Biomedical subjects

C Gu

Publications and source records attributed to C Gu.

At least 19 recordsLinked to original sources

PubMed2001

The nonhelical tail domain of keratin 14 promotes filament bundling and enhances the mechanical properties of keratin intermediate filaments in vitro.

Keratin filaments arise from the copolymerization of type I and II sequences, and form a pancytoplasmic network that provides vital mechanical support to epithelial cells. Keratins 5 and 14 are expressed as a pair in basal cells of stratified epithelia, where they occur as bundled arrays of filaments. In vitro, bundles of K5-K14 filaments can be induced in the absence of cross-linkers, and exhibit enhanced resistance to mechanical strain. This property is not exhibited by copolymers of K5 and tailless K14, in which the nonhelical tail domain has been removed, or copolymers of K5 and K19, a type I keratin featuring a short tail domain. The purified K14 tail domain binds keratin filaments in vitro with specificity (kD approximately 2 microM). When transiently expressed in cultured cells, the K14 tail domain associates with endogenous keratin filaments. Utilization of the K14 tail domain as a bait in a yeast two-hybrid screen pulls out type I keratin sequences from a skin cDNA library. These data suggest that the tail domain of K14 contributes to the ability of K5-K14 filaments to self-organize into large bundles showing enhanced mechanical resilience in vitro.

Animals↗

PubMed2001

Sterically stabilized polyplex: ligand-mediated activity.

Synthetic vectors have been considered as a safer and more versatile alternative to viral-based gene delivery systems. A variety of very simple synthetic vector systems, e.g., cationic lipid- and polymer-complexed plasmid DNA have activity in vivo but it appears to be mediated by non-specific electrostatic interactions limiting targeting. In order to avoid these problems, we designed a sterically stabilized layered colloidal system. The steric polymer coating reduces non-specific interactions. We have synthesized a PEG conjugate of PEI that complexes DNA to form small, stable colloids with a steric polymer coat on their surface. The polymer enhances colloidal stability and reduces non-specific binding and toxicity. It also renders the complex inactive presumably due to reduced binding. Ligands are then appended to the distal end of the steric polymer to restore cell binding and expression at target cells. We prepared conjugates with RGD peptide ligands appended to the distal end of the steric polymer. The resulting conjugates also form complexes but with ligands exposed on their surface restoring binding and activity. Labeled oligonucleotides and DNA were used to measure intracellular distribution. Oligonucleotides are found localized in the nucleus, whereas the labeled plasmid DNA remained in the cytoplasm. Import of plasmid DNA into the nucleus appears to be very inefficient yet sufficient for expression.

Chemical Phenomena↗

PubMed2001

Persistent interactions between the two transmembrane clusters dictate the targeting and functional assembly of adenylyl cyclase.

Adenylyl cyclases possess complex structures like those of the ATP binding cassette (ABC) transporter family, which includes the cystic fibrosis transmembrane regulator, the P-glycoprotein, and ATP-sensitive K(+) channels [1-4]. These structures comprise a cytosolic N terminus followed by two tandem six-transmembrane cassettes, each associated with a highly homologous (ATP binding) cytosolic loop [5-8]. The catalytic domains, which are located in the two large cytoplasmic loops, are highly conserved and well studied. The crystal structure of these domains has even been described recently [9, 10]. However, nothing is known of the function or organization of the 12 transmembrane segments. In the present study we adopted a range of strategies including live-cell fluorescence resonance energy transfer (FRET) microscopy, coimmunoprecipitation, and functional assays of various truncated and substituted, fluorescently-tagged molecules to analyze the trafficking and activity of this molecule. When expressed as individual peptides, the two transmembrane domains - largely independently of any cytosolic region - formed a tight complex that was delivered to the plasma membrane. This cooperation between the two intact transmembrane domains was essential and sufficient to target the enzyme to the plasma membrane of the cell. The extracellular loop between the ninth and tenth transmembrane segments, which contains an N-glycosylation site, was also necessary. Furthermore, the interaction between the two transmembrane clusters played a critical role in bringing together the cytosolic catalytic domains to express functional adenylyl cyclase activity in the intact cell.

Adenylyl Cyclases↗

PubMed2001

Meta-analysis for model-free methods.

