1,4-Cyclohexanecarboxylates: potent and selective inhibitors of phosophodiesterase 4 for the treatment of asthma

Evaluation of a variety of PDE4 inhibitors in a series of cellular and in vivo assays suggested a strategy to improve the therapeutic index of PDE4 inhibitors by increasing their selectivity for the ability to inhibit PDE4 catalytic activity versus the ability to compete for high affinity [3H]rolipram-binding sites in the central nervous system. Use of this strategy led ultimately to the identification of cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxyl ic acid (1, SB 207499, Ariflo), a potent second-generation inhibitor of PDE4 with a decreased potential for side effects versus the archetypic first generation inhibitor, (R)-rolipram.     

Methohexital-induced changes in spectral power of neuromagnetic signals: beta augmentation is smaller over the hemisphere containing the epileptogenic focus

cAMP levels in GH3gsp cells (Q227L mutation of Gs alpha), in comparison with uninfected GH3 and GH3vt (with vector alone) cells, were two to three fold (P< 0.01) higher (basal), and 10-20 fold (P<0.001) higher (in the presence of isobutyl methylxanthine, (IBMX)). Proliferation of GH3gsp cells after 7 days in culture, as determined by cell number and [(3)H]thymidine incorporation, were up to 25% (respectively P <0.001 and P < 0.02) higher. After chronic (4 days) but not acute (15 min) exposure to forskolin (10 microM) or dibutyryl cAMP (50 microM) all cell types showed a greater than 200% (P < 0.001) increase in [(3)H]thymidine incorporation. Secretion of prolactin and growth hormone by GH3gsp cells were two to four fold (P <0.001) higher than GH3 and GH3vt cells after 4 h and 10-12 fold (P <0.001) higher after 8 h. In conclusion GH3 cells possessing Q227L have a higher proliferation rate and secrete higher levels of prolactin and growth hormone which are associated with higher levels of cAMP.     

An amphotropic retroviral vector expressing a mutant gsp oncogene: effects on human thyroid cells in vitro

Point mutations of the gsp protooncogene (encoding the alpha-subunit of the Gs protein) that constitutively activate the cAMP signaling pathway are a common feature of and a plausible causative mechanism for thyroid hyperfunctioning adenomas (hot nodules). To investigate the extent to which mutant gsp acting alone can induce proliferation of thyroid follicular cells, we generated an amphotropic retroviral vector (based on the pBABE-neo plasmid and psi-CRIP packaging line) to permit stable introduction of a hemagglutinin-tagged Gln227-->Leu mutant gsp gene into normal human thyrocytes in vitro. The biological activity of the vector was confirmed by detection of HA-tagged Gsp protein expression and induction of cAMP synthesis in selected target cells. Normal human thyroid follicular cells in primary monolayer culture were infected with the gsp retroviral vector or with corresponding vectors expressing mutant H-ras or neo only as positive and negative controls, respectively. Although, as before, mutant ras generated 10-20 well differentiated epithelial colonies/dish of 10(5) infected cells, with an average lifespan of 15-20 population doublings, only small groups of no more than 15-50 differentiated thyrocytes were observed with the gsp vector. In addition to standard conditions (10% FCS), infections were performed in reduced serum (1% FCS, TSH, and insulin), in the presence of isobutylylmethylxanthine, or in the presence of agents capable of closing gap junctions, with no significant difference in outcome. Although little or no proliferative response was observed regardless of the conditions, there was clear evidence of morphological response (rearrangement of the actin cytoskeleton and increased cell size). The results suggest that gsp mutation may not be a sufficient proliferogenic stimulus by itself to account for hot nodule formation.     

