The baroreflex bandwagon: time to get off?
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Animals↗
Biomedical subjects
C J Dickinson
Publications and source records attributed to C J Dickinson.
Why are strokes related to hypertension? Classic studies and hypotheses revisited.
Although it seems obvious that excessive intravascular pressure is the cause of spontaneous intracerebral haemorrhage, the available evidence instead suggests that haemorrhage arises from previous ischaemic damage to the walls of small blood vessels. This interpretation unifies the aetiology of cerebral infarction and intracerebral haemorrhage. It is supported by much pathological evidence and also fits with observations on spontaneous stroke-prone hypertensive rats, which have smaller cerebral arteries than Wistar-Kyoto rats. Ischaemic damage to the brain probably occurs during spontaneous dips in aortic pressure in the presence of atheromatous arterial lesions and arteriolar narrowing by lipohyaline deposits. It may also follow long-lasting arterial spasm provoked by sudden pressure elevations. Local factors, especially unevenness of cerebral perfusion, probably determine the site of an infarct and whether it becomes haemorrhagic or not. In the long term, hypotensive drugs will lessen atheroma deposition. In the short term, they may act by reducing or preventing damaging arteriolar spasm.
Animals↗
Molecular mechanisms for the antiapoptotic action of gastrin.
Gastrin (G17) has a CCK-B receptor-mediated growth-promoting effect on the AR42J rat acinar cell line. We examined whether G17 inhibits apoptosis induced by serum withdrawal of AR42J cells and CHO-K1 cells stably expressing CCK-B receptors (CHO-K1/CCK-B cells). Cellular apoptosis was measured by flow cytometry and the terminal deoxynucleotidyltransferase-mediated dUTP-FITC nick end-labeling method. Serum withdrawal induced AR42J and CHO-K1/CCK-B cell apoptosis. Addition of 10 nM G17 reversed these effects. We examined the action of G17 (10 nM) on phosphorylation and activation of protein kinase B/Akt, a kinase known to promote cell survival. Akt phosphorylation and activation were measured by kinase assays and Western blots with an anti-phospho-Akt antibody. G17 stimulated Akt phosphorylation and activation. G17 induction of Akt phosphorylation was inhibited by the phosphoinositide 3-kinase (PI 3-kinase) inhibitors LY-294002 (10 microM) and wortmannin (200 nM) but not by the mitogen-activated protein kinase kinase 1 inhibitor PD-98059 (50 microM). To study the role of p38 kinase in G17 signaling to Akt, we examined the effect of G17 on p38 kinase activation and phosphorylation using kinase assays and Western blots with an anti-phospho-p38 kinase antibody. G17 induced p38 kinase activity at doses and with kinetics similar to those observed for Akt induction. The p38 kinase inhibitor SB-203580 inhibited G17 induction of Akt phosphorylation and activation at a concentration (10 microM) 10-fold higher than necessary to block p38 kinase (1 microM), suggesting the possible involvement of kinase activities other than p38 kinase. Transduction of AR42J cells with the adenoviral vector Adeno-dn Akt, which overexpresses an inhibitor of Akt, reversed the antiapoptotic action of G17. In conclusion, G17 promotes AR42J cell survival through the induction of Akt via PI 3-kinase and SB-203580-sensitive kinase activities.
Animals↗
Molecular determinants of ligand binding to the human melanocortin-4 receptor.
To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.
Agouti-Related Protein↗
Diminished prohormone convertase 3 expression (PC1/PC3) inhibits progastrin post-translational processing.
Gastrin is initially synthesized as a large precursor that requires endoproteolytic cleavage by a prohormone convertase (PC) for bioactivation. Gastric antral G-cells process progastrin at Arg(94)Arg(95) and Lys(74)Lys(75) residues generating gastrin heptadecapeptide (G17-NH(2)). Conversely, duodenal G-cells process progastrin to gastrin tetratriacontapeptide (G34-NH(2)) with little processing at Lys(74)Lys(75). Both tissues express PC1/PC3 and PC2. Previously, we demonstrated that heterologous expression of progastrin in an endocrine cell line that expresses PC1/PC3 and little PC2 (AtT-20) resulted in the formation of G34-NH(2). To confirm that PC1/PC3 was responsible for progastrin processing in AtT-20 cells and capable of processing progastrin in vivo we coexpressed either human wild-type (Lys(74)Lys(75)) or mutant (Arg(74)Arg(75), Lys(74)Arg(75), and Arg(74)Lys(75)) progastrins in AtT-20 cells with two different antisense PC1/PC3 constructs. Coexpression of either antisense construct resulted in a consistent decrease in G34-NH(2) formation. Gastrin mRNA expression and progastrin synthesis were equivalent in each cell line. Although mutation of the Lys(74)Lys(75) site within G34-NH(2) to Lys(74)Arg(75) resulted in the production of primarily G17-NH(2) rather than G34-NH(2), inhibition of PC1/PC3 did not significantly inhibit processing at the Lys(74)Arg(75) site. We conclude that PC1/PC3 is a progastrin processing enzyme, suggesting a role for PC1/PC3 progastrin processing in G-cells.
