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A C Howlett

Publications and source records attributed to A C Howlett.

84 records · Page 5Linked to original sources
PubMed1982

Effects of NaCl concentration on adenylate cyclase regulation by prostaglandins and guanine nucleotides.

Na+ has been implicated as a requirement for the inhibition of adenylate cyclase by hormones and neurotransmitters. This study examines effects of salt concentration on neuroblastoma plasma membranes that occur in the absence of an inhibitory hormone. The adenylate cyclase response to stimulatory agonists (GTP plus PGE1 (3), PGI2 or PGE2) was influenced by NaCl. As the [NaCl] increased to 150 mM, an increase in maximal activity and a decrease in apparent affinity was observed. At concentrations above 150 mM, NaCl decreased prostaglandin affinity and progressively decreased maximal activation. The GTP requirement was not altered by 30 or 150 mM NaCl in the presence of PGE1 or PGI2. The rate of Gpp(NH)p stimulated activity increased as the [NaCl] was increased in the assay. This increased rate was conserved when membranes activated in the presence of Gpp(NH)p and NaCl were reassayed in the absence of guanine nucleotide or salt. The salt evoked rate increase was proportionally greater at submaximal MgCl2 concentrations. The concentration requirement for Mg2+ was reduced by salt for adenylate cyclase in the presence of GTP or Gpp(NH)p. However, the enzyme stimulated by hormone exhibited a Mg2+ requirement that was low in the absence of salt and could not be further reduced by increased [NaCl]. Alternative monovalent cations (150 mM Li+, K+, Cs+, but not choline or tetramethylammonium) and anions (SO4=) substituted for NaCl. The observed effects were reversible upon washing the membranes and neither ouabain nor tetrodotoxin altered the response. These effects may result from a conformational alteration of a protein particularly sensitive to neutral salts in the assay.

Adenylyl Cyclases↗
PubMed1980

Hydrodynamic properties of the regulatory component of adenylate cyclase.

Hydrodynamic parameters of the regulatory component of adenylate cyclase, G/F, have been estimated by gel filtration and sucrose density gradient centrifugation. In solutions containing Lubrol 12A9, the protein has an apparent molecular weight of 130,000. G/F from various sources and resolved from the catalytic moiety of the enzyme by different techniques behaves similarly. Consistent with our previous proposal that this protein is the site of action of both guanine nucleotides and fluoride, treatment with these activating ligands causes a reduction in both the sedimentation coefficient and the Stokes radius of G/F. These changes suggest a loss of mass of approximately 40,000 daltons. Nevertheless, this alteration is fully reversible when ligands are removed, even if the liganded protein is first fractionated by gel filtration or sucrose density gradient centrifugation.

Adenylyl Cyclases↗
PubMed1979

Identification and partial characterization of some components of hormone-stimulated adenylate cyclase.

Adenylate cyclase can be resolved into at least two protein components, neither of which by itself catalyzes the formation of cyclic AMP with Mg-ATP as substrate. Mixture of the two reconstitutes Mg-ATP-dependent, fluoride- and Gpp(NH)p-stimulable activity. One, a heat-labile, N-ethylmaleimide-sensitive protein of molecular weight 190,000 can catalyze cyclic AMP formation with Mn-ATP as substrate, and is therefore proposed to be the catalytic moiety of the adenylate cyclase complex. The other protein (or proteins) is more resistant to heating or N-ethylmaleimide, and is proposed to confer upon the catalyst the ability to ultilize Mg-ATP as substrate. It is also required for the regulation of that activity by guanine nucleotides, hormones, and probably fluoride ion. The catalytic protein is found in a phenotypically adenylate cyclase-deficient (AC-) variant of S49 lymphoma cells. The thermostable regulatory protein can be resolved from the catalyst by heat treatment or N-ethylmaleimide treatment of plasma membranes of wild-type S49 cells, rat or rabbit liver, or avian erythrocytes, and is also found in a phenotypically adenylate cyclase-deficient hepatoma cell line. Mixture of AC- S49 membranes, which contain the beta-adrenergic receptor, with a crude detergent-solubilized preparation of the regulatory protein reconstitutes hormone-stimulable adenylate cyclase activity. Binding of the regulatory protein to the membranes is a time- and temperature-dependent process that requires an activating ligand of the adenylate cyclase system [fluoride, Gpp(NH)p].

Adenylyl Cyclases↗
PubMed1978

Reconstitution of hormone-sensitive adenylate cyclase activity with resolved components of the enzyme.