Activating mutations of the Gs alpha-gene in nonfunctioning pituitary tumors

The majority of pituitary tumors are of monoclonal origin; however, the molecular basis for their formation is poorly understood. Somatic mutations in the alpha-subunit of the GTP-binding protein, Gs alpha (gsp oncogene) have been found in about one third of GH-secreting tumors. Mutations in another alpha-subunit of a GTP-binding protein, Gi2 alpha (gip mutations) have been described in other endocrine tumors. In this study, we examined 21 nonfunctioning pituitary tumors and 4 macroprolactinomas for gsp mutations and 27 nonfunctioning tumors and 4 macroprolactinomas for gip mutations. Using the polymerase chain reaction and denaturing gradient gel electrophoresis, 2 nonfunctioning pituitary tumors displayed migration abnormalities when the Gs alpha-gene was analyzed. Sequence analysis of these abnormally migrating polymerase chain reaction products revealed two previously known gsp mutations: arginine at codon 201 altered to cysteine, and glutamine at codon 227 changed to leucine. No gip mutations could be demonstrated. These findings emphasize the monoclonal origin of nonfunctioning pituitary tumors and suggest that cAMP may play a role in tumorigenesis of nonfunctioning pituitary tumors.     

Urinary cyclic adenosine 3',5'-monophosphate response in McCune-Albright syndrome: clinical evidence for altered renal adenylate cyclase activity

The recent finding of an activating mutation in the Gs alpha protein, the protein that couples receptors to stimulation of adenylate cyclase, from endocrine and nonendocrine tissues of patients with McCune-Albright syndrome (MAS) suggests that alterations in adenylate cyclase activity may account for the clinical abnormalities in these patients. Many patients with MAS have hypophosphatemia. This may result from the presence of the activating Gs alpha mutation in proximal renal tubules or the elaboration of a phosphaturic factor from fibrous dysplasia. We, therefore, sought to characterize renal cAMP generation and phosphate handling in MAS patients. Intravenous infusion of PTH is a classic clinical test used to evaluate hormonal responsiveness of renal proximal tubule adenylate cyclase and examine PTH-dependent phosphate clearance. We performed PTH infusion in 6 MAS patients, 10 normal subjects, and 6 patients with pseudohypoparathyroidism (PHP). The basal urinary cAMP (UcAMP) level in the MAS group [5.5 +/- 2.6 nmol/dL glomerular filtration (GF)] was elevated (P < 0.05) compared to those in both normal subjects (3.2 +/- 1.2 nmol/dL GF) and patients with PHP (1.9 +/- 0.6 nmol/dL GF). However, PTH-stimulated peak UcAMP (15.0 +/- 7.0 nmol/dL GF) and the peak/basal UcAMP ratio (3.1 +/- 1.7) in MAS were significantly lower than the respective values in normal subjects (30.8 +/- 16.9 nmol/dL GF and 9.3 +/- 2.9; P < 0.05 for both) and were statistically similar to the blunted levels in PHP (respectively, 3.1 +/- 1.5 nmol/dL GF and 2.0 +/- 1.7). By contrast, the PTH-induced phosphaturic response in MAS patients was similar to that in the normal subjects. Our study provides clinical evidence that MAS patients have altered renal adenylate cyclase activity, manifested by an elevated basal UcAMP, but a blunted UcAMP response to PTH stimulation. These observations are presumably due to a mutation in the Gs alpha protein in the renal tubules. Despite the blunted UcAMP excretion, the phosphaturic response to PTH in MAS patients is intact.     

Functional activity and expression of the myocardial postreceptor adenylyl cyclase system in pressure overload hypertrophy in rat

