2-fold Stimulation Research Articles

Somatostatin type 2 receptor (SSTR2) inhibition of histidine decarboxylase (HDC) transcription is not mediated through a phosphatase pathway in the human gastric cancer cell line AGS-B

Background: Somatostatin (SS) inhibits both basal and gastrin stimulated histamine release from ECL cells in gastric mucosa. This is mediated through the SSTR2 receptor in human and rat antral mucosal strips. Histidine decarboxylase (HDC) is the enzyme responsible for histamine production in ECL cells and has previously been shown to be transcriptionally regulated through a gastrin response element. We postulated that SS may regulate the HDC promoter. The mechanisms through which SS may alter HDC transcription have not been elucidated. Methods: AGS-B cells were transfected with a plasmid containing 1.8 kb of the 5' promoter region of hHDC upsteam of the firefly luciferase reporter (1.8 kb hHDC-luc). Plasmids containing the cDNA of either human or rat SSTR2 were co-transfected. The cells were stimulated with 10 -7 M of gastrin, phothol ester (PMA) and dose responses with SS or octreotide were done. In addition overexpression of phosphatase SHP-1 and the G protein subunits Gictl and Gict3 were done using plasmids with the respective cDNA. Results.: Overexpression of both human and rat SSTR2 resulted in a dose dependent inhibition of hHDC transcription as measured by a luminometer. Maximal inhibition caused a 80% reduction in HDC transcription. This was seen in basal conditions, as well as with gastrin and PMA stimulated cells. Co-transfection with SHP-1 or its mutant did not block this effect. This inhibitory effect was not overcome with the phosphatase inhibitors sodium orthovanadate or okadaic acid. Addition of receptor agonists SS or octreotide resulted in a dose dependent 2-3 fold stimulation of HDC-Iuc activity. SS mediated stimulation in stably transfected cells was not altered by overexpression of GiQtl and Gia 3, suggesting that this paradoxical effect was not due to a lack of these subunits in AGS-B cells. Conclusions: SSTR2 has an inhibitory effect on HDC transcription in basal and stimulated AGS-B cells. This inhibition by the receptor is not mediated through a phosphatase pathway. Addition of agonists SS and octreotide produces a dose dependent stimulation in transcription of HDC. This is not due to a lack of the appropiate G i subunits in AGS-B cells. SS stimulates HDC transcription independently of G i pathway in AGS-B cells.

Dual regulation of the AMP-activated protein kinase provides a novel mechanism for the control of creatine kinase in skeletal muscle.

The AMP-activated protein kinase (AMPK) is activated by a fall in the ATP:AMP ratio within the cell in response to metabolic stresses. Once activated, it phosphorylates and inhibits key enzymes in energy-consuming biosynthetic pathways, thereby conserving cellular ATP. The creatine kinase-phosphocreatine system plays a key role in the control of ATP levels in tissues that have a high and rapidly fluctuating energy requirement. In this study, we provide direct evidence that these two energy-regulating systems are linked in skeletal muscle. We show that the AMPK inhibits creatine kinase by phosphorylation in vitro and in differentiated muscle cells. AMPK is itself regulated by a novel mechanism involving phosphocreatine, creatine and pH. Our findings provide an explanation for the high expression, yet apparently low activity, of AMPK in skeletal muscle, and reveal a potential mechanism for the co-ordinated regulation of energy metabolism in this tissue. Previous evidence suggests that AMPK activates fatty acid oxidation, which provides a source of ATP, following continued muscle contraction. The novel regulation of AMPK described here provides a mechanism by which energy supply can meet energy demand following the utilization of the immediate energy reserve provided by the creatine kinase-phosphocreatine system.

The Regulation of Glycogen Synthase by Protein Phosphatase 1 in 3T3-L1 Adipocytes: EVIDENCE FOR A POTENTIAL ROLE FOR DARPP-32 IN INSULIN ACTION

