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PubMed1984

The effect of phenformin on insulin-stimulated isoaminobutyric acid (AIB) transport by isolated hepatocytes.

Hepatocytes isolated from adult rat livers by collagenase perfusion were used to investigate the effect of phenformin on amino acid transport by measuring the uptake of aminoisobutyric acid (AIB). At concentrations between 0.01 and 100 micrograms/ml, phenformin hydrochloride did not affect AIB uptake, but concentrations of 500 and 1000 micrograms/ml were inhibitory. Incubating hepatocytes with insulin increased their accumulation of AIB. Inclusion of up to 100 micrograms/ml, phenformin hydrochloride during maximal and submaximal stimulation by insulin did not affect AIB uptake, but inhibition occurred with 500 and 1000 micrograms/ml. The continued presence of phenformin after addition of AIB was not required in order to produce inhibition. Furthermore, increasing the duration of exposure of hepatocytes to phenformin increased the degree of inhibition, suggesting that it is a non-competitive inhibitor. We conclude that phenformin does not enhance the sensitivity of hepatocytes to insulin and that at higher concentrations it may exert an inhibitory effect on basal and insulin stimulated aminoacid transport.

Aminoisobutyric Acids↗
PubMed1988

Insulin and insulin-like growth factor I regulate the same biological functions in HEP-G2 cells via their own specific receptors.

The receptors for insulin and insulin-like growth factor I (IGF-I) are closely related molecules, with an extracellular binding domain and an intracellular tyrosine kinase domain. The interaction of insulin and IGF-I with their respective receptors activates the receptor kinase domain, leading to the biological actions of the hormones. Since insulin generally regulates metabolic events and IGF-I generally regulates growth events, it is believed that structural differences in the tyrosine kinase domains of the two respective receptors may elicit different biological responses via different transmembrane signaling mechanisms. We studied the regulation of glycogen metabolism and amino acid uptake in human cultured HEP-G2 hepatoma cells, which have distinct receptors for both insulin and IGF-I. The receptor specificity of these responses was probed with specific monoclonal antibodies to both the insulin and IGF-I receptors. Stimulation of both [3H]glucose incorporation into glycogen and alpha-[3H]aminoisobutyric acid uptake by insulin was half-maximal at concentrations of 1-5 nmol/L. These effects were blocked by the insulin receptor monoclonal antibody MA-10, but not by the IGF-I receptor antibody alpha IR-3. Stimulation of both functions by IGF-I was half-maximal at concentrations of 1-5 nmol/L, and these effects were inhibited by alpha IR-3, but not by MA-10. These studies indicate that in HEP-G2 cells both insulin and IGF-I, via their own receptors, stimulate the same biological responses.

Amino Acids↗
PubMed1990

Effect of sepsis on amino acid transport system A and its response to insulin in incubated rat skeletal muscle.

The effect of sepsis on neutral amino acid transport systems A, ASC, and L, was studied in incubated rat soleus (SOL) muscles. We also examined the effects of plasma from septic rats and of varying concentrations of insulin (10 to 10(5) microU/mL), added in vitro to incubated muscles, on system A amino acid transport. Sepsis was induced by cecal ligation and puncture (CLP) in rats weighing 40 to 60 g. Control rats were sham-operated. System A activity was assessed by determining uptake of 2-(methylamino)isobutyrate (MeAIB) 16 hours after CLP or sham-operation. System ASC was studied by measuring uptake of alpha-aminoisobutyric acid (AIB) in the presence of 25 mmol/L MeAIB and 25 mmol/L 2-amino-2-norbornane carboxylic acid (BCH) to inhibit uptake by systems A and L. System L activity was defined as sodium-independent uptake of cycloleucine. MeAIB uptake was reduced by 28% in muscles of septic rats, while amino acid transport by systems ASC and L was almost identical in muscles from control and septic rats. Addition of plasma from septic rats to incubated normal SOL muscles inhibited MeAIB uptake by 31%. Addition of insulin to the incubation medium resulted in increased uptake of MeAIB, both in nonseptic and septic muscle. The lowest hormone concentration tested that significantly enhanced MeAIB uptake in nonseptic muscle was 10(2) microU/mL and in septic muscle 10 microU/mL. The results suggest that sepsis in rats specifically inhibits amino acid transport system A and that reduced muscle amino acid uptake may be caused by a circulating factor in sepsis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1991

Thyroid autoregulation: evidence for an action of iodoarachidonates and iodide at the cell membrane level.