The intricate nature of complex genetic traits dictates that novel methodologies be developed and utilized to achieve better power, better accuracy, and more favorable balance between type I and type II errors than could be achieved by the traditional methods as they are used in mapping Mendelian traits. Meta-analysis provides one such method for synthesizing information from multiple studies. This has the advantage of being able to pool relatively weak signals from individual studies into a collectively stronger evidence of genetic effects, while at the same time providing a quantitative framework for modeling variability among studies. The traditional lod score measures significance level of a linkage effect in an individual study, and its additive property make it a natural candidate for combining results across independent studies. To incorporate the within-study variation of the linkage effect into the pooled overall measure of genetic effect, the effect sizes (such as the proportion of genes shared identical-by-descent, IBD) should be pooled directly across studies. Traditional regression models and mixed effects models can be used to estimate the overall genetic effect size and its variance, and to test heterogeneity among studies. Our simulation studies show that designing studies with moderate power and pooling their results via meta-analysis may be more cost-effective than large dedicated studies. We believe that, as a newly emerging methodology, the meta-analysis approach has the potential to become an integral part of our toolbox that will expedite the search for complex human disease genes.

Alleles↗

PubMed2001

Optimum study designs.

Because simplistic designs will lead to prohibitively large sample sizes, the optimization of genetic study designs is critical for successfully mapping genes for complex diseases. Creative designs are necessary for detecting and amplifying the usually weak signals for complex traits. Two important outcomes of a study design--power and resolution--are implicitly tied together by the principle of uncertainty. Overemphasis on either one may lead to suboptimal designs. To achieve optimality for a particular study, therefore, practical measures such as cost-effectiveness must be used to strike a balance between power and resolution. In this light, the myriad of factors involved in study design can be checked for their effects on the ultimate outcomes, and the popular existing designs can be sorted into building blocks that may be useful for particular situations. It is hoped that imaginative construction of novel designs using such building blocks will lead to enhanced efficiency in finding genes for complex human traits.

Case-Control Studies↗

PubMed2001

False positives and false negatives in genome scans.

It is emphasized that two types of errors are made in the testing of a hypothesis, false positive (type I) and false negative (type II). Genome-wide scans involving many markers give rise to the problem of multiple testing, which results in an increased number of false positives, thus necessitating a correction in the nominal significance level. While the literature has concentrated reasonably heavily on controlling false positives in genomic scans, the need to control false negatives has been largely neglected. This chapter highlights this need and attempts to strike a balance between the two error types. The need to develop alternative methods for discriminating between false positives and true positives is also stressed.

Chromosome Mapping↗

PubMed2001

Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptoms.

Eight botanical preparations that are commonly used for the treatment of menopausal symptoms were tested for estrogenic activity. Methanol extracts of red clover (Trifolium pratense L.), chasteberry (Vitex agnus-castus L.), and hops (Humulus lupulus L.) showed significant competitive binding to estrogen receptors alpha (ER alpha) and beta (ER beta). With cultured Ishikawa (endometrial) cells, red clover and hops exhibited estrogenic activity as indicated by induction of alkaline phosphatase (AP) activity and up-regulation of progesterone receptor (PR) mRNA. Chasteberry also stimulated PR expression, but no induction of AP activity was observed. In S30 breast cancer cells, pS2 (presenelin-2), another estrogen-inducible gene, was up-regulated in the presence of red clover, hops, and chasteberry. Interestingly, extracts of Asian ginseng (Panax ginseng C.A. Meyer) and North American ginseng (Panax quinquefolius L.) induced pS2 mRNA expression in S30 cells, but no significant ER binding affinity, AP induction, or PR expression was noted in Ishikawa cells. Dong quai [Angelica sinensis (Oliv.) Diels] and licorice (Glycyrrhiza glabra L.) showed only weak ER binding and PR and pS2 mRNA induction. Black cohosh [Cimicifuga racemosa (L.) Nutt.] showed no activity in any of the above in vitro assays. Bioassay-guided isolation utilizing ER competitive binding as a monitor and screening using ultrafiltration LC-MS revealed that genistein was the most active component of red clover. Consistent with this observation, genistein was found to be the most effective of four red clover isoflavones tested in the above in vitro assays. Therefore, estrogenic components of plant extracts can be identified using assays for estrogenic activity along with screening and identification of the active components using ultrafiltration LC-MS. These data suggest a potential use for some dietary supplements, ingested by human beings, in the treatment of menopausal symptoms.

Binding, Competitive↗

PubMed2001

Equine estrogen metabolite 4-hydroxyequilenin induces DNA damage in the rat mammary tissues: formation of single-strand breaks, apurinic sites, stable adducts, and oxidized bases.

