Adenosine stimulates adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate accumulation in rat pinealocytes: evidence for a role for adenosine in pineal neurotransmission

Adenosine produces a concentration-dependent increase in pinealocyte cAMP (EC50, approximately 0.3 nM) and cGMP accumulation (EC50, approximately 0.7 nM). Maximal increases in both nucleotides are evident 10 min after treatment; 1 h later values return to pretreatment levels. Concentration-dependent effects on cAMP are also observed with N6-(L-2-phenylisopropyl)adenosine (EC50, approximately 0.75 nM), 5'-N-ethylcarboxy aminoadenosine (EC50, approximately 0.75 nM), and 2-chloroadenosine (EC50, approximately 2.0 nM); the EC50 values for stimulation of cGMP with these agents are higher by a factor of 2-10. In the case of 5'-N-ethylcarboxy amidoadenosine, the concentration-response curve is biphasic, with a significant effect evident within the range of 1-100 pM. The stimulatory nature of this response and the relative potency of the agonists tested are consistent with the involvement of an A2-like adenosine receptor. Comparison of adenosine and the selective beta-adrenergic agonist isoproterenol indicated that their maximal EC50 values were generally similar. Studies with antagonists revealed that both 8-(p-sulfophenyl)theophylline (1 microM) and the xanthine amine congener (8-[4-[[[(2-aminoethyl)carbonyl]methyl]oxy]phenyl]1,3- dipropylxanthine (1 microM) inhibited the effects of adenosine (1 nM to 1 microM), but xanthine amine congener was more potent; the latter was markedly effective at 0.1 nM, whereas 8-(p-sulfophenyl)theophylline was nearly ineffective at this concentration. It was also determined that pineal cells generate extracellular adenosine from extracellular ATP. ATP is thought to be released along with catecholamines during neurotransmission. Hence, these studies support the view that adenosine could participate in the transsynaptic regulation of pineal function.     

See-saw signal processing: reciprocal effects of stimulus deprivation on vasoactive intestinal peptide-stimulated adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate accumulation in rat pinealocytes.

In the present study the effects of stimulus deprivation on vasoactive intestinal peptide (VIP)- and alpha 1-adrenergically mediated amplification of VIP-stimulated cAMP and cGMP accumulation were examined. Dispersed pinealocytes were prepared from either Sprague-Dawley rats maintained for 2 weeks in a normal lighting schedule providing 14 h of light/day (LD cells) or from animals maintained in constant lighting (LL cells). LL treatment enhanced the VIP-stimulated cAMP response up to 2-fold, while reducing the peak VIP-stimulated cGMP by 70%. In LL cells, phenylephrine potentiated the VIP-stimulated cAMP response, but did not potentiate the VIP-stimulated cGMP response. Potentiation of the cAMP response to VIP can be produced in LD cells by treatment with agents that elevate intracellular Ca2+ (depolarizing concentrations of K+ or A23187) or an activator of protein kinase-C [14 beta-phorbol 12-myristate 13-acetate (PMA)]. LL treatment abolished the potentiating effects of K+ or A23187 on cAMP and cGMP responses in VIP-treated cells. In contrast, LL treatment augmented the PMA potentiation of VIP-stimulated cAMP response. The potentiation effects of PMA and K+ on the cGMP response in VIP-treated cells, however, were suppressed by LL treatment. To further investigate the involvement of postreceptor mechanisms, forskolin was used to stimulate pineal cAMP and cGMP accumulation. LL treatment had similar effects on the forskolin-stimulated cyclic nucleotide responses, with one exception. Depolarizing concentrations of K+ potentiated the forskolin-stimulated cAMP response while having no effect on the VIP-stimulated cAMP responses. These findings suggest that LL treatment results in a larger VIP-stimulated cAMP response, while its effect on the cGMP response is inhibitory. LL treatment appears to inhibit a step distal to elevation of intracellular Ca2+ which is of importance to the alpha 1-adrenergic potentiation of VIP-stimulated cAMP and cGMP responses.     

