Receptor number determines latency and amplitude of the thyrotropin-releasing hormone response in Xenopus oocytes injected with pituitary RNA

TRH evokes depolarizing membrane electrical responses in Xenopus laevis oocytes injected with RNA from pituitary cells. We have shown previously that the amplitude of this response is directly proportional to the dose of TRH and the amount of RNA injected. Herein we show that the number of TRH receptors expressed on oocytes after injection of rat pituitary (GH3) cell RNA or mouse thyrotropic (TtT) tumor RNA determines the latency as well as the amplitude of the response. In oocytes injected with a maximally effective amount of GH3 cell RNA, the latency of the response decreased from a maximal duration of 103 +/- 16 to 10 +/- 1 sec when the TRH concentration was increased from 5 to 3000 nM. When oocytes injected with different amounts of GH3 cell RNA were stimulated with 3000 nM TRH, the latency decreased from 31 +/- 4 to 11 +/- 0.5 sec when the amount of RNA injected was increased from 30 to 400 ng. Specific binding of [3H]methylhistidine-TRH increased when increasing amounts of TtT poly(A)+ RNA was injected, and binding correlated with increased response amplitude. To show that these effects were caused by mRNA for the TRH receptor and did not depend on other mRNAs, TtT poly(A)+ RNA was fractionated on a sucrose gradient. Using RNA from each fraction, there was an inverse correlation between response amplitude and latency. For size-fractionated RNA, as for unfractionated RNA, there was a direct correlation between specific [3H]methylhistidine-TRH binding and response amplitude.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repopulation and colonization by birds in the Agmon Wetland,Israel

Lake Hula and its surrounding wetland in northern Israel were drained in the late 1950s and the dried wetlands were transformed into a diverse agricultural region with a 0.3 km2 nature reserve. A portion of the extinct Hula wetland was re-flooded in April 1994 by constructing a small lake, Agmon, and 90 km of canals. The purposes of this study were (a) to document the re-population and colonization of the new Agmon wetland by birds after its flooding, (b) to evaluate bird species richness in this new wetland in comparison to that in a nearby mature Hula Nature Reserve, and, (c) to investigate if the species originally present in the Hula Valley before the drainage had been re-established. The new lake has succeeded in attracting a large variety of water birds to the newly formed habitats, especially ducks, egrets and herons, plovers, waders and snipes. The distribution of bird species among the different habitats was not random. The northern section of the lake, which is shallow and has an open shore, had the largest number of species during 1995 and 1996 and was preferred by plovers, waders and snipes. A large roosting and breeding colony of thousands of egrets and herons was formed in the dense cattails ( Typha domingensis) in the southern section of the lake. Unlike the nearby Hula Nature Reserve, the new wetland lacks a large seasonally flooded area and is less attractive for feeding and breeding plovers, waders and stilts. The new wetland also has very little muddy area without vegetation and attracts few of the species that regularly feed in this habitat in the reserve. The new wetland enlarges the area of only a few of the habitats found in the nearby reserve and therefore attracts fewer species than it might otherwise. Recommendations for management of the new wetland include increased diversity of habitats, restriction of human activities and optimization of conflicts that have arisen between the needs of the wetland and those of nearby agriculture.     

Nonopioid Effect of Morphine on Electrically Evoked Acetylcholine Release from Torpedo Electromotor Neurons

The release of acetylcholine from Torpedo electric organ slices following their electrical stimulation was modulated by morphine, by the muscarinic antagonist atropine, and by the nicotinic antagonist tubocurarine. Addition of either atropine or tubocurarine in the presence of the acetylcholinesterase inhibitor phospholine iodide enhanced acetylcholine release. The effects of the two antagonists were additive, a result suggesting that the secreted acetylcholine regulates its own release by activating both muscarinic and nicotinic cholinergic receptors and that these receptors inhibit acetylcholine release by different mechanisms. The effects of opiates on acetylcholine release were examined under conditions in which the cholinergic modulation of release is blocked, i.e., in the presence of atropine and tubocurarine. These experiments revealed that electrically evoked release of acetylcholine is blocked by the opiate agonists morphine and levorphanol. However, the inhibitory effect of morphine on acetylcholine release was not reversed by the opioid antagonist naloxone. Furthermore, dextrorphan, the nonopioid stereoisomer of levorphanol, had the same inhibitory effect as its opioid counterpart. These findings suggest that the effects of opiates on electrically evoked release of acetylcholine are not mediated by opioid receptors. The possible mechanisms underlying these nonopioid effects of morphine and levorphanol are discussed.     

Activation of two different receptors mobilizes calcium from distinct stores in Xenopus oocytes.

Acetylcholine (ACh) and thyrotropin-releasing hormone (TRH) utilize inositol 1,4,5-trisphosphate (IP3) as a second messenger and evoke independent depolarizing membrane electrical responses accompanied by characteristic 45Ca efflux profiles in Xenopus laevis oocytes injected with GH3 pituitary cell mRNA. To determine whether this could be accounted for by mobilization of calcium from functionally separate stores, we measured simultaneously 45Ca efflux and membrane electrical responses to ACh and TRH in single oocytes. We found that depletion of ACh-sensitive calcium store did not affect the membrane electrical response to TRH and the TRH-evoked 45Ca efflux. Our data suggest that ACh and TRH mobilize calcium from distinct cellular stores in the oocyte. This is the first demonstration in a single cell of strict subcellular compartmentalization of calcium stores coupled to two different populations of cell membrane receptors that utilize the same second messenger.     

