Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase.

The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase involves a cyclic AMP-dependent lesion that is after the G-protein.     

Current recording from sensory cilia of olfactory receptor cells in situ. II. Role of mucosal Na+, K+, and Ca2+ ions

Action potential-driven current transients were recorded from sensory cilia and used to monitor the spike frequency generated by olfactory receptor neurons, which were maintained in their natural position in the sensory epithelium. Both basal and messenger-induced activities, as elicited with forskolin or cyclic nucleotides, were dependent on the presence of mucosal Na+. The spike rate decreased to approximately 20% when mucosal Na+ was lowered from 120 to 60 mM (replaced by N-methyl-D-glucamine+), without clear changes in amplitude and duration of the recorded action potential-driven transients. Mucosal Ca2+ and Mg2+ blocked spike discharge completely when increased from 1 to 10 mM in Ringer solution. Lowering mucosal Ca2+ below 1 mM increased the spike rate. These results can be explained by the presence of a cyclic nucleotide-dependent, Ca(2+)-sensitive cation conductance, which allows a depolarizing Na+ inward current to flow through the apical membrane of in situ receptor cells. A conductance with these properties, thought to provide the receptor current, was first described for isolated olfactory cells by Nakamura and Gold (1987. Nature (Lond.). 325:442-444). The forskolin-stimulated spike rate decreased when l-cis-diltiazem, a known blocker of the cyclic nucleotide-dependent receptor current, was added to the mucosal solution. Spike rate also decreased when the mucosal K+ concentration was lowered. Mucosal Ba2+ and 4-aminopyridine, presumably by means of cell depolarization, rapidly increased the spike rate. This suggests the presence of apical K+ channels that render the receptor cells sensitive to the K+ concentration of the olfactory mucus. With a slower time course, mucosal Ba2+ and 4-aminopyridine decreased the amplitude and caused rectification of the fast current transients (prolongation of action potentials). Abolishment of the apical Na+ current (by removal of mucosal Na+), as indicated by a strong decrease in spike rate, could be counteracted by adding 10 mM Ba2+ or 1 mM 4-aminopyridine to the mucosal solution, which re-established spiking. Similarly, blockage of the apical cation conductance with 10 mM Ca could be counteracted by adding 10 mM Ba2+ or by raising the mucosal K+ concentration. Thus mucosal concentrations of Na+, K+, and Ca2+ will jointly affect the sensitivity of odor detection.     

Insulin modifies the properties of glucose transporters in rat brown adipose tissue.

The influence of cyclic AMP on cartilage degradation was investigated by using phosphodiesterase inhibitors [theophylline and 3-isobutyl-1-methylxanthine (IBMX)], forskolin (which activates the catalytic subunit of adenylate cyclase) and cyclic AMP analogues (dibutyryl and 8-bromo). Breakdown was assessed by quantification of proteoglycans released into the media of 8-day bovine nasal-septum cartilage cultures. Theophylline (1-20 mM), IBMX (0.01-2 mM) and dibutyryl cyclic AMP (0.1-2 mM) had little or no influence on the rate of proteoglycan release from unstimulated (no-endotoxin) cartilages. A small but detectable increase in breakdown was observed with 8-bromo cyclic AMP (0.5-2 mM) and forskolin (50-75 micrograms/ml). To examine potential inhibitory influences of these agents, the cyclic AMP modulators were added to cultures simultaneously treated with Salmonella typhosa endotoxin (12-25 micrograms/ml), a potent stimulator of cartilage degradation. The 3-4-fold stimulation of breakdown by endotoxin was strikingly inhibited by all three classes of cyclic AMP regulators. Optimal inhibition was found at 10-20 mM-theophylline, 1-2 mM-IBMX, 50-75 micrograms of forskolin/ml, 2 mM-dibutyryl cyclic AMP and 2 mM-8-bromo cyclic AMP. Inhibition was shown to be reversible, indicating that cartilages were viable after treatment. Sepharose CL-2B chromatography of proteoglycan products released from treated cartilages showed that the endotoxin-stimulated shift to lower average Mr was significantly prevented by cyclic AMP analogues and phosphodiesterase inhibitors. Together, these results show that agents which increase cyclic AMP inhibit both quantitative and qualitative aspects of endotoxin-mediated cartilage degradation.     

