Biomedical subjects

Y Zhang

Publications and source records attributed to Y Zhang.

At least 649 records · Page 36Linked to original sources

PubMed2001

A comparison of ovariectomy models for estrogen studies.

Many estrogen-replacement studies use ovariectomized animals as controls. However, ovariectomy greatly increases body weight and can enhance the peripheral synthesis of estrogen. Tamoxifen is commonly used as an antiestrogen, but it may elicit mixed agonist or antagonist actions. The aim of our study was to compare vascular function in mesenteric arteries among groups of rats with low estradiol levels. The groups (n = 5, each) of Sprague-Dawley rats were cycling (diestrus), ovariectomized (OVX), OVX + tamoxifen (OVX-T), OVX + 4-hydroxyandrostene-3,17-dione, an aromatase inhibitor (OVX-A) to prevent peripheral synthesis of estrogen, and control-fed OVX to prevent excess weight gain. Body weight was significantly elevated in only the non-control-fed OVX group. Estrogen levels were significantly greater in the cycling rats compared with the other groups, whereas uterine weights were significantly reduced in only the OVX-A and control-fed OVX groups. Methacholine relaxation was blunted only in the OVX-A and control-fed OVX groups, suggesting a possible estrogenic influence in the non-control-fed OVX and OVX-T groups. These data indicate the potential for confounding factors to decrease the efficacy of OVX controls.

Animals↗

PubMed2001

Vascular function in alcohol-treated pregnant and nonpregnant mice.

The effect of alcohol on maternal vascular adaptations to pregnancy is unknown. This study was designed to determine the effect of alcohol consumption on nitric oxide-mediated vascular function in mice during pregnancy. Female pregnant or nonpregnant C57BL/6J mice were fed a control diet or a liquid diet of 25% ethanol-derived calories for 13 days (from gestational days 6-18). Phenylephrine vasoconstriction was blunted in pregnancy compared with the nonpregnant state due to enhanced nitric oxide modulation, which was impaired by ethanol exposure. Although the EC50 and maximal responses to methacholine were not different in nonpregnant vs. pregnant mice, the nitric oxide component to methacholine-induced vasorelaxation was greater in the pregnant mice. Interestingly, alcohol affected only the pregnant animals in their response to methacholine. These data indicate that alcohol reduced the nitric oxide modulation of vascular response, which was more pronounced during pregnancy. These studies provide novel information regarding the effects of alcohol on the maternal vascular system during pregnancy and thereby contribute to further understanding of the adverse effects associated with prenatal alcohol exposure.

Animals↗

PubMed2001

Peroxisome proliferator-activated receptor-gamma activity is associated with renal microvasculature.

Peroxisome proliferator-activated receptor-gamma (PPAR gamma) is a nuclear transcription factor and the pharmacological target for antidiabetic thiazolidinediones (TZDs). TZDs ameliorate diabetic nephropathy and have direct effects on cultured mesangial cells (MCs); however, in situ hybridization failed to detect expression of PPAR gamma in glomeruli in vivo. The purpose of this study was to determine whether PPAR gamma is expressed in renal glomeruli. Two rabbit PPAR gamma isoforms were cloned. Nuclease protection assays demonstrate that both PPAR gamma isoforms are expressed in freshly isolated glomeruli. Treatment of rabbits with the TZD troglitazone selectively induced expression of an endogenous PPAR gamma target gene, adipocyte fatty acid-binding protein (A-FABP), in renal glomerular cells and renal medullary microvascular endothelial cells, demonstrated by both in situ hybridization and immunostain. Troglitazone also dramatically increased A-FABP expression in cultured MCs. Constitutive PPAR gamma expression was detected in cultured rabbit MCs. Endogenous MC PPAR gamma can also drive PPAR gamma reporter. Troglitazone and 15-deoxy-Delta 12,14 prostaglandin J(2) at low concentrations reduced mesangial cell [(3)H]thymidine incorporation without affecting viability. These data suggest that constitutive PPAR gamma activity exists in renal glomeruli in vivo and could provide a pharmacological target to directly modulate glomerular injury.