Aspartic Acid Endopeptidases↗
The possible role of osteoclastogenic oral bacterial products in etiology of Paget's disease.
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Administration, Oral↗
Contribution of melanocortin receptor exoloops to Agouti-related protein binding.
Agouti-related protein (AGRP) is an endogenous antagonist of melanocortin action that functions in the hypothalamic control of feeding behavior. Although previous studies have shown that AGRP binds three of the five known subtypes of melanocortin receptor, the receptor domains participating in binding and the molecular interactions involved are presently unknown. The present studies were designed to examine the contribution of extracytoplasmic domains of the melanocortin-4 receptor (MC4R) to AGRP binding by making chimerical receptor constructs of the human melanocortin-1 receptor (MC1R; a receptor that is not inhibited by AGRP) and the human MC4R (a receptor that is potently inhibited by AGRP). Substitutions of the extracytoplasmic NH2 terminus and the first extracytoplasmic loop (exoloop) of the MC4R with homologous domains of the MC1R had no effect on AGRP (87-132) binding affinity or inhibitory activity (the ability to inhibit melanocortin-stimulated cAMP generation). In contrast, cassette substitutions of exoloops 2 and 3 of the MC4R with the homologous exoloops of the MC1R resulted in a substantial loss of AGRP binding affinity and inhibitory activity. Conversely, the exchange of exoloops 2 and 3 of the MC1R with the homologous exoloops of the MC4R was found to confer AGRP binding and inhibitory activity to the basic structure of the MC1R. Importantly, these substitutions did not affect the ability of the alpha-melanocyte stimulating hormone analogue [Nle4,D-Phe7] melanocyte stimulating hormone to bind or activate the chimeric receptors. These data indicate that exoloops 2 and 3 of the melanocortin receptors are important for AGRP binding.
Agouti-Related Protein↗
Effects of somatostatin, somatostatin analogs, and endothelial cell somatostatin gene transfer on smooth muscle cell proliferation in vitro.
OBJECTIVE: Somatostatin analogs inhibit neointimal hyperplasia and smooth muscle cell (SMC) proliferation in vivo. The gene transfer of somatostatin to endothelial cells (ECs) represents a potential means of local delivery of somatostatin to areas of arterial injury. This study tested the hypothesis that the retroviral gene transfer of somatostatin to ECs would inhibit SMC proliferation in vitro and evaluated the effects of somatostatin analogs on DNA synthesis and the growth of SMCs. METHODS: Media transfer and coculture were used to determine the effects of somatostatin-producing ECs on SMC proliferation in vitro. The effects of a variety of somatostatin isoforms and analogs on the proliferation of SMCs, mitogenesis of serum-restimulated quiescent SMCs, and arterial explants were measured. RESULTS: Despite the production of biologically relevant concentrations of somatostatin by ECs, no inhibition of SMC proliferation was noted. Somatostatin analogs inhibited DNA synthesis in arterial explants but did not inhibit either DNA synthesis or growth of cultured SMCs, which showed a likely effect of somatostatin on the initial transition in SMC phenotype. CONCLUSION: Somatostatin exerts inhibitory effects on SMC proliferation only during the early transition to a proliferative phenotype. There are significant differences between this in vivo transition and the standard serum-restimulated model of cultured SMCs. These differences may account for the failure of somatostatin to inhibit SMC proliferation in the standard in vitro models.
Animals↗
Mouth bacteria as the cause of Paget's disease of bone.
The many viruses associated with Pagetic osteoclasts could be opportunistic rather than causative. Some mouth bacteria can lyse bone. One (Actinobacillus actinomycetemcomitans) can grow and even multiply inside human cell lines in culture, producing osteolytic materials -- one 62 kDa protein having a potency in the picomolar range. A small focus of this, or of one of the other periodontitis-causing bacteria, in a bone might gradually spread its influence to activate osteoclasts -- the first stage in Paget's disease. The focus in each bone might be small and easily overlooked, as other intracellular bacteria have been in the past.
Aggregatibacter actinomycetemcomitans↗
Could tight garments cause endometriosis?