Cyclic AMP-Elevating Agents Prevent Oligodendroglial Excitotoxicity

Abstract: Previously, we have demonstrated that cells of the oligodendroglial lineage express non-NMDA glutamate receptor genes and are damaged by kainate-induced Ca²⁺ influx via non-NMDA glutamate receptor channels, representing oligodendroglial excitotoxicity. We find in the present study that agents that elevate intracellular cyclic AMP prevent oligodendroglial excitotoxicity. After oligodendrocyte-like cells, differentiated from the CG-4 cell line established from rat oligodendrocyte type-2 astrocyte progenitor cells, were exposed to 2 mM kainate for 24 h, cell death was evaluated by measuring activity of lactate dehydrogenase released into the culture medium. Released lactate dehydrogenase increased about threefold when exposed to 2 mM kainate. Kainate-induced cell death was prevented by one of the following agents: adenylate cyclase activator (forskolin), cyclic AMP analogues (dibutyryl cyclic AMP and 8-bromo-cyclic AMP), and cyclic AMP phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine, pentoxifylline, propentofylline, and ibudilast). Simultaneous addition of both forskolin and phosphodiesterase inhibitors prevented the kainate-induced cell death in an additive manner. A remarkable increase in Ca²⁺ influx (～5.5-fold) also was induced by kainate. The cyclic AMP-elevating agents caused a partial suppression of the kainate-induced increase in Ca²⁺ influx, leading to a less prominent response of intracellular Ca²⁺ concentration to kainate. The suppressing effect of forskolin on the kainate-induced Ca²⁺ influx was partially reversed by H-89, an inhibitor of cyclic AMP-dependent protein kinase. In contrast to this, okadaic acid, an inhibitor of protein phosphatases 1 and 2A, brought about a decrease in the kainate-induced Ca²⁺ influx. We therefore concluded that cyclic AMP-elevating agents prevented oligodendroglial excitotoxicity by cyclic AMP-dependent protein kinase-dependent protein phosphorylation, resulting in decreased kainate-induced Ca²⁺ influx.     

Effects of phosphodiesterase inhibitors and forskolin on cyclic GMP-activated channels in intact isolated cells of the chick pineal gland

Application of the phosphodiesterase inhibitors isobutylmethylxanthine (100 μM) caused activation of low-conductance cationic channels in intact acutely-isolated chick pineal cells. This effect required continued contact between the cytoplasmic face of the patch membrane and the cytoplasm. The biophysical properties of the channels were identical to those of cyclic GMP-activated channels described previously in excised patches. Application of 40 μM forskolin did not cause activation of the low-conductance channels. A second population of spontaneously active large-conductance cationic channels was observed in some patches. Gating of large-conductance channels was not affected by phosphodiesterase inhibitors of forskolin. These results support the theory that phototransduction cascades similar to those of the vertebrate retina are also present in chick pineal cells.     

Role of GTP-binding proteins in the regulation of mammalian cardiac chloride conductance

Beta-Adrenoceptor agonists activate a time- and voltage-independent Cl- conductance in mammalian cardiac myocytes. To characterize the cellular signaling pathways underlying its regulation, wide-tipped pipettes fitted with a pipette perfusion device were used to record whole-cell current and to introduce nucleotides to the interior of guinea pig ventricular myocytes. Replacement of pipette GTP with GDP beta S prevented activation of the Cl- conductance by Iso, suggesting a requirement for G protein turnover. With GTP in the pipette, the effect of Iso could be abolished by the beta-adrenoceptor antagonist propranolol, and mimicked by histamine or forskolin. These actions of Iso and forskolin are mediated exclusively via cAMP-dependent protein kinase (PKA), because (a) maximal activation of the Cl- conductance by forskolin or pipette cAMP occluded the effect of Iso, and (b) switching to pipette solution containing a synthetic peptide inhibitor (PKI) of PKA completely abolished the Cl- conductance activated by Iso and prevented the action of forskolin, but had no further effect. These results argue against basal activation of the Cl- conductance, and make it extremely unlikely that the stimulatory G protein, Gs, has any direct, phosphorylation-independent influence. The muscarinic receptor agonists acetylcholine (ACh) and carbachol diminished, in a reversible manner, Cl- conductance activated by Iso or forskolin, but not that elicited by cAMP. The muscarinic inhibition was abolished by replacing pipette GTP with GDP beta S, or by preincubating cells with pertussis toxin (PTX), and was therefore mediated by an inhibitory G protein, presumably Gi, influencing adenylyl cyclase activity. Nonhydrolyzable GTP analogues (GTP gamma S or GppNHp) applied via the pipette did not themselves activate Cl- conductance, but rendered Cl- current activation by brief exposures to Iso or histamine, but not to forskolin, irreversible. The Cl- conductance persistently activated by Iso was insensitive to propranolol or ACh, but could still be abolished by pipette application of PKI. The data indicate that stimulation of beta-adrenergic or histaminergic receptors in the presence of nonhydrolyzable GTP analogues causes persistent activation of Gs and uncouples it from the receptors. We conclude that autonomic regulation of cardiac Cl- conductance reflects accurately the underlying modulation of adenylyl cyclase activity and, hence, that this system is a suitable mammalian model for in situ studies of the interactions between adenylyl cyclase, Gs, Gi, and forskolin.     