Constitutive signaling by Kaposi's sarcoma-associated herpesvirus G-protein-coupled receptor desensitizes calcium mobilization by other receptors

We coexpressed Kaposi's sarcoma-associated herpesvirus G protein-coupled receptors (KSHV-GPCRs) with thyrotropin-releasing hormone (TRH) receptors or m1-muscarinic-cholinergic receptors in Xenopus oocytes and in mammalian cells. In oocytes, KSHV-GPCR expression resulted in pronounced (81%) inhibition (heterologous desensitization) of Ca(2+)-activated chloride current responses to TRH and acetylcholine. Similar inhibitions of cytoplasmic free Ca(2+) responses to TRH were observed in human embryonic kidney HEK 293 EM cells and in mouse pituitary AtT20 cells. Further study of oocytes showed that this inhibition was partially reversed by interferon-gamma-inducible protein 10 (IP-10), an inverse agonist of KSHV-GPCR. The basal rate of (45)Ca(2+) efflux in oocytes expressing KSHV-GPCRs was 4.4 times greater than in control oocytes, and IP-10 rapidly inhibited increased (45)Ca(2+) efflux. In the absence of IP-10, growth-related oncogene alpha caused a further 2-fold increase in (45)Ca(2+) efflux. In KSHV-GPCR-expressing oocytes, responses to microinjected inositol 1,4,5-trisphosphate were inhibited by 74%, and this effect was partially reversed by interferon-gamma-inducible protein 10. Treatment with thapsigargin suggested that the pool of calcium available for mobilization by TRH was decreased in oocytes coexpressing KSHV-GPCRs. These results suggest that constitutive signaling by KSHV-GPCR causes heterologous desensitization of responses mediated by other receptors, which signal via the phosphoinositide/calcium pathway, which is caused by depletion of intracellular calcium pools.     

PAR-3 Knockdown Enhances Adhesion Rate of PANC-1 Cells via Increased Expression of Integrinαv and E-Cadherin

The balance between the adhesion of cancer cells to extracellular matrix and their migratory potential, as well as their proteolytic activity, are important parameters that determine cancer cells invasiveness and metastasis. Since thrombin has been implicated in cancer progression, we studied the role(s) of thrombin-activated receptors in the adhesion process. We stably knocked down proteinase-activated receptors (PARs) -1, or -3 in human pancreatic adenocarcinoma PANC-1 cells. PANC-1 cells exhibit rapid adhesion to cell culture treated plastic and much faster kinetics of adhesion to Matrigel coated surface. Knockdown of PAR-1 had no effect on cells'' adhesiveness, while PAR-3 knockdowns (KDs) exhibited much faster adhesion kinetics. PAR-3 KDs also exhibited slower in vitro wound closure than vector-control and PAR-1 KD cells. To study the molecular mechanism(s) of PAR-3 KD cells'' enhanced rate of adhesion, we assayed the expression of the molecules that mediate cell-surface and cell-cell adhesion. ITGαv, as well as ITGα6 and ITGα10 mRNAs, were greatly enriched (>40-fold) in a rapidly-adhering sub-population of PAR-3 KD cells. The whole population of both PAR-1 and -3 KDs exhibited enhanced expression of a number of integrins (ITGs) mRNAs. However, ITGαv mRNA and protein expression was increased in PAR-3 KD and markedly decreased in PAR-1 KD. PAR-3 KD cells also expressed more E-cadherin mRNA and protein. The enhanced adhesion kinetics of PAR-3 KDs was almost fully inhibited by calcium chelation, or by a HAV-motive decapeptide that affects E-cadherin intermolecular interactions. We propose that the enhanced rate of adhesion of PAR-3 KDs results from enhanced expression of E-cadherin, leading to a greater adhesion of free-floating cells to cells rapidly bound to the surface via their integrins, and particularly ITGαv.     

Plasminogen-induced aggregation of PANC-1 cells requires conversion to plasmin and is inhibited by endogenous plasminogen activator inhibitor-1

PANC-1 cells express proteinase-activated receptors (PARs)-1, -2, and respond to their activation by transient elevation of cytosolic [Ca(2+)] and accelerated aggregation (Wei et al., 2006, J Cell Physiol 206:322-328). We studied the effect of plasminogen (PGN), an inactive precursor of the PAR-1-activating protease, plasmin (PN) on aggregation of pancreatic adenocarcinoma (PDAC) cells. A single dose of PGN time- and dose-dependently promoted PANC-1 cells aggregation in serum-free medium, while PN did not. PANC-1 cells express urokinase plasminogen activator (uPA), which continuously converted PGN to PN. This activity and PGN-induced aggregation were inhibited by the uPA inhibitor amiloride. PGN-induced aggregation was also inhibited by alpha-antiplasmin and by the PN inhibitor epsilon-aminocaproic acid (EACA). Direct assay of uPA activity revealed very low rate, markedly enhanced in the presence of PGN. Moreover, in PGN activator inhibitor 1-deficient PANC-1 cells, uPA activity and PGN-induced aggregation were markedly potentiated. Two additional human PDAC cell lines, MiaPaCa and Colo347, were assayed for PGN-induced aggregation. Both cell lines responded by aggregation and exhibited PGN-enhanced uPA activity. We hypothesized that the continuous conversion of PGN to PN by endogenous uPA is limited by PN's degradation and negatively controlled by endogenously produced PAI-1. Indeed, we found that PANC-1 cells inactivate PN with t1/2 of approximately 7 h, while the continuous addition of PN promoted aggregation. Our data suggest that PANC-1 cells possess intrinsic, PAI-1-sensitive mechanism for promotion of aggregation and differentiation by prolonged exposure to PGN and, possibly, additional precursors of PARs agonists.