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Clothing↗
Gastrin induces c-fos gene transcription via multiple signaling pathways.
We previously observed that the trophic actions of gastrin (G17) on the AR42J rat acinar cell line are mediated by mitogen-activated protein kinase (MAPK)-induced c-fos gene transcription via protein kinase C (PKC)-dependent and -independent pathways. In this study, we further investigated the signaling pathways that target c-fos in response to G17. G17 led to a sixfold induction in luciferase activity in cells transfected with plasmids containing the -356+109 sequence of the murine c-fos promoter, which includes the Sis-inducible element (SIE), serum response element (SRE), and the Ca2+/cAMP response element (CRE) regulatory elements. Addition of either the selective PKC inhibitor GF-109203X or the MAPK/extracellular signal-regulated kinase inhibitor PD-98059 resulted in an 80% reduction in luciferase activity. G17 induced the transcriptional activity of both Elk-1 and Sap-1a, transcription factors that bind to the E26 transformation specific (Ets) DNA sequence of the SRE, and this effect was inhibited by both GF-109203X and PD-98059. Point mutations in the Ets sequence led to a 4-fold induction of c-fos transcription stimulated by G17 and to a 1.3-fold induction in response to epidermal growth factor (EGF). In contrast, mutations in the CA rich G (CArG) sequence of the SRE prevented transcriptional activation by both G17 and EGF. G17 induction of the Ets mutant construct was unaffected by either GF-109203X or PD-98059. Because activation of the SRE involves the small GTP-binding protein Rho A, we examined the role of Rho A in G17 induction of c-fos transcription. Inactivation of Rho A by either the specific inhibitor C3 or by expression of a dominant negative Rho A gene inhibited G17 induction of both the wild-type and the Ets mutant constructs by 60%. C3 also inhibited G17-stimulated AR42J cell proliferation. Thus G17 targets the c-fos promoter CArG sequence via Rho A-dependent pathways, and Rho A appears to play an important role in the regulation of the trophic action of G17.
Animals↗
Cell type-specific requirement of the MAPK pathway for the growth factor action of gastrin.
Gastrin (G17) has a CCKB receptor-mediated growth-promoting effect on the AR42J rat acinar cell line that is linked to induction of both mitogen-activated protein kinase (MAPK) and c-fos gene expression. We investigated the mechanisms that regulate the growth factor action of G17 on the rat pituitary adenoma cell line GH3. Both AR42J and GH3 cells displayed equal levels of CCKB receptor expression and similar binding kinetics of 125I-labeled G17. G17 stimulation of cell proliferation was identical in both cell lines. G17 stimulation of GH3 cell proliferation was completely blocked by the CCKB receptor antagonist D2 but not by the MEK inhibitor PD-98059 or the protein kinase C inhibitor GF-109203X, which completely inhibited G17 induction of AR42J cell proliferation. G17 induced a c-fos SRE-luciferase reporter gene plasmid more than fourfold in the AR42J cells, whereas it had no effect in the GH3 cells. In contrast to what we observed in the AR42J cells, G17 failed to stimulate MAPK activation and Shc tyrosyl phosphorylation and association with the adapter protein Grb2. Epidermal growth factor induced the MAPK pathway in the GH3 cells, demonstrating the integrity of this signaling system. G17 induced Ca2+ mobilization in both the GH3 and AR42J cells. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide inhibited AR42J cell proliferation by 20%, whereas it completely blocked G17 induction of GH3 cell growth. The Ca2+ ionophore ionomycin stimulated GH3 cell proliferation to a level similar to that observed in response to G17, but it had no effect on AR42J cell proliferation. Thus there are cell type specific differences in the requirement of the MAPK pathway for the growth factor action of G17. Whereas in the AR42J cells G17 stimulates cell growth through activation of MAPK and c-fos gene expression, in the GH3 cells, G17 fails to activate MAPK, and it induces cell proliferation through Ca2+-dependent signaling pathways. Furthermore, induction of Ca2+ mobilization in the AR42J cells appears not to be sufficient to sustain cell proliferation.
Adaptor Proteins, Signal Transducing↗
Glycine-extended gastrin regulates HEK cell growth.