Atypical synergistic alpha 1- and beta-adrenergic regulation of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate in rat pinealocytes

The adrenergic control of cAMP and 3',5'-cyclic GMP (cGMP) in dispersed adult rat pinealocytes was investigated. Norepinephrine treatment increased cAMP and cGMP content 60- and 400-fold, respectively; both alpha- and beta-adrenoceptors had to be activated for these responses to occur. Beta-Adrenergic stimulation alone produced only about 6- and 2-fold increase in cAMP and cGMP content, respectively. Alpha-Adrenergic stimulation, which alone had no effect on either cyclic nucleotide concentration, markedly amplified the beta-adrenergic stimulation of both cAMP and cGMP. The relative potency of alpha-adrenergic agonists and antagonists indicates the alpha 1-subclass of adrenoceptors is involved. A role of alpha 1-adrenoceptors in the control of pineal cAMP is consistent with published evidence of the presence of alpha 1-adrenoceptors on pinealocytes and their role in the regulation of N-acetyltransferase activity and melatonin production.     

Serotonin stimulates adenosine 3',5'-monophosphate accumulation in parathyroid adenoma

The effect of serotonin on cAMP accumulation in parathyroid adenoma tissue from patients with primary hyperparathyroidism was studied in vitro. Incubation with 10(-5) M serotonin elicited a marked increase (of 90--150%) in cAMP content in slices of parathyroid adenoma tissue. This stimulatory effect of serotonin was already apparent after 2 min of incubation; stimulation by serotonin was dose dependent, with the highest stimulation being achieved at 10(-4) M serotonin. The serotonin antagonists, methylsergide and cinanserin, in concentrations equimolar to serotonin completely blocked the stimulatory effect of serotonin on cAMP increase. The serotonin content in surgically removed parathyroid adenoma tissue, as determined by fluorometric assay, was 6.4 +/- 1.2 pmol/mg wet wt (approximately 0.8 x 10(-5) M). The present observations demonstrate that parathyroid adenoma tissue has a high content of serotonin, and serotonin stimulates cAMP accumulation in this tissue. Since cAMP acts as a mediator of parathyroid hormone (PTH) release, our results suggest that serotonin could be one of the factors regulating PTH secretion and/or contributing to PTH hypersecretion in various forms of primary hyperparathyroidism.     

Stimulation of prostaglandin accumulation in preovulatory rat follicles by adenosine 3',5'-monophosphate.

LH stimulates an increase in prostaglandins in vitro in preovulatory follicles from rats pretreated with PMS gonadotropin. The role of cAMP in this action of LH was examined by incubating preovulatory follicles with various substances and determining the resultant prostaglandin (PG) E accumulation by radioimmunoassay. LH (5 microgram/ml) increased PGE accumulation to approximately 4 times the control (181 +/- 23 to 886 +/- 83 pg/follicle). The addition of 20 mM cAMP also stimulated PGE accumulation, and the addition of 20 mM cAMP in the presence of 0.5 mM 1-methyl-3-isobutylxanthine was as effective as LH. Other nucleotides such as ATP, ADP, 3'-AMP, 5'-AMP, cGMP, and O2'-monobutyryl-cAMP did not stimulate PGE accumulation. On the other hand, (Bu)2cAMP, 8-bromo-cAMP, and N6-monobutyryl-cAMP produced an increase in PGE accumulation similar to that observed with LH. In addition, 10 microgram/ml cholera toxin was shown to increase both cAMP and PGE accumulation in preovulatory follicles. These results indicate that the prostaglandin response of follicles is specific for cAMP-like nucleotides or substances capable of increasing intracellular cAMP. The data support the concept that cAMP mediates the effect of LH on PGE accumulation in preovulatory follicles in the rat.     

Inhibitory effects of amilorides on pinealocyte adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate accumulation: possible involvement of postreceptor mechanisms

