cGMP Phosphodiesterase Activity Research Articles

The helical domain of a G protein alpha subunit is a regulator of its effector.

The alpha subunit (Galpha) of heterotrimeric G proteins is a major determinant of signaling selectivity. The Galpha structure essentially comprises a GTPase "Ras-like" domain (RasD) and a unique alpha-helical domain (HD). We used the vertebrate phototransduction model to test for potential functions of HD and found that the HD of the retinal transducin Galpha (Galphat) and the closely related gustducin (Galphag), but not Galphai1, Galphas, or Galphaq synergistically enhance guanosine 5'-gamma[-thio]triphosphate bound Galphat (GalphatGTPgammaS) activation of bovine rod cGMP phosphodiesterase (PDE). In addition, both HDt and HDg, but not HDi1, HDs, or HDq attenuate the trypsin-activated PDE. GalphatGDP and HDt attenuation of trypsin-activated PDE saturate with similar affinities and to an identical 38% of initial activity. These data suggest that interaction of intact Galphat with the PDE catalytic core may be caused by the HD moiety, and they indicate an independent site(s) for the HD moiety of Galphat within the PDE catalytic core in addition to the sites for the inhibitory Pgamma subunits. The HD moiety of GalphatGDP is an attenuator of the activated catalytic core, whereas in the presence of activated GalphatGTPgammaS the independently expressed HDt is a potent synergist. Rhodopsin catalysis of Galphat activation enhances the PDE activation produced by subsaturating levels of Galphat, suggesting a HD-moiety synergism from a transient conformation of Galphat. These results establish HD-selective regulations of vertebrate retinal PDE, and they provide evidence demonstrating that the HD is a modulatory domain. We suggest that the HD works in concert with the RasD, enhancing the efficiency of G protein signaling.

Cyclic GMP and cGMP-binding Phosphodiesterase Are Required for Interleukin-1-induced Nitric Oxide Synthesis in Human Articular Chondrocytes

This study addressed the role of guanylyl cyclase (GC) and phosphodiesterase (PDE) in interleukin (IL)-1 activation of human articular chondrocytes. The GC inhibitors LY83583 and methylene blue dose-dependently inhibited IL-1-induced nitric oxide (NO) production, inducible NO synthase (iNOS) protein, and mRNA expression. These effects of GC inhibition were consistent with the rapid induction of cGMP by IL-1, which reached maximal levels after 5 min. The effects of GC inhibitors were selective as they did not reduce IL-1-induced cyclooxygenase II protein and mRNA. An inhibitor specific for soluble GC did not affect IL-1-induced NO production, and activators of soluble GC did not induce NO. However, the expression of iNOS mRNA was induced by atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP), activators of particulate GC, indicating that particulate rather than soluble guanylyl cyclases were involved in iNOS induction. The expression of iNOS mRNA and the production of NO were induced by a slowly hydrolyzable analog of cGMP, 8-bromo-cGMP, but not by nonhydrolyzable analog, dibutyryl cGMP, suggesting that PDE rather than cGMP-dependent protein kinase mediates the cGMP effects. Chondrocytes contained extensive cGMP PDE activity. This had PDE5 biochemical features and an inhibitor profile consistent with PDE5. Furthermore, the nonisoformspecific PDE inhibitor IBMX and PDE5-specific inhibitors suppressed IL-1-induced NO release and iNOS mRNA expression. PDE5 mRNA was constitutively expressed in chondrocytes. In addition to increasing PDE5 activities, IL-1 treatment reduced the sensitivity of PDE5 to several pharmacological inhibitors by up to 50-fold. In summary, inhibitors of either GC or PDE5 prevented IL-1 induction of iNOS; IL-1 increased the rates of both cGMP generation and hydrolysis; and exogenous PDE hydrolyzable cGMP analog induced iNOS and NO. These results suggest that increased cGMP metabolic flux is sufficient to induce iNOS, and GC and PDE5 activities are required for IL-1 induction of iNOS expression via increases in coupled cGMP synthesis and hydrolysis.