Amino Acid Sequence↗

PubMed2001

Urogenital distribution of a mouse membrane-associated prostaglandin E(2) synthase.

PGE(2) plays a critical role in regulating renal function and facilitating reproduction. One of the rate-limiting biosynthetic enzymes in PGE(2) synthesis is the terminal PGE(2) synthase (PGES). In the present studies, we report the functional expression of a membrane-associated murine PGES (mPGES) and its expression in urogenital tissues. Two independent cDNA clones sharing an identical open reading frame of 459 bp and encoding a peptide of 153 amino acids, but differing in the 3'-untranslated region, were identified. Assays for enzymatic activity, using microsomes prepared from cells transfected with mPGES cDNA, showed that these cDNA sequences encode a functional protein that catalyzes the conversion of PGH(2) to PGE(2). Constitutive expression of mPGES was highest in the mouse kidney, ovary, and urinary bladder but was also expressed at lower levels in uterus and testis. Renal mPGES expression was predominantly localized to epithelia of distal tubules and medullary collecting ducts. High expression was also seen in transitional epithelial cells of bladder and ureter and in the primary and secondary follicles in the ovary. In conclusion, mPGES is constitutively expressed along the urogenital tract, where it may have important roles in normal physiology and disease.

Amino Acid Sequence↗

PubMed2001

Hemodynamic characterization of arterialized and nonarterialized liver transplants in the rat.

Persistent hyperkinetic circulation after liver transplantation has been described in humans, but similar changes have not been well characterized in the rat model. This study aimed to investigate the hemodynamics of the systemic and splanchnic circulations in both arterialized and nonarterialized hepatic allografts. Orthotopic liver transplantation was performed in four groups of Sprague-Dawley rats. Group A comprised sham-operated rats with hepatic artery ligation that did not receive transplants; group B comprised rats that received transplants without arterialization; group C comprised sham-operated rats with intact hepatic artery that did not receive transplants; and group D comprised rats that received transplants with arterialization. Blood flow measurements were performed three weeks after the surgical procedure, using the radioactive microsphere method. The results showed that rats that received transplants exhibited a significantly higher cardiac index and lower systemic vascular resistance than the control rats. Splanchnic hyperemia was also present with increased mesenteric blood flow. However, there was no difference in hemodynamics between rats that received arterialized transplants and those that received nonarterialized transplants. Arterial collateral vessels from adjacent tissues were observed in the nonarterialized grafts; this was confirmed histologically. It is concluded that rats that undergo orthotopic liver transplantation exhibit hyperdynamic circulation, regardless of the arterial reconstruction procedure, possibly due to extensive collateral formation in the hepatosplanchnic circulation.

Animals↗

PubMed2001

Vitamin E inhibits proliferation of primary cultured human mesangial and endothelial cells.

BACKGROUND: Vitamin E (VE) has been used as an antioxidant and has been suggested to inhibit the proliferation of mesangial cells in rat and vascular endothelial cells. The direct effect of VE on primary cultures of mesangial cells (MC) and endothelial cells (EC) from the human glomerulus was studied. METHODS: (1) MC (in 17 or 2.5% FCS DMEM) or EC (in 10 or 5% FCS CSC) at 5,000 cells/well was incubated with serial concentrations of VE from 0.05 to 50 microg/ml (0.06 to 60 IU/l). (2) MC was cocultured with 160, 80, 40 or 20 microg/ml of low-density lipoprotein (LDL) or oxidized LDL (ox-LDL) in 17 or 2.5% FCS DMEM with or without VE. After 3 days of incubation at 37 degrees C in 5% CO(2), cell proliferation was measured by the Premix WST-1 Assay System. RESULTS: The concentration of VE that significantly inhibited the proliferation of MC cultured in 17 or 2.5% FCS DMEM was 50 or 2.5 microg/ml (60 or 3.0 IU/l), respectively, and that of EC in 10 or 5% FCS medium was 50 or 25 microg/ml (60 or 30 IU/l). VE at 25 microg/ml (30 IU/l) inhibited the LDL proliferative effect on MC cultured in 2.5 FCS DMEM by 21.79-93.21% in a LDL concentration-dependent manner. There was little difference between the effects of LDL and ox-LDL on the VE inhibitory effect on MC under our experimental conditions. CONCLUSION: VE at low concentrations had no effect on the proliferation of both MC and EC, but at high concentrations, it showed an inhibitory effect on both cells.

