Monophosphate Phosphodiesterase Research Articles

Possible mechanisms of inhibition with atropine against noradrenaline-induced contraction in the rabbit aorta.

1. In the rabbit isolated aorta, atropine (3 x 10(-6) M-10(-4) M) inhibited contractile response to noradrenaline without affecting contraction to KCl. 2. In the presence of contraction to noradrenaline, atropine (3 x 10(-7) M-10(-4) M) caused concentration-dependent relaxation. Pretreatment with theophylline (10(-3) M) potentiated the relaxant action of atropine. Relaxation to atropine was not affected by the specific guanosine 3':5'-cyclic monophosphate phosphodiesterase inhibitor, M & B 22,948 (10(-4) M), tetraethylammonium (10 mM), indomethacin (10(-5) M), propranolol (10(-7) M), nifedipine (10(-6) M) or removal of the endothelium. 3. Relaxation to either atropine or prazosin was not affected by preincubation with prazosin and atropine, respectively. 4. In Ca(2+)-free medium containing EGTA and nifedipine, atropine (10(-7) M-10(-4) M) inhibited the residual noradrenaline response more than the subsequent Ca(2+)-induced contraction. Pretreatment with either theophylline (10(-3) M), forskolin (3 x 10(-7) M) or a low concentration of prazosin (3 x 10(-9) M) also inhibited the residual contraction to noradrenaline and Ca2+. The effect of combined treatment of atropine and any of these agents was much greater than with each individual agent. 5. Atropine (10(-6) M-10(-4) M) also inhibited increases in the level of inositol monophosphates (IP) in response to noradrenaline. Theophylline (10(-3) M) and a low concentration of prazosin (3 x 10(-9) M) also inhibited IP formation. Combined with atropine, the effect was much greater than with each of these agents individually. 6. Atropine did not affect adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in the aorta and also failed to displace specific [3H]-prazosin binding.7. These results suggest the possibility that smooth muscle relaxation to atropine may be due to the inhibition of phosphoinositide metabolism. The relaxation is not apparently due to an action of atropine on ax-adrenoceptors, or a change in the level of cyclic AMP.

Identification and molecular characterization of a novel circulating protein in pars planitis

Purpose: The γ-subunit of cyclic guanosine monophosphate phosphodiesterase (PDEγ) plays an important role in the phototransduction process of rod photoreceptors. A previous report indicated that experimental autoimmune uveoretinitis (EAU) could be induced in Lewis rats by immunization with PDEγ. In this study, we identified the uveitopathogenic site of PDEγ synthetic peptides and identified pivotal amino acid residues using analogue peptides.Methods: Several synthetic peptides derived from PDEγ plus adjuvants were injected in Lewis rats. The induction of EAU was examined clinically and histologically. In addition, humoral and cellular immunity against peptides was investigated.Results: The smallest uveitopathogenic peptide was identified as PDEγ 64-76 (ITVICPWEAFNHL), which consists of 13 amino acid residues, and the core sequence was identified as PDEγ 70-76 (WEAFNHL), which consists of 7 amino acid residues. The lowest dose of peptide to induce EAU was 0.03 nmol. The pivotal amino acid residues for eliciting EAU are at 70(W), 71(E), 73(F), and 75(H).Conclusion: Our findings demonstrated the presence of a potent uveitopathogenic site in PDEγ whose potency in Lewis rats was comparable to that of interphotoreceptor retinoid-binding protein.

Autoradiographic analysis of cyclic adenosine monophosphate phosphodiesterase using [ 3H]rolipram in the postischemic rat brain

Postischemic alteration of cyclic adenosine monophosphate phosphodiesterase was investigated in the rat brain, using [ 3H]rolipram in vitro autoradiography. After 90 min of middle cerebral artery (MCA) occlusion, [ 3H]rolipram binding sites decreased rapidly in the brain areas supplied by the occluded MCA. Moreover, 3 days after the ischemia, significant decreases of [ 3H]rolipram binding sites were observed in the thalamus and the substantia nigra, both areas had not been directly affected by the original ischemic insult. These results suggest that alteration of second messenger pathways may be involved in neuronal degeneration caused by transsynaptic process and that alteration of intracellular signal transduction may precede the neuronal damage in the exo-focal postischemic brain areas.