The stimulation of glycogen-targeted protein phosphatase 1 (PP1), glycogen synthase, and glycogen synthesis by insulin was examined during the differentiation of 3T3-L1 fibroblasts into adipocytes. Insulin treatment barely changed the low levels of glycogen synthesis measured in fibroblasts. Following differentiation into adipocytes, insulin increased glycogen synthesis up to 40-fold. After further culturing of the adipocytes for a week, insulin stimulated glycogen accumulation 700-fold. Differentiation of 3T3-L1 cells also resulted in the increased expression of glycogen synthase and in increases in both total glycogen synthase activity and -fold stimulation by insulin. While the levels of PP1 protein were unchanged by differentiation, PP1 specific activity decreased over 60%, although sensitivity to insulin treatment was augmented. Concurrently, levels of the PP1 inhibitor protein DARPP-32 were dramatically induced upon 3T3-L1 adipogenesis. DARPP-32 in both 3T3-L1 and primary rat adipocytes was exclusively localized to the particulate fractions, including the glycogen-enriched pellet. PP1 activity from 3T3-L1 adipocytes exhibited a kinetic lag in vitro, which was not present in fibroblast extracts. Insulin pretreatment of the adipocyte cells overcame the in vitro lag in PP1 activity, resulting in up to 5-fold stimulation of PP1 activity being measured at early assay time points. These results suggest that in 3T3-L1 adipocytes, DARPP-32 may maintain glycogen-targeted PP1 activity in a low basal state, priming the phosphatase for stimulation by insulin.

The multi-drug resistance reversal agent SR33557 and modulation of vinca alkaloid binding to P-glycoprotein by an allosteric interaction.

1. The interaction of the indolizin sulfone SR33557 with the multidrug resistance P-glycoprotein (P-gp), was used to explore the nature of drug binding site(s) on this transporter. The steady-state accumulation of [3H]-vinblastine in P-gp expressing CHrB30 cells was increased by SR33557 with greater potency than verapamil. Furthermore, SR33557 potentiated the affinity of verapamil to modulate vinblastine transport when added simultaneously. 2. Verapamil elicited a 1.5 to 2.5 fold stimulation of basal ATPase activity in CHrB30 membranes, whereas SR33557 and vinblastine inhibited activity, but only at relatively high concentrations. However, SR33557 and vinblastine decreased the Vmax but not the Km for verapamil stimulation of ATPase activity. This is indicative of a non-competitive interaction, most likely at distinct sites. 3. The specific [3H]-vinblastine binding to P-gp in CHrB30 cell membranes was displaced by SR33557 with an IC50 of 8.3 +/- 4.5 nM. Moreover, SR33557 caused a 3 fold increase in the dissociation rate of vinblastine binding to P-gp indicating a negative allosteric effect on the vinca alkaloid acceptor site. 4. These results demonstrate that SR33557 interacts with a site on P-gp which is distinct from, but allosterically linked to the vinca alkaloid site. The apparent broad substrate specificity displayed by P-gp may be explained by a multiple drug binding site model.

The cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors.

The tissue-restricted GATA-4 transcription factor and Nkx2-5 homeodomain protein are two early markers of precardiac cells. Both are essential for heart formation, but neither can initiate cardiogenesis. Overexpression of GATA-4 or Nkx2-5 enhances cardiac development in committed precursors, suggesting each interacts with a cardiac cofactor. We tested whether GATA-4 and Nkx2-5 are cofactors for each other by using transcription and binding assays with the cardiac atrial natriuretic factor (ANF) promoter_the only known target for Nkx2-5. Co-expression of GATA-4 and Nkx2-5 resulted in synergistic activation of the ANF promoter in heterologous cells. The synergy involves physical Nkx2-5-GATA-4 interaction, seen in vitro and in vivo, which maps to the C-terminal zinc finger of GATA-4 and a C-terminus extension; similarly, a C-terminally extended homeodomain of Nkx2-5 is required for GATA-4 binding. Structure/function studies suggest that binding of GATA-4 to the C-terminus autorepressive domain of Nkx2-5 may induce a conformational change that unmasks Nkx2-5 activation domains. GATA-6 cannot substitute for GATA-4 for interaction with Nkx2-5. This interaction may impart functional specificity to GATA factors and provide cooperative crosstalk between two pathways critical for early cardiogenesis. Given the co-expression of GATA proteins and NK2 class members in other tissues, the GATA/Nkx partnership may represent a paradigm for transcription factor interaction during organogenesis.

