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M D Houslay

Publications and source records attributed to M D Houslay.

At least 91 records · Page 5Linked to original sources
PubMed1995

Effects of lactation on the signal transduction systems regulating lipolysis in sheep subcutaneous and omental adipose tissue.

The effect of lactation on the regulation of lipolysis by beta- and alpha 2-adrenergic agents and by adenosine has been investigated. When changes in adipocyte mean cell volume (which decreases with lactation) are allowed for, lactation increased the maximum response both to beta-adrenergic agents and to the adenosine analogue N6-phenylisopropyladenosine, but had no apparent effect on the responsiveness of the alpha 2-adrenergic system in both subcutaneous and omental adipocytes. For subcutaneous adipocytes, there was no significant change in the number of beta-adrenergic or alpha 2-adrenergic receptors, but the amount of Gs and the maximum (forskolin-stimulated) adenylate cyclase activity were increased by lactation. In contrast, in omental adipocytes, the number of beta- (but not alpha 2-) adrenergic receptors and the amount of Gs were increased, whereas forskolin-stimulated adenylate cyclase activity was unchanged by lactation. In both types of adipocyte, cyclic AMP phosphodiesterase and total protein kinase A activities were unchanged. Lactation had no effect on the number of adenosine receptors but increased the amounts of the Gi isoforms expressed in both types of adipocyte. These various adaptations differ markedly in a number of respects from those described previously in the rat. Lactation, then, while having a similar overall effect on the response to beta-adrenergic agonists of adipocytes, achieves this by depot-specific mechanisms. In contrast, changes in response to adenosine appear to involve the same mechanism in the two depots investigated.

Adenylyl Cyclases↗
PubMed1995

A role for protein kinase C-mediated phosphorylation in eliciting glucagon desensitization in rat hepatocytes.

An immobilized hepatocyte preparation was used to show that both vasopressin and glucagon could desensitize the ability of glucagon to increase intracellular cyclic AMP concentrations. This process was not dependent on any influx of extracellular Ca2+ and was not mediated by any rise in the intracellular level of Ca2+. The protein kinase C-selective inhibitors chelerythrine, staurosporine and calphostin C acted as potent inhibitors of the desensitization process but with various degrees of selectivity regarding their ability to inhibit the desensitizing actions of glucagon and vasopressin. The protein phosphatase inhibitor okadaic acid was just as potent as vasopressin and glucagon in causing desensitization. Treatment of hepatocyte membranes with alkaline phosphatase restored to near control levels the ability of glucagon to stimulate adenylate cyclase activity in membranes from both glucagon- and vasopressin-treated (desensitized) hepatocytes. It is suggested that the desensitization of glucagon-stimulated adenylate cyclase activity involves a reversible phosphorylation reaction with the likely target being the glucagon receptor itself.

Adenylyl Cyclases↗
PubMed1995

Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum.