Iodolipids are the possible mediators of excess iodide in thyroid autoregulation. Previous work from our laboratory has shown that 14-iodo-15-hydroxy-5,8,11 eicosatrienoic acid (I-HO-A) and its omega lactone (IL-w) mimic the inhibitory action of excess iodide upon several parameters of thyroid metabolism. The present experiments were performed in order to study the mechanism of the inhibitory effect of I-HO-A and IL-w on 2-deoxy-D-glucose (DOG) and aminoisobutyric acid (AIB) uptake by calf slices. I-HO-A, IL-w and KI 0.1 mM caused a 33, 31 and 25% inhibition, respectively, of AIB uptake. The presence of 0.1 mM methimazole (MMI) only reversed the effect of KI. The transport of DOG was inhibited by both compounds: I-HO-A caused a 62% decrease, while IL-w produced a 64% inhibition; and MMI failed to relieve their action. On the contrary, the 33% inhibition caused by KI disappeared when MMI was present. Taking into account that AIB and DOG transport across the membrane requires energy, supplied by Na-K-ATPase, changes in its activity were studied. TSH (10 mU/ml) produced a 74% increase in the enzyme activity which was significantly blocked by KI (82%), I-HO-A (100%) and IL-w (100%). Basal enzyme activity was impaired by IL-w (33%), but not by KI. These results were correlated with the decrease of DOG uptake produced by 1 mM ouabain. Tissue specificity effect of iodoarachidonates was demonstrated by the absence of action on DOG transport in kidney and liver.(ABSTRACT TRUNCATED AT 250 WORDS)

8,11,14-Eicosatrienoic Acid↗
PubMed1994

Intracarotid infusion of RMP-7, a bradykinin analog: a method for selective drug delivery to brain tumors.

The bradykinin analog, RMP-7, was investigated for its ability to selectively increase uptake of molecular tracers in RG2 glial tumors. When infused in low doses (0.1 microgram/kg/min) through the intracarotid artery ipsilateral to RG2 gliomas in rats, RMP-7 significantly increased the permeability of tumor capillaries to methotrexate and to four other tracers of varying molecular weights, compared to intracarotid infusion of vehicle alone. Tracers used to examine permeability included radiolabeled alpha-aminoisobutyric acid (M(r) 103 D), sucrose (M(r) 342 D), methotrexate (M(r) 454.5 D), inulin (M(r) 5000 D), and dextran (M(r) 70,000 D). Permeability was expressed as the unidirectional transfer constant, Ki (microliters/gm/min). The permeability (Ki) of tumors in the RMP-7 group compared to the vehicle control group was as follows: alpha-aminoisobutyric acid, 35.3 +/- 9.11 versus 12.7 +/- 4.56 (p < 0.001); sucrose, 16.5 +/- 3.83 versus 9.28 +/- 3.12 (p < 0.05); methotrexate, 26.3 +/- 10.3 versus 8.98 +/- 6.78 (p < 0.005); inulin, 13.5 +/- 3.23 versus 6.55 +/- 4.32 (p < 0.005); dextran, 15.2 +/- 3.42 versus 1.47 +/- 1.24 (p < 0.001). The permeability of RG2 gliomas to high-molecular-weight dextran (70,000 D) was 10.3-fold higher in the RMP-7 group than in the vehicle control group. Intracarotid infusion of RMP-7 did not significantly increase the blood volume in tumor or brain tissue. The permeability of normal brain capillaries was unaffected by intracarotid infusion of 0.1 microgram/kg/min RMP-7 relative to that achieved in tumor. These data support the idea that intracarotid infusion of RMP-7 will be a useful technique for selective delivery of antitumor compounds to brain tumors.