cGMP-gated conductance in retinal bipolar cells is suppressed by the photoreceptor transmitter.

Transmitter release from photoreceptors is decreased by light, resulting in a conductance increase in depolarizing bipolar cells. Addition of exogenous cGMP through a patch pipette to depolarizing bipolar cells from slices of dark-adapted tiger salamander retina resulted in an enhancement of the light response. This enhancement was blocked by GTP-gamma-S and dipyridamole, an inhibitor of phosphodiesterase. GTP-gamma-S and dipyridamole also blocked responses to exogenously applied 2-amino-4-phosphonobutyrate (APB), the glutamate agonist selective for this receptor. These data support the hypothesis that the postsynaptic receptor is linked via a G protein to a phosphodiesterase. The binding of glutamate or APB to the receptor suppresses a cGMP-activated current by increasing the rate of cyclic nucleotide hydrolysis.     

Roles of GRK and PDE4 activities in the regulation of beta2 adrenergic signaling

An important focus in cell biology is understanding how different feedback mechanisms regulate G protein-coupled receptor systems. Toward this end we investigated the regulation of endogenous beta(2) adrenergic receptors (beta2ARs) and phosphodiesterases (PDEs) by measuring cAMP signals in single HEK-293 cells. We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels. This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 muM isoproterenol triggered transient increases in cAMP levels near the plasma membrane. Pretreatment of cells with 10 muM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 muM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 muM 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included. Taken together, these data indicate that either GRK-mediated desensitization of beta2ARs or PKA-mediated stimulation of PDE4 activity is sufficient to cause declines in cAMP signals. In addition, the data indicate that GRK-mediated desensitization is primarily responsible for a sustained suppression of beta2AR signaling. To better understand the interplay between receptor desensitization and PDE4 activity in controlling cAMP signals, we developed a mathematical model of this system. Simulations of cAMP signals using this model are consistent with the experimental data and demonstrate the importance of receptor levels, receptor desensitization, basal adenylyl cyclase activity, and regulation of PDE activity in controlling cAMP signals, and hence, on the overall sensitivity of the system.     

Vasoactive-intestinal-polypeptide-stimulated adenosine 3'',5''-cyclic monophosphate accumulation in GH3 pituitary tumour cells. Reversal of desensitization by forskolin.

The interaction between forskolin and vasoactive intestinal polypeptide (VIP) in the regulation of cyclic AMP production in GH3 pituitary tumour cells was investigated. Both forskolin (10nM-10 microns) and VIP (10pM-10nM) increased the cyclic AMP content of GH3 cells. Forskolin (50-100nM) was additive with VIP in stimulating cyclic AMP accumulation when low concentrations (less than 1 nM) of the peptide were used, but exhibited a synergistic interaction with higher VIP concentrations (10-100 nM). These effects on cyclic AMP accumulation were reflected in a leftward shift in the concentration-response curve for VIP-stimulated prolactin release from GH3 cells, a process known to be regulated by intracellular cyclic AMP concentrations. The synergy observed did not appear to be related to changes in cyclic nucleotide phosphodiesterase activity, since it was even more marked in the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor. Studies of the time-course of VIP-induced changes in GH3-cell cyclic AMP content revealed that, with high concentrations of VIP, production ceased within 2 min of addition. This attenuation of cyclic AMP synthesis was still observed in the presence of isobutylmethylxanthine, but was markedly inhibited by low concentrations of forskolin (50-100nM). The results suggest that VIP-induced cyclic AMP production rapidly becomes desensitized. This process, which is prevented by forskolin, may be related to changes in the ability of the guanine nucleotide regulatory protein to couple receptor occupancy to activation of adenylate cyclase.     