Growth Hormone-Induced Reorganization of the Actin Cytoskeleton Is Not Required for STAT5 (Signal Transducer and Activator of Transcription-5)-Mediated Transcription

We have investigated the effect of GH on the organization of the actin cytoskeleton within the cell. Human GH (hGH) treatment (50 nm) of Chinese hamster ovary (CHO) cells stably transfected with the complementary DNA for the rat GH receptor (CHO-GHR1–638) resulted in a reorganization of actin filaments in the cell that was not observed upon GH treatment of the untransfected parental CHO cell line. hGH initially induced depolymerization of actin stress fibers similar in magnitude to that induced by treatment of the cells with 100 nm human insulin-like growth factor I. This loss of stress fibers was observed as early as 30 sec after addition of hGH to the medium, and maximal depolymerization of stress fibers was observed between 1–4 min after addition of hGH. This was followed by a slow, but submaximal, repolymerization of the stress fibers and the formation of localized focal filamentous actin containing complexes. Similar cytoskeletal changes were observed after hGH treatment in Swiss 3T3 fibroblasts and BRL cells stably transfected with rat GH receptor complementary DNA (BRL-GHR1–638). Pretreatment of CHO-GHR1–638 cells with wortmannin (a phosphatidylinositol 3-kinase inhibitor) and verapamil (a calcium channel antagonist) both inhibited the hGH-induced actin reorganization. The integrity of the actin cytoskeleton was not required for GH-induced STAT5 (signal transducer and activator of transcription-5)-mediated transcription, as treatment of cells with cytochalasins B and D did not alter the fold stimulation of the STAT5-mediated transcriptional response to GH. We conclude that GH induces a rapid reorganization of the actin cytoskeleton by a process requiring phosphatidylinositol 3-kinase activation and calcium influx, but this cytoskeletal reorganization is not required for the STAT5-mediated transcriptional response to GH.

Growth hormone-induced reorganization of the actin cytoskeleton is not required for STAT5 (signal transducer and activator of transcription-5)-mediated transcription.

We have investigated the effect of GH on the organization of the actin cytoskeleton within the cell. Human GH (hGH) treatment (50 nM) of Chinese hamster ovary (CHO) cells stably transfected with the complementary DNA for the rat GH receptor (CHO-GHR(1-638)) resulted in a reorganization of actin filaments in the cell that was not observed upon GH treatment of the untransfected parental CHO cell line. hGH initially induced depolymerization of actin stress fibers similar in magnitude to that induced by treatment of the cells with 100 nM human insulin-like growth factor I. This loss of stress fibers was observed as early as 30 sec after addition of hGH to the medium, and maximal depolymerization of stress fibers was observed between 1-4 min after addition of hGH. This was followed by a slow, but submaximal, repolymerization of the stress fibers and the formation of localized focal filamentous actin containing complexes. Similar cytoskeletal changes were observed after hGH treatment in Swiss 3T3 fibroblasts and BRL cells stably transfected with rat GH receptor complementary DNA (BRL-GHR(1-6381)). Pretreatment of CHO-GHR(1-638) cells with wortmannin (a phosphatidylinositol 3-kinase inhibitor) and verapamil (a calcium channel antagonist) both inhibited the hGH-induced actin reorganization. The integrity of the actin cytoskeleton was not required for GH-induced STAT5 (signal transducer and activator of transcription-5)-mediated transcription, as treatment of cells with cytochalasins B and D did not alter the fold stimulation of the STAT5-mediated transcriptional response to GH. We conclude that GH induces a rapid reorganization of the actin cytoskeleton by a process requiring phosphatidylinositol 3-kinase activation and calcium influx, but this cytoskeletal reorganization is not required for the STAT5-mediated transcriptional response to GH.

The insulinotropic potency of fatty acids is influenced profoundly by their chain length and degree of saturation.

Lowering of the elevated plasma FFA concentration in 18- 24-h fasted rats with nicotinic acid (NA) caused complete ablation of subsequent glucose-stimulated insulin secretion (GSIS). Although the effect of NA was reversed when the fasting level of total FFA was maintained by coinfusion of soybean oil or lard oil (plus heparin), the more saturated animal fat proved to be far more potent in enhancing GSIS. We therefore examined the influence of individual fatty acids on insulin secretion in the perfused rat pancreas. When present in the perfusion fluid at 0.5 mM (in the context of 1% albumin), the fold stimulation of insulin release from the fasted pancreas in response to 12.5 mM glucose was as follows: octanoate (C8:0), 3.4; linoleate (C18:2 cis/cis), 5.3; oleate (C18:1 cis), 9.4; palmitate (C16:0), 16. 2; and stearate (C18:0), 21.0. The equivalent value for palmitoleate (C16:1 cis) was 3.1. A cis--> trans switch of the double bond in the C16:1 and C18:1 fatty acids had only a modest, if any, impact on their potency. A similar profile emerged with regard to basal insulin secretion (3 mM glucose). When a subset of these fatty acids was tested in pancreases from fed animals, the same rank order of effectiveness at both basal and stimulatory levels of glucose was seen. The findings reaffirm the essentiality of an elevated plasma FFA concentration for GSIS in the fasted rat. They also show, however, that the insulinotropic effect of individual fatty acids spans a remarkably broad range, increasing and decreasing dramatically with chain length and degree of unsaturation, respectively. Thus, for any given level of glucose, insulin secretion will be influenced greatly not only by the combined concentration of all circulating (unbound) FFA, but also by the makeup of this FFA pool. Both factors will likely be important considerations in understanding the complex interplay between the nature of dietary fat and whole body insulin, glucose, and lipid dynamics.