In rat pinealocytes, alpha 1-adrenergic receptor activation increases intracellular pH (pHi) through Ca2+/protein kinase-C-dependent activation of the Na+/H+ antiporter. Using a series of amiloride analogs, norepinephrine stimulation of cGMP accumulation is also found to be pHi dependent. In this study, we examined the postreceptor mechanisms involved in the amiloride effects on cyclic nucleotide accumulation using agents that simulate alpha 1-adrenoceptor activation. Four amiloride analogs, with a 500-fold difference in their inhibitory potency of the Na+/H+ antiporter, were used. 5-(N,N-Hexamethylene)amiloride (HA), the most active inhibitor of the Na+/H+ antiporter, had a stimulatory effect on isoproterenol (ISO)-stimulated cAMP, while its effect on cGMP was inhibitory. The other three amiloride derivatives had no effect on the ISO-stimulated cAMP or cGMP responses. All four amilorides (at 10 microM) had no effect on the phenylephrine potentiation of cAMP responses in beta-adrenergically stimulated cells, while they inhibited the potentiation of cGMP accumulation according to their inhibitory potency on the Na+/H+ antiporter. Using depolarizing concentrations of K+, it was found that HA was additive to the submaximal potentiation by K+ on ISO-stimulated cAMP, while its effect on cGMP was inhibitory. Amiloride hydrochloride dihydrate, the amiloride that is least potent in its inhibitory action on the Na+/H+ antiporter, had no effect on the K+ potentiation of either cAMP or cGMP. Using 4 beta-phorbol 12-myristate 13-acetate in cells treated with 10 mM K+ and ISO, it was found that HA was additive to phorbol 12-myristate 13-acetate and K+ potentiation of the cAMP response, while its effect on the cGMP response was inhibitory. Amiloride hydrochloride had no effect on either the cAMP or cGMP response. It can be concluded from these studies that 1) HA has a stimulatory effect on the beta-adrenoceptor-Gs-adenylate cyclase pathway that is independent of inhibition of the Na+/H+ antiporter; 2) postreceptor mechanisms are involved in HA's effects on cAMP and cGMP accumulation; and 3) the action of HA on cGMP is likely to be related to its effect on the Na+/H+ antiporter.     

Light and agonist alter vasoactive intestinal peptide binding and intracellular accumulation of adenosine 3',5'-monophosphate in the rat pineal gland

We examined the effects of environmental light and prior treatment with an agonist on vasoactive intestinal polypeptide (VIP) binding and VIP stimulation of cAMP accumulation in the rat pineal gland. VIP binding to pinealocytes and cAMP accumulation in response to VIP were significantly increased in animals kept exposed to constant light compared to those in animals experiencing a dark night before the experiments. Scatchard analysis of [125I]VIP binding indicated the presence of two classes of binding sites: high affinity, low capacity sites and low affinity, high capacity sites. The increased VIP binding to pinealocytes in rats maintained in constant light was attributed to an increase in the number of available VIP receptors at both high and low affinity sites. The affinity of VIP binding to cells was not affected by exposure of the animals to light. VIP stimulation of cAMP accumulation was not inhibited by d,l-propranolol. Prior treatment of pinealocytes with VIP decreased [125I]VIP binding by reducing the number of receptors and significantly inhibited subsequent VIP stimulation of cAMP accumulation. Prior treatment with norepinephrine did not alter the number of VIP receptors. Our results strongly suggest that VIP is a neuromodulator of pineal function.     

Histamine stimulates progesterone synthesis and cyclic adenosine 3',5'-monophosphate accumulation in isolated preovulatory rat follicles

The effect of histamine on progesterone synthesis and cyclic adenosine 3',5'-monophosphate (cAMP) accumulation was studied in superfused and incubated follicles dissected free from immature rats treated with pregnant mare serum gonadotrophin (PMSG). Histamine, like LH, increased the progesterone synthesis, but to a smaller extent. The H2-antagonist, cimetidine, inhibited completely the histamine-induced progesterone increase while the H1-antagonist, pyrilamine, as well as propranolol and atropine did not affect the initial response but modified its duration. The specific H2-agonist, 4-methylhistamine, but not the H1-agonist, 2-methylhistamine, mimicked the effect of histamine on progesterone synthesis. In the presence of the phosphodiesterase inhibitor, IBMX, histamine increased tissue levels of cAMP. These results suggest that histamine stimulates progesterone synthesis via the H2-receptor with cAMP acting as secondary intracellular messenger.     

Forskolin inhibition of glucose metabolism in rat adipocytes independent of adenosine 3',5'-monophosphate accumulation and lipolysis

Addition of forskolin (1 microM) to rat adipocytes incubated with insulin and 0.2 mM glucose inhibited glucose metabolism in the absence of any significant stimulation of either cAMP accumulation or lipolysis. The action of forskolin was in contrast to that of isoproterenol, since forskolin inhibited [1-14C]glucose conversion to total lipid, whereas isoproterenol had the opposite effect. Elevation of the medium glucose concentration to 10 mM reversed the inhibitory effects of forskolin on glucose metabolism. The ability of forskolin to inhibit glucose metabolism was observed whether glucose was labeled in the 1 or the 6 position. The results of the present study suggest that forskolin affects glucose metabolism independently of adenylate cyclase activation.     

