Deficiency of 5-lipoxygenase accelerates renal allograft rejection in mice.

Acute renal allograft rejection is associated with alterations in renal arachidonic acid metabolism, including enhanced synthesis of leukotrienes (LTs). LTs, the products of the 5-lipoxygenase (5-LO) pathway, are potent lipid mediators with a broad range of biologic activities. Previous studies, using pharmacological agents to inhibit LT synthesis or activity, have implicated these eicosanoids in transplant rejection. To further investigate the role of LTs in acute graft rejection, we transplanted kidneys from CByD2F1 mice into fully allogeneic 129 mice that carry a targeted mutation in the 5lo gene. Unexpectedly, allograft rejection was significantly accelerated in 5-LO-deficient mice compared with wild-type animals. Despite the marked reduction in graft survival, the 5lo mutation had no effect on the hemodynamics or morphology of the allografts. Although LTB4 levels were reduced, renal thromboxane B2 production and cytokine expression were not altered in 5-LO-deficient allograft recipients. These findings suggest that, along with their proinflammatory actions, metabolites of 5-LO can act to enhance allograft survival.

Domains of the parathyroid hormone (PTH) receptor required for regulation by G protein-coupled receptor kinases (GRKs).

To investigate the domains of the parathyroid hormone (PTH) receptor required for regulation by G protein-coupled receptor kinases (GRKs), we created mutant PTH receptors lacking potential GRK-phosphorylation sites. Mutant #1 was truncated at amino acid 544 and, therefore, lacked nine hydroxyl group-containing amino acids at the C-terminus. In mutant #2, we replaced threonines 392 and 399 in the third intracellular loop with glycines. Co-transfection of HEK293 cells with the wild-type receptor and either GRK2, GRK3, or GRK5 inhibited PTH-induced cyclic (cAMP) generation; co-transfection of GRK4 or GRK6 had no effect on PTH receptor responsiveness. GRK2-mediated inhibition of PTH receptor signaling was associated with enhanced phosphorylation receptor proteins. Co-expression of GRK2 similarly reduced PTH-induced cAMP generation by the wild-type receptor and mutant #1, and caused phosphorylation of receptor proteins to a similar extent. Co-expression of GRK2 had little effect on PTH-induced cAMP generation by mutant #2 but enhanced agonist-induced phosphorylation of mutant #2 compared with that of either the wild-type receptor or mutant #1. Enhanced phosphorylation of mutant #2 was associated with a reduction in agonist-induced internalization of mutant #2 compared with the wild-type receptor. Thus, phosphorylation of mutant #2 failed to cause receptor desensitization and inhibited receptor internalization. These data are consistent with the notion that: (a) GRKs contribute to regulating PTH receptor responsiveness, and (b) domains in the third intracellular loop are not required for agonist-induced phosphorylation of PTH receptors, but are critical for both agonist-induced internalization of PTH receptors and GRK2-mediated regulation of PTH receptor signaling.

The prostaglandin E2 EP1 receptor mediates pain perception and regulates blood pressure.

The lipid mediator prostaglandin E2 (PGE2) has diverse biological activity in a variety of tissues. Four different receptor subtypes (EP1-4) mediate these wide-ranging effects. The EP-receptor subtypes differ in tissue distribution, ligand-binding affinity, and coupling to intracellular signaling pathways. To identify the physiological roles for one of these receptors, the EP1 receptor, we generated EP1-deficient (EP1-/-) mice using homologous recombination in embryonic stem cells derived from the DBA/1lacJ strain of mice. The EP1-/- mice are healthy and fertile, without any overt physical defects. However, their pain-sensitivity responses, tested in two acute prostaglandin-dependent models, were reduced by approximately 50%. This reduction in the perception of pain was virtually identical to that achieved through pharmacological inhibition of prostaglandin synthesis in wild-type mice using a cyclooxygenase inhibitor. In addition, systolic blood pressure is significantly reduced in EP1 receptor-deficient mice and accompanied by increased renin-angiotensin activity, especially in males, suggesting a role for this receptor in cardiovascular homeostasis. Thus, the EP1 receptor for PGE2 plays a direct role in mediating algesia and in regulation of blood pressure.

Sensing of extracellular cations in CasR-deficient osteoblasts. Evidence for a novel cation-sensing mechanism.