An antiserum was generated against a dodecapeptide whose sequence is found at the C-terminus of a cyclic AMP (cAMP)-specific, type-IVA phosphodiesterase encoded by the rat 'dunc-like' cyclic AMP phosphodiesterase (RD1) cDNA. This antiserum identified a single approximately 73 kDa protein species upon immunoblotting of cerebellum homogenates. This species co-migrated upon SDS/PAGE with a single immunoreactive species observed in COS cells transfected with the cDNA for RD1. Native RD1 in cerebellum was found to be predominantly (approximately 93%) membrane-associated and could be found in isolated synaptosome populations, in particular those enriched in post-synaptic densities. Fractionation of lysed synaptosomes on sucrose density gradients identified RD1 as co-migrating with the plasma membrane marker 5'-nucleotidase. Laser scanning confocal and digital deconvolution immunofluorescence studies done on intact COS cells transfected with RD1 cDNA showed RD1 to be predominantly localized to plasma membranes but also associated with the Golgi apparatus and intracellular vesicles. RD1-specific antisera immunoprecipitated phosphodiesterase activity from solubilized cerebellum membranes. This activity had the characteristics expected of the type-IV cAMP phosphodiesterase RD1 in that it was cAMP specific, exhibited a low Km cAMP of 2.3 microM, high sensitivity to inhibition by 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) (Ki approximately 0.7 microM) and was unaffected by Ca2+/calmodulin and low concentrations of cyclic GMP. The phosphodiesterase activities of RD1 solubilized from both cerebellum and transfected COS cell membranes showed identical first-order thermal denaturation kinetics at 50 degrees C. Native RD1 from cerebellum was shown to be an integral protein in that it was solubilized using the non-ionic detergent Triton X-100 but not by either re-homogenization or high NaCl concentrations. The observation that hydroxylamine was unable to cause the release of RD1 from either cerebellum or COS membranes and that [3H]palmitate was not incorporated into the RD1 protein immunoprecipitated from COS cells transfected with RD1 cDNA, indicated that RD1 was not anchored by N-terminal acylation. The engineered deletion of the 25 residues forming the unique N-terminal domain of RD1 caused both a profound increase in its activity (approximately 2-fold increase in Vmax) and a profound change in intracellular distribution. Thus, immunofluorescence studies identified the N-terminal truncated species as occurring exclusively ion the cytosol of transfected COS cells. The cDNA for RD1 thus appears to encode a native full-length type-IVA phosphodiesterase that is expressed in cerebellum.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Identification of two splice variant forms of type-IVB cyclic AMP phosphodiesterase, DPD (rPDE-IVB1) and PDE-4 (rPDE-IVB2) in brain: selective localization in membrane and cytosolic compartments and differential expression in various brain regions.

In order to detect the two splice variant forms of type-IVB cyclic AMP phosphodiesterase (PDE) activity, DPD (type-IVB1) and PDE-4 (type-IVB2), anti-peptide antisera were generated. One set ('DPD/PDE-4-common'), generated against a peptide sequence found at the common C-terminus of these two PDEs, detected both PDEs. A second set was PDE-4 specific, being directed against a peptide sequence found within the unique N-terminal region of PDE-4. In brain, DPD was found exclusively in the cytosol and PDE-4 exclusively associated with membranes. Both brain DPD and PDE-4 activities, isolated by immunoprecipitation, were cyclic AMP-specific (KmcyclicAMP: approximately 5 microM for DPD; approximately 4 microM for PDE-4) and were inhibited by low rolipram concentrations (K1rolipram approximately 1 microM for both). Transient expression of DPD in COS-1 cells allowed identification of an approx. 64 kDa species which co-migrated on SDS/PAGE with the immunoreactive species identified in both brain cytosol and membrane fractions using the DPD/PDE-4-common antisera. The subunit size observed for PDE-4 (approx. 64 kDa) in brain membranes was similar to that predicted from the cDNA sequence, but that observed for DPD was approx. 4 kDa greater. Type-IV, rolipram-inhibited PDE activity was found in all brain regions except the pituitary, where it formed between 30 and 70% of the PDE activity in membrane and cytosolic fractions when assayed with 1 microM cyclic AMP, PDE-4 formed 40-50% of the membrane type-IV activity in all brain regions save the midbrain (approx. 20%). DPD distribution was highly restricted to certain regions, providing approx. 35% of the type-IV cytosolic activity in hippocampus and 13-21% in cortex, hypothalamus and striatum with no presence in brain stem, cerebellum, midbrain and pituitary. The combined type-IVB PDE activities of DPD and PDE-4 contributed approx. 10% of the total PDE activity in most brain regions except for the pituitary (zero) and the mid-brain (approx. 3%. The isolated cDNAs for DPD and PDE-4 appear to reflect transcription products which are expressed in vivo in brain. The unique N-terminal domain of PDE-4 is suggested to target this PDE to membranes in brain. Type-IVB PDEs are differentially expressed in various brain regions, indicating that there are tissue-specific controls on both the expression of the gene and the splicing of its products.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Purification, characterization and analysis of rolipram inhibition of a human type-IVA cyclic AMP-specific phosphodiesterase expressed in yeast.