Identification and Characterization of a Novel Family of Cyclic Nucleotide Phosphodiesterases

We report the cloning, expression, and characterization of a new family of cyclic nucleotide phosphodiesterase (PDE) that has unique kinetic and inhibitor specificities. A clone corresponding to the C terminus of this PDE was initially identified by a bioinformatic approach and used to isolate a cDNA that is likely full-length. This novel PDE, designated as MMPDE9A1, shows highest mRNA expression in kidney with lower levels in liver, lung, and brain. The mRNA size by Northern blot analysis is approximately 2.0 kilobases, and the cDNA encoding PDE9A1 is 1929 base pairs in length. The largest open reading frame predicts a protein of 534 amino acids with a molecular mass of 62,000 Da. When expressed in COS-7 cells, PDE9A1 activity was not inhibited well by either the nonselective inhibitor 3-isobutyl-1-methyl-xanthine or the new selective PDE5 inhibitor, sildenafil, but it is inhibited by the PDE1/5 inhibitor (+)-cis-5,6a, 7,8,9 hyl] phenylmethyl]-5-methyl-cylopent[4,5]imidao[2, 1-b]purin-49(3H)one (SCH51866) with an IC50 of 1.55 microM. This new phosphodiesterase is highly specific for cGMP. Its Km of approximately 0.07 microM for cGMP is the lowest yet reported for a PDE, being at least 40-170 times lower than that of PDE5 and PDE6, respectively.

Probing functional interfaces of rod PDE gamma-subunit using scanning fluorescent labeling.

In the dark, the activity of the rod cGMP phosphodiesterase (PDE) catalytic alpha- and beta-subunits (P alpha beta) is inhibited by two gamma-subunits (P gamma). On light stimulation of the photoreceptor cells, the GTP-bound alpha-subunit of visual G-protein transducin (GtaGTP) displaces the P gamma-subunits from their inhibitory sites on P alpha beta, leading to the effect or enzyme activation. We designed a number of P gamma mutants, each with a single cysteine residue evenly distributed at a different position along the P gamma polypeptide chain. These cysteine residues served as sites for the introduction of the environmentally sensitive fluorescent probe, 3-(bromoacetyl)-7-diethyl aminocoumarin (BC). Analysis of the interactions of P alpha beta and Gta with the fluorescently labeled P gamma mutants suggests two distinct functional interfaces of P gamma. The P alpha beta/P gamma interface is formed essentially by the C-terminus of P gamma and by the N-terminal portion of the P gamma polycationic region, P gamma-24-45, whereas the P gamma/Gta interface includes the C-terminal portion of P gamma-24-45 and the region surrounding P gamma Cys68. Such functional organization of P gamma may represent an important element for the PDE activation mechanism during transduction of visual signals.

In Memoriam. ELAINE RUTH BERMAN (1923–1997)

Rod-cone dysplasia types 1 (rcd1; Irish setter) and 2 (rcd2; collie) in dogs are early onset forms of progressive retinal atrophy (PRA) which serve as models of retinitis pigmentosa (RP) in humans. As bothrcd1 and rcd2 result from abnormal retinal cGMP metabolism associated with a deficiency in cGMP-phosphodiesterase (PDE) activity, and a nonsense mutation in the PDE6B subunit gene has been shown to cause rcd1, the genes encoding the four subunits of the PDE complex (PDE6A, PDE6B, PDE6G and PDE6D) make compelling candidates for the rcd2 locus. We adopted diverse strategies to evaluate causal association of the four PDE subunit genes with the rcd2 phenotype. Identification in an informative pedigree of obligate recombinations between intragenic polymorphisms within PDE6A and PDE6D and the rcd2 locus unequivocally excludes these two genes. PDE6B was excluded by a breeding strategy demonstrating nonallelism of rcd1 and rcd2. Direct sequencing of PDE6G from an rcd2 -homozygous collie dog revealed no abnormality in the entire genomic sequence. To evaluate cosegregation between PDE6G and rcd2, advantage was taken of prior knowledge that PDE6G and Galactokinase 1 (GALK1) localize to the same canine-rodent somatic hybrid cell line. Linkage analysis using a single nucleotide polymorphism (SNP) in the PDE6G gene, and a (CA)n repeat polymorphism in the GALK1 gene, which were both segregating in an unrelated pedigree, established close linkage of these two genes (θ=0; Z=4.21). Identification of obligate recombinations between GALK1 and the rcd2 locus in an informative rcd2 pedigree thus excluded PDE6G as a candidate gene forrcd2 ; the exclusion distance between GALK1 and rcd2 is at least 0.35 cM. These results therefore exclude the entire set of genes coding for the rod PDE complex as candidates for rcd2.