Animals↗
PubMed1992

Insulin-like growth factor-I stimulates amino acid accumulation in cultured human granulosa cells.

In order to study the effect of insulin-like growth factor (IGF)-I on amino acid transport, granulosa-luteal cells were obtained from stimulated cycles in women undergoing in-vitro fertilization. The cells were precultured for 1-3 days in serum-containing medium. The medium was then changed to serum-free with added IGF-I and/or human chorionic gonadotrophin (HCG). Following 24 h culture, the cellular uptake of [14C]alpha-aminoisobutyric acid AIB (0.1 mM, 0.6 mCi/ml) was studied during a 2-6 h incubation. The results showed that IGF-I (10-100 ng/ml) consistently stimulated AIB uptake to levels which were 44 +/- 7% above the control (n = 11 experiments). The stimulatory effect of IGF-I was abolished by HCG and was reduced by IGF-binding protein. In conjunction with previous findings that IGF-I stimulates the uptake and incorporation of thymidine into DNA, these results suggest that IGF-I is involved in growth of human granulosa-luteal cells.

Amino Acids↗
PubMed1986

Effect of endotoxic shock on kinetics of system A amino acid transport in rat soleus muscle.

This study evaluated the kinetics of system A amino acid transport in soleus muscle during endotoxic shock. Fasted male rats (80-100 g) were killed by decapitation 5 h after the intravenous injection of saline (control) or 20 mg/kg Salmonella enteritidis endotoxin. Soleus muscles (40 mg) from control or endotoxic rats were incubated in oxygenated Krebs-Ringer-bicarbonate buffer (pH 7.4) at 37 degrees C for 20, 40, or 60 min. Initial rates of entry of alpha-aminoisobutyric acid (AIB) into the inulin-inaccessible space of muscles were measured over a wide range of AIB concentrations (0.1-20.0 mM). Initial rates of AIB transport were separated into two parallel transport processes, a saturable carrier-mediated component and a nonsaturable passive diffusive process. The diffusion constant for the nonsaturable process in the endotoxic muscles (0.36 h-1) was 38% greater than control muscles (0.26 h-1). The maximum rate of AIB transport decreased from 55.6 +/- 3.3 nmol . g dry wt-1 . min-1 in controls to 18.3 +/- 1.9 nmol . g dry wt-1 . min-1 in endotoxic muscles. The apparent Km for saturable AIB transport was not different between control (1.68 +/- 0.12 mM) and endotoxic muscles (1.64 +/- 0.19 mM). Alterations of system A amino acid transport in endotoxic soleus muscles were due to an increase in passive AIB diffusion and a decrease in the number and/or activity of existing carriers and not to a change in carrier affinity for AIB. These results elucidate the mechanism for the decreased amino acid transport (system A) by soleus muscle in endotoxic shock.

Amino Acids↗
PubMed1979

Classification of microbial, plant and animal cytolysins based on their membrane-damaging effects of human fibroblasts.

38 cytolytic agents of mainly microbial origin were investigated with respect to membrane-damaging activity on human diploid fibroblasts. Increased plasma membrane permeability was measured as leakage of three defined cytoplasmic markers of various sizes: alpha-aminoisobutyric acid, uridine nucleotides and ribosomal RNA. The relative leakages of these markers, caused by different concentrations of the various cytolysins, yielded a leakage pattern for each substance. Five distinct types of leakage patterns were obtained. These were transformed into numerical expressions by calculating the ratios between the amounts of cytolysin needed to release 50% of the nucleotide and ribosomal RNA markers and the amounts required to release 50% of the alpha-aminoisobutyric acid marker (ED50 ratios). A classification of the cytolysins into five groups was arrived at on the basis of the different types of leakage patterns with the aid of reference cytolysins with well-known mechanisms of membrane interaction. These groups comprised: (1) detergent-like agents, (2) agents interacting with only certain constituents of the cell membrane, (3) agents interacting with specific receptor molecules in the membrane, (4) agents inducing small functional holes of a definable size, and (5) agents inducing only a very limited increase in plasma membrane permeability. The system may be useful for characterization and differentiation of new cytolytic agents of various sources as it divides membrane-damaging agents into separate groups on the basis of their principal function on intact human cells.