Epidemiological data strongly suggest that a woman's risk of developing breast cancer is directly related to her lifetime estrogen exposure. Estrogen replacement therapy in particular has been correlated with an increased cancer risk. Previously we showed that the equine estrogens equilin and equilenin, which are major components of the estrogen replacement formulation Premarin (Wyeth-Ayerst), are metabolized to the catechol, 4-hydroxyequilenin which autoxidizes to an o-quinone causing oxidation and alkylation of DNA in vitro [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113-1227]. In the present study, we injected 4-hydroxyequilenin into the mammary fat pads of Sprague-Dawley rats. Analysis of cells isolated from the mammary tissue for DNA single-strand breaks and oxidized bases using the comet assay showed a dose-dependent increase in both types of lesions. In addition, LC-MS-MS analysis of extracted mammary tissue showed the formation of an alkylated depurinating guanine adduct. Finally, extraction of mammary tissue DNA, hydrolysis to deoxynucleosides, and analysis by LC-MS-MS showed the formation of stable cyclic deoxyguanosine and deoxyadenosine adducts as well as oxidized bases. This is the first report showing that 4-hydroxyequilenin is capable of causing DNA damage in vivo. In addition, the data showed that 4-hydroxyequilenin induced four different types of DNA damage that must be repaired by different mechanisms. This is in contrast to the endogenous estrogen 4-hydroxyestrone where only depurinating guanine adducts have been detected in vivo. These results suggest that 4-hydroxyequilenin has the potential to be a potent carcinogen through the formation of variety of DNA lesions in vivo.

Animals↗

PubMed2001

Familial resemblance for glucose and insulin metabolism indices derived from an intravenous glucose tolerance test in Blacks and Whites of the HERITAGE Family Study.

Type 2 diabetes mellitus (T2DM), characterized by hyperglycemia, is a complex disease primarily caused by impairment in insulin sensitivity (SI) and insulin secretion. While a strong genetic component for T2DM is well established, there are few reports on racial differences in the magnitude of the genetic effects of T2DM and indices of glucose and insulin metabolism. We report here on the familial resemblance for traits related to glucose metabolism at pre-exercise training levels in 492 members from 99 sedentary White families and 259 members from 108 Black families participating in the multicenter HERITAGE Family Study. All these traits were obtained from the frequently sampled intravenous glucose tolerance test (IVGTT). They include glucose disappearance index (Kg), an overall index for glucose tolerance, acute insulin response to glucose (AIR(Glucose)) which is an index for insulin secretion, and those derived from the minimal model including SI and the disposition index (DI). DI, derived as the product of SI and AIR(Glucose), is a measure of the activity of the B-cells adjusted for insulin resistance. After adjustment for age, sex, and body mass index, the maximal heritability estimates in Blacks (Whites) are 48+/-14% (25+/-8%) for Kg, 44+/-14% (46+/-8%) for AIR(Glucose), 38+/-12% (44+/-8%) for SI and 32+/-14% (24+/-8%) for DI. Interestingly, Blacks have higher heritability for overall glucose tolerance than Whites but there is no race difference in heritability estimates for insulin sensitivity or insulin secretion.

Adult↗

PubMed2001

The EphA8 receptor regulates integrin activity through p110gamma phosphatidylinositol-3 kinase in a tyrosine kinase activity-independent manner.

Recent genetic studies suggest that ephrins may function in a kinase-independent Eph receptor pathway. Here we report that expression of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to fibronectin via alpha(5)beta(1)- or beta(3) integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly promoted cell attachment to fibronectin in these cell lines. Using a panel of EphA8 point mutants, we have demonstrated that EphA8 kinase activity does not correlate with its ability to promote cell attachment to fibronectin. Analysis using EphA8 extracellular and intracellular domain mutants has revealed that enhanced cell adhesion is dependent on ephrin A binding to the extracellular domain and the juxtamembrane segment of the cytoplasmic domain of the receptor. EphA8-promoted adhesion was efficiently inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we found that EphA8 had associated PI 3-kinase activity and that the p110gamma isoform of PI 3-kinase is associated with EphA8. In vitro binding experiments revealed that the EphA8 juxtamembrane segment was sufficient for the formation of a stable complex with p110gamma. Similar results were obtained in assay using cells stripped of endogenous ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110gamma mutant was demonstrated to stably associate with EphA8 and suppress EphA8-promoted cell adhesion to fibronectin. Taken together, these results suggest the presence of a novel mechanism by which the EphA8 receptor localizes p110gamma PI 3-kinase to the plasma membrane in a tyrosine kinase-independent fashion, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion.