Forskolin stimulates adenylate cyclase and iodine metabolism in thyroid

Forskolin is a potent activator of the cyclic AMP-generating system in many tissues. In dog thyroid slices, the enhancement of cyclic AMP level was rapid, sustained in the presence of forskolin, but easily reversible after its withdrawal. Contrary to TSH, forskolin induced little apparent desensitization. Forskolin potentiated the effects of TSH, PGE1 and cholera toxin. However, the forskolin-induced cyclic AMP accumulation was still sensitive to inhibitors of dog thyroid adenylate cyclase such as iodide, norepinephrine and adenosine. As fluoride, but contrary to TSH and PGE1, forskolin stimulated adenylate cyclase in a medium where Mg2+ was replaced by Mn2+. This suggests that in thyroid, as in other tissues, forskolin acts beyond the receptor level but, as it potentiates hormone action and does not impair modulation by inhibitors, it may interact with the nucleotide-binding regulatory proteins. Forskolin mimicked the effect of TSH on iodide organification and secretion.     

Forskolin, phosphodiesterase inhibitors, and cyclic AMP analogs inhibit proliferation of cultured bovine aortic endothelial cells

The role of cyclic AMP on endothelial cell proliferation was investigated, since these cells can be exposed to high concentrations of physiological and pharmacological agents that alter cyclic AMP metabolism. Cloned bovine aortic endothelial cells were plated at 25,000 cells/35mm dish and grown for 5 days in the presence of phosphodiesterase (PDE) inhibitors, forskolin, or cyclic AMP analogs. The PDE inhibitors dipyridamole, ZK 62 711, isobutylmethylxanthine (IBMX) and theophylline inhibited cell growth in a concentration-dependent manner. Dipyridamole produced a 30% and a 50% inhibition at 5 microM and 12.5 microM, while higher concentrations were cytotoxic. At its therapeutic plasma concentration range (50-100 microM) theophylline inhibited cell proliferation by 15-25%, while IBMX and the highly specific cyclic AMP phosphodiesterase inhibitor, ZK 62 711 inhibited growth by 60-80% and 40-50%, respectively. Forskolin (5 microM) increased cyclic AMP levels and cyclic AMP-kinase activity ratios by 2.5-fold and 2-fold. In the absence of PDE inhibitors forskolin produced a 20% growth inhibition at 0.5 microM and a 60% inhibition at 10 microM. The forskolin dose-response curve was not altered by theophylline, but was shifted to the left by approximately 10-fold with dipyridamole and ZK 62 711 and 5-fold with IBMX. Forskolin (5 microM), by itself produced a 1.8-fold increase in cyclic AMP. In the presence of 5 microM theophylline, dipyridamole, IBMX, and ZK 62 711, cyclic AMP was increased by forskolin 2.0, 2.6, 3.5, and 6.6-fold, respectively. 8-Bromo cyclic AMP and dibutyryl cyclic AMP produced a 55% and 60% growth inhibition at 100 microM. The cyclic GMP analogs were less effective inhibitors of growth (15-30%). Our results demonstrate that cyclic AMP analogs and pharmacological agents that elevate intracellular cyclic AMP levels inhibit cell growth and suggest that cyclic AMP may be an important endogenous regulator of endothelial cell proliferation.     

Characterization of the MRP4- and MRP5-mediated transport of cyclic nucleotides from intact cells

Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3',5'-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20-600 microm, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A(1) and E(1), the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.     

Agents which increase cyclic AMP have diverse effects on low-density-lipoprotein-receptor function in human vascular smooth-muscle cells and skin fibroblasts.

Receptor-mediated binding and metabolism of low-density lipoproteins (LDL) in cultured human vascular smooth-muscle cells and skin fibroblasts are altered by increased cellular cyclic AMP concentrations. However, the LDL receptor does not respond to changes in cyclic AMP concentration in a simple manner. The activation of adenylate cyclase with forskolin, or the addition of membrane-permeant cyclic AMP analogues, initially decreases the expression of the LDL receptor, but is followed by a substantial increase in receptor expression after 24 h. This increase does not occur in the presence of inhibitors of RNA or protein synthesis, and is due to doubling of the Bmax. of the LDL receptor, without alteration of its affinity for LDL. By contrast, elevation of cyclic AMP concentration by inhibition of phosphodiesterases results in decreased receptor expression throughout the 24 h period. These two response patterns are reproducible phenomena, consistently observed in low-passaged cells derived from seven unrelated individuals.     