Animals↗
PubMed1976

Development of responsiveness of young normal rats to growth hormone.

Growth hormone (GH) exerts a biphasic effect on the membrane transport of amino acids and sugars in diaphragms from hypophysectomized rats. A stimulatory (insulin-like) effect of GH is observed for approximately 3 hr after the administration of the hormone, and then the diaphragm becomes "refractory" to further administration of the hormone for 24-48 hr. In the present study the in vitro responsiveness of diaphragms from young normal rats of different ages to bovine growth hormone (bGH) was studied by measuring the accumulation of alpha-aminoisobutyric acid (AIB), cycloleucine, and 3-0-methyl-glucose (3-OMG) and by determining the incorporation of phenylalanine into diaphragm proteins. bGH increased the uptake of AIB in 6, 10, 14, 18, and 22-day old and of cycloleucine in 10, 14, and 18-day old rats, respectively. The maximal stimulatory effect was seen in 18-day old rats and no effects were seen in 26 and 30-day old animals. A similar pattern of age-related responsiveness was also seen when the uptake of 3-OMG and the incorporation of phenylalanine were studied. The rate of accumulation of the nonutilizable amino acids, as well as the rate of incorporation of phenylalanine, decreased markedly with age. Fasting the rats for 20 hr did not change the principal age-related responsiveness of AIB transport to GH, but the magnitude of the hormone effect increased due to a lower uptake of AIB in control diaphragms. bGH had a dose-dependent effect on the accumulation of AIB in fasted 18-day old rats, the threshold concentration of bGH being 0.1 mug/ml. When diaphragms from 18-day old rats were incubated with bGH for various periods, the uptake of AIB was stimulated between 0 and 60 and 120 and 180 min, but not between 60 and 120 min after the start of the incubation. This observation indicated that the muscles were "refractory" to GH between 60 and 120 min. Preincubation of diaphragms from 21-day old rats with bGH for 3 hr did not inhibit the stimulatory effect of bGH added subsequently indicating that the duration of the "refractory phase" did not exceed 270 min in this group of rats. When diaphragms from fasted rats of different ages were preincubated in Krebs bicarbonate buffer for 3 hr, a stimulatory effect of bGH on AIB uptake was also observed in 26 and 30-day old rats, possibly because the influence of endogenous GH subsided during the preincubation period. These results suggest that diaphragms from "old" normal rats are insensitive to exogenous GH because the "refractory phases" become considerably prolonged with age.

Age Factors↗
PubMed2000

Hypoxia inhibits amino acid uptake in human lung fibroblasts.

Hypoxia and amino acid deprivation downregulate expression of extracellular matrix genes in lung fibroblasts. We examined the effect of hypoxia on amino acid uptake and protein formation in human lung fibroblasts. Low O(2) tension (0% O(2)) suppressed incorporation of [(3)H]proline into type I collagen without affecting [(35)S]methionine labeling of other proteins. Initial decreases in intracellular [(3)H]proline incorporation occurred after 2 h of exposure to 0% O(2), with maximal suppression of intracellular [(3)H]proline levels at 6 h of treatment. Hypoxia significantly inhibited the uptake of radiolabeled proline, 2-aminoisobutyric acid (AIB), and 2-(methylamino)isobutyric acid (methyl-AIB) while inducing minor decreases in leucine transport. Neither cycloheximide nor indomethacin abrogated hypoxia-related suppression of methyl-AIB uptake. Efflux studies demonstrated that hypoxia inhibited methyl-AIB transport in a bidirectional fashion. The downregulation of amino acid transport was not due to a toxic effect; function recovered on return to standard O(2) conditions. Kinetic analysis of AIB transport revealed a 10-fold increase in K(m) accompanied by a small increase in maximal transport velocity among cells exposed to 0% O(2). These data indicate that low O(2) tension regulates the system A transporter by decreasing transporter substrate affinity.