Analyses were done on a human type-IV cyclic AMP (cAMP) phosphodiesterase (hPDE-IVA-h6.1) expressed in an engineered strain of Saccharomyces cerevisiae. This strain (YMS6) expressed soluble PDE activity, together with an insoluble activity which was not released by re-homogenization, treatment with high-ionic-strength solutions or with the detergent Triton X-100. Pellet and soluble PDE activities were typical of type-IV PDE. They were cAMP-specific, insensitive to the addition of either cGMP (1 microM) or Ca2+/calmodulin, and inhibited by rolipram. Thermostability studies showed both activities to decay as single exponentials, indicating the presence of homogeneous PDE protein species in each fraction. Pellet PDE activity was more thermostable than the soluble enzyme. Mg2+ and Mn2+ dose-dependently increased PDE activity and reversed the inactivating effect of EDTA.h6.1 was engineered to express a C-terminal five-histidine motif (h6.1his5). This allowed purification of the PDE to apparent homogeneity in a simple two-step process involving a rolipram affinity column and a Ni2(+)-chelate column. A single monomeric protein of subunit molecular mass approximately 73 kDa and native molecular mass approximately 74 kDa resulted after a approximately 53000-fold purification. This exhibited a Km for cAMP of 8 microM, a true Vmax. of 0.8 mumol of cAMP hydrolysed/min per mg of PDE protein, a kcat. of 3702 s-1, and a value of the specificity constant kcat/Km of 4.6 x 10(8) M-1.s-1, the last implying a diffusion controlled reaction. Rolipram (Ki 0.4 soluble; 0.7 microM pellet) and 3-isobutyl-1-methylxanthine (Ki 15 soluble; 19 microM pellet) served as simple competitive inhibitors for both soluble and pellet forms of h6.1, respectively.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Multi-site phosphorylation of the inhibitory guanine nucleotide regulatory protein Gi-2 occurs in intact rat hepatocytes.

A phosphorylated form of alpha-Gi-2 (the alpha-subunit of Gi-2), immunoprecipitated from hepatocytes under basal conditions, migrated as a single species of pI approximately 5.7, the labelling of which increased approximately 2-fold in cells challenged with either vasopressin or phorbol 12-myristate 13-acetate (PMA); agents which activate protein kinase C. In contrast, treatment of hepatocytes with 8-bromo-cyclic AMP produced a more acidic species of phosphorylated alpha-Gi-2 having a pI of approximately 5.4 and whose labelling was increased approximately 3-fold. Trypsin digestion of labelled alpha-Gi-2 isolated from hepatocytes under basal conditions identified, on two-dimensional peptide analyses, three positively charged phosphoserine-containing peptides (C1, C2 and C3), with only peptides C1 and C2 being evident upon less extensive digestion with trypsin. These are suggested to reflect a single site of phosphorylation, with proteolysis by trypsin being incomplete, and where C2 is larger than C1, which is larger than C3. An identical pattern of tryptic phosphopeptides was seen in hepatocytes treated with either vasopressin or PMA, although labelling of this group of peptides was increased by approximately 2-fold compared with the basal state. In contrast, treatment of hepatocytes with glucagon, 8-bromo-cyclic AMP or forskolin not only resulted in increased labelling of the 'basal' sites approximately 3-fold, but identified a novel positively charged tryptic phosphoserine-containing peptide (AN). All four tryptic peptides were susceptible to proteolysis by V8 protease. Treatment of labelled alpha-Gi-2 from basal and PMA-treated cells produced a pattern of peptides which was identical with those found when the tryptic phosphopeptide was treated with V8 protease. We tentatively suggest that, on alpha-Gi-2, Ser144 is phosphorylated through the action of protein kinase C and Ser207 is phosphorylated upon elevation of the intracellular concentrations of cyclic AMP.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Analysis of the adenylate cyclase signalling system, and alterations induced by culture with insulin, in a novel SV40-DNA-immortalized hepatocyte cell line (P9 cells).