Role of cGMP mechanisms in response of rat pulmonary arteries to hypoxia

We have demonstrated previously that in response to hypoxia, isolated rat pulmonary arteries show an initial endothelium-dependent relaxation followed by an endothelium-independent transient contraction. In the presence of increased extracellular Ca2+, both of these responses were enhanced in endothelium-intact arteries. Nitro-L-arginine, a blocker of the biosynthesis of endothelium-derived relaxing factor (EDRF), abolished the initial endothelium-dependent relaxation and Ca(2+)-induced enhancement of hypoxic contraction in endothelium-intact arteries but did not alter responses in endothelium-denuded vessels. Inhibition of prostaglandin formation with indomethacin had no effect on the hypoxia-elicited responses. Preincubation with LY 83583, an inhibitor of guanylate cyclase activation, abolished the initial hypoxia-elicited relaxation and subsequent contraction. M & B 22948, a guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor, decreased tone under O2 but not under N2, causing an apparent enhancement of the contraction to hypoxia. Thus the modulation of hypoxic responses by the endothelium is dependent on changes in EDRF production, and a decrease in smooth muscle cGMP not involving an EDRF mechanism appears to mediate the endothelium-independent hypoxic contraction observed in the isolated rat pulmonary artery.

Stimulatory effect of vanadate on 3',5'-cyclic guanosine monophosphate- inhibited low Michaelis-Menten constant 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in isolated rat fat pads

When isolated rat fat pads were incubated with vanadate, the low Michaelis-Menten constant (Km) cAMP phosphodiesterase (PDE) activity in the microsomal fraction was increased in a time- and dose-dependent manner with vanadate. 3',5'-Cyclic GMP inhibited the vanadate-stimulated PDE activity with similar profile to the insulin-stimulated one. The stimulatory effect of vanadate was inhibited by inhibitors of tyrosine kinases such as amiloride, biochanin A, and genistein to various extents. Vanadate and insulin both showed the full effect in the absence of either K+, N+, or Ca2+ in the medium, while preincubation of the fat pads with a chelator of intracellular Ca2+ inhibited the vanadate action in a dose-dependent manner. The insulin action was not inhibited by it at tested concentrations. These results suggest that the vanadate action, in contrast to the insulin one, is dependent on the intracellular level of Ca2+. Preincubation of the fat pads with inhibitors of protein kinase C such as 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7) and staurosporine inhibited, in part, the vanadate action but did not inhibit the insulin one. Furthermore, vanadate increased the protein kinase C activity in fat pads but insulin did not increase. H-7 and amiloride showed a significant inhibition of stimulation of protein kinase C activity by vanadate. These results suggest that vanadate stimulates, in part, the 3',5'-cyclic GMP-inhibited low Km cAMP PDE activity in the microsomal fraction of fat pads through the activation of tyrosine kinase and protein kinase C-mediated processes.

Influence of an impeller centrifugal pump on blood components in chronic animal experiments.

A chronic animal experiment was designed to examine the changes in blood components induced by the use of a centrifugal pump (CP). In the pump, an impeller spins in a blood chamber by magnetic coupling with a rotating magnet outside the blood chamber. A pulsatile ventricular assist device was implanted between the left atrium and the descending aorta in four goats weighing from 63 to 75 kg; the CP was installed to replace the assist device, without surgery and anesthesia, more than 2 weeks later when the influences of implantation surgery were diminished. Antithrombotic therapy was performed with oral administration of an antiplatelet agent, cilostazol, a cyclic adenosine monophosphate phosphodiesterase at a dose of 30 mg/kg/day. No significant differences were observed in any of the following parameters: 1) hematocrit, 2) plasma free hemoglobin, 3) lactic acid dehydrogenase, 4) adenosine diphosphate, 5) platelet count, 6) fibrinogen, and 7) antithrombin III, between the data before and after the use of the CP, nor were deformation or pseudopods of platelets seen. The CP developed in the authors' institute and evaluated in this study did not damage blood components, and it proved to be a promising device for long-term use.

Stimulatory effect of vanadate on 3',5'-cyclic guanosine monophosphate-inhibited low Michaelis-Menten constant 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in isolated rat fat pads.

