against both wild-type KIT and the KITD816V mutation in the nanomolar range in in vitro and cell-based kinase assays. Additionally, dasatinib leads to growth inhibition of a KITD816V-harboring human masto-cytosis cell line. Significantly, dasatinib selectively kills primary neoplastic bone marrow mast cells from patients with systemic mastocytosis while sparing other hematopoietic cells. Computer modeling suggests that the KITD816V mutation destabilizes the inactive conformation of the KIT activation loop to which imatinib binds, but it is not predicted to impair binding of KIT by dasatinib. Based upon our results, further evaluation of dasatinib for the treatment of systemic masto-cytosis in clinical trials is warranted. Moreover, dasatinib may be of clinical utility in other disease settings driven by activating KIT mutations.     

Comparison of imatinib mesylate, dasatinib (BMS-354825), and nilotinib (AMN107) in an N-ethyl-N-nitrosourea (ENU)-based mutagenesis screen: high efficacy of drug combinations

BMS-354825 (dasatinib) and AMN107 (nilotinib) are potent alternate Abl inhibitors with activity against many imatinib mesylate-resistant BCR-ABL kinase domain (KD) mutants, except T315I. We used N-ethyl-N-nitrosourea (ENU)-exposed Ba/F3-p210(BCR-ABL) cells to compare incidence and types of KD mutants emerging in the presence of imatinib mesylate, dasatinib, and nilotinib, alone and in dual combinations. Although ENU is expected to induce mutations in multiple proteins, resistant clones were almost exclusively BCR-ABL KD mutant at relevant concentrations of nilotinib and dasatinib, consistent with a central role of KD mutations for resistance to these drugs. Twenty different mutations were identified with imatinib mesylate, 10 with nilotinib (including only 1 novel mutation, E292V) and 9 with dasatinib. At intermediate drug levels the spectrum narrowed to F317V and T315I for dasatinib and Y253H, E255V, and T315I for nilotinib. Thus, cross-resistance is limited to T315I, which is also the only mutant isolated at drug concentrations equivalent to maximal achievable plasma trough levels. With drug combinations maximal suppression of resistant clone outgrowth was achieved at lower concentrations compared with single agents, suggesting that such combinations may be equipotent to higher-dose single agents. However, sequencing uniformly revealed T315I, consistent with the need for a T315I inhibitor, to completely block resistance.     

Oral imatinib treatment reduces early fibrogenesis but does not prevent progression in the long term

BACKGROUND/AIMS: Transactivated hepatic stellate cells (HSCs) represent the key source of extra cellular matrix (ECM) in fibrotic liver. Imatinib, a potent inhibitor of the PDGF receptor tyrosine kinase, reduces HSC proliferation and fibrogenesis when treatment is initiated before fibrosis has developed. We tested the antifibrotic potential of imatinib in ongoing liver injury and in established fibrosis. METHODS: BDL-rats were gavage fed with 20 mg/kg/d imatinib either early (days 0-21) or late (days 22-35) after BDL. Untreated BDL-rats served as controls. ECM and activated HSCs were quantified by morphometry. Tissue activity of MMP-2 was determined by gelatin zymography. mRNA expression of TIMP-1 and procollagen alpha1(I) were measured by RT-PCR. Liver tissue concentration of imatinib was measured by tandem mass spectrometry. RESULTS: Early imatinib reduced ECM formation by 30% (P=0.0455) but left numbers of activated HSCs and procollagen I expression unchanged. MMP-2 activity and TIMP-1 expression were reduced by 50%. Late imatinib treatment did not alter histological or molecular markers of fibrogenesis despite high imatinib tissue levels. CONCLUSIONS: The antifibrotic effectiveness of imatinib is limited to the early phase of fibrogenesis. In ongoing liver injury other mediators most likely compensate for the inhibited PDGF effect.     