Cell Division↗

PubMed2001

Effects of alcohol on blood pressure and production of vascular aldosterone and corticosterone.

AIMS: The aims of this study were to search for the role of alcohol in blood pressure regulation in rats and to investigate the effects of alcohol on the production of vascular aldosterone and corticosterone. METHODS: Male Wistar rats received alcohol 0.7 g x kg(-1) x day(-1) (alcohol-treated group 1) or 1.4 g x kg(-1) x day(-1) (alcohol-treated group 2) or 2.1 g x kg(-1) x day(-1) (alcohol-treated group 3), orally, for 3 months, and blood pressure was monitored by a pressure transducer. Systolic blood pressure increased in Wistar rats treated with alcohol compared to control rats. Mesenteric artery perfusion ex vivo was performed and pressor responses to norepinephrine were determined. The pressor responses to norepinephrine in mesenteric arteries treated with alcohol were significantly increased. The perfusate from the mesenteric arteries was collected and applied to a Sep-Pak C 18 cartridge column for reverse-phase high-performance liquid chromatography and aldosterone and corticosterone levels were determined by radioimmunoassay, aldosterone was decreased but corticosterone was increased in the perfusate from arteries treated with alcohol. RESULTS: Reverse transcriptase polymerase chain reaction showed that alcohol inhibited the expression of 11beta-HSD2 and CYP11B2 mRNA in mesenteric arteries. CONCLUSIONS: These results reveal that alcohol is able to induce hypertension and provide evidence that alcohol inhibits the transcriptions of both 11beta-HSD2 and CYP11B2 in the vasculature, leading to lower aldosterone and higher corticosterone production in vessels, and increased vasoconstrictor responses to norepinephrine.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

PubMed2001

Effects of long-term enalapril and losartan therapy of hypertension on cardiovascular aldosterone.

BACKGROUND: Plasma aldosterone escape is found during long-term angiotensin-converting enzyme inhibitor therapy. Evidence for aldosterone production in cardiovascular tissues raised the question of whether or not aldosterone escape occurs in these tissues. METHOD: Spontaneously hypertensive rats were treated with enalapril (20 mg/kg/day) and losartan (50 mg/kg/day) for 20 weeks; untreated spontaneously hypertensive and Wistar rats were used as positive and normal controls, respectively. Ex vivo mesenteric artery and heart perfusion, high-performance liquid chromatography, and radioimmunoassay for aldosterone were performed. RESULTS: The results showed that enalapril failed to significantly inhibit aldosterone production in mesenteric artery, myocardium and plasma. Losartan significantly inhibited aldosterone production to that of Wistar rats in the mesenteric artery, myocardium and plasma. CONCLUSION: This study provides the first evidence that long-term angiotensin-converting enzyme inhibition therapy induces aldosterone escape in hypertensive cardiovascular tissues, and angiotensin II subtype 1 receptor antagonist does not induce aldosterone escape in mesenteric artery, myocardium and plasma of spontaneously hypertensive rats.

Aldosterone↗

PubMed2001

Localization, genomic organization, and alternative transcription of a novel human SAM-dependent methyltransferase gene on chromosome 2p22-->p21.