Amino Acids↗
PubMed1985

Distinct biologically active receptors for insulin, insulin-like growth factor I, and insulin-like growth factor II in cultured skeletal muscle cells.

The expression of insulin-like growth factor (IGF) receptors at the cell surface and the changes in IGF responsiveness during differentiation were studied in the L6 skeletal muscle cell line. Throughout the entire developmental sequence, distinct receptors for IGF I and IGF II that differed in structure and peptide specificity could be demonstrated. During differentiation, both 125I-IGF I and 125I-IGF II binding to the L6 cells decreased as a result of a 3-4-fold reduction in receptor number, whereas 125I-insulin binding increased. Under nonreducing conditions, disuccinimidyl suberate cross-linked 125I-IGF I and 125I-IGF II to two receptor complexes with apparent Mr greater than 300,000 (type I) and 220,000 (type II). Under reducing conditions, the apparent molecular weight of the type I receptor changed to Mr 130,000 (distinct from the 120,000 insulin receptor) and the type II receptor changed to 250,000. IGF I and IGF II both stimulated 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake in the L6 cells with a potency close to that of insulin, apparently through interaction with their own receptors. The stimulatory effects of IGF II correlated with its affinity for the type II but not the type I IGF receptor, as measured by inhibition of affinity labeling, whereas the effects of IGF I correlated with its ability to inhibit labeling of the type I receptor. In spite of the decrease in type I and type II receptor number, stimulation of 2-deoxy-glucose and alpha-aminoisobutyric acid uptake by the two IGFs increased during differentiation.

Affinity Labels↗
PubMed1982

Distribution of some non-physiological amino acids (AIB, DAB, MeAIB) in mice.

The distribution of labelled 2-aminoisobutyric acid (AIB), 2,4-diaminobutyric acid (DAB) and 2-(methylamino)-isobutyric acid (MeAIB) was studied by whole body autoradiography in mice. All the amino acids were rapidly cleared from the blood and were taken up in most of the organs in the body. The results suggest the presence of operating transport systems for these amino acids into the majority of different cell types in mice. In the central nervous system, small amounts could be registered and in the pancreas there was a marked difference between DAB, which was not taken up, and the other two amino acids which were heavily accumulated. In an early embryo only MeAIB was observed in a high concentration.

Aminobutyrates↗
PubMed1990

Interference with hepatocellular substrate uptake by 1,1,1-trichloroethane and tetrachloroethylene.

The effects of chlorinated aliphatic hydrocarbon solvent exposure on hepatocellular transport of some model substrates have been investigated. Exposure of isolated hepatocytes to 1,1,1-trichloroethane or tetrachloroethylene resulted in suppression of uptake of taurocholate, ouabain, and 2-aminoisobutyric acid but not CdCl2 or 3-O-methyl-D-glucose. The effect was clearly evident at noncytotoxic concentrations, as indicated by the lack of intracellular enzyme leakage and unaltered intracellular K+ ion concentration. Moreover, the ultrastructure of solvent-exposed hepatocytes was similar to that of control cells, except for a reduction in membrane microvilli. The suppression of uptake was reversible provided that sufficient time was allowed for the cells to recover. The mechanism of this inhibition may be associated with energy-linked processes, as uptake of taurocholate, ouabain, and 2-aminoisobutyric acid is energy requiring while uptake of CdCl2 and 3-O-methyl-D-glucose is not. Cellular ATP was reduced in a dose-dependent manner, but a marked depletion occurred only at cytotoxic concentrations. Na(+)-K(+)- and Mg2(+)-ATPase activities in hepatocyte plasma membrane preparations were also inhibited by solvent exposure. The data suggest that 1,1,1-trichloroethane and tetrachloroethylene interfere specifically with energy-dependent hepatic transport functions and that a decrease in ATP levels and/or inhibition of cell membrane ATPases may be the mechanism.