Measurement of plasma adenosine 3',5'-monophosphate.

Currently used methods for plasma cAMP measurements are either tedious (chromatographic preparation of sample) or potentially inaccurate (direct assay of plasma samples). A rapid, simple, and accurate competitive binding assay for plasma cAMP, which does not require chromatographic preparation of the sample, has been developed. This procedure prevents destruction of plasma cAMP by utilizing both theophylline and EDTA in the collection of the blood sample. Human plasma contains variable amounts of cAMP-binding activity which interfere with the measurement of cAMP by the standard competitive binding assay. Our assay procedure removes this binding activity by precipitation of plasma proteins with perchloric acid. The normal fasting value (+/- SD) of plasma cAMP using this technique is 17.6 +/- 4.3 pmol/ml, which is identical to values obtained by methods utilizing chromatographic purification of samples (18.3 +/- 3.0). The fasting plasma cAMP of patients with hyperparathyroidism is normal (16.2 +/- 3.4), but patients with maturity-onset diabetes mellitus have fasting values significantly below normal (12.3 +/- 2.4).     

Effects of adenosine 3',5'-monophosphate, dibutyryl adenosine 3',5'-monophosphate, aminophylline, and imidazole on renal cellular calcium metabolism.

The effects of cAMP, dibutyryl cAMP (DBcAMP), aminophylline, and imidazole on total cell calcium, calcium transport, and distribution were studied in cultured kidney cells by kinetic analysis of 45Ca uptake and desaturation curves. Low concentrations of the cyclic nucleotides (10(-7) and 10(-5) M) increase the total cell calcium, all intracellular exchangeable pools, and calcium transport between all cellular compartments. Aminophylline (1 mM) has effects qualitatively similar to cAMP and DBcAMP, while imidazole has opposite effects. At concentrations of 15 and 40 mM, imidazole depresses the total cell calcium and the cellular exchangeable calcium. Compared to the effects of parathyroid hormone (PTH), the changes obtained with 10(-7) and 10(-5) M cAMP are relatively modest, but higher concentrations (10(-3) M) of both cAMP and DBcAMP produce stimulations as marked as with 15 ng/ml PTH. The most dramatic changes are seen in the mitochondrial calcium pool and in the mitochondrial calcium exchange, which increase between 20- and 40-fold. These experiments show that cAMP mimics the effect of PTH on kidney cells and support the theory that cAMP is the mediator of PTH action on renal cell calcium transport.     

Release of growth hormone from purified somatotrophs: role of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate.

Studies were carried out to simultaneously measure cAMP and cGMP accumulation and GH release from acutely dispersed purified somatotrophs obtained from rat adenohypophyses. cAMP accumulation was dramatically increased by both prostaglandin E2 (10(-6) M) and 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor, 0.5 mM) within 1 min of their addition, while there was a delay of 8--16 min before a significant increase in GH release was seen. SRIF (100, 10, or 1 ng/ml) completely blocked the stimulated release of GH. SRIF also consistently decreased the elevation of cAMP induced by the two secretagogues, but this decrease was small and not always significant. cGMP was unmeasurable (less than 0.02 fmol/1000 cells) in all of our experiments, while basal cAMP levels were about 1 fmol/1000 cells. We conclude that cAMP plays a role in the intracellular mechanisms governing GH release and that SRIF primarily acts subsequent to cAMP elevation, with a possible secondard or minor action on cAMP formation.     

Inhibition of adenosine 3',5'-monophosphate accumulation and parathyroid hormone release by sodium nitroprusside.

Sodium nitroprusside effected a significant reduction in intracellular cAMP accumulation and parathyroid hormone release in dispersed bovine parathyroid cells. The inhibition was apparent at 3 x 10-4 M and maximal at 10-2 M nitroprusside. The effect was rapid and reversible and could be demonstrated in both the presence and absence of stimulating agonists [i.e. (-)isoproterenol, dopamine, and cholera toxin]. The inhibition was additive with that previously described for alpha-adrenergic agonists and prostaglandin F2 alpha and was not affected by phentolamine, suggesting that nitroprusside does not act through the inhibitory receptors previously described in this system. The nitroprusside effect on cAMP accumulation and parathyroid hormone release was present at virtually all concentrations of extracellular calcium tested; 2mM EGTA failed to prevent the inhibition. While extracellular calcium may play some role in this inhibition, it is not required for demonstration of the effect.     

