Absence Of Phosphodiesterase Inhibitor Research Articles

Angiotensin II potentiates prostaglandin stimulation of cyclic AMP levels in intact bovine adrenal medulla cells but not adenylate cyclase in permeabilized cells.

The level of cyclic AMP in primary cultures of bovine adrenal medulla cells is elevated by prostaglandin E1. Angiotensin II is commonly reported to act on receptors linked to phosphoinositide metabolism or to inhibition of adenylate cyclase. We have investigated the effect of angiotensin II on prostaglandin E1-stimulated cyclic AMP levels in these primary cultures. Rather than reducing cyclic AMP levels, we have found that angiotensin II powerfully potentiates prostaglandin E1-stimulated cyclic AMP accumulation in intact cells, both in the presence and absence of phosphodiesterase inhibitors. The 50% maximal response was similar to that for stimulation of phosphoinositide breakdown by angiotensin II in these cultures. The potentiation of stimulated cyclic AMP levels was seen, although to a smaller maximum, with the protein kinase C (Ca2+/phospholipid-dependent enzyme) activating phorbol ester tetradecanoyl phorbolacetate and with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol; pretreatment (24 h) with active phorbol ester, which would be expected to diminish protein kinase C levels, attenuated the angiotensin II potentiation of cyclic AMP. Using digitonin-permeabilized cells we showed that adenylate cyclase activity was stimulated by prostaglandin E1 with the same dose-response relationship as was cyclic AMP accumulation in intact cells, but the permeabilized cells showed no response to angiotensin II. The results are discussed with respect to the hypothesis that the angiotensin II influence on cyclic AMP levels is mediated, in part, by diacylglycerol stimulation of protein kinase C.

N 6-phenylisopropyladenosine stimulates in normal and inhibits in adrenalectomized rats the low KM cyclic AMP phosphodiesterase in the brain

In crude rat brain membranes, apparent adenylate cyclase activity tested in the absence of phosphodiesterase inhibitors was dose-dependently inhibited by the adenosine ‘R-site’ agonist N 6-phenylisopropyladenosine (N 6-PIA). In membranes from adrenalectomized rats, however, N 6-PIA induced, under the same conditions, an activation. However, in the presence of the phosphodiesterase inhibitor Ro-20-1724, brain adenylate cyclase responsiveness to N 6-PIA resulted in a dose-dependent inhibition in both sham-operated and adrenalectomized rats. Thus, the low K M cyclic AMP phosphodiesterase activity present in these brain membranes was investigated. Although this activity (tested in the presence of GTP) was unaltered by adrenalectomy, the dose-response curve of this enzyme to low concentrations of N 6-PIA showed an activation in sham-operated and an inhibition in adrenalectomized rats, two effects which were suppressed by sodium (80 mM). These results showing that N 6-PIA modulates both adenylate cyclase and cyclic AMP phosphodiesterase in the brain, provide an additional argument for a potential role of adenosine in the regulation of cyclic AMP metabolism in normal as well as in pathological brain.

Mononuclear Leukocyte Cyclic Adenosine Monophosphate Responses in Psoriasis Are Normal

It has been proposed that immune dysfunction in psoriasis is a consequence of aberrant cyclic nucleotide metabolism. We have examined cyclic AMP responses to isoprenaline, histamine, and prostaglandin E2 in peripheral blood mononuclear leukocytes from patients with psoriasis, in the presence and absence of a potent cyclic AMP phosphodiesterase inhibitor. Stimulated and basal cyclic AMP levels in mononuclear leukocytes from psoriatics did not differ from those observed in mononuclear leukocytes from normal subjects, irrespective of the stimulant employed, either in the presence or in the absence of the phosphodiesterase inhibitor. These findings do not support the hypothesis that psoriasis is associated with either impaired beta-adrenergic reactivity or a more generalized abnormality of mononuclear leukocyte cyclic nucleotide metabolism.

Relationship Between Fertilizing Ability and cAMP in Human Spermatozoa

The goal of this study was to investigate the relationship between cAMP and the fertilizing ability of human spermatozoa. Levels of cAMP were measured in human spermatozoa during 6‐hour in vitro incubation in capacitation medium, in both the presence and absence of phosphodiesterase (PDE) inhibitor. The fertilizing ability of the same samples was assayed with zona‐free hamster eggs. In the absence of PDE inhibitor, no relationship was apparent between mean cAMP levels, which remained at 3–4 pmol cAMP/107 spermatozoa, and mean fertilizing ability, which increased from 4% at the start of the incubation to 33% at 6 hours. In the presence of PDE inhibitor (7 mM caffeine or 10 mM theophylline), cAMP concentrations increased within minutes to 3–4 times control levels. Despite this increase in cAMP, there was no immediate change in fertilizing ability. This was true whether PDE inhibitor was present from the start or added to control sperm after 6 or 22 hours of incubation. However, once the sperm were exposed to PDE inhibitor for 4–6 hours, they fertilized a significantly greater proportion of eggs than did control samples. These results suggest that PDE inhibitors, or elevated cAMP levels, do not immediately induce the acrosome reaction, but rather appear to reduce the amount of time required for capacitation to occur in vitro.