3T3 Cells↗

PubMed2001

Loss of secondary esophageal peristalsis is not a contributory pathogenetic factor in posterior laryngitis.

Secondary esophageal peristalsis helps prevent the entry of gastric acid into the pharynx by clearing the refluxed gastric contents back into the stomach. Because the loss of this mechanism may contribute to the pathogenesis of reflux-induced laryngeal disorders, our aim was to study the frequency of stimulation and parameters of secondary esophageal peristalsis in patients with posterior laryngitis (PL). We studied 14 patients (45 +/- 5 years) with PL documented by videolaryngoscopy and 11 healthy controls (46 +/- 6 years). The upper esophageal sphincter (UES) pressure was monitored by a sleeve assembly incorporating an injection port 5 cm distal to the sleeve. The esophageal body and lower esophageal sphincter (LES) pressures were measured by an LES sleeve assembly. Primary esophageal peristalsis was induced by 5-mL water swallows. Secondary esophageal peristalsis was induced by abrupt injection of volumes of air, incrementally increased by 5 mL, into the esophagus. Secondary esophageal peristalsis could not be elicited by injection of any volume (up to 60 mL) in 3 PL patients and 2 controls. These 5 subjects had normal primary peristalsis. The threshold volume of air required to stimulate secondary esophageal peristalsis in PL patients (median, 15 mL) was similar to that of controls (median, 10 mL). The parameters of the secondary esophageal peristaltic pressure wave were similar in both groups, and in both groups, they were similar to those of primary peristalsis. The UES response to the injection of the threshold volume that induced secondary esophageal peristalsis in PL patients was contraction in 58% of the trials, partial relaxation in 3%, and no response in 39%. The findings were similar to those in the controls. The LES response to injection of the threshold volume was complete relaxation in both the PL patients and the controls. We conclude that the integrity of secondary esophageal peristalsis is preserved in PL patients.

Adolescent↗

PubMed2001

[Fas and ICE expression in the hepatocytes of tupaia with HDV/HBV infection].

OBJECTIVE: To investigate the relationship between Fas and ICE expression and HDV infection. METHODS: HDAg, Fas, and ICE were determined in 45 liver tissue specimens of tupaia with HDV/HBV infection by immunohistochemistry. RESULTS: Fas was detected in 39 out of 45 liver tissue specimens (86.7%), and ICE in 43 (95.6%). Fas and ICE were located in the cytoplasm or/and the membrane of hepatocytes, especially in the cytoplasm. There was significant correlation between the expression of HDAg and the expression of Fas and ICE (chi(2)=29.2 and 36.2, respectively, P<0.01). CONCLUSIONS: The expression of Fas and ICE may be induced by HDAg in hepatocytes.

Animals↗

PubMed2001

The role of sexual related Y gene detection in the diagnosis of patients with gonadal dysgenesis.

OBJECTIVE: To clarify the role of sexual related Y (SRY) gene detection in the diagnosis of gonadal dysgenesis. METHODS: Sixteen cases of gonadal dysgenesis were included in this study: 5 with androgen insensitivity syndrome, 1 with 17-alpha-hydroxylase deficiency, 4 with true hermaphrodite, 2 with 45, X/46, XY gonadal dysgenesis, 1 with 45, X gonadal dysgenesis, 1 with XY pure gonadal dysgenesis, 1 with testicular regression, and 1 XY female who gave birth to a normal baby. SRY gene was detected by using polymerase chain reaction (PCR) in blood and gonad samples and by direct sequencing of the SRY motif. RESULTS: Among the 16 cases, 15 were blood SRY positive, among which 13 (86.7%) showed the presence of testicular tissue, and 2 showed ovaries without testicular tissue. One SRY negative case showed the presence of testicular tissue. In 3 cases, SRY detection in gonadal tissue correlated with pathological findings but not with blood karyotype. The correlation between peripheral blood SRY and the pathology of the gonads was 81.25% and the correlation between the presence of peripheral blood Y chromosome and pathology of the gonads was 68.75%. Sequencing of the SRY motif in an XY female who gave birth to a normal baby showed no mutation. CONCLUSIONS: SRY detection is more sensitive and specific than blood karyotype in the prediction of the presence of testicular tissue. Peripheral blood karyotype does not necessarily reflect gonadal type. There may be testicular related factors other than the SRY gene.