Posttranslational processing of progastrin to a carboxy terminally amidated form (G-NH(2)) is essential for its effect on gastric acid secretion and other biological effects mediated by gastrin/CCK-B receptors. The immediate biosynthetic precursor of G-NH(2), glycine-extended gastrin (G-Gly), does not stimulate gastric acid secretion at physiological concentrations but is found in high concentrations during development. G-NH(2) and G-Gly have potent growth stimulatory effects on gastrointestinal tissues, and G-NH(2) can stimulate proliferation of human kidney cells. Thus we sought to explore the actions of G-NH(2) and G-Gly on the human embryonic kidney cell line HEK 293. HEK 293 cells showed specific binding sites for (125)I-labeled Leu(15)-G17-NH(2) and (125)I-Leu(15)-G(2-17)-Gly. Both G-NH(2) and G-Gly induced a dose-dependent increase in [(3)H]thymidine incorporation, and both peptides together significantly increased [(3)H]thymidine incorporation above the level of either peptide alone. G-NH(2) and G-Gly were detected by radioimmunoassay in serum-free conditioned media. Antibodies directed against G-NH(2) and G-Gly lead to a significant reduction in [(3)H]thymidine incorporation. G-NH(2) but not G-Gly increased intracellular Ca(2+) concentration. We conclude that G-NH(2) and G-Gly act cooperatively via distinct receptors to stimulate the growth of a nongastrointestinal cell line (HEK 293) in an autocrine fashion.
Binding Sites↗
Characterization of Agouti-related protein binding to melanocortin receptors.
Agouti-related protein (AGRP) is a naturally occurring antagonist of melanocortin action that is thought to play an important role in the hypothalamic control of feeding behavior. The exact mechanism of AGRP and Agouti protein action has been difficult to examine, in part because of difficulties in producing homogeneous forms of these molecules that can be used for direct binding assays. In this report we describe the application of chemical protein synthesis to the construction of two novel AGRP variants. Examination of the biological activity of the AGRP variants demonstrates that a truncated variant, human AGRP(87-132), a 46-amino acid variant based on the carboxyl-terminal cysteine-rich domain of AGRP, is equipotent to an 111-amino acid variant, mouse [Leu127Pro]AGRP (mature AGRP minus its signal sequence), in its ability to dose dependently inhibit alpha-MSH-generated cAMP generation at the cloned melanocortin receptors. Furthermore, deletion of the amino-terminal portion of the full-length variant did not alter the MCR subtype specificity of AGRP(87-132). Finally, iodination of human AGRP(87-132) provided a useful reagent with which the binding properties of AGRP could be analyzed. In both conventional and photoemulsion binding studies [125I]AGRP(87-132) was observed only to bind to cells expressing melanocortin receptors MC3R, MC4R, and MC5R. These results demonstrate that the residues critical for receptor binding, alpha-MSH inhibition, and melanocortin receptor subtype specificity are all located in the carboxyl terminus of the molecule. Because [Nle4, D-Phe7] (NDP)-MSH displaces the binding of [125I]AGRP(87-132) to MCRs and AGRP(87-132) displaces the binding of [125I]NDP-MSH, we conclude that these molecules bind in a competitive fashion to melanocortin receptors.
Agouti Signaling Protein↗
Glycine-extended gastrin exerts growth-promoting effects on human colon cancer cells.
BACKGROUND: Since human colon cancers often contain significant quantities of progastrin-processing intermediates, we sought to explore the possibility that the biosynthetic precursor of fully processed amidated gastrin, glycine-extended gastrin, may exert trophic effects on human colonic cancer cells. MATERIALS AND METHODS: Binding of radiolabeled glycine-extended and amidated gastrins was assessed on five human cancer cell lines: LoVo, HT 29, HCT 116, Colo 320DM, and T 84. Trophic actions of the peptides were assessed by increases in [3H]thymidine incorporation and cell number. Gastrin expression was determined by northern blot and radioimmunoassay. RESULTS: Amidated gastrin did not bind to or stimulate the growth of any of the five cell lines. In contrast, saturable binding of radiolabeled glycine-extended gastrin was seen on LoVo and HT 29 cells that was not inhibited by amidated gastrin (10(-6) M) nor by a gastrin/CCKB receptor antagonist (PD 134308). Glycine-extended gastrin induced a dose-dependent increase in [3H]thymidine uptake in LoVo (143 +/- 8% versus control at 10(-10) M) and HT 29 (151 +/- 11% versus control at 10(-10) M) cells that was not inhibited by PD 134308 or by a mitogen-activated protein (MAP) or ERK kinase (MEK) inhibitor (PD 98509). Glycine-extended gastrin did stimulate jun-kinase activity in LoVo and HT 29 cells. The two cell lines expressed the gastrin gene at low levels and secreted small amounts of amidated gastrin and glycine-extended gastrin into the media. CONCLUSIONS: Glycine-extended gastrin receptors are present on human colon cancer cells that mediate glycine-extended gastrin's trophic effects via a MEK-independent mechanism. This suggests that glycine-extended gastrin and its novel receptors may play a role in colon cancer cell growth.