Animals↗
PubMed1983

[Patterns of urinary aminoacid excretion in exceptional children and patients with mental disorders in Costa Rica].

The amino acid profile was studied in individual random samples of urine from 1147 normal schoolchildren and 1074 exceptional children: 628 with mental retardation, 332 with hearing and speech defects and 114 with visual defects as well as in 673 patients with mental disorders. Laboratory procedures included chemical tests and one-dimension paper- electro- and column-chromatography. Phenylketonuria was found in a mentally retarded girl and in one of her brothers; iminoglycinuria in a mentally retarded boy and heterozygote cystinuria in a man with manic-depressive psychosis. The percentage of high excretors of beta-aminoisobutyric acid (B-AIB) in the controls (4.88%) was similar to previous findings in the Caucasian race. The children with hearing and speech defects showed a number of high excretors of B-AIB significantly lower (X2 = 5.32; p less than 0.025) and the children with visual defects a number of hyperglycinurias significantly higher (X2 = 9.19; p less than 0.05). Previous non-consistent findings on the excess of high excretors of B-AIB in Down's syndrome were not confirmed in this study. These results suggest a relationship between transport defects in the plasma membrane and pathological disorders in some of the cases screened.

Alanine↗
PubMed1989

Preferential uptake of cytotoxic porphyrins from hematoporphyrin derivative in murine L929 fibroblasts and Chinese hamster ovary K1 epithelial cells.

Photodynamically induced loss of clonogenicity of murine L929 fibroblasts and Chinese hamster ovary K1 epithelial cells was determined with two different assays. It appeared that the loss of clonogenicity was much higher when 20 cells/cm2 were incubated with hematoporphyrin derivative (HPD) and illuminated, than when confluent cell layers were incubated with the same amount of HPD and illuminated prior to plating out. This dependency of cell killing on the experimental protocol was also observed when protoporphyrin (90-95% pure) was used as photosensitizer, but not when the cells were photodynamically treated with rose bengal or exposed to mitomycin C. Further, when cell layers were incubated with the residual solution that remained after the previous incubation of a confluent cell layer with HPD, illumination of these layers appeared to be almost non-toxic, although the overall porphyrin concentration in the residual solution was only slightly lower than in HPD. These results indicate that the porphyrins, responsible for loss of clonogenicity, are present in relatively small amounts in HPD and unpurified protoporphyrin and are preferentially taken up by the cells. Although 2-aminoisobutyric acid transport and DNA synthesis are among the most photosensitive targets with HPD, photodynamic treatment of L929 cells with the residual solution did not result in inhibition of the transport system and DNA synthesis. In contrast, the K+ content of the cells still decreased considerably, when utilizing the porphyrins, remaining in the residual solution as sensitizer. This indicates that under the present experimental conditions the disturbance of the membrane barrier function does not contribute to loss of clonogenicity of these cells and, moreover, that the photodynamically induced K+ leakage is caused by a component of HPD other than inhibition of 2-aminoisobutyric acid transport and DNA synthesis.

Animals↗
PubMed1984

Effects of ethanol and/or caffeine on fetal development and placental amino acid uptake in rats.

The effects of ethanol alone or in combination with caffeine on fetal viability, growth and placental amino acid uptake function in the rat were examined. Compared to pair-fed control values, chronic ethanol exposure reduced fetal survival by 24%, fetal weight by 17%, along with weight decreases of fetal brain (16%), heart (31%), and kidney (48%) as compared to pair-fed control values. Placental weight was significantly increased by 17%. Concomitant caffeine intake generally exacerbated these effects with a further reduction in fetal survival, fetal body and visceral weights. Caffeine intake alone had no consistent effect on these parameters. Acute in vitro (3 mg/ml) and chronic in vivo ethanol exposure reduced placental net uptake of alpha-aminoisobutyric acid (AIB) by 32 and 45%, respectively. Neither acute in vitro (10 micrograms/ml) nor prior chronic caffeine exposure altered villous AIB uptake. Concomitant ethanol and caffeine treatment increased AIB uptake as compared to ethanol alone. However, AIB uptake continued to be reduced (by 22%) as compared to pair-fed control values.