Light-induced cGMP-phosphodiesterase activity in intact rat retinal rod cells as revealed by rapid-freezing enzyme cytochemistry.

Cyclic guanosine monophosphate (cGMP)-phosphodiesterase (PDE) in the outer segment of vertebrate retinal rod cells is one of key enzymes mediating phototransduction. We report here on light-induced PDE activity in intact rat retinal rod cells processed by rapid-freezing enzyme cytochemistry, a new morphological technique that is a combination of rapid-freezing, freeze-substitution fixation, and subsequent enzyme cytochemistry. This technique quickly immobilizes and preserves both enzyme activity and the cell ultrastructure in a state approximating living conditions; consequently, it has proved useful for cytochemical detection of light-induced PDE activity. Using this technique we observed that the catalytic site of PDE molecules in rapid-frozen outer segments was predominantly located in the extradiscal spaces; PDE activity was significantly greater in light than in darkness; and illumination elicited marked increases in PDE activity in dark-adapted cells. Light-induced PDE activity was first cytochemically detected after 3 s of illumination and reached a peak within 5 s, at which time it was in virtually the same as that seen in fully light adapted cells. In addition, cytochemical guanosine triphosphatase (GTPase) activity in dark-adapted cells, as well as corresponding PDE activity, increased in a time-dependent manner with illumination duration; this acceleration in GTPase activity closely paralleled the PDE activity. Thus, our results suggest, in part, the existence of reciprocal regulation of PDE and activated transducin alpha subunit, thereby modulating light adaptation in rod cells.

Transcriptional activation of the human rod cGMP-phosphodiesterase beta-subunit gene is mediated by an upstream AP-1 element.

During photoactivation retinal cGMP-phosphodiesterase (PDE) mediates signal transduction in the photoreceptor outer segments. Mutations in the beta-subunit gene of rod-specific PDE (beta-PDE) have been associated with inherited retinal degeneration in a number of species, including human. Here we have investigated the proximal upstream sequences that participate in transcriptional activation of this gene. Transient transfections demonstrated that the sequence from -72 to +53 bp contained sufficient information to direct high levels of gene expression in cells of retinal origin. Deletion or mutagenesis of an AP-1 motif present in this region caused 90-95% reduction in reporter gene expression. By gel mobility shift assay we demonstrated specific interactions between putative nuclear transcription factors and this AP-1 element. These findings indicate that the proximal AP-1 site in the human beta-PDE promoter is functionally relevant and necessary for transcriptional activation of this gene.

Arrestin with a single amino acid substitution quenches light-activated rhodopsin in a phosphorylation-independent fashion.

Arrestins are members of a superfamily of regulatory proteins that participate in the termination of G protein-mediated signal transduction. In the phototransduction cascade of vertebrate rods, which serves as a prototypical G protein-mediated signaling pathway, the binding of visual arrestin is stimulated by phosphorylation of the C-terminus of photoactivated rhodopsin (Rh*). Arrestin is very selective toward light-activated phosphorhodopsin (P-Rh*). Previously we reported that a single amino acid substitution in arrestin, Arg175Gln, results in a dramatic increase in arrestin binding to Rh* [Gurevich, V. V., & Benovic, J. L. (1995) J. Biol. Chem. 270, 6010-6016]. Here we demonstrate that a similar mutant, arrestin(R175E), binds to light-activated rhodopsin independent of phosphorylation. Arrestin(R175E) binds with high affinity not only to P-Rh* and Rh* but also to light-activated truncated rhodopsin in which the C-terminus phosphorylation sites have been proteolytically removed. In an in vitro assay that monitored rhodopsin-dependent activation of cGMP phosphodiesterase (PDE), wild type arrestin quenched PDE response only when ATP was present to support rhodopsin phosphorylation. In contrast, as little as 30 nM arrestin(R175E) effectively quenched PDE activation in the absence of ATP. Arrestin(R175E) had no effect when the lifetime of Rh* no longer contributed to the time course of PDE activity, suggesting that it disrupts signal transduction at the level of rhodopsin-transducin interaction.

Hyporesponsiveness to inhaled nitric oxide in isolated, perfused lungs from endotoxin-challenged rats.