OBJECTIVE: The aim of the present study was to investigate the functional regulation of the myocardial postreceptor adenylyl cyclase (AC) system in compensated left ventricular hypertrophy (LVH) and the effect of long-term angiotensin converting enzyme (ACE) inhibition. METHODS: Pressure overload LVH was induced in rats by supravalvular aortic banding for 12 weeks. At 12 weeks left ventricular function and inner diameters were analyzed by echocardiography of anesthetized animals, and responsiveness to forskolin (systolic developed pressure) was determined in isolated perfused hearts. Functional activities of AC and the stimulatory G protein Gs were measured as well as mRNA expression (quantitative slot blot analyses) of AC type V, isoforms of Gs alpha and Gi alpha 2. G protein alpha-subunits were also quantified by immunoblotting. Rats were treated with ramipril (Ram, 10 mg/kg per day p.o.) during weeks 7 to 12 to induce regression of LVH or with vehicle (Veh, tap water). RESULTS: Pressure overload induced severe LVH (3.2 +/- 0.09 g/kg in Veh vs. 1.8 +/- 0.03 in sham; P < 0.05) which was significantly reduced by ramipril (2.7 +/- 0.09; P < 0.05 vs. Veh). In-vivo left ventricular function and diameters were unchanged in LVH. In contrast, in hearts with LVH, responsiveness of left ventricles to forskolin was attenuated and basal, GTP gamma S and forskolin as well as manganese chloride-stimulated adenylyl cyclase activity was significantly downregulated by approximately 40% (basal 20.8 +/- 1.9 pmol cAMP/mg per min vs. 34.0 +/- 2.2 in sham; P < 0.01). However, no significant changes of AC type V mRNA were found in hypertrophied left ventricles. Functional activity of the stimulatory G protein Gs was reduced in LVH (48 +/- 7 pmol cAMP/mg per min in Veh vs. 68 +/- 3 in sham), whereas mRNA expression of long and short Gs alpha-isoforms was not altered and that of Gi alpha 2 was only slightly increased in ramipril-treated animals. Western analysis showed no significant differences of Gs alpha or Gi alpha 2 subunits. Long-term blockade of the renin-angiotensin system had no effect on the activity of the adenylyl cyclase system. CONCLUSIONS: Functional desensitization of adenylyl cyclase and stimulatory G protein occurred in rat adaptive LVH prior to the onset of severe left ventricular dysfunction which was not restored by ACE-inhibitor treatment. The desensitization seems not to be mediated by significant changes of mRNA expression of AC type V or abundance of regulatory G proteins.     

Signal transduction through trimeric G proteins in megakaryoblastic cell lines

The biogenesis of trimeric G proteins was investigated by measurement of the expression of alpha-subunits in the megakaryoblastic cell lines MEG-01, DAMI, and CHRF-288-11, representing stages of increasing maturation, and compared with platelets. Megakaryoblasts and platelets contained approximately equal amounts of Gi alpha-1/2, Gi alpha-3, Gq alpha, and G12 alpha protein. Maturation was accompanied by (1) downregulation of mRNA for Gs alpha and disappearance of iloprost-induced Ca2+ mobilization, (2) upregulation of the long form of Gs alpha protein (Gs alpha-L) and an increase in iloprost-induced cAMP formation, and (3) upregulation of G16 alpha mRNA and G16 alpha protein and appearance of thromboxane A2-induced signaling (Ca2+ mobilization and stimulation of prostaglandin I2-induced cAMP formation). Gz alpha protein was absent in the megakaryoblasts despite weak expression of Gz alpha mRNA in DAMI and relatively high levels of Gz alpha mRNA and Gz alpha protein in platelets. These findings reveal major changes in G protein-mediated signal transduction during megakaryocytopoiesis and indicate that G16 alpha couples the thromboxane receptor to phospholipase C beta.     

Recruitment curve of the soleus H reflex in patients with neurogenic claudication

An abnormal stimulation of the cAMP pathway has been recognized as the primary event in various pathological situations that lead to goitrogenesis or thyroid tumors. Thyroid adenomas are monoclonal neoplasms that become independent of thyroid stimulating hormone (TSH) in their secretory function and growth. Mutated forms of the TSH receptor (TSHR) and the adenylyl cyclase-activating Gs alpha protein, which confer a constitutive activity on these proteins, have been observed in human adenomas. The FRTL-5 rat thyroid cell line is a permanent but untransformed line; the growth of which depends on the presence of TSH, and at least in part, on the stimulation of the cAMP pathway. In order to compare the oncogenic potential of the activated mutant Gs alpha protein and the constitutively activated TSHR, we have transfected FRTL-5 cells with an expression vector bearing either the cDNA of the Gs alpha gene carrying the A201S mutation or the cDNA of the TSH receptor carrying the M453T mutation recently identified in a case of congenital hyperthyroidism. The expression of these two cDNAs was driven by the bovine thyroglobulin gene promoter. We show that, although the expression of both the Gs alpha or TSHR mutant proteins leads to TSH-independent proliferation and to constitutive cAMP accumulation in FRTL-5 cells, only the mutant TSHR is able to induce neoplastic transformation, as demonstrated by growth in semi-solid medium and tumorigenesis in nude mice.     