As part of our studies to identify the gene responsible for hereditary gingival fibromatosis, GINGF (OMIM 135300), we have identified and cloned a novel human gene that contains the highly conserved methyltransferase domain characteristic of S-adenosylmethionine-dependent methyltransferases. We localized this gene (C2orf8 encoding 288L6 SAM-methyltransferase) to chromosome 2p22-->p21 by FISH, and sublocalized it to BAC RP11 288L6 flanked by D2S2238 and D2S2331. Computational analysis of aligned ESTs identified ten exons in the hypothetical C2orf8 gene. Results of RACE analyses in placenta identified multiple transcripts of this gene with heterogeneity at the 5'-UTR. Alternative transcription and tissue specific expression of C2orf8 were detected by RT-PCR and Northern blot analyses. C2orf8 is expressed in a variety of tissues including brain, colon, gingiva, heart, kidney, liver, lung, placenta, small intestine, spleen, and thymus. Open reading frame analysis of the alternative transcripts identified a shared coding region spanning exons 6-10. This ORF consists of 732 nucleotides encoding a putative 244 amino acid protein. Bioinformational searches of both C2orf8 and the putative protein product identified three methyltransferase motifs conserved across many prokaryotic and eukaryotic species. Sequence analyses of C2orf8 excluded coding region mutations as causative of GINGF.

Chromosomes, Human, Pair 2↗

PubMed2001

Neuroprotection in transient focal brain ischemia after delayed intravenous administration of brain-derived neurotrophic factor conjugated to a blood-brain barrier drug targeting system.

BACKGROUND AND PURPOSE: Neuroprotection with brain-derived neurotrophic factor (BDNF) requires direct injection into the brain owing to poor transport of the neurotrophin through the blood-brain barrier (BBB) in vivo. The present studies investigate whether BDNF alone or conjugated to a BBB drug targeting system is neuroprotective in focal, reversible brain ischemia after delayed intravenous administration at 60 or 120 minutes after middle cerebral arterial occlusion. METHODS: BDNF was conjugated to the OX26 murine monoclonal antibody to the rat transferrin receptor, which undergoes transport into brain from blood via the BBB transferrin receptor transcytosis system. After a 1-hour occlusion of the middle cerebral artery in nitrous oxide- ventilated animals with normal blood sugar, the brain was reperfused, and either BDNF or the BDNF/OX26 conjugate was administered as a single intravenous injection at a dose of 50 microg per rat. RESULTS: After the intravenous administration of unconjugated BDNF, there was no neuroprotection on the basis of analysis of brain at either 24 hours or 7 days after a 1-hour middle cerebral arterial occlusion. In contrast, there was a 68% and 70% reduction in cortical stroke volume at 24 hours and 7 days, respectively, after intravenous administration of 50 microg per rat of the BDNF conjugate (P<0.01). No effects on subcortical stroke volume were observed. CONCLUSIONS: These studies demonstrate marked neuroprotection in focal, transient brain ischemia with a single, delayed intravenous injection of BDNF if the neurotrophin is conjugated to a BBB drug targeting system. The neuroprotection is long lasting and persists for at least 7 days after a 1-hour middle cerebral artery occlusion.

Animals↗

PubMed2001

Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression.