Effects of fish oil and n-3 fatty acids on the regulation of Δ 9-fatty acid desaturase mRNA and -activity in rat liver

This study was designed to study the effects of treatment with dietary fish oil, or with long-chain n-3 polyunsaturated fatty acids, on Δ 9-desaturase in rat liver. The desaturase activity was measured in isolated microsomes in parallel with measurements of liver content of Δ 9-desaturase mRNA. Fish oil-diet [20% ( w w ) of dietary fat] led to an 85% decrease in Δ 9-desaturase activity, whereas the decrease in Δ 9-desaturase mRNA was about 90%. In comparison, soybean oil diet [20% ( w w )] decreased Δ 9-desaturase activity by 50%, and the hepatic mRNA content by about 60%. Treatment with docosahexaenoic acid per os decreased the Δ 9 desaturase mRNA by 70%, whereas treatment with eicosapentaenoic acid decreased the mRNA level by 15%. Clofibrate treatment, in contrast, caused about a six fold stimulation of Δ 9-desaturase activity, and about two fold increase in mRNA concentration. At the same time the amounts of acyl-CoA oxidase- and acyl-CoA dehydrogenase mRNA did not vary significantly with high-fat diets or long-chain n-3 fatty acids, but increased two and two and-a-half-fold, respectively, with clofibrate. The experiments show that fish oil has a strong decreasing effect on the amount of Δ 9-desaturase mRNA, and that docosahexaenoic acid in the oil may be one potent component in this respect.

The expression of functionally-coupled B2-bradykinin receptors in human corneal epithelial cells and their pharmacological characterization with agonists and antagonists.

1. Bradykinin (BK) and Lys-BK are peptides which are released at high nanomolar concentrations into the tear-film of ocular allergic patients. We hypothesized that these peptides may activate specific receptors on the ocular surface, especially the corneal epithelium (CE) and thus the CE cells may represent a potential target tissue for these kinins. 2. The purpose of the present studies, therefore, was to determine the presence of and the pharmacological characteristics of bradykinin receptors on normal cultured primary and SV40 virus-transformed human corneal epithelial (CEPI) cells by use of the accumulation of [3H]-inositol phosphates ([3H]-IPs) as a bioassay. 3. Bradykinin (BK) induced a maximal 1.95 +/- 0.24 fold (n = 17) and 2.51 +/- 0.29 fold (n = 26) stimulation of [3H]-IPs accumulation in normal, primary (P-CEPI) and SV40-immortalized (CEPI-17-CL4) cells, respectively. This contrasted with a maximal 3.2-4.5 fold and 2.0-2.9 fold stimulation by histamine (100 microM) and platelet activating factor (100 nM) in both cell-types, respectively. 4. The molar potencies of BK and some of its analogues in the CEPI-17-CL4 cells were as follows: BK (EC50 = 3.26 +/- 0.61 nM, n = 18), Lys-BK (EC50 = 0.95 +/- 0.16 nM, n = 5), Met-Lys-BK (EC50 = 2.3 +/- 0.42 nM, n = 5), Ile-Ser-BK (EC50 = 5.19 +/- 1.23 nM, n = 6), Ala3-Lys-BK (EC50 = 12.7 +/- 2.08 nM, n = 3), Tyr8-BK (EC50 = 19.3 +/- 0.77 nM, n = 3), Tyr5-BK (EC50 = 467 +/- 53 nM, n = 4) and des-Arg9-BK (EC50 = 14.1 +/- 2.7 microM, n = 4). The potencies of BK-related peptides in normal, P-CEPI cells were similar to those found in transformed cells, thus: BK, EC50 = 2.02 +/- 0.69 nM (n = 7), Tyr8-BK, EC50 = 14.6 +/- 2.7 nM (n = 3), Tyr5 = BK, EC50 = 310 +/- 70 nM (n = 4) and des-Arg9-BK, EC50 = 12.3 +/- 3.8 microM (n = 3). 5. The bradykinin-induced responses were competitively antagonized by the B2-receptor selective BK antagonists, Hoe-140 (D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]BK; Icatibant; molar antagonist potency = 2.9 nM; pA2 = 8.54 +/- 0.06, n = 4; and slope = 1.04 +/- 0.08) and D-Arg0[Hyp3,Thi5,8, DPhe7]-BK (KB = 371 nM; pKB = 6.43 +/- 0.08, n = 4) in CEPI-17-CL4 cells. The antagonist potency of Hoe-140 against BK in normal, P-CEPI cells was 8.4 +/- 1.8 nM (pKi = 8.11 +/- 0.12, n = 4), this being similar to the potency observed in the immortalized cells. 6. This rank order of potency of agonist BK-related peptides, coupled with the antagonism of the BK-induced [3H]-IPs by the specific B2-receptor antagonists, strongly suggests that a B2-receptor subtype is involved in mediating functional phosphoinositide (PI) responses in the CEPI-17-CL4 and P-CEPI cells. 7. In conclusion, these data indicate that the P-CEPI and CEPI-17-CL4 cells express BK receptors of the B2-subtype coupled to the PI turnover signal transduction pathway. The CEPI-17-CL4 cells represent a good in vitro model of the human corneal epithelium in which to study further the role of BK receptors in its physiology and pathology, such as in allergic/inflammatory conditions, potential wound healing and other functions of the cornea.