An immortalized cell line, called P9, was derived from hepatocytes by transfection with SV40 DNA. These cells expressed enzyme activities characteristic of hepatocytes, namely glucose-6-phosphatase, glycogen phosphorylase, bilirubin glucuronyltransferase and both glucagon- and prostaglandin E1 (PGE1)-stimulated adenylate cyclase activities, albeit at decreased levels compared with native hepatocytes. Levels of the G-protein subunits alpha-Gi-2, alpha-Gi-3, G beta and the 'long' form of alpha-G2 (45 kDa) were approximately 4-fold higher relative to native hepatocytes, whereas those of the 'short' form of alpha-G2 (42 kDa) were lower by approximately 40%. Associated with this were marked alterations in the guanine nucleotide regulation of adenylate cyclase. Receptor-mediated stimulation, achieved by either PGE1 or glucagon, was apparent in P9 cells, although the latter was only evident upon amplification with forskolin. Glucagon-stimulated cyclic AMP accumulation in P9 cells did not exhibit desensitization, as in hepatocytes, nor was the phosphorylation of alpha-Gi-2 evident. Culture of P9 cells with insulin led to a dose-dependent decrease (EC50 0.2 +/- 0.1 nM) in the ability of PGE1 to stimulate adenylate cyclase activity, with the maximum effect attained after approximately 6 h. A comparable attenuation of stimulation was seen for glucagon- and guanine-nucleotide-stimulated adenylate cyclase activities. In cells cultured with insulin, lower levels of GTP were required to stimulate adenylate cyclase, ADP-ribosylation of the 45 kDa form of alpha-Gs with cholera toxin was attenuated, and the expression of both alpha Gi-2 and alpha-Gi-3 was increased. It is suggested that the expression of alpha-Gi-2 and alpha-Gi-3 may be directly regulated by the action of insulin in hepatocytes and P9 cells.

Adenosine Diphosphate Ribose↗
PubMed1994

Phosphorylation of calmodulin on Tyr99 selectively attenuates the action of calmodulin antagonists on type-I cyclic nucleotide phosphodiesterase activity.

Tyr99 phosphorylation of calmodulin appears to induce a distinct conformational change as is evident from the profound attenuation of the Ca(2+)-induced enhancement of calmodulin's mobility seen during SDS/PAGE. The effect of this conformational change appears to be localized, in that both calmodulin and P-Tyr99-calmodulin show identical dose-dependent activation profiles for stimulation of a physiological effector, type-I (Ca2+/calmodulin-stimulated) cyclic nucleotide phosphodiesterase (PDE) activity and their presence engenders similar dose-dependent PDE activation by Ca2+. In marked contrast with this, with P-Tyr99-calmodulin there were 3-4-fold increases in the IC50 values for inhibition of type-I PDE activity by the calmodulin antagonists TFP and W7, together with increased values for Hill coefficients for inhibition. The polybasic compound poly(L-lysine) potently augmented the action of calmodulin as a PDE activator, causing an approx. 7-fold decrease in the EC50 value for activation of PDE. It is suggested (i) that the Tyr99 phosphorylation of calmodulin, which occurs within a high-affinity Ca(2+)-binding domain, induces a localized conformational change in this peptide which can selectively attenuate the action of calmodulin antagonists on type-I PDE activity while leaving unaffected Ca(2+)-dependent activation, and (ii) that polybasic substances on complexing with calmodulin may serve to enhance the sensitivity of type-I PDE to activation by this regulatory peptide.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Growth hormone decreases the response to anti-lipolytic agonists and decreases the levels of Gi2 in rat adipocytes.