Excessive airway mucus is an important cause of morbidity and mortality in asthma, but the relationship between accumulation of mucus and goblet cell size, number, and function is incompletely understood. To address these questions, stored mucin in the epithelium and goblet cell size and number were measured morphometrically, and mucin gene expression was measured by polymerase chain reaction and immunohistochemistry in endobronchial biopsies from 13 subjects with mild and moderate asthma and from 12 healthy control subjects. Secreted mucin was measured in induced sputum. We found that stored mucin in the airway epithelium was three times higher than normal in the subjects with asthma (p < 0.005). Goblet cell size was similar in both groups, but goblet cell number was significantly higher in the subjects with asthma (93,043 +/- 15,824 versus 41,959 +/- 9,230/mm3, p < 0.05). In mild asthma (FEV1 > or = 80% pred, n = 7), the level of stored mucin was as high as in moderate asthma (FEV1 < 80% pred, n = 6), but the level of secreted mucin was significantly lower (28.4 +/- 6.3 versus 73.5 +/- 47.5 microg/ml, p < 0.05). Secreted mucin was inversely correlated with stored mucin for the whole asthma group (rs = -0.78, p = 0.007). MUC5AC was the predominant mucin gene expressed in healthy subjects and subjects with asthma, and MUC5AC protein was increased in the subjects with asthma. We conclude that even mild asthma is associated with goblet cell hyperplasia and increased stored mucin in the airway epithelium, whereas moderate asthma is associated with increased stored mucin and secreted mucin. These findings suggest that acute degranulation of hyperplastic goblet cells may represent a mechanism for asthma exacerbations in mild and moderate asthma and that chronic degranulation of goblet cells may contribute to chronic airway narrowing in moderate asthma.

Adult↗

PubMed2001

New models of the tracheal airway define the glandular contribution to airway surface fluid and electrolyte composition.

Antibacterial defenses in the airway are dependent on multifactorial influences that determine the composition of both fluid and/or electrolytes at the surface of the airway and the secretory products that aid in bacterial killing and clearance. In cystic fibrosis (CF), these mechanisms of airway protection may be defective, leading to increased colonization with Pseudomonas aeruginosa. Submucosal glands, a predominant site of cystic fibrosis transmembrane conductance regulator (CFTR) protein expression in the airway, have been hypothesized to play an important role in protection of the airway. Furthermore, recent studies have suggested that the salt concentration at the airway surface may be a key factor in regulating the activity of antibacterial substances in the airway. To explore these issues, we have used a new model of the ferret tracheal airway to evaluate the contribution of submucosal glands in regulating airway surface fluid and electrolyte composition. Using tracheal xenograft models with and without submucosal glands, we have characterized several aspects of airway physiology that may be important in defining antibacterial properties. These endpoints included the contribution of submucosal glands in defining bioelectric properties of the surface airway epithelium, airway surface fluid (ASF) chloride composition, ASF volumes, and secretion of the antibacterial factor lysozyme. Findings from these studies demonstrate a significantly elevated secreted fluid volume (Vs) and chloride concentration ([Cl](s)) in ASF from airways with submucosal glands (Vs = 47 +/- 4 microl; [Cl](s) = 128 +/- 5 mM), as compared with xenograft airways without glands (Vs = 36 +/- 2 microl; [Cl](s) = 103 +/- 6 mM). Furthermore, a temperature labile factor secreted by submucosal glands appears to alter the baseline activation of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and/or diphenylamine-2-carboxylic acid-sensitive chloride channels in the surface airway epithelium. Lastly, the lysozyme content of tracheal airways with submucosal glands was 8.5-fold higher than were airways without glands. These studies demonstrate that submucosal glands affect both the ionic composition and bioelectric properties of the airway and suggest that models evaluating antibacterial properties of the airway in CF should take into account the contribution of glands in airway physiology.

Animals↗

PubMed2001

Antibacterial activity of apical surface fluid from the human airway cell line Calu-3: pharmacologic alteration by corticosteroids and beta(2)-agonists.

Calu-3 cells, a human lung carcinoma cell line with properties like serous cells of the upper airway, were used to develop an in vitro model for airway antibacterial activity. Calu-3 cell monolayers were cultured on permeable supports at an air-liquid interface. Apical surface fluid (ASF) was collected by washing; antibacterial activity was assayed by incubating ASF washings with bacteria for 18 h and counting surviving colony-forming units. ASF washings killed Escherichia coli and Pseudomonas aeruginosa. Antibacterial activity was salt sensitive and dependent on protein concentration. After washing, approximately 30 h were required before antibacterial activity recovered to its initial level. After culturing with topical corticosteroids (budesonide, triamcinolone, or beclomethasone, 0.1 microg/ml for 48 h), ASF antibacterial activity was 4- to 10-fold greater than the ASF from control monolayers. The increase in antibacterial activity was dose-dependent. The beta(2)-agonists salbutamol and terbutaline (100 microg/ml for 48 h) decreased ASF antibacterial activity by 5- to 8-fold. The nonsteroidal anti-inflammatory agents ibuprofen and cromolyn sodium had no effect. Our results are most consistent with agonist-dependent changes in the composition of ASF antibacterial proteins. We conclude that Calu-3 cells synthesize and secrete antibacterial proteins and that clinical agents can alter these functions.