Amino Acids↗
PubMed1994

Incorporation of a potentially helix breaking D-Phe-Pro sequence into the center of a right handed 16 residue peptide helix.

Non protein amino acids with strong secondary structure preferences are potentially useful in peptide design. alpha-Aminoisobutyric acid (Aib) is a powerful 'stereochemical director' of polypeptide chain folding, stabilizing helical conformations in diverse oligopeptide sequences. In an approach to the de novo design of alpha,alpha motifs, the 16 residue peptides Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Xxx-Pro-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe (Xxx = D-Phe 1; Xxx = L-Phe 2) have been spectroscopically studied in solution. Analysis of nuclear Overhauser effects, delineation of solvent shielded NH groups and circular dichroism spectra establish helical conformations in both the peptides. Despite the presence of a potentially helix breaking, central, (D)Phe-(L)Pro segment, peptide 1 is forced into a continuous helical fold presumably as a consequence of the overriding stereochemical dominance of the Aib residues.

Amino Acid Sequence↗
PubMed2004

Novel parathyroid hormone (PTH) antagonists that bind to the juxtamembrane portion of the PTH/PTH-related protein receptor.

Current antagonists for the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (PTHR) are N-terminally truncated or N-terminally modified analogs of PTH(1-34) or PTHrP(1-34) and are thought to bind predominantly to the N-terminal extracellular (N) domain of the receptor. We hypothesized that ligands that bind only to PTHR region comprised of the extracellular loops and seven transmembrane helices (the juxtamembrane or J domain) could also antagonize the PTHR. To test this, we started with the J domain-selective agonists [Gln(10),Ala(12),Har(11),Trp(14),Arg(19) (M)]PTH(1-21), [M]PTH(1-15), and [M]PTH(1-14), and introduced substitutions at positions 1-3 that were predicted to dissociate PTHR binding and cAMP signaling activities. Strong dissociation was observed with the tri-residue sequence diethylglycine (Deg)(1)-para-benzoyl-l-phenylalanine (Bpa)(2)-Deg(3). In HKRK-B7 cells, which express the cloned human PTHR, [Deg(1,3),Bpa(2),M]PTH(1-21), [Deg(1,3),Bpa(2),M]PTH(1-15), and [Deg(1,3),Bpa(2),M]PTH(1-14) fully inhibited (IC(50)s = 100-700 nm) the binding of (125)I-[alpha-aminoisobutyric acid(1,3),M]PTH(1-15) and were severely defective for stimulating cAMP accumulation. In ROS 17/2.8 cells, which express the native rat PTHR, [Deg(1,3),Bpa(2),M]PTH(1-21) and [Deg(1,3),Bpa(2),M]PTH(1-15) antagonized the cAMP-agonist action of PTH(1-34), as did PTHrP(5-36) (IC(50)s = 0.7 microm, 2.6 microm, and 36 nm, respectively). In COS-7 cells expressing PTHR-delNt, which lacks the N domain of the receptor, [Deg(1,3),Bpa(2), M]PTH(1-21) and [Deg(1,3),Bpa(2),M]PTH(1-15) inhibited the agonist actions of [alpha-aminoisobutyric acid(1,3)]PTH(1-34) and [M]PTH(1-14) (IC(50)s approximately 1 microm), whereas PTHrP(5-36) failed to inhibit. [Deg(1,3),Bpa(2),M]PTH(1-14) inhibited the constitutive cAMP-signaling activity of PTHR-tether-PTH(1-9), in which the PTH(1-9) sequence is covalently linked to the PTHR J domain, as well as that of PTHR(cam)H223R. Thus, the J-domain-selective N-terminal PTH fragment analogs can function as antagonists as well as inverse agonists for the PTHR. The new ligands described should be useful for further studies of the ligand binding and activation mechanisms that operate in the critical PTHR J domain.

Animals↗