Desensitization of canonical transient receptor potential channel 5 by protein kinase C

The classic type of transient receptor potential channel (TRPC) is a molecular candidate for Ca(2+)-permeable cation channel in mammalian cells. TRPC5 is desensitized rapidly after activation by G protein-coupled receptor. Herein we report our investigation into the desensitization of mTRPC5 and localization of the molecular determinants of this desensitization using mutagenesis. TRPC5 was initially activated by muscarinic stimulation using 100 microM carbachol (CCh) and then decayed rapidly even in the presence of CCh (desensitization). Increased EGTA or omission of MgATP in the pipette solution slowed the rate of this desensitization. The protein kinase C (PKC) inhibitors, 1 microM chelerythrine, 100 nM GF109203X, or PKC peptide inhibitor (19-36), inhibited this desensitization of TRPC5 activated by 100 microM CCh. When TRPC5 current was activated by intracellular GTPgammaS, PKC inhibitors prevented TRPC5 desensitization and the mutation of TRPC5 T972 to alanine slowed the desensitization process dramatically. We conclude that the desensitization of TRPC5 occurs via PKC phosphorylation and suggest that threonine at residue 972 of mouse TRPC5 might be required for its phosphorylation by PKC.     

The time-course of cyclic AMP signaling is critical for leukemia U-937 cell differentiation

The regulation of the cAMP signaling is intimately involved in several cellular processes, including cell differentiation. Here, we provide strong evidence supporting that the time-course of cAMP signal is critical for leukemia U-937 cell differentiation. Three stimulating-cAMP agents were used to analyze the correlation between cAMP time-course and cell differentiation. All three agents denoted similar cAMP maximal responses in dose-response experiments. The kinetic of desensitization showed differential characteristics, while H2 receptor desensitized homologously without affecting PGE2 or forskolin effect, PGE2 response showed mixed desensitization characterized by a homologous initial phase followed by a heterologous phase. Regarding forskolin, long-term stimuli attenuated PGE2 and H2 agonist response without affecting adenylyl cyclase activity. In the absence of phosphodiesterase inhibitors, the three agents induced similar maximal cAMP levels after 5 min, but only that induced by the H2 agonist returned to basal levels. Consistent with this observation, H2 agonist was not able to induce U-937 cell maturation in contrast to PGE2 and forskolin, supporting the importance of time-course signaling in the determination of cell behavior.     

Forskolin and phosphodiesterase inhibitors release adenosine but inhibit morphine-evoked release of adenosine from spinal cord synaptosomes.

The effects of forskolin, Ro 20-1724, rolipram, and 3-isobutyl-1-methylxanthine (IBMX) on morphine-evoked release of adenosine from dorsal spinal cord synaptosomes were evaluated to examine the potential involvement of cyclic AMP in this action of morphine. Ro 20-1724 (1-100 microM), rolipram (1-100 microM), and forskolin (1-10 microM) increased basal release of adenosine, and at 1 microM inhibited morphine-evoked release of adenosine. Release of adenosine by Ro 20-1724, rolipram, and forskolin was reduced 42-77% in the presence of alpha,beta-methylene ADP and GMP, which inhibits ecto-5'-nucleotidase activity by 81%, indicating that this adenosine originated predominantly as nucleotide(s). Significant amounts of adenosine also were released from the ventral spinal cord by these agents. Ro 20-1724 and rolipram did not significantly alter the uptake of adenosine into synaptosomes. Although Ro 20-1724 and rolipram had only limited effects on the extrasynaptosomal conversion of added cyclic AMP to adenosine, IBMX, a phosphodiesterase inhibitor with a broader spectrum of inhibitory activity for phosphodiesterase isoenzymes, significantly inhibited the conversion of cyclic AMP to adenosine and resulted in recovery of a substantial amount of cyclic AMP. As with the non-xanthine phosphodiesterase inhibitors, IBMX increased basal release of adenosine and reduced morphine-evoked release of adenosine. Adenosine released by IBMX was reduced 70% in the presence of alpha,beta-methylene ADP and GMP, and release from the ventral spinal cord was 61% of that from the dorsal spinal cord. Collectively, these results indicate that forskolin and phosphodiesterase inhibitors release nucleotide(s) which is (are) converted extrasynaptosomally to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane currents elicited by porcine vasoactive intestinal peptide (VIP) in follicle-enclosed Xenopus oocytes