Adrenal Cortex Hormones↗

PubMed2001

The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation.

Atherosclerosis causes most acute coronary syndromes and strokes. The pathogenesis of atherosclerosis includes recruitment of inflammatory cells to the vessel wall and activation of vascular cells. CD44 is an adhesion protein expressed on inflammatory and vascular cells. CD44 supports the adhesion of activated lymphocytes to endothelium and smooth muscle cells. Furthermore, ligation of CD44 induces activation of both inflammatory and vascular cells. To assess the potential contribution of CD44 to atherosclerosis, we bred CD44-null mice to atherosclerosis-prone apoE-deficient mice. We found a 50-70% reduction in aortic lesions in CD44-null mice compared with CD44 heterozygote and wild-type littermates. We demonstrate that CD44 promotes the recruitment of macrophages to atherosclerotic lesions. Furthermore, we show that CD44 is required for phenotypic dedifferentiation of medial smooth muscle cells to the "synthetic" state as measured by expression of VCAM-1. Finally, we demonstrate that hyaluronan, the principal ligand for CD44, is upregulated in atherosclerotic lesions of apoE-deficient mice and that the low-molecular-weight proinflammatory forms of hyaluronan stimulate VCAM-1 expression and proliferation of cultured primary aortic smooth muscle cells, whereas high-molecular-weight forms of hyaluronan inhibit smooth muscle cell proliferation. We conclude that CD44 plays a critical role in the progression of atherosclerosis through multiple mechanisms.

Animals↗

PubMed2001

Quantitative effects of Nf1 inactivation on in vivo hematopoiesis.

The NF1 tumor-suppressor gene is frequently inactivated in juvenile myelomonocytic leukemia, and Nf1 mutant mice model this myeloproliferative disorder (MPD). Competitive repopulation assays were performed to quantify the proliferative advantage of Nf1(-/-) hematopoietic cells in vivo. Nf1 mutant stem cells demonstrated a growth advantage that was greatest in myeloid lineage cells and least pronounced in T lymphocytes. Surprisingly, although low numbers of Nf1-deficient cells consistently outcompeted wild-type cells, levels of chimerism were stable over months of observation, and MPD was not observed unless threshold numbers of mutant cells were injected. These data showing that normal competitor cells can strongly modulate the growth of mutant populations in vivo have general implications for modeling cancer in the mouse. In particular, strains in which cancer-associated mutations are expressed in fields of target cells may not accurately model early events in tumorigenesis because they eliminate the requirement for a mutant clone to outcompete resident normal cells.

Adoptive Transfer↗

PubMed2001

The influence of food on the bioavailability of a twice-daily controlled release carbamazepine formulation.

Carbatrol, a new dosage form of carbamazepine (CBZ), was developed consisting of three different types of pellets (immediate release, controlled release, and enteric release). The objective of this study was to explore the influence of food on absorption of CBZ. This was a randomized, open-label, single-dose crossover study conducted in 12 healthy volunteers. Treatments were 2 x 200 mg Carbatrol with a high-fat meal, fasted, or sprinkled over applesauce (but otherwise fasted). Each subject received one dose of each treatment separated by a washout period of at least 2 weeks. CBZ bioequivalence was established based on the equivalence of AUC (extent of absorption) in all three conditions. Carbatrol may be taken with or without food or the capsule opened and sprinkled on food.