Aging affects but does not eliminate the enzymatic antioxidative response to hypoxia/reoxygenation in cerebral cortex

The effect of aging on basal and hypoxia/reoxygenation levels of both oxidative stress (protein carbonyl and TBARS) and antioxidative-enzyme activity (Cu/Zn-SOD; Mn-SOD; Catalase, CAT; Se-independent and Se-dependent glutathione peroxidase, GPX; glutathione transferase, GST and glutathione reductase, GR) has been studied in the cerebral cortex of adult and old rats. Oxidative stress markers increased with aging and show an age-dependent post-hypoxic response. Moreover, aging caused either no change (GST, GR and CAT) or an increase (Se-GPX, Cu/Zn-SOD, Mn-SOD) in the basal activity of the enzymes analysed. Only Se-independent GPX activity decreases. However, we detected an age-dependent response of SODs to the hypoxic injury. The early and sustained Cu/Zn-SOD activity rise in adult animals became late and weak in aged animals. Meanwhile, aging slowed the Mn-SOD post-hypoxic response although this activity was consistently higher in aged rats. Aging eliminated the post-hypoxic CAT response, but, perhaps offset by increased GPX activity, did not affect the GST response and slightly reduced post-hypoxic GR activity. In conclusion, aging rise basal ROS production, does not diminish or even increase the antioxidative-enzyme activity, and may slow but does not usually eliminate the enzymatic antioxidant response to the increased post-hypoxic ROS generation.     

Leptin deficiency reduces but does not eliminate the development of hepatic fibrosis in mice infected with Schistosoma mansoni

AIMS/BACKGROUND: Leptin, a product of the obese (ob) gene, was demonstrated previously in activated hepatic collagen-producing stellate cells, but not in quiescent retinol-storing stellate cells. The role of leptin in fibrogenesis is unknown. This study investigated the possible influence of leptin in the pathogenesis of fibrosis by determination of the amount of fibrosis produced by Schistosoma mansoni infection in leptin deficient male ob/ob mice as compared to control mice. METHODS: The mice were infected percutaneously with cercaria of Schistosoma mansoni and the amount of liver fibrosis determined 12 weeks after infection. The amount of hepatic collagen deposited was quantified by morphometric analysis of liver sections stained with sirius red and by hydroxyproline content. RESULTS: The amount of histologically detectable fibrosis was greater in the infected controls than in the infected ob/ob mice. In the infected control mice, but not in the ob/ob mice, the fibrosis surrounding the granuloma was broad and extended beyond the portal tracts into the lobule with the formation of fibrous septa. CONCLUSIONS: This study shows that leptin is a potentiating, but not an essential factor, for the development of hepatic fibrosis, because leptin deficiency reduces but does not prevent the development of hepatic fibrosis.     

Pretreatment with anticatabolic agents blunts but does not eliminate the skeletal anabolic response to parathyroid hormone in oophorectomized mice

Previous studies have indicated that bisphosphonate pretreatment can inhibit the anabolic actions of PTH. We examined the capacity of two anticatabolic agents with different mechanisms of action, alendronate and osteoprotegerin (OPG), to influence the anabolic activity of PTH. Oophorectomized mice were pretreated for 30 d with alendronate or OPG and then treated for 30 d with the respective anticatabolic alone or the respective anticatabolic plus PTH(1-34). Bones were analyzed by bone mineral density (BMD), microcomputed tomography, histology and histomorphometry, and biochemical bone markers. OPG pretreatment produced a greater inhibition of bone turnover and a greater increase in bone than alendronate. Increases in bone were sustained during subsequent treatment with vehicle or continued administration of the anticatabolic. Pretreatment with each anticatabolic blunted the capacity of PTH to increase BMD and bone volume and continued treatment with each anticatabolic agent also reduced the effectiveness of PTH. Although both anticatabolics decreased the maximal PTH effect, BMD and bone volume increased more when PTH was added than when only anticatabolics were used. These results demonstrate that mechanistically distinct anticatabolics may reduce PTH efficacy, that the characteristics of this inhibition may reflect the different modes of action of the anticatabolics, but that the addition of PTH still provides a skeletal benefit even if the anabolic effect is submaximal.     