The effect of growth hormone (GH) in vivo on the Gi-mediated anti-lipolytic signalling system of rat adipocytes has been investigated. Lowering of serum GH levels, by treatment of rats with an antiserum (anti-rGH) specific for rat GH, increased the sensitivity of adipocytes to the anti-lipolytic agonists N6-phenylisopropyladenosine (PIA) and prostaglandin E1. This occurred in the absence of any change in PIA binding to adipocyte membranes. Immunoblot analysis of adipocyte membranes revealed that lowering of serum GH resulted in at least a 3-fold increase in the levels of alpha-subunit of Gi2, but had no effect on the alpha-subunits of Gi1 and Gi3 nor on the 42 and 45 kDa forms of the alpha-subunit of Gs. Replenishment of serum GH, by concurrent administration of ovine GH to rats, prevented all of these effects of anti-rGH. It is concluded that GH down-regulates the amount of Gi2 alpha-subunit in adipocyte membranes, resulting in a decrease in the sensitivity of the cells to anti-lipolytic agonists.

Adipose Tissue↗
PubMed1994

Regulation of hepatocyte adenylate cyclase by amylin and CGRP: a single receptor displaying apparent negative cooperatively towards CGRP and simple saturation kinetics for amylin, a requirement for phosphodiesterase inhibition to observe elevated hepatocyte cyclic AMP levels and the phosphorylation of Gi-2.

Challenge of intact hepatocytes with amylin only succeeded in elevating intracellular cyclic AMP levels and activating phosphorylase in the presence of the cAMP phosphodiesterase inhibitor IBMX. Both amylin and CGRP similarly activated adenylate cyclase, around 5-fold, although approximately 400-fold higher levels of amylin were required to elicit half maximal activation. Amylin activated adenylate cyclase though apparently simple Michaelien kinetics whereas CGRP elicited activation by kinetics indicative of apparent negative co-operativity. Use of the antagonist CGPP(8-37) showed that both CGRP and amylin activated hepatocyte adenylate cyclase through a common receptor by a mnemonical mechanism where it was proposed that the receptor co-existed in interconvertible high and low affinity states for CGRP. It is suggested that this model may serve as a paradigm for G-protein linked receptors in general. Amylin failed to both stimulate inositol phospholipid metabolism in hepatocytes and to elicit the desensitization of glucagon-stimulated adenylate cyclase. Amylin did, however, elicit the phosphorylation of the inhibitory guanine nucleotide regulatory protein Gi-2 in hepatocytes and prevented the action of insulin in reducing the level of phosphorylation of this G-protein.

Adenylyl Cyclases↗
PubMed1994

Molecular cloning and expression, in both COS-1 cells and S. cerevisiae, of a human cytosolic type-IVA, cyclic AMP specific phosphodiesterase (hPDE-IVA-h6.1).

Screening a human T lymphocyte cDNA library with a phosphodiesterase (PDE) specific probe resulted in the isolation of two overlapping cDNA clones, h2.2 and h6.1, that encode a type IV, rolipram inhibited cAMP-specific PDE. Clones h2.2 and h6.1 were 1015 bp and 2288 bp in length, respectively, and overlapped for 984 bp with only one nucleotide difference. The h6.1 cDNA was extended at the 5'-end by 1304 bp, with respect to h2.2, and encoded an incomplete ORF (lacking an initiation codon) of 668 amino acids. The merged nucleotide sequence of h6.1/h2.2 exhibited 99.5% homology in the ORF (ten nucleotide changes resulting in six amino acid changes), and 95% homology in the 3'-untranslated region, with the previously reported human PDE-IVA cDNA [Livi G. P., Kmetz P., Mchale M. M., Cieslinski L. B., Sathe G. M., Taylor D. P., Davis R. L., Torphy T. J. and Balcarek J. M. (1990) Mol. Cell Biol. 10, 2678-2686]. The sequence reported for h6.1/h2.2 matched that found for IVA clones isolated from three other human cDNA libraries, a human genomic cosmid clone and pcr amplified products of the exon covering these differences in two individuals. The h6.1 cDNA was engineered to generate a complete ORF by building in the 56 bp, including the initiation codon, present in hPDE-IVA-Livi and missing from the 5'-end of h6.1, producing a cognate ORF encoding a protein of 687 amino acids but differing in five amino acids which lay in or adjacent to the putative catalytic domain. The complete h6.1 ORF was engineered for expression in both Saccharomyces cerevisiae and in COS-1 cells. Integration of a single copy of the engineered ORF of h6.1, under the transcriptional control of a constitutive yeast promoter, at the pep4 locus of a S. cerevisiae strain lacking both yeast PDE genes resulted in functional complementation of the yeast pde-phenotype. Yeast strains with functional PDE were a light creamy white colour, while strains devoid of PDE activity were a dull brown colour. Expression of h6.1 in COS-1 cells led to the production of a typical type IV PDE activity in that cAMP, but not cGMP, served as substrate and its activity was insensitive to either Ca2+/CaM or cGMP but was inhibited by low concentrations of rolipram.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Dietary linoleic acid-induced changes in respiratory beta-adrenergic receptor function and the form of arrhenius plots of isoprenaline- and prostaglandin E2-stimulated adenylate cyclase activity in a model for atopy.