Leptin stimulates glucose transport and glycogen synthesis in C2C12 myotubes: evidence for a P13-kinase mediated effect.

It was recently shown that leptin impairs insulin signalling, i.e. insulin receptor autophosphorylation and insulin-receptor substrate (IRS)-1 phosphorylation in rat-1 fibroblasts, NIH3T3 cells and HepG2 cells. To evaluate whether leptin might impair the effects of insulin in muscle tissue we studied the interaction of insulin and leptin in a muscle cell system, i.e. C2C12 myotubes. Preincubation of C2C12 cells with leptin (1-500 ng/ml) did not significantly affect insulin stimulated glucose transport and glycogen synthesis (1.8 to 2 fold stimulation); however, leptin by itself (1 ng/ml) was able to mimic approximately 80-90% of the insulin effect on glucose transport and glycogen synthesis. Both glucose transport as well as glycogen synthesis were inhibited by the phosphatidylinositol-3 (PI3)-kinase inhibitor wortmannin and the protein kinase C inhibitor H7 while no effect was observed with the S6-kinase inhibitor rapamycin. We determined whether the effect of leptin occurs through activation of IRS-1 and PI3-kinase. Leptin did not stimulate PI3-kinase activity in IRS-1 immunoprecipitates; however, PI3-kinase activation could be demonstrated in p85 alpha immunoprecipitates (3.04 +/- 1.5 fold of basal). In summary the data provide the first evidence for a positive crosstalk between the signalling chain of the insulin receptor and the leptin receptor. Leptin mimics in C2C12 myotubes insulin effects on glucose transport and glycogen synthesis most likely through activation of PI3-kinase. This effect of leptin occurs independently of IRS-1 activation in C2C12 cells.

Bradykinin-induced contractions of canine saphenous veins: mediation by B2 receptors and involvement of eicosanoids.

1. Experiments were designed to determine the subtype of kinin-receptors mediating the contraction of venous smooth muscle to bradykinin and to investigate the involvement of metabolites of arachidonic acid in this response. 2. Bradykinin (10(-9) to 10(-6) M) caused concentration-dependent contractions of the canine isolated saphenous vein without endothelium, which were potentiated by indomethacin (10(-5) M, an inhibitor of cyclo-oxygenase). The concentration-response curve was biphasic, reaching an asymptote at 10(-8) M and a secondary maximal response at 10(-6) M. 3. Bradykinin (10(-8) M to 3 x 10(-6) M) caused a three fold stimulation in the release of the vasodilator prostaglandin E2 (PGE2) and a two fold stimulation of that of the vasodilator prostacyclin, measured by the production of 6-keto-PGF1 alpha (its stable breakdown product). 4. Under control conditions, nordihydroguaiaretic acid (NDGA, 10(-5) M), an inhibitor of lipoxygenase, did not affect the response to bradykinin. In the presence of indomethacin (10(-5) M), NDGA reduced contractions to bradykinin, suggesting the involvement of lipoxygenase metabolites in the potentiation evoked by the inhibitor of cyclo-oxygenase. 5. The selective B1 receptor agonist [des-Arg9]-bradykinin, in the concentration-range 10(-6) to 10(-5) M, induced contractions, which were abolished by the B2 receptor antagonist D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]-bradykinin (Hoe 140; 10(-6) M). The selective B1 receptor antagonist [des-Arg9, Leu8]-bradykinin, (10(-7) to 10(-5) M) had no significant effect on bradykinin-induced contractions. 6. The B2 receptor antagonists Hoe 140 (10(-8) to 10(-6) M) and D-Arg [Hyp3, D-Phe7]-bradykinin (10(-7) to 10(-5) M) shifted the concentration-response curve to bradykinin to the right in a concentration-dependent manner. 7. These results indicate that, in the canine saphenous vein, bradykinin causes contraction by activating B2 receptors. This results in the production of metabolites of arachidonic acid, which play a key role in the contraction of canine saphenous venous smooth muscle.