DNA↗

PubMed2000

Selective gas-phase cleavage at the peptide bond C-terminal to aspartic acid in fixed-charge derivatives of Asp-containing peptides.

This study focuses on the molecular level interpretation of the selective gas-phase cleavage at aspartic acid residues (Asp) in protonated peptides. A phi3P+CH2C(=O)group (phi = 2,4,6-trimethoxyphenyl) is attached to the N-terminal nitrogen of the selected peptides LDIFSDF and LDIFSDFR, via solid-phase synthesis, to "mimic" the tightly held charge of a protonated arginine (Arg) residue. Collision-induced dissociation in a quadrupole ion trap instrument and surface-induced dissociation in a dual quadrupole instrument were performed for electrospray-generated ions of the fixed-charge peptide derivatives. Selective cleavages at Asp-Xxx are observed for those ions with charge provided only by the fixed charge or for those with a fixed charge and one Arg plus one added proton. This supports a previously proposed mechanism which suggests that the cleavages at Asp-Xxx, initiated by the acidic hydrogen of the Asp residue, become significant when ionizing protons are strongly bound by Arg in the protonated peptides. It is clear that the fixed charge is indeed serving as a "mimic" of protonated Arg and that a protonated Arg side chain is not required to interact with the Asp to induce cleavage at Asp-Xxx. When the number of protons exceeds the number of Arg in a peptide containing Arg and Asp, nonselective cleavages occur. The fragmentation efficiency of the peptides is consistent with the idea that these nonselective cleavages are promoted by a mobile proton. The peptide with a fixed charge and one added proton, [phi3P+CH2C(=O)-LDIFSDF + H]2+, fragments much more efficiently than the corresponding peptide with a fixed charge, an Arg and one added proton, [phi3P+CH2C(=O)-LDIFSDFR + H]2+; both of these fragment more efficiently than the peptide with a fixed charge and no added proton, phi3P+CH2C(=O)-LDIFSDF. MS/MS/MS (i.e., MS3) experimental results for bn ions formed at Asp-Xxx from phi3P+CH2C(=O)-LDIFSDF and its H/D exchange derivative, phi3P+CH2C(=O)-LDIFSDF-d11, are consistent with the bn ions formed at Asp-Xxx having a succinic anhydride cyclic structure. MS/MS experiments were also carried out for phi3P+CH2C(=O)-AAAA, a peptide derivative containing active hydrogens only at amide nitrogens plus the C-terminus, and its active H/D exchange product, phi3P+CH2C(=O)-AAAA-d5. The results show that a hydrogen originally located at an amide nitrogen is transferred away in the formation of a cyclic charge remote b ion.

Aspartic Acid↗

PubMed2000

Use of high-performance liquid chromatography-tandem mass spectrometry to distinguish Panax ginseng C. A. Meyer (Asian ginseng) and Panax quinquefolius L. (North American ginseng).

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed to distinguish Asian ginseng (Panax ginseng C. A. Meyer) and North American ginseng (Panax quinquefolius L.). The method is based on the baseline chromatographic separation of ginsenoside Rf and 24(R)-pseudoginsenoside F11, two potential chemical markers present in ginseng root methanolic extracts, and their unambiguous on-line identification using tandem mass spectrometry. Consistent with the literature, 24(R)-pseudoginsenoside F11 was detected in abundance in North American ginseng roots in excess of 0.1% (w/w) of the dried root. In contrast to some reports, 24(R)-pseudoginsenoside F11 was also identified in Asian ginseng roots at trace levels using LC-MS-MS but at less than 0.0001% (w/w). Besides showing identical tandem mass spectra to authentic 24(R)-pseudoginsenoside F11, the corresponding compound in Asian ginseng root coeluted with standard under different HPLC conditions, thus confirming this compound as 24(R)-pseudoginsenoside F11. Another ginsenoside often used to distinguish Asian and North American ginseng, ginsenoside Rf, was found in abundance in Asian ginseng roots at more than 0.021% (w/w). In Asian ginseng roots, the ratio of ginsenoside Rf to 24(R)-pseudoginsenoside F11 exceeded 700:1. The limit of detection of ginsenoside Rf or 24(R)-pseudoginsenoside F11 was 120 pg injected on-column, and the limit of quantification was 240 pg on-column. In summary, LC-MS-MS analysis of ginseng products for the presence and ratio of ginsenoside Rf and 24(R)-pseudoginsenoside F11 may be used for the unambiguous identification of Asian and North American ginsengs.