Spontaneous diabetic BB rat: studies of cyclic adenosine 3',5'-monophosphate phosphodiesterase and calmodulin

Uncontrolled diabetes in man is associated with increased plasma and tissue levels of cAMP and decreased cAMP phosphodiesterase (PDE) activity. Spontaneously diabetic BB rats (SDR) were used in these experiments. Specific tissues (i.e. liver and epididymal fat) were studied without therapeutic insulin. Another group of normal animals were rendered diabetic by streptozotocin (STZ) and killed without benefit of insulin therapy. Calmodulin (CM), a small molecular weight protein essential for activation of specific cAMP PDE was assayed. STZ diabetes is associated with a decrease (58%) in CM biological activity and in immunoreactive CM in fat (69%) and liver (13%) tissues. Similarly, SDR rats and the nondiabetic genetic controls (NDR) demonstrate decreased CM bioactivity in fat (76% and 56%, respectively) and decreased CM immunoreactivity in liver (68% and 74%, respectively) compared to normal control rats. In addition, maximum velocity (Vmax) of the low Michaelis-Menten constant (Km) cAMP PDE is decreased in SDR animals, as compared to controls in both fat (42%) and liver (39%) tissues. Similar data are presented for NDR animals. STZ diabetes is also associated with a reduction in Vmax of the low Km cAMP PDE in both liver (70%) and fat (70%) tissues. These changes found in the NDR animals suggests that the diabetic defect may be under dual regulation: genetic and environmental.     

Somatomedin-C as an amplifier of follicle-stimulating hormone action: enhanced accumulation of adenosine 3',5'-monophosphate

Somatomedin-C (Sm-C) has recently been found to amplify the FSH-mediated acquisition of granulosa cell progestin biosynthetic capacity, aromatase activity, and LH receptors, an effect distinct from its established replicative property. To further characterize the cellular mechanism(s) underlying the synergistic interaction of Sm-C with FSH, we have set out to evaluate the intermediary role of cAMP in this regard. Isolated granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were cultured for up to 3 days under serum-free conditions. The basal extracellular accumulation of cAMP remained unchanged in response to treatment with highly purified Sm-C (50 ng/ml). However, concurrent treatment with increasing concentrations (0.3-50 ng/ml) of Sm-C, produced dose- and time-dependent increments in the FSH-stimulated accumulation of cAMP, with an apparent median effective dose (ED50; mean +/- SE) of 5.1 +/- 0.6 ng/ml, a maximal response 8.8-fold greater than that induced by FSH alone, and a minimal time requirement of 1-2 days. Given increasing concentrations of FSH, treatment with a constant concentration (50 ng/ml) of Sm-C resulted in 1.7-, 5.8-, and 4.3-fold increases in cAMP accumulation for 10, 30, and 100 ng/ml FSH, respectively. The ability of Sm-C to augment FSH-stimulated cAMP accumulation was evident and, in fact, enhanced by ZK62711 (Rolipram; 3 X 10(-6) M)-induced blockade of cAMP-phosphodiesterase activity. Decreasing dilutions (1:64,000 to 1:1,000) of a monoclonal antibody raised against Sm-C (sm 1.2) produced progressive and complete immunoneutralization of the synergistic interaction of Sm-C with FSH, suggesting specificity of action. Taken together, these findings suggest that Sm-C, acting at nanomolar concentrations compatible with its granulosa cell receptor binding affinity (0.6-2.0 nM), is capable of amplifying FSH-stimulated cAMP accumulation in a time- and dose-dependent manner. These observations suggest that the synergistic action of Sm-C is exerted, at least in part, at a site(s) proximal to cAMP generation.     

Effects of ouabain on insulin release, adenosine 3',5'-monophosphate and guanine 3',5'-monophosphate in pancreatic islets.

Isolated pancreatic islets of noninbred ob/ob mice were used to test the hypothesis that adenylate cyclase responds to changes of the transmembrane milieu or electric field in intact beta-cells. In the presence of a phosphodiesterase inhibitor, ouabainstimulated both the release of insulin and the islet content of cAMP. Ouabain had no noticeable effect on the islet content of cGMP. These results support the hypothesis at test. However, because ouabain also had some stimulatory effect on cAMP in islet homogenates, a direct action of ouabain on adenylate cyclase cannot be ruled out.