Inhaled nitric oxide (iNO) causes selective pulmonary vasodilation and improves oxygenation in patients with the adult respiratory distress syndrome (ARDS). Approximately 30% of ARDS patients fail to respond to iNO. Because sepsis syndrome often accompanies a decreased response to iNO, we investigated NO responsiveness in isolated, perfused lungs from rats exposed to lipopolysaccharide (LPS). Eighteen hours after intraperitoneal injection of 0.5 mg/kg LPS, rat lungs were isolated, perfused, and preconstricted with U-46619. Ventilation with 0.4, 4, and 40 parts per million by volume NO vasodilated LPS-pretreated lungs 75, 47, and 42% less than control lungs (P < 0.01 value differs at each concentration). The diminished vasodilatory response to iNO was associated with decreased NO-stimulated guanosine 3',5'-cyclic monophosphate (cGMP) release into the perfusate. Soluble guanylate cyclase activity did not differ in lung extracts from LPS-pretreated and control rats. LPS increased pulmonary cGMP-phosphodiesterase (PDE) activity by 40%. The PDE-sensitive cGMP analogue 8-bromoguanosine 3',5'-cyclic monophosphate vasodilated lungs from LPS-pretreated rats less than lungs from control rats. In contrast, the PDE-insensitive 8-para-chlorophenylthioguanosine 3',5'-cyclic monophosphate vasodilated lungs equally from both groups. After LPS challenge, the rat pulmonary vasculature becomes hyporesponsive to iNO. Hyporesponsiveness to iNO appears partly attributable to increased pulmonary cGMP-PDE activity.

Molecular origin of continuous dark noise in rod photoreceptors

Noise in the rod photoreceptors limits the ability of the dark-adapted visual system to detect dim lights. We investigated the molecular mechanism of the continuous component of the electrical dark noise in toad rods. Membrane current was recorded from intact, isolated rods or truncated, internally dialyzed rod outer segments. The continuous noise was separated from noise due to thermal activation of rhodopsin and to transitions in the cGMP-activated channels. Selectively disabling different elements of the phototransduction cascade allowed examination of their contributions to the continuous noise. These experiments indicate that the noise is generated by spontaneous activation of cGMP phosphodiesterase (PDE) through a process that does not involve transducin. The addition of recombinant gamma, the inhibitory subunit of PDE, did not suppress the noise, indicating that endogenous gamma does not completely dissociate from the catalytic subunit of PDE during spontaneous activation. Quantitative analysis of the noise provided estimates of the rate constants for spontaneous PDE activation and deactivation and the catalytic activity of a single PDE molecule in situ.

Equivalence of background and bleaching desensitization in isolated rod photoreceptors of the larval tiger salamander.

Psychophysical experiments have shown an equivalence between sensitivity reduction by background light and by bleaches for the human scotopic system. We have compared the effects of backgrounds and bleaches on the light-sensitive membrane-current responses of isolated rod photoreceptors from the salamander Ambystoma tigrinum. The quantum catch loss was factored out from the desensitization due to bleaching to give the fraction of "extra" desensitization due to adaptation. For backgrounds, desensitization is well described by the Weber/Fechner equation. The extra desensitization after bleaches can also be described by the Weber/Fechner equation, if an "equivalent" background produced by bleaching is made linearly proportional to the fraction of pigment bleached. A background which produces an extra desensitization of a factor of two is equivalent to a fractional bleach of approximately 6%. Equivalent background and bleaching desensitizations were associated with similar reductions in circulating current. There is a linear relation between log flash sensitivity and decrease in circulating current. Equivalent background and bleaching desensitizations were associated with similar increases in cGMP phosphodiesterase and guanylate cyclase activity. These were inferred from membrane current changes after steps into lithium or IBMX solutions. There were also similar reductions in the integration times of dim flash responses for equivalent desensitizations produced by backgrounds and bleaches. These results suggest that the equivalence between background and bleaching found psychophysically may arise at the very earliest stages of visual processing and that these two processes of desensitization have similar underlying mechanisms.

