Endogenous Responsiveness Research Articles

Platelet-derived growth factor (PDGF) in human acute myelogenous leukemia: PDGF receptor expression, endogenous PDGF release and responsiveness to exogenous PDGF isoforms by in vitro cultured acute myelogenous leukemia blasts.

We investigated effects of Platelet-derived growth factor (PDGF) and Platelet factor 4 (PF-4) on the functional characteristics of native, human acute myelogenous leukemia (AML) blasts. AML blast expression of the PDGF-receptor alpha-chain was detected for a subset of patients (45%), whereas PDGF-receptor beta-chain expression was detected for most patients (90%). Constitutive AML blast release of the PDGF-AB isoform (the major form also derived from normal platelets) was detected for 43% of patients, whereas PDGF-BB release was not detected for any patient. The PDGF isoforms AA, AB and BB had dose-dependent and divergent effects on spontaneous and cytokine-dependent AML blast proliferation, whereas for constitutive cytokine secretion (IL-1beta, IL-6, TNF-alpha) inhibitory effects were rare and all three isoforms usually had no effect or enhanced the constitutive secretion. The PDGF effects were caused by a direct effect on the AML blasts and were not dependent on the presence of serum. The PDGF effects could also be detected after in vitro culture of AML cells in the presence of IL-4+granulocyte-macrophage colony stimulating factor. PF-4 had divergent effects on proliferation and cytokine secretion by native AML blasts. Our results suggest that exogenous (e.g. platelet-secreted) PDGF and PF-4 can function as regulators of leukemic hematopoiesis and possibly also modulate the function of residual AML cells in peripheral blood stem cell grafts. On the other hand, endogenous release of PDGF-AB by native blasts may modulate the function of normal cells in the bone marrow microenvironment (e.g. bone marrow stromal cells).

Dual signaling and ligand selectivity of the human PTH/PTHrP receptor.

Parathyroid hormone (PTH) activates PTH/PTH-related peptide-related receptors (PTHRs) to stimulate both adenylyl cyclase (AC) and phospholipase C (PLC). How these parallel signals mediate specific cellular and tissue responses to PTH, such as the complex anabolic versus catabolic actions of PTH on bone, remains unsettled. Previous studies of PTHR signaling and function employed mainly rodent or other cell lines that express endogenous PTHRs and, possibly, alternate species of PTH receptors. To preclude confounding effects of such receptors, we stably expressed recombinant human PTHRs (hPTHRs) at different levels of surface density in LLC-PK1 porcine renal epithelial cells that lack endogenous PTH responsiveness. hPTH(1-34) induced concentration-dependent activation of both AC and PLC via transfected hPTHRs. Maximal intensity of each signal increased with receptor density, but more hPTHRs were required for PLC than for AC activation. Coupling to AC was saturated at receptor densities too low to detect sustained PLC activation. hPTH(3-34), found by others to be a PLC/protein kinase C (PKC)-selective peptide in rat cells, did not activate PLC via human (or rat) PTHRs under conditions (1 microM peptide, 106 hPTHRs/cell) where hPTH(1-34) stimulated PLC severalfold. Other cellular responses that require PKC activation in these cells, such as sodium-dependent phosphate transport and cAMP-independent secretion of plasminogen activator, were induced by PTH(1-34) but not by hPTH(3-34) or hPTH(7-34). We conclude that amino-truncated PTH analogs reported to activate PKC cannot directly activate phosphatidylinositol-specific PLC via the human or rat PTHR and therefore that PTH receptors may access alternate, PLC-independent pathways of PKC activation in some target cells. The relative intensity of AC and PLC signaling via the hPTHR may be strongly regulated by changes in its surface expression.

Alterations in endogenous gonadotropin secretion and pituitary responsiveness to gonadotropin-releasing hormone in adult ewes, following indirect selection in prepubertal male lambs.