Diverse effects of anti-CD44 antibodies on the stromal cell-mediated support of normal but not leukaemic (CML) haemopoiesis in vitro

We have identified three non-cross-reacting anti-human CD44 monoclonal antibodies that have significant positive or negative (or no) effects on normal human haemopoiesis in the long-term culture (LTC) system. These effects manifested as increases or decreases in the number of LTC-initiating cells (LTC-IC), and the number of colony-forming cells (CFC) recovered from cultures in which either unseparated or highly purified CD34⁺CD38⁻ normal marrow cells were placed on pre-established normal marrow feeder layers in the presence or absence of each antibody. The effects seen were rapid and sustained, and dependent on the presence of a preformed feeder layer. Interestingly, the same anti-CD44 antibodies had no effect on the maintenance of leukaemic (Ph⁺) progenitors (from patients with chronic myeloid leukaemia) when these cells were cultured on preformed feeder layers established from normal marrow. CD44 appears to be part of a mechanism by which stromal elements can regulate primitive normal haemopoietic cells but not their leukaemic (Ph⁺) counterparts.     

Overexpression of P-glycoprotein in K562 cells does not confer resistance to the growth inhibitory effects of imatinib (STI571) in vitro

Elevated expression of multidrug efflux pumps such as P-glycoprotein (Pgp) have been associated with resistance to cytotoxic drugs used in the treatment of leukemias and other cancers. Imatinib mesylate (STI-571 or Gleevec) is a potent inhibitor of the BCR/ABL and c-KIT tyrosine kinases. It has displayed considerable efficacy in treatment of patients with Philadelphia-positive acute lymphoblastic leukemia and chronic myelogenous leukemia and those with gastrointestinal stromal tumors (GISTs). However, recently imatinib-resistant relapse has emerged as a significant problem. Although a major cause of resistance appears to be point mutation in the kinase domain of the target enzyme, the potential contribution of elevated multidrug efflux activity has not been systematically evaluated. The imatinib-sensitive human leukemic cell line K562, which is dependent on the activity of BCR/ABL for survival and growth, provides a convenient system for evaluating modulation of drug activity. By expressing Pgp at high levels in these cells, we have demonstrated that this pump provides minimal protection against cell growth inhibition and apoptosis induced by imatinib. In contrast, overexpression of Bcl-xL, which blocks apoptosis, resulted in partial protection against the drug. We conclude that Pgp up-regulation is not likely to be a significant contributor to imatinib resistance.     

Bcl-x(L) prevents apoptosis of late-stage erythroblasts but does not mediate the antiapoptotic effect of erythropoietin

The long form of B-cell lymphoma-x (Bcl-x(L)), an outer mitochondrial membrane protein, has been proposed to mediate the antiapoptotic action of erythropoietin on erythroid progenitor cells and to be necessary for heme synthesis in erythroblasts. Mice with conditional knockout of Bcl-x(L) (conditional bcl-x(-/-) mice) develop severe anemia that has been attributed to hemolysis and is accompanied by splenomegaly. We characterized further the anemia of conditional bcl-x(-/-) mice and investigated the role of Bcl-x(L) in the action of erythropoietin and in heme synthesis. We analyzed peripheral blood cells and cultured splenic erythroblasts of conditional bcl-x(-/-) mice and littermates that were rendered anemic by bleeding. Although they had massive splenic erythroblastosis, conditional bcl-x(-/-) mice had decreased circulating reticulocytes compared to littermates even prior to bleeding the littermates. Compared to erythroblasts of bled littermates, bcl-x(-/-) erythroblasts cultured with erythropoietin underwent apoptosis during the later, hemoglobin-synthesizing stages of differentiation. The bcl-x(-/-) erythroblasts synthesized heme, but at reduced rates compared to bled littermate erythroblasts. When cultured without erythropoietin, bcl-x(-/-) erythroblasts underwent apoptosis at early stages of differentiation, prior to hemoglobin synthesis. Bcl-x(L) is not required for heme synthesis and does not mediate the antiapoptotic effects of erythropoietin, but it prevents ineffective erythropoiesis due to apoptosis in late-stage, hemoglobin-synthesizing erythroblasts.     