Effect of relaxin on bound cAMP in rat uterus.

Relaxin was able to elevate uterine bound cAMP in the presence of 3-isobutyl-1-xanthine in buffers of low or high (0.5 M NaCl) ionic strength. Significant elevation was observed 10 min after exposure to the hormone. The peak in bound cAMP preceded that in total cAMP (15 vs 20 min, respectively, under isometric conditions). The response was concentration-dependent, with maximal elevation in bound cAMP observed at 0.1-0.4 microgram/ml relaxin. The effect of relaxin on bound cAMP represented a change in a sizable proportion of the total available binding sites. However, relaxin did not elevate bound cAMP in the absence of phosphodiesterase inhibitors under conditions where it clearly inhibited contractile activity. These data suggest that although relaxin can alter the level of bound cAMP in the rat uterus under some conditions, these effects may not be involved in the acute changes elicited by this hormone which result in the rapid inhibition of spontaneous contractile activity.

The interaction of relaxin with the rat uterus. I. Effect on cyclic nucleotide levels and spontaneous contractile activity.

Three different preparations of relaxin elevated cAMP levels in isometrically suspended uterine strips obtained from estrogen-primed female rats in the presence of phosphodiesterase inhibitors. The effect was time and dose dependent, with maximal response at 20 min and 1 microgram/ml, respectively. No changes in cGMP levels were observed. No significant response was observed in ileum, vas deferens, epididymis, or testicular capsule. In the absence of phosphodiesterase inhibitors, relaxin markedly suppressed spontaneous contractile activity at doses of 0.36 and 0.96 microgram/ml but did not elevate cAMP in this dose range. While the beta-adrenergic antagonist propranolol suppressed both the relaxant action and cAMP stimulation promoted by isoproterenol, it did not interfere with the effect of relaxin on these same parameters. Relaxin is therefore capable of relaxing uterine muscle and elevating cAMP by mechanisms which probably do not require beta-adrenergic mediation. It is not clear at present whether the effects of relaxin on cAMP levels and contractile activity are causally related.

Receptor-mediated gonadotropin action in the ovary. Regulatory role of cyclic nucleotide phosphodiesterase(s) in intracellular adenosine 3′:5′-cyclic monophosphate turnover and gonadotropin-stimulated progesterone production by rat ovarian cells

The regulatory role of cyclic nucleotide phosphodiesterase(s) and cyclic AMP metabolism in relation to progesterone production by gonadotropins has been studied in isolated rat ovarian cells. Low concentrations of choriogonadotropin (0.4-5ng/ml) increased steroid production without any detectable increase in cyclic AMP, when experiments were carried out in the absence of phosphodiesterase inhibitors. The concentration of choriogonadotropin (10ng/ml) that stimulated progesterone synthesis maximally resulted in a minimal increase in cyclic AMP accumulation and choriogonadotropin binding. Choriogonadotropin at a concentration of 10ng/ml and higher, however, significantly stimulated protein kinase activity and reached a maximum between 250 and 1000ng of hormone/ml. Higher concentrations (50-2500ng/ml) of choriogonadotropin caused an increase in endogenous cyclic AMP, and this increase preceded the increase in steroid synthesis. Analysis of dose-response relationships of gonadotropin-stimulated cyclic AMP accumulation, progesterone production and protein kinase activity revealed a correlation between these responses over a wide concentration range when experiments were performed in the presence of 3-isobutyl-1-methylxanthine. The phosphodiesterase inhibitors papaverine, theophylline and 3-isobutyl-1-methylxanthine each stimulated steroid production in a dose-dependent manner. Incubation of ovarian cells with dibutyryl cyclic AMP or 8-bromo cyclic AMP mimicked the steroidogenic action of gonadotropins and this effect was dependent on both incubation time and nucleotide concentration. Maximum stimulation was obtained with 2mm-dibutyryl cyclic AMP and 8-bromo cyclic AMP, and this increase was close to that produced by a maximally stimulating dose of choriogonadotropin. Other 8-substituted derivatives such as 8-hydroxy cyclic AMP and 8-isopropylthio cyclic AMP, which were less susceptible to phosphodiesterase action, also effectively stimulated steroidogenesis. The uptake and metabolism of cyclic [(3)H]AMP in ovarian cells was also studied in relation to steroidogenesis. When ovarian cells were incubated for 2h in the presence of increasing concentrations of cyclic [(3)H]AMP, the radioactivity associated with the cells increased almost linearly up to 250mum-cyclic [(3)H]AMP concentration in the incubation medium. The (3)H label in the cellular extract was recovered mainly in the forms ATP, ADP, AMP, adenosine and inosine, with cyclic AMP accounting for less than 1% of the total tissue radioactivity. Incubation of cyclic AMP in vitro with ovarian cells resulted in a rapid breakdown of the nucleotide in the medium. The degradation products in the medium have been identified as AMP, adenosine and inosine. The rapid degradation of cyclic AMP by phosphodiesterase(s) makes it difficult to correlate changes in cyclic AMP concentrations with steroidogenesis. These observations thus provide an explanation for the previously observed lack of cyclic AMP accumulation under conditions in which low doses of choriogonadotropin stimulated steroidogenesis without any detectable changes in cyclic AMP accumulation.