Minimally modified low density lipoproteins induce aortic smooth muscle cell proliferation via the activation of mitogen activated protein kinase.

We have investigated the effects of modifying LDL by Cu++ and various hemoglobin preparations on aortic smooth muscle cell proliferation and on the activation of mitogen activated protein kinase. We found that at very low concentrations (10 microg/ml), LDL modified by all of the above agonists markedly stimulated cell proliferation (5-10 fold). This was accompanied by a 2-3 fold stimulation in mitogen activated protein kinase (MAPK) activity. We conclude that modification of LDL under situations that are closer to those found in vivo (i.e. hypoxic conditions), may involve the activation of MAPK as a common biochemical mechanism of action. This in turn, contributes to aortic smooth muscle cell proliferation.

Pharmacological Characterization of Bradykinin Receptors Coupled to Phosphoinositide Turnover in SV40-Immortalized Human Trabecular Meshwork Cells

This study sought to pharmacologically characterize bradykinin receptors on SV40-immortalized human trabecular meshwork (HTM3) cells. Phosphoinositide (PI) turnover studies were conducted using [ 3H]myo-inositol-labeled HTM3 cells and anion exchange chromatography to quantify [ 3H]inositol phosphates generated in response to bradykinin (BK) and various BK analogs. The blockade of these responses was studied using two potent and receptor-subtype selective antagonists. BK and T-kinin (Ile-Ser-BK; TK) induced a 4.2–4.4 fold stimulation of PI turnover above base levels at 1–10 μ M. Several other peptides unrelated to BK, including angiotensin II, endothelin, cholecystokinin, bombesin and peptide YY tested at 1–10 μ Mwere essentially inactive. The molar potencies (EC 50) of BK, TK and close analogs were: BK=4.5±0.5 n M( n=6), Lys-BK=6.5±0.7 n M( n=3), TK=38.8±6.6 n M( n=8), Met-Lys-BK=41.5±13.4 n M( n=4), Des-Arg 9-BK=2093±626 n M( n=4). All the latter BK-related peptides>were full agonists. The actions of BK and TK were potently and competitively antagonized by Hoe-140 (molar potency=0.6–1 n M;pA 2 n=8.97–9.21, n=3–4) and by D-Arg 0[Hyp 3,-Thi 5,8,-DPhe 7]-BK (molar potency=251 n M;-log potency, pK b=6.6), two selective B 2-type BK antagonists. In conclusion, rank order of potency of BK agonists and the blockade of BK- and TK-induced PI turnover by the selective antagonists are consistent with the classification of the BK receptors on HTM3 cells as the B 2-receptor subtype.

Characterization of the human brain putative A2B adenosine receptor expressed in Chinese hamster ovary (CHO.A2B4) cells.

1. An [3H]-adenine pre-labelling methodology was employed to assay cyclic AMP generation by adenosine analogues in Chinese hamster ovary (CHO.A2B4) cells, transfected with cDNA which has been proposed to code for the human brain A2B adenosine receptor, and in guinea-pig cerebral cortical slices. 2. Adenosine analogues showing the following rank order of potency in the CHO.A2B4 cells (pD2 value): 5'-N-ethylcarboxamidoadenosine (NECA, 5.91) > adenosine (5.69) > 2-chloroadenosine (5.27) > N6-(2-(4-aminophenyl)-ethylamino)adenosine (APNEA, 4.06). The purportedly A2A-selective agonist, CGS 21680, failed to elicit a significant stimulation of cyclic AMP generation at concentrations up to 10 microM in CHO.A2B4 cells. In the guinea-pig cerebral cortex, NECA was more potent than APNEA with pD2 values of 5.91 and 4.60, respectively. 3. Of these agents, NECA was observed to exhibit the greatest intrinsic activity in CHO.A2B4 cells (ca. 10 fold stimulation of cyclic AMP), while, in comparison, maximal responses to adenosine (32% NECA response), 2-chloroadenosine (61%), and APNEA (73%) were reduced. 4. Antagonists of NECA-evoked cyclic AMP generation showed the rank order of apparent affinity (apparent pA2 value in CHO.A2B4 cells: guinea-pig cerebral cortex): XAC (7.89: 7.46) > CGS 15943 (7.75: 7.33) > DPCPX (7.16: 6.91) > PD 115,199 (6.95: 6.39) > 8FB-PTP (6.52: 6.55) > 3-propylxanthine (4.63: 4.59). 5. We conclude that, using the agents tested, the A2B adenosine receptor cloned from human brain expressed in Chinese hamster ovary cells exhibits an identical pharmacological profile to native A2B receptors in guinea-pig brain.