Gs alpha mutation may be uncommon in patients with multiple endocrine neoplasia type 1

Activating mutations of the Gs alpha gene, termed gsp, have been identified in various endocrine tumors. Recently, a high frequency of gsp mutation in patients with multiple endocrinopathies was reported, and a family with both McCune-Albright syndrome and multiple endocrine neoplasia type 1 was described. Each suggests that the oncogenic mutations of Gs alpha may play an important role in tumorigenesis in patients with multiple neoplastic endocrinopathies, and a search for the gsp mutation in multiple endocrine neoplasia type 1 (MEN1) should be undertaken. We, therefore, reevaluated the frequency of gsp mutations in endocrine tumors of patients with MEN1. Of 18 tumors from 13 patients with MEN1, we found no gsp mutations regardless of heredity. We conclude that the gsp mutation may be uncommon in endocrine tumors of MEN1 patients, and thus, this mutation plays little, if any, role in their tumorigenesis.     

Heterogeneous mutations in the gene encoding the alpha-subunit of the stimulatory G protein of adenylyl cyclase in Albright hereditary osteodystrophy

Albright hereditary osteodystrophy (AHO) is an inherited disorder associated with deficient activity of the alpha-subunit of the guanine nucleotide-binding regulatory protein (Gs alpha) that couples receptors to adenylyl cyclase. To identify mutations that lead to Gs alpha deficiency, we isolated genomic DNA from patients with AHO and used the polymerase chain reaction to amplify exons of the Gs alpha genes. DNA was amplified using intron-specific oligonucleotide primers flanking exons of the Gs alpha gene. To optimize our ability to detect mutations, one oligonucleotide from each primer pair was synthesized with a 5' GC-clamp. Amplified Gs alpha gene fragments were analyzed by denaturing gradient gel electrophoresis in order to detect mutations that alter the melting point of the double-stranded DNA fragment. Using this technique, we have identified and characterized three mutations and one neutral polymorphism. The polymorphism, located in exon 5, consisted of a T-->C substitution that conserves the isoleucine residue at codon 131 (ATT-->ATC). Two mutations were missense mutations, which in one family consisted of a nucleotide substitution (T-->C) in exon 4 that results in replacement of Leu by Pro at codon 99 of the Gs alpha molecule. Affected subjects in a second family had a single base (C-->T) mutation in exon 6 that resulted in replacement of Arg by Cys at codon 165. A 4-base pair deletion (GTGG) in exon 8 at position +214 was identified in one Gs alpha allele from each affected subject in the third family. This mutation causes a frameshift after the codon for Gln213 that results in a premature stop codon 81 base pair after the deletion. Immunoblot analysis of plasma membranes prepared from cultured fibroblasts or erythrocytes indicated that levels of immunoactive Gs alpha protein were decreased in all affected subjects. We conclude that heterogeneous mutations in the gene encoding Gs alpha, including deletions and single amino acid substitutions, are responsible for Gs alpha deficiency in AHO.     

Somatic mutations in the thyrotropin receptor gene and not in the Gs alpha protein gene in 31 toxic thyroid nodules

Studies on frequency and distribution pattern of TSH receptor (TSHR) and Gs alpha protein (gsp) mutations in toxic thyroid nodules (TTNs) reported conflicting results, most likely also related to the different screening methods applied and the investigation of only part of exon 10 of the TSHR. Therefore, we screened a consecutive series of 31 TTNs for both TSHR and gsp mutations by direct sequencing of exon 9 and the entire exon 10 of the TSHR gene and exons 7-10 of the gsp gene. Somatic TSHR mutations were identified in 15 of 31 TTNs. TSHR mutations were localized in the third intracellular loop (Asp619Gly and Ala623Val), the sixth transmembrane segment (Phe631Leu and Thr632Ile, Asp633Glu) and the second extracellular loop (Ile568Thr). One mutation was found in the extracellular TSHR domain (Ser281Asn). Two new TSHR mutations were identified. One involves codon 656 in the third extracellular loop (Val656Phe). The other new mutation is a 27-bp deletion in the third intracellular loop resulting in deletion of 9 amino acids at codons 613-621. Transient expression of the new TSHR mutations in COS-7 cells demonstrated their constitutive activity. No mutation was found in exons 7-10 of the gsp gene. This finding was confirmed by an allele-specific PCR for mutations in gsp codons 201 (Arg-->His, Cys) and 227 (Gln-->His, Arg). Our data indicate that constitutively activating TSHR mutations can be found in 48% of TTNs and thus currently represent the most frequent molecular mechanism known in the etiopathogenesis of TTNs. Moreover, the absence of gsp mutations in our series argues for an only minor role of these mutations in TTNs. Constitutive activation of the TSHR by a deletion in a region that might be involved in G protein coupling of the TSHR offers new insights into TSHR activation.     