When isolated rat fat pads were incubated with vanadate, the low Michaelis-Menten constant (Km) cAMP phosphodiesterase (PDE) activity in the microsomal fraction was increased in a time- and dose-dependent manner with vanadate. 3',5'-Cyclic GMP inhibited the vanadate-stimulated PDE activity with similar profile to the insulin-stimulated one. The stimulatory effect of vanadate was inhibited by inhibitors of tyrosine kinases such as amiloride, biochanin A, and genistein to various extents. Vanadate and insulin both showed the full effect in the absence of either K+, N+, or Ca2+ in the medium, while preincubation of the fat pads with a chelator of intracellular Ca2+ inhibited the vanadate action in a dose-dependent manner. The insulin action was not inhibited by it at tested concentrations. These results suggest that the vanadate action, in contrast to the insulin one, is dependent on the intracellular level of Ca2+. Preincubation of the fat pads with inhibitors of protein kinase C such as 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7) and staurosporine inhibited, in part, the vanadate action but did not inhibit the insulin one. Furthermore, vanadate increased the protein kinase C activity in fat pads but insulin did not increase. H-7 and amiloride showed a significant inhibition of stimulation of protein kinase C activity by vanadate. These results suggest that vanadate stimulates, in part, the 3',5'-cyclic GMP-inhibited low Km cAMP PDE activity in the microsomal fraction of fat pads through the activation of tyrosine kinase and protein kinase C-mediated processes.

Purification and characterization of guanosine 3',5'-monophosphate- inhibited low K(m) adenosine 3',5'-monophosphate phosphodiesterase from human placental cytosolic fractions

Purification and characterization of guanosine 3',5'-monophosphate- inhibited low K(m) adenosine 3',5'-monophosphate phosphodiesterase from human placental cytosolic fractions

Purification and characterization of guanosine 3',5'-monophosphate-inhibited low K(m) adenosine 3',5'-monophosphate phosphodiesterase from human placental cytosolic fractions.

We previously characterized human placental cytosolic cAMP phosphodiesterase (PDE) and found that two low K(m) cAMP PDE isoforms that were very sensitive to inhibition by cGMP and cilostamide were activated by insulin. As a first step toward understanding the mechanisms by which insulin activates this enzyme, we purified the cGMP-inhibited low K(m) cAMP PDE (cGI-PDE) from human placentas. The enzyme was purified 11,700-fold from a pool of 100,000 x g supernatant fractions of 10-15 placentas by ammonium sulfate precipitation, diethylaminoethyl-cellulose chromatography, and affinity chromatography, using an isothiocyanate derivative of cilostamide (CIT-agarose). The specific activity of the affinity-purified enzyme was 432 +/- 17 nmol/min.mg (mean +/- SD; n = 4). Gel permeation chromatography of the CIT-agarose eluates revealed one protein peak that coincided with PDE activity at an elution position of 135,000 daltons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of this protein peak and CIT-agarose eluates revealed the same patterns, indicating that the purified PDE preparations contained multiple proteins with apparent mol wt of 138K, 83K, 72K, 67K, 63K, and 44K. The 138K form appears to be an intact enzyme; an analogous approximately 135K form has recently been identified in rat adipocyte particulate fractions by specific immunoprecipitation or Western immunoblots. In addition, other smaller forms eluted at 135,000 daltons on gel permeation chromatography, suggesting that, although proteolyzed, they must have been associated by either noncovalent interactions or disulfide bonds. All of the protein bands observed on the sodium dodecyl sulfate-polyacrylamide electrophoresis gel reacted with rabbit antibodies raised against human platelet cGI-PDE. Ten peptides from endoproteinase Lys-C-digests of the affinity-purified placental cGI-PDE were isolated and sequenced; sequences of eight peptides were identical to the deduced amino acid sequences in the C-terminal half of a human heart cGI-PDE cDNA, while those of two peptides were not found in the heart enzyme. The sequences of the eight peptides also matched peptide sequences derived from a purified human platelet cGI-PDE. These results provide evidence that the catalytic C-terminal half domain of the placental insulin-sensitive cGI-PDE shares homology with those of human heart and platelet cGI-PDEs. K(m) and maximum velocity values for cAMP and cGMP were 0.57 microM and 862 nmol/min.mg, and 15 microM and 467 nmol/min.mg, respectively. ED50 values for cGMP, cilostamide, and Ro 20-1724 were 0.12, 0.22, and 120 microM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Bronchodilatory activity and pharmacokinetics of new xanthines in guinea-pigs.