Stimulation of sodium pump by vasoactive intestinal peptide in guinea-pig isolated trachea: potential contribution to mechanisms underlying relaxation of smooth muscle.

1. Relaxation of airway smooth muscle induced by vasoactive intestinal peptide (VIP) is mediated by adenosine 3':5' cyclic monophosphate (cyclic AMP). An interaction between the synthesis of cyclic AMP and enzymic activity of the plasmalemmal sodium pump (Na(+)-K(+)-ATPase) exists in certain isolated cell systems. This study sought to determine the contribution of Na(+)-K(+)-ATPase activity to relaxation of airway smooth muscle evoked by VIP. 2. All experiments were performed on isolated strips of guinea-pig trachea from which the epithelium had been removed. VIP was a more potent relaxant in tissues that were contracted with carbachol than those contracted with an equi-effective depolarizing concentration of K+. 3. Ouabain (0.1 microM-10 microM) induced contraction of tracheal strips. Contraction to ouabain (5 microM) was abolished following incubation of tissues with K(+)-free, or Ca(2+)-free (+ EGTA, 0.1 mM) physiological solutions. The contractile response to ouabain (5 microM) was not influenced significantly by exposure of the tissues to atropine (1 microM), phentolamine (5 microM) and diphenhydramine (1 microM) for 60 min. 4. Tissues were incubated with ouabain (5 microM; 60 min) or K(+)-free physiological solution (60 min) to inhibit Na(+)-K(+)-ATPase activity. These procedures reduced relaxation induced by VIP, peptide histidine isoleucine, forskolin, isoprenaline and sodium nitroprusside. 5. Relaxation to VIP was impaired significantly following exposure of tissues to a low Na+ solution (30 min) or amiloride (500 microM; 30 min). 6. Ouabain-sensitive uptake of 86Rb was measured in tracheal strips (devoid of epithelium and cartilage) as an index of Na(+)-K(+)-ATPase activity. VIP (1 microM; 2 min) caused a 4.7 fold stimulation of ouabain-sensitive uptake of 86Rb. This effect was impaired significantly by low Na+ solution. 7. The results suggest that (i) relaxation of tracheal smooth muscle to VIP is sensitive to procedures that inhibit activity of Na(+)-K(+)-ATPase and invoke a role for altered sodium pump function in the mechanisms that underlie cyclic AMP-dependent relaxation; and (ii) VIP stimulates ouabain-sensitive uptake of 86Rb in airway smooth muscle in a Na(+)-dependent manner.

The gene encoding for the novel transacting factor proopiomelanocortin corticotropin-releasing hormone responsive element binding protein 1 (PCRH-REB-1) is constitutively expressed in rat pituitary and in discrete brain regions containing CRH or CRH receptors: pathophysiological implications.

The nucleotide sequence CTGTGCGCGCAG is a core corticotropin-releasing hormone (CRH) responsive element in the proopiomelanocortin (POMC) promoter that gives 5-7 fold stimulation of POMC transcription by CRH. This region binds a novel transacting factor, proopiomelanocortin corticotropin-releasing hormone responsive element binding protein 1 (PCRH-REB-1), recently cloned from CRH-stimulated pituitary cells. We conducted in situ hybridization histochemistry experiments, using a species-specific, antisense, 35S-labeled PCRH-REB-1 riboprobe, to determine the pattern of PCRH-REB-1 gene expression in the brain, and showed that PCRH-REB-1 mRNA is localized not only in pituitary, but it is also found in discrete brain regions such as septal nuclei, hypothalamus, cortex, hippocampus, amygdala, and cerebellum. Those regions contain CRH or CRH receptors, and respond to CRH. We hypothesize that PCRH-REB-1 may be a marker of cellular responsiveness to CRH in the brain and pituitary, and might therefore be useful in the evaluation of CRH function and in the identification of clinically effective CRH agonists and antagonists.