Development of decompensated dilated cardiomyopathy is associated with decreased gene expression and activity of the milrinone-sensitive cAMP phosphodiesterase PDE3A

BACKGROUND: Phosphodiesterase III (PDE3) inhibitors are inotropic agents used to treat congestive heart failure (CHF) and are less effective in patients with severe CHF. Little is known about relative changes in PDE3 activity or gene expression during the evolution of cardiomyopathy. METHODS AND RESULTS: In the present study, we evaluated temporal changes in PDE3A gene expression before and after pacing-induced CHF in nine mongrel dogs. Three weeks of left ventricular (LV) pacing produced LV end-diastolic pressures of 15+/-1.7 mm Hg, whereas overt CHF at 4 to 5 weeks was associated with LV end-diastolic pressures of 24+/-1.7 mm Hg; prepacing values were 6.6+/-0.6 mm Hg. Total RNA isolated from LV tissues was analyzed on Northern blots; 10 unpaced normal hearts served as tissue controls. Signals for PDE3A mRNAs (7, 8, and 10 kb) or PDE4D (7.6 kb) were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or ribosomal 18S RNA. Before the onset of CHF, PDE3A/GAPDH ratios were not different between the control and 3-week paced groups. In contrast, all PDE3A/GAPDH ratios were selectively reduced by 52%, and PDE3A/18S was reduced by 70% (P<.05) in CHF; PDE4D/GAPDH (or 18S) was unchanged. LV tissues from four control and four CHF dogs were also processed to isolate cytosolic and microsomal membrane protein for cAMP PDE3 activity assays. CHF was associated with a significant 54% reduction (P<.05) in microsomal but not cytosolic PDE3 activity. CONCLUSIONS: Selective downregulation of PDE3A may account in part for the ineffectiveness of milrinone in the treatment of severe CHF.     

A single mutation Asp229 --> Ser confers upon Gs alpha the ability to interact with regulators of G protein signaling

RGS proteins (regulators of G protein signaling) are GTPase activating proteins (GAPs) for Gi and Gq families of heterotrimeric G proteins but have not been found to interact with Gs alpha. The Gs alpha residue Asp229 has been suggested to be responsible for the inability of RGS proteins to interact with Gs alpha [Natochin, M., and Artemyev, N. O. (1998) J. Biol. Chem. 273, 4300-4303]. To test this hypothesis, we have investigated the possibility of generating an interaction between Gs alpha and RGS proteins by substituting Gs alpha Asp229 with Ser and replacing the potential Gs alpha Asp229 contact residues in RGS16, Glu129 and Asn131, by Ala and Ser, respectively. RGS16 and its mutants failed to interact with Gs alpha. A single mutation of Gs alpha, Asp229Ser, rendered the Gs alpha subunit with the ability to interact with RGS16 and RGS4. Like RGS protein binding to Gi and Gq alpha-subunits, RGS16 preferentially recognized the AlF4--bound conformation of Gs alpha Asp229Ser. In a single-turnover assay, RGS16 maximally stimulated GTPase activity of Gs alpha Asp229Ser by approximately 5-fold with an EC50 value of 7.5 microM. Our findings demonstrate that Asp229 of Gs alpha represents a major barrier for Gs alpha interaction with known RGS proteins.     