Varying dietary linoleic acid altered lung membrane fatty acid composition with linoleic acid content increasing from approximately 6% total in those on 3 en% diet to approximately 14% total fatty acid in those on a 12 en% diet. Accompanying this were two- to three-fold increases in the levels of the elongation products of linoleic acid, namely 20:2 (n-6) and 22:5 (n-6) and a decrease in 18:1 oleic acid from approximately 26% to approximately 19% total. Administration of Haemophilus influenzae, to animals on 6 en% linoleic acid, serving as a model for atopy, effected a small increase in the levels of 22:5 (n-3) and doubled those of 22:6 (n-3). beta-Adrenergic-induced tracheal relaxation and stimulation of lung adenylate cyclase were elevated by increasing dietary linoleic acid from 3 to 6 en%, although such differences were abolished in the atopic model and when dietary linoleic acid was increased to 12 en%. Arrhenius plots of NaF-stimulated lung adenylate cyclase activities exhibited a break (t1) at approximately 26 degrees C in all dietary groups with unchanged activation energies and activity. In contrast, whilst both isoprenaline and PGE2-stimulated adenylate cyclase activities showed similar break-points in their Arrhenius plots, dietary linoleic acid manipulation markedly altered their form. As with NaF-stimulated activities then, irrespective of dietary manipulation and induction of atopy, these plots showed an invariant break occurring at approximately 26 degrees C. But, for animals on 3 and 6 en% diets, a second break was apparent at approximately 15 degrees C, which was slightly decreased to approximately 12 degrees C upon induction of atopy and completely abolished on increasing dietary linoleic acid to 12 en%. Accompanying such changes were marked alterations in activation energies. We suggest that profound changes in lung plasma membrane bilayer properties occur upon both altering dietary linoleic acid levels and in atopy. These selectively perturb adenylate cyclase activity when it is receptor-stimulated but not when it is activated by direct G-protein stimulation with NaF. We suggest that atopy and dietary challenge elicit an asymmetric perturbation of the plasma membrane that predominantly affects the outer half of the lipid bilayer.

Adenylyl Cyclases↗
PubMed1993

Diabetes induces selective alterations in the expression of protein kinase C isoforms in hepatocytes.

Membrane and cytosol fractions from hepatocytes of both normal and streptozotocin-induced diabetic animals were probed with a panel of polyclonal anti-peptide antisera in order to identify protein kinase C (PKC) isoforms. Immunoreactive species were noted with antisera specific for alpha (approximately 81 kDa), beta-II (approximately 82 kDA), epsilon (approximately 95 kDa) and epsilon (approximately 79 kDa). In addition, a species migrating with an apparent size of approximately 94 kDa was also detected in cytosol fractions using an antiserum specific for PKC-alpha. Each of these species was specifically displaced when the PKC-isoform specific peptide was included in the immunodetection system. No immunoreactive species consistent with the presence of the beta-I, gamma, delta and eta isoforms of protein kinase C was observed. Induction of diabetes using streptozotocin invoked selective alterations in the expression of PKC isoforms which were reversed upon insulin therapy. In the cytosol fraction, marked increases of approximately 3-fold occurred in levels of the beta-II isoform and the approximately 90 kDa (upper) form of PKC-alpha, with no apparent/little change in the levels of the approximately 81 kDa (lower) form of PKC-alpha and those of PKC-zeta. Diabetes induction also appeared to have elicited the translocation of PKC-beta-II and the approximately 81 kDa (lower) form of PKC-alpha to the membrane fraction where immunoreactivity for these species was now apparent. The level of PKC-epsilon, which was noted only in membrane fractions, was also increased upon induction of diabetes. It is suggested that the selective alterations in the expression of PKC isoforms occurring upon streptozotocin-induced diabetes may lead to altered cellular functioning and underly defects in inhibitory G-protein functioning and insulin action which characterise this animal model of diabetes.