Responses to iron deficiency in Arabidopsis thaliana: the Turbo iron reductase does not depend on the formation of root hairs and transfer cells.

Arabidopsis thaliana (L.) Heynh. Columbia wild type and a root hair-less mutant RM57 were grown on iron-containing and iron-deficient nutrient solutions. In both genotypes, ferric chelate reductase (FCR) of intact roots was induced upon iron deficiency and followed a Michaelis-Menten kinetic with a Km of 45 and 54 microM FeIII-EDTA and a Vmax of 42 and 33 nmol Fe2+.(g FW)-1.min-1 for the wild type and the mutant, respectively. The pH optimum for the reaction was around pH 5.5. The approximately four fold stimulation of FCR activity was independent of formation of root hairs and/or transfer cells induced by iron deficiency. Iron-deficiency-induced chlorosis and the development of a rigid root habit disappeared when ferric chelate was applied to the leaves, while FCR activity remained unchanged. The time course of the responses to iron deficiency showed that morphological and physiological responses were controlled separately.

Regulation by hypoxia of endothelin‐1‐stimulated phospholipase D activity in sheep pulmonary artery cultured smooth muscle cells

1. The aim of the study was to characterize the effects of hypoxia on agonist-stimulated phospholipase D (PLD) and phospholipase C activity of sheep pulmonary artery cultured smooth muscle cells. 2. Endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT) and the protein kinase C (PKC) activator tetradecanoylphorbol acetate (TPA), stimulated a time- and concentration-dependent increase in [3H]-phosphatidylbutanol accumulation. This was abolished by pretreatment of the cells with the PKC inhibitor, Ro-318220, suggesting that agonist-stimulated phospholipase D activity is dependent upon the activation of PKC. 3. Hypoxia (PO2 20 mmHg for 30 min) stimulated basal [3H]-phosphatidylbutanol accumulation by approximately 2 fold and this activity was abolished by preincubation of the cells with 10 microM Ro-318220. 4. In cells preincubated in low O2 containing medium for 30 min, the subsequent agonist-stimulated accumulation of [3H]-phosphatidylbutanol was reduced. However, the decrease in stimulation was greater for ET-1 and 5-HT than for TPA. 5. ET-1 and TPA stimulated a time-dependent increase in protein kinase C- mediated psuedosubstrate phosphorylation. Following preincubation for 30 min in low O2 containing media, basal pseudosubstrate phosphorylation increased whilst the fold stimulation by TPA and ET-1 decreased. 6. In cells preincubated in low O2 containing medium, ET-1-stimulated [3H]-inositol phosphate accumulation was reduced by approximately 30-40%. This reduction was reversed by preincubation of the cells with Ro-318220. 7. These results suggest a role for PKC in the effects of hypoxia on PLD in pulmonary artery smooth muscle cells.

Glucose activates the multifunctional Ca2+/calmodulin-dependent protein kinase II in isolated rat pancreatic islets.

The influence of the insulin secretagogues, glucose and K+, to activate the multifunctional, Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in isolated rat pancreatic islets has been examined. Glucose (28 mM) and K+ (40 mM) were demonstrated to induce a 1.89 +/- 0.19- and 1.75 +/- 0.15-fold increase, respectively, in phosphorylation of a subunit of CaM kinase II immunoprecipitated by an anti-CaM kinase II alpha antibody. In intact islets, glucose and K+ also induced the generation of an autonomous, Ca2+/calmodulin-independent protein kinase II activity characteristic of autophosphorylated enzyme. Maximal activation, 2.9 +/- 0.2- and 3.0 +/- 0.5-fold for glucose and K+, respectively, relative to basal glucose control, was achieved at 2.5-5 min followed by a decline to near basal levels by 20 min. Glucose induced the production of autonomous CaM kinase II activity that, in terms of -fold stimulation, correlated closely with the extent of insulin release over a glucose concentration range of 3-28 mM. This stimulated activity was completely prevented by an inhibitor of glucose metabolism, mannoheptulose. These data demonstrate that the exposure of islets to stimulatory glucose concentrations activates CaM kinase II. The close correlation of enzyme activation with insulin secretion is consistent with the hypothesis that CaM kinase II plays an important role in the regulation of insulin secretion or related beta-cell processes.