Ca2+Induces an Increase in cGMP-Phosphodiesterase Activity in Squid Retinal Photoreceptors

In invertebrate photoreceptors, light elicits the opening of cationic channels to produce a depolarizing receptor potential. One hypothesis is that cGMP is the agent that gates the channels. It has been previously proposed that the light-induced rise in intracellular Ca2+down-regulates phosphodiesterase activity, thereby eliciting an increase in intracellular cGMP concentration. cGMP-phosphodiesterase activity from squid photoreceptors was monitored both by a continuous fluorescence assay using 2′-o-(N-methylanthraniloyl)-cGMP and by hydrolysis of [3H]cGMP. The activity of cGMP-phosphodiesterase was found to be increased by Ca2+in the physiological range of concentrations. These findings suggest that the previously known light-induced increase in Ca2+in invertebrate photoreceptors cannot directly account for light-elicited down-regulation of cGMP-phosphodiesterase.

Elevation of cGMP with normal expression and activity of rod cGMP-PDE in photoreceptor degenerate labrador retrievers.

Cyclic guanosine 3',5'-monophosphate (cGMP) levels were determined in retinas from a strain of Labrador Retrievers with inherited retinal dystrophy manifesting at early stages of retinal differentiation. The cGMP contents of dystrophic retinas of dogs from 1 to 4 months of age (n = 7) were significantly higher (p = 0.001) than in age-matched controls of the same breed (n = 11). Ultrastructure along the vertical retinal meridian was studied in developing retinas and findings were related to those of age-matched wild-type controls of the same breed. Slow central to peripheral progression of degeneration was observed in affected dogs. No differences were found in total cGMP-phosphodiesterase (PDE) activity, in PDE subunit composition as determined by Western blotting of 2-month-old homozygote affected retinas, or in the amino acid sequence deduced from the nucleotide sequence of the PDE beta-subunit as compared to controls. This model of photoreceptor degeneration thus is the first case of an apparent abnormality of cGMP metabolism that is not associated with a defect in the PDE catalytic subunits, and it is also the first reported model not associated with severe developmental abnormalities and rapid degeneration.

Phototransduction: modeling the primate cone flash response.

We have developed a new model of phototransduction that accounts for the dynamics of primate and human cone flash responses in both their linear and saturating range. The model incorporates many of the known elements of the phototransduction cascade in vertebrate photoreceptors. The input stage is a new analytic expression for the activation and inactivation of cGMP-phosphodiesterase (PDE). Although the Lamb and Pugh (1992) model (of a delayed ramp for the rising phase of the PDE response in amphibian rods) provided a good fit for the first 2 log units of stimulus intensity without parameter adjustments, the remaining 4 log units of the data required nonlinear modifications of both delay and gain (slope). We show that this nonlinear behavior is a consequence of the delay approximation and develop a completely linear model to account for the rising phase of amphibian rod photocurrent responses over the full intensity range (approximately 6 log units). We use the same dynamic model to account for primate cone responses by decreasing the time constants of PDE activation and introducing an enhanced inactivation process. This PDE response activates a nonlinear calcium feedback stage that modulates guanylate cyclase synthesis of cyclic GMP. By adjustment of the throughput and feedback parameters, the full model successfully captures most of the features of the primate and human cone flash responses throughout their dynamic range. Our analysis suggests that initial processes in the transduction cascade may be qualitatively different from comparable processes in rods.

The cGMP-phosphodiesterase and its contribution to sensitivity regulation in retinal rods

We have used the truncated outer segment preparation to measure rod cGMP-phosphodiesterase activity, as well as its modulation by Ca2+, in darkness and in light. The basal enzyme activity in darkness was approximately 0-3 s-1, and was largely independent of Ca2+ concentration from 10 nM to 10 microM. The steady state activity elicited by a step of light (lambda = 520 nm) was strongly enhanced by Ca2+, increasing from approximately 0.005 s-1/(h nu micron-2 s-1) at 10 nM Ca2+ to approximately 0.16 s-1/h nu micron-2 s-1) at 10 microM Ca2+. Based on these measurements, as well as previous measurements on the effects of Ca2+ on rod guanylate cyclase and the cGMP-gated channel, we have calculated the step response-intensity relation for the rod cell in steady state. This relation agrees reasonably well with the relation directly measured from intact rods. We have also evaluated the relative contributions from the three Ca2+ effects to rod sensitivity. At low background light intensities, the Ca2+ modulation of the guanylate cyclase appears to be the most important for sensitivity regulation. At higher light intensities, especially above half-saturation of the response, the Ca2+ modulation of the light-stimulated phosphodiesterase shows a progressively important influence on the light response; it also extends the Weber-Fechner behavior of the cell to higher intensities. The contribution of the Ca2+ modulation of the cGMP-gated channel is slight throughout.