Indirect selection to improve reproductive performance in the female, through use of a physiologically related trait in prepubertal males, the LH response to a GnRH challenge, has created two lines of sheep with altered pituitary gland sensitivity to both physiological and pharmacological doses of GnRH. These lines also exhibit correlated between-line differences in female reproductive performance during the first, and at the beginning of the second, breeding season. This study was designed to determine whether the observed between-line differences in female reproductive performance were related to between-line differences in endogenous gonadotropin secretion during the luteal and follicular phases of the estrous cycle and whether the between-line difference in the LH response to a GnRH challenge was maintained in adult females. The results indicated that, despite similar mean gonadotropin and steroid concentrations during the luteal and follicular phases of the estrous cycle, significant between-line differences in LH pulse amplitude were present during the follicular phase (high line > low line). In addition, the results indicated that the between-line difference in the LH response to a GnRH challenge was maintained in adult ewes, being most apparent during the follicular phase, when the magnitude of the difference was similar to that in prepubertal female lambs. Therefore, in addition to showing maintenance of the between-line difference in the selected characteristic in postpubertal females, the results demonstrate that adult ewes from the two lines differ in additional, unselected but physiologically related traits and provide a model system with which to study steroidal regulation of gonadotropin secretion in the ewe.

Identification of multiple, widely spaced estrogen-responsive regions in the rat progesterone receptor gene.

Progesterone receptors (PRs) mediate the actions of progestin hormones and play important roles during the reproductive cycle and pregnancy. Since PR expression is known to be regulated by estrogen, we have undertaken studies to examine the mechanisms underlying this regulation. We have identified multiple distinct regions of the rat PR gene, widely spaced and spread throughout the 5'-flanking region, the 5'-untranslated region, and the first exon (between -2264 and +2241), that can form a strong estrogen-responsive enhancer when linked together. Estrogen-responsive activities for two of the regions in isolation (+461/+636 and +2176/+2241) were demonstrated in one or more homologous or heterologous promoter contexts. The contributions of the other regions (-2264/-1970, -1167/-957 and +2088/+2110) to the overall activity of the assembled enhancer were cryptic in that they were only observed in the context of the other PR gene fragments, not in isolation. We identified four weak, but functional, imperfect estrogen response elements (EREs) in these regions of the PR gene, each differing from the consensus by 2 base pairs. In addition, we identified four ERE half-sites in the PR gene, three of which are paired (i.e. < 150 base pairs away) with the EREs in the estrogen-responsive regions. Competitive gel shift assays demonstrated weak, but detectable, binding of estrogen receptor to the EREs. Of note, the estrogen-responsive enhancer assembled from the five regions of the PR gene exhibited promoter specificity; it conferred estrogen responsiveness of the distal PR gene promoter, but it failed to enhance the endogenous estrogen responsiveness of the proximal PR gene promoter. The positioning of response elements in the rat PR gene, which we show to be unique among steroid hormone-regulated genes, may have functional consequences for the regulation of the magnitude and timing of PR gene expression by estrogen.

A Trk nerve growth factor (NGF) receptor point mutation affecting interaction with phospholipase C-gamma 1 abolishes NGF-promoted peripherin induction but not neurite outgrowth.

We analyzed the function of Trk nerve growth factor (NGF) receptors containing a point mutation (Tyr-->Phe) in a major autophosphorylation site (Tyr-785). Tyr-785 is required for phospholipase C-gamma 1 to interact with Trk and to become tyrosine-phosphorylated in response to NGF. The altered receptors were transfected into a mutant subline of PC12 rat pheochromocytoma cells (designated PC12nnr5) that, unlike wild-type PC12 cells, lack expression of endogenous Trk and responsiveness to NGF. PC12nnr5 cells permanently transfected with Trk Y785F exhibit NGF-dependent autophosphorylation and normal NGF binding and internalization. Moreover, Trk Y785F mediates NGF-stimulated neurite outgrowth as well as a variety of additional responses including induction of immediate-early and late genes. However, in contrast to cells expressing wild-type Trk, cells expressing Trk Y785F lack NGF-promoted elevation of peripherin intermediate filament mRNA and protein. These observations indicate that phospholipase C-gamma 1 activation or other signaling pathways dependent on Tyr-785 autophosphorylation are selectively required for regulation of peripherin expression by NGF, but not for many other functional NGF responses. This supports the presence of multiple and separable signaling pathways in the NGF mechanism of action.