Characterization of the endothelin receptor subtype mediating epithelium-derived relaxant nitric oxide release from guinea-pig trachea

The coupling of the endogenously expressed alpha2A-adrenoceptors in human erythroleukemia cells (HEL 92.1.7) to Ca2+ mobilization and inhibition of forskolin-stimulated cAMP production was investigated. The two enantiomers of medetomidine [(+/-)-[4-(1-[2, 3-dimethylphenyl]ethyl)-1H-imidazole]HCl] produced opposite responses. Dexmedetomidine behaved as an agonist in both assays (i.e. , it caused Ca2+ mobilization and depressed forskolin-stimulated cAMP production). Levomedetomidine, which is a weak agonist in some test systems, reduced intracellular Ca2+ levels and further increased forskolin-stimulated cAMP production and therefore can be classified as an inverse agonist. A neutral ligand, MPV-2088, antagonized responses to both ligands. Several other, chemically diverse alpha2-adrenergic ligands also were tested. Ligands that could promote increases in Ca2+ levels and inhibition of cAMP production could be classified as full or partial agonists. Their effects could be blocked by the alpha2-adrenoceptor antagonist rauwolscine and by pertussis toxin treatment. Some typical antagonists such as rauwolscine, idazoxan, and atipamezole had inverse agonist activity like levomedetomidine. The results suggest that the alpha2A-adrenoceptors in HEL 92.1.7 cells exist in a precoupled state with pertussis toxin-sensitive G proteins, resulting in a constitutive mobilization of intracellular Ca2+ and inhibition of cAMP production in the absence of agonist. This constitutive activity can be antagonized by inverse agonists such as levomedetomidine and rauwolscine. Levomedetomidine can be termed a "protean agonist" because it is capable of activating uncoupled alpha2-adrenoceptors in other systems and inhibiting the constitutive activity of precoupled alpha2-adrenoceptors in HEL 92.1. 7 cells. With this class of compounds, the inherent receptor "tone" could be adjusted, which should provide a new therapeutic principle in receptor dysfunction.     

Phosphodiesterase isoforms in the pulmonary arterial circulation of the rat: changes in pulmonary hypertension

Phosphodiesterase (PDE) activity was determined in pulmonary arteries removed from control and chronic hypoxia-induced pulmonary hypertensive rats. The main, first-branch, intrapulmonary and resistance pulmonary arteries were studied. We measured total cAMP PDE activity and cGMP PDE activity, as well as that of individual isoforms (PDE1-5). cAMP PDE activity in chronic hypoxic rats was increased in first-branch and intrapulmonary arteries from hypoxic rats. No changes were observed in the main or resistance pulmonary arteries. Similarly, cGMP PDE activity was increased in the main, first-branch and intra-pulmonary arteries of the hypoxic rats. No changes in cGMP PDE activity were observed in resistance arteries. There was evidence for PDE1-5 activity in all pulmonary arteries. The increased cAMP PDE activity in first-branch and intrapulmonary vessels was associated with an increase in cilostimide-inhibited PDE (PDE3) activity. Increased total cGMP PDE in main pulmonary artery was associated with increases in Ca++/calmodulin-stimulated (PDE1) activity. An increase in zaprinast-inhibited (PDE5) activity was observed in first-branch and intrapulmonary arteries. Our results suggest that decreases in intracellular cyclic nucleotide levels in pulmonary arteries from pulmonary hypertensive rats are associated with increased PDE activity. Further, these changes may reflect alterations at the level of specific types of PDE isoforms.     

Guanine nucleotide-binding inhibitory protein-mediated inhibition of adenylyl cyclase is enhanced in spontaneously hypertensive rat preglomerular arteriolar smooth muscle cells