Asia↗

PubMed2000

Introducing a null mutation in the mouse K6alpha and K6beta genes reveals their essential structural role in the oral mucosa.

Mammalian genomes feature multiple genes encoding highly related keratin 6 (K6) isoforms. These type II keratins show a complex regulation with constitutive and inducible components in several stratified epithelia, including the oral mucosa and skin. Two functional genes, K6alpha and K6beta, exist in a head-to-tail tandem array in mouse genomes. We inactivated these two genes simultaneously via targeting and homologous recombination. K6 null mice are viable and initially indistinguishable from their littermates. Starting at two to three days after birth, they show a growth delay associated with reduced milk intake and the presence of white plaques in the posterior region of dorsal tongue and upper palate. These regions are subjected to greater mechanical stress during suckling. Morphological analyses implicate the filiform papillae as being particularly sensitive to trauma in K6alpha/K6beta null mice, and establish the complete absence of keratin filaments in their anterior compartment. All null mice die about a week after birth. These studies demonstrate an essential structural role for K6 isoforms in the oral mucosa, and implicate filiform papillae as being the major stress bearing structures in dorsal tongue epithelium.

Animals↗

PubMed2000

Apoptotic signaling through the beta -adrenergic receptor. A new Gs effector pathway.

Stimulation of beta-adrenergic receptor normally results in signaling by the heterotrimeric G protein G(s), leading to the activation of adenylyl cyclase, production of cAMP, and activation of cAMP-dependent protein kinase (PKA). Here we report that cell death of thymocytes can be induced after stimulation of beta-adrenergic receptor, or by addition of exogenous cAMP. Apoptotic cell death in both cases was observed with the appearance of terminal deoxynucleotidyl transferase-mediated UTP end labeling reactivity and the activation of caspase-3 in S49 T cells. Using thymocytes deficient in either Galpha(s) or PKA, we find that engagement of beta-adrenergic receptors initiated a Galpha(s)-dependent, PKA-independent pathway leading to apoptosis. This alternative pathway involves Src family tyrosine kinase Lck. Furthermore, we show that Lck protein kinase activity can be directly stimulated by purified Galpha(s). Our data reveal a new signaling pathway for Galpha(s), distinct from the classical PKA pathway, that accounts for the apoptotic action of beta-adrenergic receptors.

Animals↗

PubMed2000

Ca(2+), Sr(2+), and Ba(2+) identify distinct regulatory sites on adenylyl cyclase (AC) types VI and VIII and consolidate the apposition of capacitative cation entry channels and Ca(2+)-sensitive ACs.

Ca(2+)-sensitive adenylyl cyclases may act as early integrators of the two major second messenger-signaling pathways mediated by Ca(2+) and cAMP. Ca(2+) stimulation of adenylyl cyclase type I (ACI) and adenylyl cyclase type VIII (ACVIII) is mediated by calmodulin and the site on these adenylyl cyclases that interacts with calmodulin has been defined. By contrast, the mechanism whereby Ca(2+) inhibits adenylyl cyclase type V (ACV) and adenylyl cyclase type VI (ACVI) is unknown. In this study, Ca(2+), Sr(2+), and Ba(2+) were compared to probe the involvement of E-F hand-like domains in both Ca(2+) stimulation and inhibition of ACVIII and ACVI, respectively. HEK 293 cells transfected with ACVIII cDNA and C6-2B glioma cells (where the endogenous adenylyl cyclases is predominantly ACVI) were used to compare the effects of these three cations in in vitro and in vivo measurements. The in vitro data identified two Ca(2+) regulatory sites for both ACVIII and ACVI. Strikingly different potency series for these cations at mediating high affinity stimulation and inhibition of ACVIII and ACVI, respectively, effectively rule out the possibility that calmodulin or proteins utilizing similar Ca(2+)-binding motifs mediate inhibition of ACVI. On the other hand, the low affinity inhibition that is common to both ACVIII and ACVI showed virtually identical potency profiles for the IIa cation series, indicating a common site of action. Remarkably, whereas Sr(2+) was rather ineffective at regulating these cyclases (particularly ACVI) in vitro, adequate concentrations accumulated in the vicinity of these enzymes as a consequence of capacitative cation entry to partially regulate both of these activities in vivo. This latter finding consolidates earlier observations that Ca(2+)-sensitive adenylyl cyclases detect and respond to capacitative cation entry rather than global cytosolic cation concentrations.

Adenylyl Cyclase Inhibitors↗