Melatonin (an antioxidant) does not ameliorate alcohol-induced Purkinje cell loss in the developing cerebellum

BACKGROUND: One popular mechanism proposed to account for alcohol-induced brain damage is the generation of free radicals after alcohol exposure. Therefore, it is reasonable to hypothesize that administration of an antioxidant should reduce the severity of alcohol-induced brain damage. Recently, melatonin has been shown to be an effective free-radical scavenger. In this study, the ability of melatonin to attenuate alcohol-induced cerebellar Purkinje cell loss in the cerebellar vermis and lobule I was assessed. METHODS: Sprague-Dawley rat pups were used in this study. These neonatal pups were exposed to alcohol (4.5 g/kg), melatonin (10 mg/kg), both alcohol and melatonin, or control vehicle via artificial-rearing methods from postnatal day (PD) 4 to PD 9. Alcohol, melatonin, or control vehicle was mixed with milk formula in 2 of the daily 12 feedings. Pups were killed 90 min after the beginning of the second alcohol feeding on PD 9. RESULTS: Alcohol significantly reduced the Purkinje cell numbers in the vermis and lobule I, with a higher percentage of cell loss in lobule I compared with the vermis. However, melatonin, per se, neither affected the Purkinje cell number nor diminished alcohol-induced Purkinje cell loss. CONCLUSIONS: Melatonin was not effective in attenuating alcohol-induced loss of Purkinje cells in our neonatal rat model system, even though such a dosage of melatonin is capable of reversing free radical-induced damage in other tissues.     

Disruption of the Lcn2 gene in mice suppresses primary mammary tumor formation but does not decrease lung metastasis

Based largely on studies in xenograft models, lipocalin-2 (Lcn2) has been implicated in the progression of multiple types of human tumors, including breast cancer. Here we examine the role of Lcn2 in mammary tumorigenesis and lung metastasis using an in vivo molecular genetics approach. We crossed a well-characterized transgenic mouse model of breast cancer, the MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen) mouse, with two independent gene-targeted Lcn2^−/− mouse strains of the 129/Ola or C57BL/6 genetic background. The onset and progression of mammary tumor development and lung metastasis in the female progeny of these crosses were monitored over a 20-week period. Female Lcn2^−/−MMTV-PyMT mice of the 129/Ola background (Lcn2^−/−PyMT¹²⁹) showed delayed onset of mammary tumors, and both Lcn2^−/−PyMT¹²⁹ mice and Lcn2^−/−MMTV-PyMT mice of the C57BL/6 background (Lcn2^−/−PyMT^B6) exhibited significant decreases in multiplicity and tumor burden (∼2- to 3-fold), as measured by total tumor weight and volume. At the molecular level, mammary tumors derived from Lcn2^−/−PyMT^B6 females showed reduced matrix metalloproteinase-9 (MMP-9) activity and a lack of high molecular weight MMP activity. However, although increased MMP-9 activity has been linked to tumor progression, neither Lcn2^−/−PyMT^B6 nor Lcn2^−/−PyMT¹²⁹ female mice showed a reduction in lung metastases compared to Lcn2^+/+PyMT controls. Our results demonstrate, using an in vivo animal model approach, that Lcn2 is a potent inducer of mammary tumor growth but not a significant promoter of lung metastasis.     

Genome-wide high density single-nucleotide polymorphism array-based karyotyping improves detection of clonal aberrations including der(9) deletion, but does not predict treatment outcomes after imatinib therapy in chronic myeloid leukemia

The current study investigated molecular cytogenetic characteristics of chronic myeloid leukemia (CML) using genome-wide, single nucleotide polymorphism arrays (SNP-A) capable of detecting cryptic submicroscopic genomic aberrations. Genome-Wide Human SNP 6.0 Array (Affymetrix, CA, USA) was performed in 118 patients having CML, chronic phase. Thirty-nine clonal aberrations (CAs) were identified (35 losses, two gains, two copy neutral loss of heterozygosity) that were not detected by metaphase cytogenetics in 25 patients (21%). The 9q34 deletions were found in 10% of cases, while 22q11.2 deletions were observed in 12% of cases. Seven patients (6%) harbored both 5??-ABL and 3??-BCR deletions adjacent to the t(9;22) breakpoint. Copy number gains were identified at 8p and 9p, and losses at 2q, 7q, 8q, 9q, 11q, 13q, 16p, and 22q. When we compared the treatment outcome of imatinib therapy between patients with and without CAs identified by SNP-A, treatment failure and progression to advanced disease were not significantly different (p?>?0.05). In addition, according to the presence of deletions of 9q34 and/or 22q11.2 identified by SNP-A, the treatment outcome did not show any significant differences (p?>?0.05). Our data suggests that SNP-A analysis is a useful tool for detection of clonal aberrations including deletions adjacent to the t(9;22) breakpoint in the CML cancer genome. However, clonal aberrations detected by SNP-A could not improve a prognostic stratification in CML patients with chronic phase.     