Activation of protein kinase and glycogen phosphorylase in isolated rat liver cells by glucagon and catecholamines.

In liver cells isolated from fed female rats, glucagon (290nM) increased adenosine 3':5'-monophosphate (cyclic AMP) content and decreased cyclic AMP binding 30 s after addition of hormones. Both returned to control values after 10 min. Glucagon also stimulated cyclic AMP-independent protein kinase activity at 30 s and decreased protein kinase activity assayed in the presence of 2 muM cyclic AMP at 1 min. Glucagon increased the levels of glycogen phosphorylase a, but there was no change in total glycogen phosphorylase activity. Glucagon increased glycogen phosphorylase a at concentrations considerably less than those required to affect cyclic AMP and protein kinase. The phosphodiesterase inhibitor, 1-methyl-3-isobutyl xanthine, potentiated the action of glucagon on all variables, but did not increase the maximuM activation of glycogen phosphorylase. Epinephrine (1muM) decreased cyclic AMP binding and increased glycogen phosphorylase a after a 1-min incubation with cells. Although 0.1 muM epinephrine stimulated phosphorylase a, a concentration of 10 muM was required to increase protein kinase activity. 1-Methyl-3-isobutyl xanthine (0.1 mM) potentiated the action of epinephrine on cyclic AMP and protein kinase. (-)-Propranolol (10muM) completely abolished the changes in cyclic AMP binding and protein kinase due to epinephrine (1muM) in the presence of 0.1mM 1-methyl-3-isobutyl xanthine, yet inhibited the increase in phosphorylase a by only 14 per cent. Phenylephrine (0.1muM) increased glycogen phosphorylase a, although concentrations as great as 10 muM failed to affect cyclic AMP binding or protein kinase in the absence of phosphodiesterase inhibitor. Isoproterenol (0.1muM) stimulated phosphorylase and decreased cyclic AMP binding, but only a concentration of 10muM increased protein kinase. 1-Methyl-3-isobutyl xanthine potentiated the action of isoproterenol on cyclic AMP binding and protein kinase, and propranolol reduced the augmentation of glucose release and glycogen phosphorylase activity due to isoproterenol. These data indicate that both alpha- and beta-adrenergic agents are capable of stimulating glycogenolysis and glycogen phosphorylase a in isolated rat liver cells. Low concentrations of glucagon and beta-adrenergic agonists stimulate glycogen phosphorylase without any detectable increase in cyclic AMP or protein kinase activity. The effects of alpha-adrenergic agents appear to be completely independent of changes in cyclic AMP protein kinase activity.

Control of cyclic AMP levels in isolated fat cells from hamsters

1. 1. Isolated epididymal fat cells from the golden hamster were prepared by a method involving minimal exposure to collagenase. 2. 2. β-Adrenergic stimuli or adrenocorticotropic hormone (ACTH) caused increases in cyclic adenosine 3′,5′-monophosphate (cyclic AMP) levels of as much as 40-fold in the absence of phosphodiesterase inhibitors. 3. 3. Norepinephrine lowered cyclic AMP levels in isolated fat cells exposed to ACTH, either in the presence (by 80%) or absence (by 30%) of caffeine. This effect was reversed by phentolamine, indicating that it was due to the α-adrenergic activity of norepinephrine. 4. 4. Caffeine acted synergistically with ACTH or isopropylnorepinephrine but not with norepinephrine. However, synergism between norepinephrine and caffeine did occur in the presence of phentolamine.