Receptors for atrial natriuretic peptide are decreased in the kidney of rats with streptozotocin-induced diabetes mellitus.

To determine whether decreased renal responsiveness to atrial natriuretic peptide (ANP) in diabetes is mediated by alterations in the renal ANP receptor, ANP receptor density and affinity were measured 17-20 d after streptozotocin injection and compared with values in vehicle-treated controls and streptozotocin-treated rats made euglycemic with insulin. Plasma ANP concentration was significantly greater in hyperglycemic diabetic rats than in control or euglycemic diabetic rats. Both in glomeruli and inner medulla, ANP receptor dissociation constant did not differ among the three study groups, whereas the maximum binding capacity was decreased significantly in hyperglycemic diabetics in comparison with controls and euglycemic diabetics. Glomerular clearance receptors were also decreased significantly in hyperglycemic diabetic rats in comparison with control and euglycemic diabetic rats. To determine whether the decreased number of renal ANP receptors in diabetic rats was associated with a decreased biological response, we measured ANP-dependent cyclic GMP (cGMP) accumulation by isolated glomeruli and inner medullary collecting duct cells in vitro. cGMP accumulation was significantly less in hyperglycemic diabetic rats than in controls or euglycemic diabetic rats both in the presence or absence of the phosphodiesterase inhibitor zaprinast. cGMP phosphodiesterase activity in inner medullary collecting duct cells obtained from control and hyperglycemic diabetic rats did not differ. Thus, the decreased number of biologically active ANP receptors in the kidneys of diabetic rats is accompanied by decreased biological responsiveness in vitro and provides a potential explanation for the reduction in renal sensitivity to ANP in this condition.

A Mutation That Depresses cGMP Phosphodiesterase Activity in Dictyostelium Affects Cell Motility through an Altered Chemotactic Signal

The streamer F (StmF) mutant of Dictyostelium discoideum is defective in cGMP-phosphodiesterase activity. In early aggregation territories, when individual cells chemotax toward aggregation centers prior to streaming, the average periodicity of surging of StmF cells is half that of wild-type cells. In addition, in the period between surges, which has been interpreted to include the peak and back of the wave, StmF cells abnormally remain nonmotile and retain their elongate shape. In contrast, in the period between surges wild-type cells form pseudopods randomly around their cell perimeter and take on an amorphous shape. Using a newly developed protocol for vitally staining and tracking individual mutant cells in unstained wild-type aggregation territories and individual wild-type cells in unstained StmF aggregation territories, we have found that mutant cells behave normally (i.e., like their predominant wild-type neighbors) in the deduced back of waves generated by wild-type cells. Conversely, wild-type cells behave aberrantly (i.e., like their predominant StmF neighbors) in the deduced back of waves generated by mutant cells. These results suggest that the defective behavior of StmF cells in early StmF aggregation territories is not due to a single cell defect in responsiveness, but, rather, is due either to the genesis of an aberrant cAMP wave or to the accumulation of a molecule which interferes with normal behavior in the back of the wave.

Inhibition of cGMP-phosphodiesterase restores the glomerular effects of atrial natriuretic factor in low sodium diet rats.