Antiserum to rat growth hormone (GH)-releasing factor suppresses but does not abolish antisomatostatin-induced GH release in the rat

Injection of SRIF antiserum (oA-SRIF) increases serum GH and TSH levels in urethane-anesthetized rats. These responses require an intact hypothalamus with endogenous GH-releasing factor (GHRF) or TRH, respectively. We examined whether pretreatment of rats with an antiserum against rat GHRF (oA-rGHRF) would abolish the GH response to oA-SRIF, since anti-TRH serum has been shown to abolish TSH response to oA-SRIF. Prior injection of oA-rGHRF reduced oA-SRIF-induced GH response in a dose-related manner in a dose up to 1 ml antiserum, but failed to produce any further suppression at higher doses. The maximum suppression of the GH response was approximately 50%. oA-rGHRF also suppresses basal GH levels significantly. On the other hand, oA-rGHRF completely abolished the GH-releasing activity of 1 microgram synthetic rGHRF when incubated for 30 min at room temperature before injection. The results suggest two conclusions: 1) 43-residue rGHRF is a physiological regulator of both basal and stimulated GH release; 2) failure of oA-rGHRF to completely abolish the GH response to oA-SRIF suggests the presence of other physiological GHRFs in the rat.     

Nitric oxide contributes to the regulation of vasomotor tone but does not modulate O(2)-consumption in exercising swine

OBJECTIVE: The role of nitric oxide (NO) in the regulation of vasomotor tone and tissue O(2)-consumption is incompletely understood. We therefore determined the contribution of endogenous NO to regulation of systemic, pulmonary and coronary vasomotor tone and myocardial (MV(O(2))) and whole body (BV(O(2))) O(2)-consumption in exercising swine. METHODS AND RESULTS: Exercise (1-5 km/h) up to 85% of maximum heart rate in 11 swine produced a 4-fold increase in BV(O(2)), which was accommodated for by 2-fold increases in both cardiac output (CO) and body O(2)-extraction. The NO synthase inhibitor N(omega)-nitro-L-arginine (NLA, 20 mg/kg, i.v.) increased mean aortic pressure by 30 mmHg both at rest and during exercise, due to a decrease in systemic vascular conductance from 37+/-2 to 22+/-1 ml/min mmHg(-1) at rest and from 88+/-3 to 60+/-3 ml/min mmHg(-1) at 5 km/h (all P< or =0.05 versus control). NLA produced vasoconstriction at rest and at 5 km/h in virtually all regional beds but did not affect the exercise-induced redistribution of CO. NLA increased mean pulmonary artery pressure from 15+/-1 to 21+/-1 mmHg at rest and from 30+/-2 to 40+/-2 mmHg at 5 km/h, due to a decrease in pulmonary vascular conductance (all P< or =0.05). BV(O(2)) remained unchanged and consequently the decrease in CO resulted in a compensatory increase in O(2)-extraction. NLA in a dose of 40 mg/kg produced similar responses. NLA had no significant effect on myocardial O(2)-demand or MV(O(2)) either at rest or during exercise, but decreased coronary vascular conductance which resulted in a decrease in coronary venous PO(2) from 24.5+/-1.1 to 21.9+/-0.8 mmHg at rest and from 23.5+/-0.5 to 21.0+/-0.6 mmHg at 5 km/h (all P< or =0. 05). CONCLUSIONS: Endogenous NO dilates the systemic, pulmonary and coronary vascular bed, but does not modify MV(O(2)) or BV(O(2)) in swine at rest and during exercise.     