A new method for simultaneous measurement of endogenous noradrenaline release and vascular responsiveness in perfused rabbit carotid arteries in vitro

1. A new preparation for large arteries was tested for the simultaneous measurement of noradrenaline release and vasoconstrictor response induced by nerve stimulation in vitro. 2. Preparations of rabbit carotid artery with an outflow cannula were perfused with a modified Krebs-Ringer bicarbonate solution containing propranolol (3 x 10(-6) M) and cocaine (10(-6) M), to block beta-adrenoceptors and uptake-1, respectively. 3. The perfused carotid arteries were subjected to both transmural field stimulations (TFS) and exogenous noradrenaline administrations. To evaluate the preparation, we carried out the procedures in two control groups and a group with yohimbine (10(-7) M) treatment, respectively. 4. TFS over the range of 1.5-24 Hz caused a frequency-dependent pressor response. Yohimbine significantly increased the TFS-induced pressor responses. 5. TFS also caused frequency-dependent endogenous noradrenaline release. The endogenous noradrenaline release was significantly enhanced by yohimbine treatment. When noradrenaline release was expressed as release per stimulus, it was frequency-dependent in the group with yohimbine treatment but not in control groups. 6. Exogenous noradrenaline induced a dose-dependent pressor response. The exogenous noradrenaline-induced vasoconstrictor response was significantly diminished by yohimbine treatment. 7. It is concluded that this preparation can be effectively used to study drug effects on noradrenergic neurotransmission in relatively large arteries under various pathophysiological states by simultaneously measuring catecholamine release and vascular responsiveness.

The Role of Protein Kinase C and Calcium in the Regulation of Norepinephrine Release From the Vascular Adrenergic Neurons in Hypertension

This study was designed to investigate the role of protein kinase C and calcium in vascular adrenergic transmission in hypertension. In perfused mesenteric vasculatures of spontaneously hypertensive rats (SHR, 7 to 10 weeks old) and age-matched Wistar-Kyoto rats (WKY), we have examined the effects of the protein kinase C inhibitor H-7 on endogenous norepinephrine release and vascular responsiveness during nerve stimulation. Endogenous norepinephrine release and pressor responses during periarterial nerve stimulation were significantly greater in SHR than in WKY. The protein kinase C inhibitor H-7 inhibited the stimulation-induced norepinephrine release and pressor responses in a dose-dependent manner. The magnitude of these suppressive responses were more pronounced in SHR than in WKY. Calcium removal from extracellular fluid also reduced the norepinephrine release more strongly in SHR than in WKY. These results demonstrate that the regulation of norepinephrine release might be more dependent on protein kinase C and calcium in the blood vessels of SHR, which could contribute, at least partially, to the pathogenesis of this form of hypertension.

Age-related changes of in vivo β-adrenergic responsiveness in normotensive and spontaneously hypertensive rats