It was shown that atrial natriuretic factor (ANF)-induced increase in glomerular filtration rate (GFR) is blunted in low sodium diet. We investigated whether saralasin, as an angiotensin II receptor antagonist, or verapamil, as a calcium entry blocker, or theophylline and zaprinast, as inhibitors of cGMP-phosphodiesterase activity, could restore the effect of ANF on GFR increase in low sodium diet rats. ANF alone (5 micrograms/kg bolus then 0.5 micrograms/min/kg BW maintenance) increased diuresis and natriuresis to the same extend in low and normal sodium diet rats but had no GFR-increasing effect in low sodium diet rats. Infusion of ANF in the presence of verapamil, saralasin, theophylline or zaprinast induced a significant increase in GFR and filtration fraction (FF). Administration of verapamil or saralasin alone did not alter GFR and FF whereas theophylline or zaprinast alone resulted in moderate but significant increases in both parameters. The increase of nephrogenous cGMP excretion in response to ANF infusion in low sodium diet rats was markedly lower as compared to normal sodium diet rats (243.4 +/- 43.3 vs. 444.0 +/- 35.6 pmol/min, respectively, p < 0.01). Administration of a selective inhibitor of cGMP-phosphodiesterase activity (zaprinast) abolished the differences in ANF-stimulated nephrogenous cGMP excretion in low and normal sodium diet rats. Basal and peak ANF (0.5 microM)-stimulated cGMP formation by isolated glomeruli was significantly lower in low than normal sodium diet rats (1.4 +/- 0.1 vs. 2.9 +/- 0.2 and 7.1 +/- 0.5 vs. 12.5 +/- 1.1 pmol/mg protein, for basal and ANF-stimulated cGMP formation, respectively; both p < 0.05). Zaprinast both alone and in combination with ANF, potentiated cGMP formation by glomeruli isolated from both groups of rats. In the presence of zaprinast, there were no differences in both basal and peak ANF-stimulated cGMP formation by glomeruli isolated from low and normal sodium diet rats. cGMP-phosphodiesterase activity was the same in the medulla of both groups of rats but markedly higher in the renal cortex of low sodium diet rats as compared to normal sodium diet rats (82.6 +/- 6.0 vs. 59.8 +/- 4.3, respectively; p < 0.05). These data suggest that the lack of GFR-increasing response to ANF in low sodium diet rats is primarily due to the increase of cGMP hydrolysis in glomeruli.

YC-1, a Novel Activator of Platelet Guanylate Cyclase

YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole] inhibited the aggregation of and ATP release from washed rabbit platelets induced by arachidonic acid (AA), collagen, U46619, platelet-activating factor (PAF), and thrombin in a concentration-dependent manner. YC-1 also disaggregated the clumped platelets caused by these inducers. The thromboxane B2 formation caused by collagen, PAF, and thrombin was inhibited by concentrations of YC-1 that did not affect formation of thromboxane B2 and prostaglandin D2 caused by AA. YC-1 suppressed the increase of intracellular Ca2+ concentration and generation of inositol 1,4,5-trisphosphate caused by these five aggregation inducers. Both the cAMP and cGMP contents of platelets were increased by YC-1 in a concentration- and time-dependent manner. Like sodium nitroprusside, YC-1 potentiated formation of cAMP caused by prostaglandin E1 but not that by 3-isobutyl-1-methylxanthine. Adenylate cyclase and cAMP phosphodiesterase activities were not altered by YC-1. Activity of cGMP phosphodiesterase was unaffected by YC-1. Activities of guanylate cyclase in platelet homogenate and cytosolic fraction were activated by YC-1, whereas particulate guanylate cyclase activity was unaffected. The antiplatelet effect of sodium nitroprusside but not that of YC-1 was blocked by hemoglobin and potentiated by superoxide dismutase. After intraperitoneal administration for 30 minutes, YC-1 prolonged the tail bleeding time of conscious mice. These data indicate that YC-1 is a direct soluble guanylate cyclase activator in rabbit platelets. It may also possess antithrombotic potential in vivo.

Molecular cloning of G proteins and phosphodiesterases from rat taste cells

To identify and characterize those proteins involved in taste transduction, we cloned G proteins and phosphodiesterases from rat taste tissue. Using degenerate primers corresponding to conserved regions of G protein α subunits, the polymerase chain reaction was used to amplify and clone eight distinct cDNAs: α i-2, α i-3, α 12, α 14, α 8, α t-rod, α 1-cone and α gustducin. α i-3, α 14, α 8, and α t-rod are more highly expressed in taste tissue than in the surrounding nonsensory tissue. α gustducin is only expressed in taste cells. Rod transducin had previously been found only in the rod cells of the retina, where it converts light stimulation of rhodopsin into activation of cGMP phosphodiesterase. The primary sequence of α gustducin shows striking similarities to rod transducin in the receptor interaction domain and the phosphodiesterase activation site. We propose that gustducin and transducin regulate phosphodiesterase activity in taste cells and that this may promote bitter transduction and inhibit sweet transduction. Consistent with this proposal, we cloned two types of cAMP PDE from taste tissue: dnc-1 and PDE-3.