1. The in vitro biological activities and the effect of protein binding on the relaxant effects in vivo of N-3-alkylxanthine and N-3-alkyl-N-1-methylxanthine derivative were investigated in guinea-pigs. 2. A significantly positive correlation was observed among the in vitro muscle relaxant activity, the cyclic adenosine monophosphate (cAMP) phosphodiesterase (PDE) inhibitory activity and the protein-binding potency of xanthine derivatives. However, there was a weak relationship between these activities and affinity for adenosine receptors. 3. When theophylline, enprofylline and 1-methyl-3-propylxanthine (MPX) were injected intravenously in guinea-pigs, their ED50 values were 6.1, 3.3 and 1.0 mg/kg, respectively. Plasma concentrations of these drugs obtained following the intravenous injection of the ED50 approximated the theoretically effective concentration (EC50) predicted from both the relaxant effects in vitro and the protein binding parameters. A good linear correlation was observed between bodyweight in four species (rats, guinea-pigs, rabbits and humans) and certain pharmacokinetic parameters of enprofylline and theophylline. 4. The present study indicates that differences in the relaxant effects of these drugs in vitro and in vivo can be explained in part by protein binding, and that the protein binding of these xanthine bronchodilators is an important determinant for their pharmacological activity. Guinea-pigs provide a useful model for studying pharmacodynamic-pharmacokinetic relationships of new bronchodilators.

Mapping of second messenger and rolipram receptors in mammalian brain

Autoradiographic localizations of major second messengers and a selective cyclic adenosine monophosphate (cyclic-AMP) phosphodiesterase in the brain were visualized in the gerbil and the rat using receptor autoradiography. [ 3H]Phorbol 12,13-dibutyrate (PDBu), [ 3H]inositol 1,4,5-trisphosphate (IP 3), [ 3H]forskolin, [ 3H]cyclic-AMP, and [ 3H]rolipram were used to label protein kinase C, IP 3 receptor, adenylate cyclase, cyclic-AMP-dependent protein kinase (cyclic-AMP-DPK), and Ca 2+/calmodulin-independent cyclic-AMP phosphodiesterase (PDE), respectively. Most second messengers and rolipram binding activities were especially found in the limbic system, basal ganglia, and cerebellum. Marked differences were noted in the hippocampus, where cyclic-AMP and rolipram binding activities were very low in gerbils but high in rats. In contrast, regional localization in the binding sites of PDBu, IP 3, and forskolin in gerbil brain was relatively similar to that in rat brain. Further, alteration of the cyclic-AMP and rolipram binding sites was studied in the gerbil hippocampus 7 days after 10-min cerebral ischemia. The results suggest that the gerbil differs from the rat with respect to the characteristic neurons or interneurons, especially in the hippocampal formation. This finding may help further elucidate the relationship or difference between gerbils and rats for brain function and behavioral pharmacology. Furthermore, our results suggest that cyclic-AMP and rolipram binding sites are predominantly distributed on the pyramidal cell layer of the hippocampal CA1 sector and that transient cerebral ischemia can cause marked reduction in these binding sites in the hippocampus.

Unique adenosine 3',5' cyclic monophosphate phosphodiesterase messenger ribonucleic acids in rat spermatogenic cells: evidence for differential gene expression during spermatogenesis.