The variations of plasma cyclic AMP concentration caused by propranolol and isoproterenol were studied in order to investigate endogenous stimulation and responsiveness of the β-adrenoceptor-cyclic AMP system in vivo, in 7 and 18 week old unanaesthetized male SHR and WKY. In both age groups, the basal cyclic AMP level was higher in SHR than in controls but was significantly reduced to a comparable value in the two strains after intraperitoneal injection of 2.5 mg · kg −1 propranolol, a dose which markedly depressed or even abolished the effect of isoproterenol. Cumulative dose-response curves of plasma cyclic AMP concentration obtained in another group of rats after successive subcutaneous injections of isoproterenol showed that the ED 50 value of this drug was higher in SHR than in WKY and increased in a parallel manner in the two strains between 7 and 18 weeks of age (respectively from 0.21 ± 0.01 to 0.34 ± 0.02 μmol · kg −1 in WKY and from 0.32 ± 0.02 to 0.52 ± 0.03 μmol · kg −1 in SHR). At the same time blood pressure increased much less markedly in WKY (from 115 ± 4 to 119 ± 2 mm Hg) than in SHR (from 134 ± 3 to 179 ± 3 mm Hg). Altogether these results show that endogenou β-adrenergic stimulation of the cyclic AMP system was higher in SHR of both ages in spite of a diminished responsiveness of this system to exogenous isoproterenol.

Serum somatomedin activity and cartilage metabolism in acutely fasted, chronically malnourished, and refed rats.

Serum somatomedin activity (SM-act) and cartilage metabolism were compared in acutely fasted, marasmic (M), and marasmic kwashiorkor (MK) rats. SM-act was estimated in the porcine bioassay. In vitro uptake of [35S]sulfate and [3H]methylthymidine in costal cartilage of the experimental animals during an incubation in medium immediately after sacrifice, called endogenous activity, and the effect of incubation in 20% normal human plasma after a preincubation of 22 h in medium only, called plasma responsiveness", were determined. Acutely fasted rats had lowered SM-act and a circulating heat-labile inhibitor. Endogenous activity and responsiveness of cartilage were depressed. MK rats (fed ad libitum a 0.5% casein, isocaloric food) showed a profound depression of growth and cartilage endogenous activity despite only partially reduced SM-act and increased responsiveness. M rats received normal food and were pair-fed with MK rats, consuming approximately 0.08 g/g BW . day. They showed very depressed SM-act and low endogenous activity, and responsiveness was increased, though less than in the MK rats. On refeeding M rats, SM-act and cartilage responsiveness increased, followed by an increase of endogenous activity. Catch-up growth was best related to [3H]methylthymidine incorporation by cartilage (endogenous activity). In conclusion, these two types of experimental chronic malnutrition induce a more diversified pattern than does acute fasting. During malnutrition, cartilage metabolism does not reflect bioassayable SM-act of serum but rather the other effects of the nutritional insult. On refeeding, the expected relationship of SM-act and cartilage metabolism is rapidly restored.

12: Serum somatomedin (SM) and cartilage metabolism in malnourished and refed rats

SM and inhibitors were estimated by the porcine bio-assay in acutely fasted, marasmic (M) and marasmic-kwas-hiorkor (MK) rats. In vitro endogenous activity and responsiveness to SM was measured in their costal cartilage using s35-Sulfate and H3-thymidine. Acute fasting for 3-4 days resulted in complete loss of SM activity, partly caused by a heat-labile, non-dialysable inhibitor. Cartilage endogenous activity and responsiveness were depressed. M-rats (pairfed with MK, normal food, ± 4 g/day, from 25 days age) after 1, 2 and 5 weeks had SM potency ratios compared to controls of 0.8, 0, 0.15, with diminished cartilage endogenous activity. MK rats (ad lib. isocaloric 0,5% casein food) showed a profounder depression of growth and cartilage endogenous activity despite SM ratios (tos controls) of 0.34, 0.75 and 0.45 after 1, 2, and 5 weeks. Evidence for a circulating inhibitor was found. On refeeding M rats, SM and cartilage responsiveness increased by 24 hrs and cartilage endogenous activity by 48 hrs. However catch-up growth in length seen after 1 and 2 weeks was best reflected by H3-thymidine uptake. Clearly differences in SM activity and target organ metabolism follow different nutritional insults. Further studies of these models may clarify the relative roles of SM and inhibitors in malnutrition.