Adenylate cyclase can be resolved into at least two proteins, a thermolabile, N-ethylmaleimide-sensitive component and a second protein (or proteins) that is more stable to either of these treatments. Neither component by itself catalyzes the formation of cyclic AMP using MgATP as substrate. However, mixture of the two reconstitutes MgATP-dependent fluoride- and guanyl-5'-yl imidodiphosphate (Gpp(NH)p)-stimulatable adenylate cyclase activity. The more stable component can be resolved from the first in various tissues or cultured cells by treatment of membrnes or detergent extracts with heat or N-ethylmaleimide. The two proteins have also been resolved genetically in two clonal cell lines that are deficient in adenylate cyclase activity. An adenylate cyclase-deficient variant of the S49 lymphoma cell (AC-) contains only the thermolabile activity, while the activity of the more stable protein is found in a complementary hepatoma cell line (HC-1). In addition, AC-S49 cell plasma membranes contain MnATP-dependent adenylate cyclase activity. The protein that catalyzes this reaction appears to be the same as that which can combine with the thermostable component to reconstitute Mg2+-dependent enzyme activity because both activities co-fractionate by gel exclusion chromatography and sucrose density gradient centrifugation, both activities have identical denaturation kinetics at 30 degrees C, and both activities are stabilized at 30 degrees C and labilized at 0 degree C by various nucleotides and divalent cations with similar specificity. It is thus hypothesized that the thermolabile factor is the catalytic subunit of the physiological adenylate cyclase and that the Mn2+-dependent activity is a nonphysiological expression of the catalytic protein. The thermostable moiety of the enzyme, which is proposed to serve a regulatory function, appears to consist of two functional components, based upon differential thermal lability of its ability to reconstitute hormone-, NaF-, or Gpp(NH)p-stimulated adenylate cyclase activity. These components have not, however, been physically separated. The thermolabile and thermostable components can interact in detergent solution or in a suitable membrane. Mixing of the detergent-solubilized regulatory component with AC-membranes that contain only the catalytic protein and beta-adrenergic receptors reconstitutes catecholamine-stimulatable adenylate cyclase activity; however, addition of the catalytic protein to membranes that contain receptor and the regulatory component yields MgATP-dependent enzymatic activity that is unresponsive to hormone.

Adenylyl Cyclases↗
PubMed1978

S49 lymphoma wild type and variant clones contain normal calcium dependent regulator.

Calcium dependent regulator is present in wild-type S49 lymphoma cells, in the variant that is deficient in adenylate cyclase activity (AC-), and in the uncoupled variant (UNC). The electrophoretic mobility and the ability to stimulate cyclic nucleotide phosphodiesterase of the calcium dependent regulator from each of these three clones are indistinguishable from those of the modulator protein isolated from bovine brain. Calcium dependent regulator does not appear to be involved in the defect responsible for the UNC or AC- variants.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMedDate not supplied

Synthesis and release of vasoactive intestinal polypeptide (VIP) by mouse neuroblastoma cells: modulation by cyclic nucleotides and ascorbic acid.

The mouse neuroblastoma cell line N18TG2 synthesizes and secretes a VIP-like immunoreactive material. The majority of this VIP-like material from both cell and media extracts elutes on HPLC in the same position as porcine or rat VIP. Several additional peaks which appear in the media extracts may represent variant forms or degradation products of VIP. The synthesis and release of VIP was significantly enhanced by agents which elevate cAMP levels directly (dbcAMP and forskolin) or through a receptor mediated process (secretin). These agents are also known to promote differentiation of these cells. The synthesis and release of VIP was also enhanced by ascorbate (thought to be a co-factor for the enzyme which amidates the carboxyl-terminal of VIP) [11]. In the presence of forskolin, ascorbate had a synergistic effect on the release of VIP, suggesting that forskolin and ascorbate are elevating VIP levels by different mechanisms; forskolin through a possible effect on VIP mRNA synthesis or translation, and ascorbate by increasing the rate of VIP processing. These results suggest that VIP synthesis and release is controlled by more than one process, whose rate can be altered with pharmacological agents.

Animals↗
PubMedDate not supplied

The ontogeny of cannabinoid receptors in the brain of postnatal and aging rats.

It is recognized that a number of the biological effects of delta 9-tetrahydrocannabinol (THC) can be attributed to a cannabinoid receptor found in abundance in the brain. Due to observations that cannabinoid drugs exert some developmental toxicity, it was of interest to examine the developmental pattern of cannabinoid receptors in the brain of neonatal rats through young adulthood, and then to further examine the cannabinoid receptor during the aging process in the brain of rats 3 to 32 months of age. Using radioligand binding assays, this study demonstrated that cannabinoid receptor binding capacity increases progressively from birth to postnatal day (PND) 60. Within the striatum, a significant increase in binding occurred between PNDs 14 and 21. In the cerebellum, cannabinoid receptor binding capacity doubled at 7-day postnatal intervals until adulthood. Cannabinoid receptor binding in the cortex doubled between PNDs 7 and 14. Within the hippocampus, there were small incremental increases until the final adult level was reached at PND 21. There was no significant alteration in the affinity for CP-55940 during development. These findings might reflect an increased differentiation of neurons into cells possessing cannabinoid receptors, or an increase in the number of cannabinoid receptors on cell bodies or projections in regions undergoing developmental changes. Once the adult cannabinoid receptor levels have been reached, binding activity in the whole brain preparation neither increased nor declined during the normal aging process.

Aging↗