Adolescent↗

PubMed2001

Differential patterns of cocaine-induced organ toxicity in murine heart versus liver.

To determine cocaine's toxicity in different organs, BALB/c mice were intraperitoneally injected daily for 15 days with either saline or cocaine: 10 mg/kg, 30 mg/kg, or 60 mg/kg. Cardiac function, hepatic pathophysiology, heart and liver apoptosis, and tumor necrosis factor (TNF-alpha) levels were analyzed. After administration of cocaine, cardiac function decreased. Inflammatory cell infiltration and eosinophilic contraction bands were visible in the hearts of mice treated with 60mg/kg cocaine. Moreover, histopathology demonstrated that cocaine caused hepatic necrosis. TdT-mediated dUTP nick end-labeling (TUNEL) staining and DNA ladder analysis indicated that cocaine caused apoptosis in both the heart and liver. Moreover, immunoassay showed that TNF-alpha levels significantly increased in the heart and liver with cocaine administration. However, our RT-PCR study showed that there was no significant difference in either the heart or liver in the levels of mRNA for TNF-alpha between cocaine-treated and saline control mice. The present study demonstrated that cocaine is toxic to multiple organs, and at low dose can induce hepatic damage without gross pathological injury to the heart. The results suggest that the liver is more sensitive than the heart to cocaine toxicity, and induction of apoptosis or TNF-alpha elevation may be a common mechanism responsible for cocaines toxicity.

Alanine Transaminase↗

PubMed2001

Phorbol ester- and growth factor-induced growth hormone (GH) receptor proteolysis and GH-binding protein shedding: relationship to GH receptor down-regulation.

GH signals by interacting with GH receptor (GHR). A substantial fraction of circulating GH complexes with GH-binding protein (GHBP), which corresponds to the GHR extracellular domain. GHBP is generated by 1) alternative splicing of a common GHR precursor messenger RNA to encode secreted GHBP (the source of the vast majority of GHBP in rodents); and 2) proteolysis of the cell-associated GHR with shedding of GHBP (a mechanism operative in rabbits and humans). We previously observed that phorbol ester (PMA)-induced activation of protein kinase C (PKC) causes metalloprotease-mediated GHR proteolysis and GHBP shedding in human IM-9 lymphocytes. We now demonstrate that PMA-induced hydroxamate (IC3)-inhibitable GHR proteolysis and GHBP shedding were also detected in murine 3T3-F442A and 3T3-L1 preadipocytes and in Chinese hamster ovary (CHO) cells stably expressing rabbit GHR (rbGHR), although the degree of GHBP shedding was much smaller for murine GHR than for rabbit or human GHRs. PMA-induced GHR proteolysis in 3T3-F442A, 3T3-L1, and CHO-rbGHR cells was significantly reduced by pretreatment with mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitors, suggesting involvement of the mitogen-activated protein kinase pathway in regulating this PKC-dependent effect. In contrast, GHR proteolysis promoted by N-ethylmaleimide, although inhibited by IC3, was unaffected by inhibition of either PKC or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1. Thus, different pathways leading to metalloprotease-mediated receptor proteolysis are accessed by PMA vs. N-ethylmaleimide. To determine whether other, possibly more physiologically relevant, stimuli induce GHR proteolysis, we tested effects of platelet-derived growth factor (PDGF) and serum. Treatment of serum-deprived cells with PDGF (in 3T3-F442A cells) or serum (in 3T3-F442A and CHO-rbGHR cells) promoted GHR proteolysis, which was inhibited by IC3. Interestingly, PMA-, PDGF-, and serum-induced GHR proteolysis was associated with substantial decreases in GH-induced activation of Janus kinase-2, which were also prevented by IC3. These findings suggest that inducible metalloprotease-mediated GHR proteolysis constitutes an important mechanism of receptor down-regulation and modulation of GH signaling.

3T3 Cells↗