We isolated osteoblastic cell lines from wild-type (CasR(+/+)) and receptor null (CasR(-/-)) mice to investigate whether CasR is present in osteoblasts and accounts for their responses to extracellular cations. Osteoblasts from both CasR(+/+) and CasR(-/-) mice displayed an initial period of cell replication followed by a culture duration-dependent increase in alkaline phosphatase activity, expression of osteocalcin, and mineralization of extracellular matrix. In addition, a panel of extracellular cations, including aluminum and the CasR agonists gadolinium and calcium, stimulated DNA synthesis, activated a transfected serum response element-luciferase reporter construct, and inhibited agonist-induced cAMP in CasR(-/-) osteoblasts. The functional responses to these cations were identical in CasR(+/+) and CasR(-/-) osteoblasts. Thus, the absence of CasR alters neither the maturational profile of isolated osteoblast cultures nor their in vitro responses to extracellular cations. In addition, CasR transcripts could not be detected by reverse transcription-polymerase chain reaction with mouse specific primers in either CasR(+/+) or CasR(-/-) osteoblasts, and immunoblot analysis with a CasR-specific antibody was negative for CasR protein expression in osteoblasts. The presence of a cation-sensing response in osteoblasts from CasR(-/-) mice indicates the existence of a novel osteoblastic extracellular cation-sensing mechanism.

Reproductive failure and reduced blood pressure in mice lacking the EP2 prostaglandin E2 receptor.

Prostaglandins (PGs) are bioactive lipids that modulate a broad spectrum of biologic processes including reproduction and circulatory homeostasis. Although reproductive functions of mammals are influenced by PGs at numerous levels, including ovulation, fertilization, implantation, and decidualization, it is not clear which PGs are involved and whether a single mechanism affects all reproductive functions. Using mice deficient in 1 of 4 prostaglandin E2 (PGE2) receptors -- specifically, the EP2 receptor -- we show that Ep2(-/-) females are infertile secondary to failure of the released ovum to become fertilized in vivo. Ep2(-/-) ova could be fertilized in vitro, suggesting that in addition to previously defined roles, PGs may contribute to the microenvironment in which fertilization takes place. In addition to its effects on reproduction, PGE2 regulates regional blood flow in various vascular beds. However, its role in systemic blood pressure homeostasis is not clear. Mice deficient in the EP2 PGE2 receptor displayed resting systolic blood pressure that was significantly lower than in wild-type controls. Blood pressure increased in these animals when they were placed on a high-salt diet, suggesting that the EP2 receptor may be involved in sodium handling by the kidney. These studies demonstrate that PGE2, acting through the EP2 receptor, exerts potent regulatory effects on two major physiologic processes: blood pressure homeostasis and in vivo fertilization of the ovum.

Aluminum is a weak agonist for the calcium-sensing receptor.

BACKGROUND: Aluminum (Al3+) has diverse biological effects mediated through activation of a putative extracellular cation-sensing receptor. A recently identified calcium-sensing receptor (CaSR), which has been identified in target tissues for Al3+, may transduce some of the biological effects of Al3+. METHODS: To test this possibility, we transfected human embryonic kidney 293 (HEK 293) cells with a cDNA encoding the rat CaSR and evaluated CaSR expression by Western blot analysis and function by measurement of intracellular calcium ([Ca2+]i) levels and inositol monophosphate (IP1) generation following stimulation with Al3+ and a panel of CaSR agonists. RESULTS: The CaSR protein was detected by immunoblot analysis in cells transfected with the CaSR cDNA but not in nontransfected HEK 293 cells. In addition, [Ca2+]i levels and IP1 generation were enhanced in a dose-dependent fashion by additions of the CaSR agonists calcium (Ca2+), magnesium (Mg2+), gadolinium (Gd3+), and neomycin only in cells transfected with CaSR. To determine if Al3+ activated CaSR, we stimulated cells transfected with rat CaSR with 10 microM to 1 mM concentrations of Al3+. Concentrations of Al3+ in the range of 10 microM to 100 microM had no effect on [Ca2+]i levels or IP1 generation. In contrast, 1 mM Al3+ induced small but significant increases in both parameters. Whereas Gd3+ antagonized calcium-mediated activation of CaSR, pretreatment with Al3+ failed to block subsequent activation of rat CaSR by Ca2+, suggesting a distinct mechanism of Al3+ action. CONCLUSION: Al3+ is not a potent agonist for CaSR. Because Al3+ affects a variety of target tissues at micromolar concentrations, it seems unlikely that CaSR mediates these cellular actions of Al3+.