Membrane currents were recorded from voltage-clamped Xenopus laevis oocytes, surrounded by their enveloping follicular and epithelial cells. Porcine vasoactive intestinal peptide (VIP) generated a membrane current due to an increase in membrane conductance to K+. The VIP current was mimicked by the adenylate cyclase activator forskolin and was potentiated by phosphodiesterase inhibitors, suggesting that adenosine 3',5'-cyclic monophosphate (cyclic AMP) plays a role in mediating the response. Though resembling the follicle's responses to catecholamines and adenosine in ionic basis and apparent mechanism, the response to VIP was not blocked by catecholaminergic or purinergic antagonists, indicating the presence of a specific VIP receptor in the follicle. Among the VIP related peptides, PHM-27 generated similar but smaller K+ currents and porcine secretin and glucagon neither elicited a response nor blocked that to VIP. After treating follicles with collagenase to remove the epithelial and follicular cells the responses to VIP were either substantially reduced or abolished, suggesting that the VIP receptors and K+ channels are both located in the follicular cells.     

Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells.

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.     

Characterization and Regulation of β1-Adrenergic Receptors in a Human Neuroepithelioma Cell Line

Intact human neuroepithelioma SK-N-MC cells bound the beta-adrenergic antagonist (-)-[3H]-CGP 12177 with a KD of 0.13 nM and a Bmax of 17,500 sites/cell. When the cells were exposed to beta-adrenergic agonists, they accumulated cyclic AMP in the following order of potency: isoproterenol much greater than norepinephrine greater than epinephrine, which is indicative of a beta 1-subtype receptor. Membranes prepared from the cells bound (-)-3-[125I]iodocyanopindolol with a KD of 11.5 pM. Inhibition of agonist-stimulated cyclic AMP production and competition binding experiments indicated that the beta 1-selective antagonists CGP 20712A and ICI 89,406 were much more potent than the beta 2-selective antagonist ICI 118,551. Analysis of the displacement curves indicated that the cells contained only beta 1-adrenergic receptors. Northern blot analysis of SK-N-MC mRNA using cDNA probes for the beta 1- and beta 2-adrenergic receptors revealed the presence of a very strong beta 1-adrenergic receptor mRNA signal, while under the same conditions no beta 2-adrenergic receptor mRNA was observed. Thus, SK-N-MC cells appear to express a pure population of beta 1-adrenergic receptors. When the cells were exposed to isoproterenol, there was no observable desensitization during the first hour. After longer exposure, desensitization slowly occurred and the receptors slowly down-regulated to 50% of control levels by 24 h. Other agents that elevate cyclic AMP levels, such as forskolin, cholera toxin, and cyclic AMP analogues, caused no or little substantial receptor loss.     

Isoproterenol-induced desensitization of mucin release in isolated rat submandibular acini

Release of [14C]glucosamine-labelled mucins was studied in vitro using well-characterised preparations of rat submandibular acini. Mucin release was stimulated by forskolin, an activator of the catalytic subunit of adenylate cyclase, and 3-isobutyl-1-methylxanthine (IBMX), a cyclic nucleotide phosphodiesterase inhibitor. Both stimulated in a dose-dependent manner to the same maximum as that seen with isoproterenol. Neither forskolin nor IBMX added in the presence of isoproterenol increased secretion above the maximum in response to isoproterenol alone, suggesting a similar mechanism of action, mediated by cyclic AMP. Prior exposure of acini to isoproterenol (10 microM) for 45 min, followed by washout resulted in (a) persistent increase in basal secretion which was abolished by propranolol and (b) reduced stimulation of mucin secretion in response to either a second isoproterenol challenge, noradrenaline or forskolin. Thus, exposure of rat submandibular acini in vitro desensitizes the cells to subsequent stimulation. Although this mimics the decreased beta-adrenergic secretory responses seen in submandibular cells from cystic fibrosis patients, results suggest that the isoproterenol-induced desensitization is at the level of beta-receptor and adenylate cyclase, rather than distal to cyclic AMP.