Four cAMP phosphodiesterase (cAMP-PDE) genes (ratPDE1, ratPDE2, ratPDE3, and ratPDE4) are expressed in the rat testis (Swinnen et al., PNAS USA 1989; 86:5325). Since multiple ratPDE1 and ratPDE2 mRNAs were present in male germ cells, their developmental expression was investigated by using purified spermatogenic cell populations. RatPDE1 mRNAs (4.0 and 2.8 kb) were found to be abundant in pachytene spermatocytes. RatPDE1 mRNA levels were decreased in round spermatids and absent from condensing spermatids/residual bodies. However, multiple ratPDE2 mRNAs (4.0, 3.5, 3.1, 2.8, and 2.4 kb) were abundant in round spermatids, and lower amounts were present in condensing spermatids/residual bodies. Transcripts related to ratPDE2 were also present in mouse round spermatids. Chromatography of germ cell cytosol identified two peaks of cAMP-PDE activity. Whereas peak A was evident in all germ cell populations examined, peak B was present in pachytene spermatocytes and round spermatids, but was at the limit of detection in condensing spermatids/residual bodies. The large decrease in peak B activity in condensing spermatids/residual bodies may be related to the drop in ratPDE1 mRNA levels observed during spermatogenesis. The sustained peak A activity in condensing spermatids/residual bodies coincides with the presence of ratPDE2 mRNA in these cells and suggests that the ratPDE2 enzyme may function during spermiogenesis and in spermatozoa.

A site on rod G protein alpha subunit that mediates effector activation.

The heterotrimeric guanine nucleotide binding proteins (G proteins) are activated by sensory or hormone receptors. In turn, the G proteins activate effector proteins such as adenylyl cyclase, cyclic guanosine 3',5'-monophosphate phosphodiesterase (cGMP PDE), phospholipase C, and potassium and calcium ion channels by mechanisms that are poorly understood. A site on the alpha subunit of the G protein transducin (alpha t) has been identified that interacts with and activates cGMP phosphodiesterase, the effector enzyme in rod photoreceptors. A 22-amino acid peptide, corresponding to residues 293 to 314 from the COOH-terminal region of alpha t, fully mimicked alpha t and potently activated PDE. This region is adjacent to the receptor activation domain; thus, the alpha subunit of this G protein has a site for interaction with both its effector and receptor that maps near the COOH-terminus.

Effect of alpha-human atrial natriuretic peptide on the synthesis of dopamine in the rat kidney.

1. The present study has examined the influence of alpha-human atrial natriuretic peptide (alpha-hANP) on the synthesis of dopamine and its deamination into 3,4-dihydroxyphenylacetic acid (DOPAC) in rat kidney slices loaded with exogenous L-dihydroxyphenylalanine (L-DOPA). 2. alpha-hANP (3.3 and 330 nM) was found to produce a marked reduction (63-78% reduction) in the time-dependent accumulation of newly-formed dopamine and of its deaminated metabolite DOPAC in kidney slices loaded with 10 microM L-DOPA. alpha-hANP (330 nM) was also found to decrease the accumulation of newly-formed dopamine (45-66% reduction) and DOPAC (38-61% reduction) in experiments in which increasing concentrations (1-100 microM) of L-DOPA were used. This inhibitory effect was found to be potentiated by zaprinast (M&B 22,948; 10 microM), a guanosine cyclic 3',5'-monophosphate (cyclic GMP) phosphodiesterase inhibitor. Alone, zaprinast also decreased the accumulation of both dopamine (54-71% reduction) and DOPAC (73-92% reduction). 3. In kidney homogenates, alpha-hANP (330 nM) was found to affect neither the formation of dopamine nor its deamination to DOPAC. 4. Both alpha-hANP (330 nM) and zaprinast (10 microM) were found not to affect the formation of dopamine and DOPAC in kidney slices obtained from rats on a high salt diet during the previous 6 weeks. A similar situation was also found to occur when kidney slices obtained from 24-months old rats were used.5. The results obtained suggest that the inhibitory effect of alpha-hANP on the renal synthesis of dopamine is dependent on the activation of a membrane-operated mechanism, coupled to the enzyme guanylate cyclase, controlling the entry of L-DOPA into the cells.

Role of nitric oxide in non-adrenergic, non-cholinergic inhibitory junction potentials in canine ileocolonic sphincter.