Animals↗
PubMed1993

A mnemonical or negative-co-operativity model for the activation of adenylate cyclase by a common G-protein-coupled calcitonin-gene-related neuropeptide (CGRP)/amylin receptor.

Both amylin and calcitonin-gene-related neuropeptide (CGRP) activated adenylate cyclase activity in hepatocyte membranes around 5-fold in a dose-dependent fashion, with EC50 values of 120 +/- 14 and 0.3 +/- 0.14 nM respectively. Whereas amylin exhibited normal activation kinetics (Hill coefficient, h approximately 1), CGRP showed kinetics indicative of either multiple sites/receptor species having different affinities for this ligand or a single receptor species exhibiting apparent negative co-operativity (h approximately 0.21). The CGRP antagonist CGRP-(8-37)-peptide inhibited adenylate cyclase stimulated by EC50 concentrations of either amylin or CGRP. Inhibition by CGRP-(8-37) was selective in that markedly lower concentrations were required to block the action of amylin (IC50 = 3 +/- 1 nM) compared with that of CGRP itself (IC50 = 120 +/- 11 nM). Dose-effect data for inhibition of CGRP action by CGRP-(8-37) showed normal saturation kinetics (h approximately 1), whereas CGRP-(8-37) inhibited amylin-stimulated adenylate cyclase activity in a fashion which was indicative of either multiple sites or apparent negative co-operativity (h approximately 0.24). Observed changes in the kinetics of inhibition by CGRP-(8-37) of CGRP, but not amylin-stimulated adenylate cyclase, at concentrations of agonists below their EC50 values militated against a model of two distinct populations of non-interacting receptors each able to bind both amylin and CGRP. A kinetic model is proposed whereby a single receptor, capable of being activated by both CGRP and amylin, obeys either a mnemonical kinetic mechanism or one of negative co-operativity with respect to CGRP but not to amylin. The relative merits of these two models are discussed together with a proposal suggesting that the activation of adenylate cyclase by various G-protein-linked receptors may be described by a mnemonical model mechanism.

Adenylyl Cyclases↗
PubMed1993

Extracellular calcium modulates insulin's action on enzymes controlling cyclic AMP metabolism in intact hepatocytes.

Absence of physiological concentrations of extracellular Ca2+ in the Krebs-Henseleit incubation buffer did not affect the ability of 10 nM glucagon (< 5%) to increase hepatocyte intracellular cyclic AMP concentrations, but severely ablated (by approximately 70%) the ability of 10 nM insulin to decrease these elevated concentrations. Cyclic AMP metabolism is determined by production by adenylate cyclase and degradation by cyclic AMP phosphodiesterase (PDE). In the absence of added extracellular Ca2+ (2.5 mM), insulin's ability to activate PDE activity was selectively compromised, showing a failure of insulin to activate two of the three insulin-stimulated activities, namely the 'dense-vesicle' and peripheral plasma-membrane (PPM) PDEs. In the absence of added Ca2+, insulin's ability to inhibit adenylate cyclase activity in intact hepatocytes was decreased dramatically. Vasopressin and adrenaline (+ propranolol) failed to elicit the activation of either the 'dense-vesicle' or the PPM-PDEs. The presence of physiological concentrations of extracellular Ca2+ in the incubation medium is shown to be important for the appropriate generation of insulin's actions on cyclic AMP metabolism.

3',5'-Cyclic-AMP Phosphodiesterases↗