Animals↗
Gastrin and colorectal cancer: a prospective study.
BACKGROUND & AIMS: Gastrin is a putative promoter of colorectal carcinomas. The aim of this study was to evaluate the temporal relationship between gastrinemia and development of colorectal malignancy. METHODS: We conducted a nested case-control study among 128,992 subscribers to a health maintenance program who had participated in a multiphasic health checkup between 1964 and 1969. Serum had been frozen since the checkup and the cohort followed up for cancer. Of 1881 incident colorectal carcinoma cases, 250 were randomly selected; 1 control without cancer was matched to each case by age, sex, education, and date of serum collection. Stored sera were tested for Helicobacter pylori immunoglobulin G and for gastrin and glycine-extended gastrin. RESULTS: Verified cases included 166 colon cancers, 58 rectal cancers, and 9 with cancer in both locations. A mean of 15.3 years had elapsed between serum collection and diagnosis of cancer. Median gastrin levels were similar in cases and controls (41.7 vs. 40.7 pg/mL). However, a gastrin level above normal was associated with increased risk for colorectal malignancy (odds ratio, 3.9; 95% confidence interval, 1.5-9.8). If this association is causal, 8.6% of colorectal cancers could be attributed to high serum gastrin level. CONCLUSIONS: Hypergastrinemia is associated with an increased risk of colorectal carcinoma.
Case-Control Studies↗
Somatostatin gene transfer and expression in endothelial cells.
PURPOSE: The antiproliferative and antisecretory effects of somatostatin have many potential uses in the clinical setting. Retroviral gene transfer of somatostatin to endothelium is a potential means of local delivery of this peptide to specific vascular beds. This investigation was designed to determine whether transduced endothelial cells (ECs) could produce and post-translationally process somatostatin. METHODS: Cultured canine venous, rat aortic, and rat microvascular ECs were transfected with retroviruses containing a human somatostatin cDNA or a control beta-galactosidase gene. Total and isoform somatostatin production and uniformity of beta-galactosidase expression were analyzed, as were the effects of somatostatin production on EC proliferation. RESULTS: Somatostatin-transduced canine venous ECs, but not rat ECs, produced approximately 10 times as much total somatostatin as did control-transfected ECs (450 +/- 32 vs 49 +/- 10 pmol/L, p < 0.05). The predominant isoform of somatostatin produced was somatostatin-14. Production of somatostatin was stable with passage and did not impair the growth of canine ECs. The failure of rat ECs to produce somatostatin correlated with nonuniform expression of beta-galactosidase, suggesting that promoter silencing was responsible for failure of transgene expression. CONCLUSION: Retroviral gene transfer of somatostatin to canine ECs results in the production of physiologically relevant concentrations of biologically active somatostatin. Significant species differences exist in EC production of somatostatin, with promoter silencing being a potential mechanism of failure of gene expression. Gene therapy strategies using retroviral transfer of somatostatin to ECs may allow somatostatin delivery to focal areas of the vasculature.
Animals↗
The determinants of long-term blood pressure stability: control of trough blood pressure during sleep.
Long-term blood pressure stability in humans is not affected by prolonged hypotensive drug therapy. After withdrawal of long-term hypotensive therapy from essential hypertensives, their blood pressures soon return to pre-treatment levels. Since the well-studied stabilizing systems all adapt to prevailing blood pressure levels, they cannot account for long-term stability of blood pressure. There is a parallel between trough blood pressure and the lower limit of cerebral autoregulation. Blood pressure goes down to a trough level once sleep has been established. This level is maintained for 3-4 h, with minor perturbations. For normal and hypertensive humans and rats, the level is almost the same as the lower limit of cerebral autoregulation. In essential hypertension the nocturnal fall of blood pressure and the lower limit for cerebral autoregulation could be set by atheromatous narrowing or occlusion of large cerebral arteries, especially those supplying the brain stem, from which the Cushing response is initiated. Constriction and maldevelopment of smaller cerebral arteries are probably the main causes of increased cerebrovascular resistance in spontaneously hypertensive rats (SHR). The structural difference between the two situations might explain the failure of prolonged hypotensive therapy to reduce the lower limit of cerebral autoregulation in humans, which it is able to achieve in young SHR. If trough blood pressure stability were the anchor which prevents casual blood pressures from drifting, a primary increase of cerebrovascular resistance would be a plausible explanation for essential hypertension. It could also make a major causal neurogenic contribution to hypertension in rat models, especially SHR and stroke-prone SHR.
Animals↗