GnRH but not warm temperature induces recrudescence of quiescent testes in the tropical lizard Calotes versicolor (Daud.) during postbreeding phase

The effects of temperature and various doses of GnRH on testicular recrudescence were studied in Calotes versicolor during the postbreeding (December-January) resting phase. Adult lizards (n = 51) were segregated into seven groups. Group I served as the initial control. Groups II, III, and IV were maintained at room temperature (23 +/- 1 degrees, range 17.5-25.5 degrees ) and natural photoperiod (12.25 h light:11.35 h dark). Groups V, VI, and VII were maintained at 30 +/- 1 degrees in an environmental chamber with 12 h light:12 h dark. Groups II and V received 0.2 ml saline, groups III and VI received 0.1 microgram GnRH/0.2 ml saline, and groups IV and VII received 0.5 microgram GnRH/0.2 ml saline on alternate days for 30 days. In the two groups that received 0.5 microgram GnRH, there was a significant increase in the diameter of the seminiferous tubules and Leydig cell nuclei, 3beta-HSDH activity in Leydig cells, and plasma testosterone level compared to those in other groups. There was no evidence of testicular recrudescence in lizards exposed to room/high temperatures treated with saline or 0.1 microgram GnRH. This study of C. versicolor shows that during the resting phase, high temperature per se does not stimulate testicular recrudescence, whereas 0.5 microgram GnRH does so. Also, the findings suggest that either higher brain centers regulating hypothalamus or hypothalamus itself become dormant, causing testicular inactivity (inactivation of hypophysial-testicular axis) during the postbreeding resting phase.     

Kisspeptin is present in ovine hypophysial portal blood but does not increase during the preovulatory luteinizing hormone surge: evidence that gonadotropes are not direct targets of kisspeptin in vivo

There is strong evidence that kisspeptin acts to regulate GnRH secretion, but whether there is also a component of action on the gonadotropes is not clear. Using quantitative RT-PCR, we found that G protein-coupled receptor-54 mRNA is expressed in ovine pituitary cell fractions enriched for gonadotropes as well as in somatotropes and lactotropes. To test whether kisspeptin acts directly on the pituitary gonadotropes, we first examined LH release from primary ovine pituitary cell cultures treated with kisspeptin. We found that kisspeptin treatment increased the concentration of LH in culture media by 80%, compared with control, but only in pituitary cultures from ewes during the follicular phase of the estrous cycle. After this, we determined whether kisspeptin acts on the pituitary gland in vivo. Using GnRH-replaced ovariectomized hypothalamo-pituitary-disconnected ewes, we were not able to achieve any effect of kisspeptin on LH under steady-state conditions or during the period of an estrogen-induced LH surge. Finally, we collected hypophysial portal blood samples from ovariectomized ewes and measured kisspeptin levels. Low but detectable amounts of kisspeptin were found in portal plasma, but levels were similar in ovariectomized ewes that were untreated or given estrogen to elicit an LH surge. Thus, although we observed an effect of kisspeptin on LH release in vitro in some situations, similar findings were not obtained in vivo. Moreover, the low concentrations of kisspeptin in hypophysial portal blood and the lack of any change during the period of an estrogen-induced GnRH/LH surge suggest that action on the pituitary gland is not of major consequence in terms of LH release.     

Protein kinase C phosphorylation sensitizes but does not activate the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1)

Protein kinase C (PKC) modulates the function of the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). This modulation manifests as increased current when the channel is activated by capsaicin. In addition, studies have suggested that phosphorylation by PKC might directly gate the channel, because PKC-activating phorbol esters induce TRPV1 currents in the absence of applied ligands. To test whether PKC both modulates and gates the TRPV1 function by direct phosphorylation, we used direct sequencing to determine the major sites of PKC phosphorylation on TRPV1 intracellular domains. We then tested the ability of the PKC-activating phorbol 12-myristate 13-acetate (PMA) to potentiate capsaicin-induced currents and to directly gate TRPV1. We found that mutation of S800 to alanine significantly reduced the PMA-induced enhancement of capsaicin-evoked currents and the direct activation of TRPV1 by PMA. Mutation of S502 to alanine reduced PMA enhancement of capsaicin-evoked currents, but had no effect on direct activation of TRPV1 by PMA. Conversely, mutation of T704 to alanine had no effect on PMA enhancement of capsaicin-evoked currents but dramatically reduced direct activation of TRPV1 by PMA. These results, combined with pharmacological studies showing that inactive phorbol esters also weakly activate TRPV1, suggest that PKC-mediated phosphorylation modulates TRPV1 but does not directly gate the channel. Rather, currents induced by phorbol esters result from the combination of a weak direct ligand-like activation of TRPV1 and the phosphorylation-induced enhancement of the TRPV1 function. Furthermore, modulation of the TRPV1 function by PKC appears to involve distinct phosphorylation sites depending on the mechanism of channel activation.