The purpose of our study was to determine whether Gi-mediated control over adenylyl cyclase in preglomerular arteriolar smooth muscle cells (PGASMC) is enhanced in the spontaneously hypertensive rat (SHR). PGASMC were cultured from preglomerular microvessels isolated from adult SHR (14-15 wk of age) and age-matched WKY rats. Confluent monolayers of cells in third passage were used for the experiments. cAMP released into the media (30 min) as well as cellular levels of cAMP were measured in the presence of a phosphodiesterase inhibitor, 1-isobutyl-3-methyl-xanthine (IBMX; 100 microM) and expressed as pmol/mg protein. Total (released + cellular) cAMP was significantly lower in SHR (14.19 +/- 2.30 pmol/mg protein) as compared with WKY (28.3 +/- 3.04 pmol/mg protein). Correspondingly, the released (4.6 +/- 0.4 pmol/mg protein) as well as cellular (9.78 +/- 2.18 pmol/mg protein) cAMP levels were also significantly lower in SHR when compared with WKY (8.85 +/- 1.26 and 18.86 +/- 2.0 pmol/mg protein, respectively). The steady-state levels of none of the Gi alpha subunits, namely Gi alpha 1, Gi alpha 2 and Gi alpha 3, were higher in the SHR PGASMC. Pertussis toxin treatment (PTX; 100 ng/ml; 24 hr) caused complete ADP-ribosylation of Gi alpha subunits in both WKY and SHR PGASMC. The same treatment of PTX also produced a significant increase in total cAMP in SHR, but not in WKY, such that the total cAMP levels after PTX treatment were not significantly different between the two strains. Interestingly, PTX significantly increased the released (20.26 +/- 0.90 pmol/mg protein) but not the cellular (13.63 +/- 1.63 pmol/mg protein) cAMP in SHR. Forskolin (1 microM) induced similar increases in total cAMP and isoproterenol (1 microM) caused greater increases in total cAMP in SHR cells compared with WKY cells. These data strongly suggest that in SHR PGASMC total adenylyl cyclase activity is not altered. Furthermore, steady-state expressions of Gi alpha-1, Gi alpha-2 and Gi alpha-3 are not increased whereas Gi-mediated inhibition of adenylyl cyclase is augmented in SHR PGASMC.     

Dual inhibition of human type 4 phosphodiesterase isostates by (R, R)-(+/-)-methyl 3-acetyl-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-3- methyl-1-pyrrolidinecarboxylate

Purified recombinant human type 4 phosphodiesterase B2B (HSPDE4B2B) exists in both a low- and a high-affinity state that bind (R)-rolipram with Kd's of ca. 500 and 1 nM, respectively [Rocque, W. J., Tian, G., Wiseman, J. S., Holmes, W. D., Thompson, I. Z., Willard, D. H., Patel, I. R., Wisely, G. B., Clay, W. C., Kadwell, S. H., Hoffman, C. R., and Luther, M. A. (1997) Biochemistry 36, 14250-14261]. Since the tissue distribution of the two isostates may be significantly different, development of inhibitors that effectively inhibit both forms may be advantageous pharmacologically. In this study, enzyme inhibition and binding of HSPDE4B2B by (R, R)-(+/-)-methyl 3-acetyl-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-3-methyl-1-pyrrolidin ecarboxylate (1), a novel inhibitor of phosphodiesterase 4 (PDE 4), were investigated. Binding experiments demonstrated high-affinity binding of 1 to HSPDE4B2B with a stoichiometry of 1:1. Inhibition of PDE activity showed only a single transition with an observed Ki similar to the apparent Kd determined by the binding experiments. Deletional mutants of HSPDE4B2B, which have been shown to bind (R)-rolipram with low affinity, were shown to interact with 1 with high affinity, indistinguishable from the results obtained with the full-length enzyme. Bound 1 was completely displaced by (R)-rolipram, and the displacement showed a biphasic transition that resembles the biphasic inhibition of HSPDE4B2B by (R)-rolipram. Theoretical analysis of the two transitions exemplified in the interaction of (R)-rolipram with HSPDE4B2B indicated that the two isostates were nonexchangeable. Phosphorylation at serines 487 and 489 on HSPDE4B2B had no effect on the stoichiometry of binding, the affinity for binding, or the inhibition of the enzyme by 1. These data further illustrate the presence of two isostates in PDE 4 as shown previously for (R)-rolipram binding and inhibition. In contrast to (R)-rolipram, where only one of the two isostates of PDE 4 binds with high affinity, 1 is a potent, dual inhibitor of both of the isostates of PDE 4. Kinetic and thermodynamic models describing the interactions between the nonexchangeable isostates of PDE 4 and its ligands are discussed.