1. Electrical field stimulation causes neurally-mediated relaxation of the ileocolonic sphincter that is due to activation of non-adrenergic and non-cholinergic (NANC) nerves. Recent studies have suggested that nitric oxide (NO) is the neurotransmitter that mediates relaxation. 2. Using intracellular recording techniques, we have tested whether NANC inhibitory junction potentials (i.j.ps) in the canine ileocolonic sphincter are also mediated by NO. 3. Electrical field stimulation elicited excitatory and inhibitory junction potentials: e.j.ps were blocked by atropine (10(-6) M) and tetrodotoxin (TTX; 10(-6) M); i.j.ps were also blocked by TTX and partially blocked by apamin (10(-6) M). I.j.ps were unaffected by atropine, phentolamine and propranolol (all at 10(-6) M). 4. The arginine analogues, L-NG-nitroarginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA), decreased the amplitude of i.j.ps and L-arginine, but not D-arginine, partially restored the i.j.ps. 5. I.j.ps were also inhibited by oxyhaemoglobin (1%), but not by methaemoglobin. 6. Exogenous NO (10(-7) M to 3 x 10(-5) M) caused concentration-dependent hyperpolarizations that were similar in amplitude to the NANC nerve-evoked i.j.ps. Hyperpolarizations to NO were unaffected by L-NAME, but were blocked by oxyhaemoglobin. 7. Tetrodotoxin, L-NAME and oxyhaemoglobin all caused depolarization of resting membrane potential. 8. The specific guanosine 3':5'-cyclic monophosphate phosphodiesterase inhibitor, M&B 22948, caused hyperpolarization, increased the maximum level of hyperpolarization reached during i.j.ps, and increased the duration of i.j.ps. 9. These data further support the hypothesis that NANC neurotransmission in the ileocolonic sphincter is mediated by NO or an NO-releasing compound. The data also suggest that tonic release of NO, possibly from spontaneous firing of NANC nerves, may regulate resting membrane potential and tone in this sphincter.

Methylxanthines reversibly inhibit tracheary-element differentiation in suspension cultures of Zinnia elegans L.

Tracheary-element (TE) differentiation in suspension-cultured mesophyll cells of Zinnia elegans L. was completely inhibited by caffeine and theophylline only when these methylxanthines were applied at least 8 h prior to the appearance of secondary cell-wall thickenings. In contrast, the calcium-channel blocker nifedipine completely inhibited TE differentiation when applied only 2-3 h prior to the onset of secondary cell-wall deposition. This indicates the involvement of a methylxanthine-inhibitable event in TE differentiation that is distinguishable from an event dependent on influx of extracellular calcium. The correlation between the time of appearance of chlorotetracycline fluorescence (an indicator of sequestered Ca(2+)) and loss of methylxanthine effectiveness indicates that inhibition by methylxanthines may result from release of Ca(2+) from intracellular stores. Methylxanthines with high potencies against adenosine 3' ∶ 5'-cyclic monophosphate (cAMP) phosphodiesterase and adenosine receptors were less effective inhibitors of TE differentiation, indicating that inhibition of differentiation by methylxanthines is independent of cAMP metabolism. The role of cAMP in transduction of the cytokinin signal, which was proposed previously on the basis of stimulation of TE differentiation by theophylline, was investigated using the non-hydrolyzable analog 8-bromo-cAMP. Although 8-bromo-cAMP stimulated differentiation in the absence of inductive concentrations of cytokinin, the non-cyclic analog 8-bromo-AMP was even more effective, indicating that 8-bromo-cAMP behaves as a cytokinin analog, rather than a second messenger, in stimulating TE differentiation.

Calmodulin content and activity in normal and coeliac duodenum.

Calmodulin is an important modulator of intracellular calcium processes and may be implicated in the calcium malabsorption of coeliac disease. The calmodulin content in extracts of duodenal biopsy specimens from 48 normal control subjects and 28 patients with coeliac disease was determined. Radioimmunoassay was used to measure immunoreactive calmodulin while a cyclic adenosine 3',5'-monophosphate phosphodiesterase activity assay was used to measure biologically active calmodulin. Calmodulin values measured by both assays were similar for control and disease groups. Mean (SEM) immunoreactive calmodulin values were 1.68 (0.09) micrograms/mg protein for controls and 1.67 (0.15) and 1.45 (0.15) micrograms/mg protein for partial and total villous atrophy respectively. These values were not significantly different. Biologically active calmodulin values were 2.77 (0.21), 1.82 (0.34), and 3.24 (0.33) micrograms/mg protein for control, partial, and total villous atrophy subjects respectively. The biologically active calmodulin values in the partial villous atrophy group were significantly lower than in controls and total villous atrophy subjects. In the phosphodiesterase assay, the calmodulin antagonist trifluoperazine inhibited the activity stimulated by purified calmodulin and by the extracts to the same extent. These results show that calmodulin values are normal in coeliac disease and provide no evidence that changes in calmodulin account for the abnormal calcium absorption in these patients.

Nitric oxide as a mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neurotransmission.

Nitric oxide has been identified as an endothelium-derived relaxing factor in blood vessels. We tried to determine whether it is involved in the relaxation of the corpus cavernosum that allows penile erection. The relaxation of this smooth muscle is known to occur in response to stimulation by nonadrenergic, noncholinergic neurons. We studied strips of corpus cavernosum tissue obtained from 21 men in whom penile prostheses were inserted because of impotence. The mounted smooth-muscle specimens were pretreated with guanethidine and atropine and submaximally contracted with phenylephrine. We then studied the smooth-muscle relaxant responses to stimulation by an electrical field and to nitric oxide. Electrical-field stimulation caused a marked, transient, frequency-dependent relaxation of the corpus cavernosum that was inhibited in the presence of N-nitro-L-arginine and N-amino-L-arginine, which selectively inhibit the biosynthesis of nitric oxide from L-arginine. The addition of excess L-arginine, but not D-arginine, largely reversed these inhibitory effects. The specific liberation of nitric oxide (by S-nitroso-N-acetylpenicillamine) caused rapid, complete, and concentration-dependent relaxation of the corpus cavernosum. The relaxation caused by either electrical stimulation or nitric oxide was enhanced by a selective inhibitor of cyclic guanosine monophosphate (GMP) phosphodiesterase (M&B 22,948). Relaxation was inhibited by methylene blue, which inhibits cyclic GMP synthesis. Our findings support the hypothesis that nitric oxide is involved in the nonadrenergic, noncholinergic neurotransmission that leads to the smooth-muscle relaxation in the corpus cavernosum that permits penile erection. Defects in this pathway may cause some forms of impotence.

Separation and some characteristics of two factors from rat liver particulate fraction which stimulate and inhibit the low-Km adenosine 3',5' cyclic monophosphate (cAMP) phosphodiesterase of rat fat cells.

Two factors were separated from rat liver particulate fraction treated with insulin, one of them having a stimulating effect on low-Km adenosine 3',5' cyclic monophosphate (cAMP) phosphodiesterase activity of crude microsomal fraction (P-2 fraction) and the other having an inhibiting effect on the activity of low-Km cAMP phosphodiesterase solubilized with 0.3% Brij 58 from P-2 fraction. Trypsin and heat treatments had essentially no effect on these two factors. The stimulating factor did not significantly change the apparent Km value of enzyme in P-2 fraction but increased the maximal velocity of the reaction. The inhibiting factor raised the Km value of solubilized enzyme without affecting the maximal velocity of the reaction. The stimulating factor level in diabetic rat was larger than that in normal rat while the inhibiting factor level in diabetic rat was smaller than that in normal rat. Possible participation of both factors in insulin action is discussed.

Inhibitors of cAMP Phosphodiesterase in Medicinal Plants. Part XVIII. Inhibitors of Adenosine 3',5'-Cyclic Monophosphate Phosphodiesterase from Schisandra chinensis and the Structure Activity Relationship of Lignans.

The structure activity relationship was studied in analogous lignans from Schisandra chinensis and their derivatives. These compounds were tested for cyclic adenosine 3', 5'-monophosphate (cAMP) phosphodiesterase inhibition. An inhibitor, nordihydroguaiaretic acid (13), was isolated from this plant, so we discussed this compound and a derivative, nordihydroguaiaretic acid tetramethyl ether, using molecular mechanics involving three-dimensional modeling and minimization of the structure using the MM2PP program. As a result, it was found that the structure of nordihydroguaiaretic acid tetrametyl ether and papaverine (30) (positive control) shared a similar low energy conformation. This fact suggested that these compounds inhibited cAMP phosphodiesterase by a similar mechanism.