Increase In Cyclic AMP Levels Research Articles

Histochemical, pharmacological, biochemical and chromatographic evidence that pituitary adenylyl cyclase activating peptide is involved in inhibitory neurotransmission in the taenia of the guinea-pig caecum

The possibility that pituitary adenylyl cyclase-activating peptide (PACAP) is an inhibitory neurotransmitter has been investigated in the taenia of the guinea-pig caecum. The action of PACAP on muscle contractility and its ability to alter levels of adenosine-3′ : 5′-cyclic monophosphate (cyclic AMP) and guanosine-3′ : 5′-cyclic monophosphate (cyclic GMP) were investigated. PACAP-1-27 was an effective agonist, giving relaxations comparable in magnitude to isoproterenol; its EC 50 was 3.4 × 10 −7 M. PACAP (10 −6 M) caused an almost two-fold increase in cyclic AMP levels; but the level of cyclic GMP was not affected. The relaxation caused by PACAP was slow in onset, with a latency of 5.8 ± 0.8 s and reached a maximum at 9.1 ± 1.1 s after onset. The relaxation was significantly reduced by apamin (10 −6 M) and suramin (10 −4 M) but was not reduced by tetrodotoxin (10 −7 M). Relaxation of the taenia coli caused by electrical stimulation of the inhibitory nerves was greatly reduced by apamin but only slightly reduced by suramin. PACAP-like immunoreactivity (-IR) was localised immunohistochemically in varicose nerve fibres within the taenia coli and in the underlying myenteric plexus and circular muscle. Approx. 50% of vasoactive intestinal peptide (VIP)-IR nerve fibres in the taenia also had immunoreactivity for PACAP; conversely, almost all PACAP-IR fibres were immunoreactive for VIP. PACAP-IR and substance P (SP)-IR were generally in separate fibres; only about 5% of SP-IR fibres were PACAP-IR. Radioimmunoassay revealed tissue concentrations of PACAP-1-27 and PACAP-1-38 of 1.0 ± 0.1 and 2.1 ± 0.3 (SEM) pmol/g wet weight of tissue, respectively. Material with PACAP-1-27 immunoreactivity co-eluted with authentic PACAP-1-27 on gel filtration chromatography, and PACAP-1-38 immunoreactivity also co-eluted with the authentic peptide. This study provides structural, chemical and pharmacological evidence that PACAP could be involved in inhibitory neurotransmission to the taenia coli of the guinea-pig caecum.

A reassessment of the modulatory role of cyclic AMP in catecholamine secretion by chromaffin cells

1. The role of adenosine 3':5'-cyclic monophosphate (cyclic AMP) in the regulation of catecholamine (CA) secretion in chromaffin cells remains equivocal from previous studies. 2. In the present study the effect of this cyclic nucleotide on basal CA secretion, as well as on intracellular calcium and membrane potential has been examined. 3. Forskolin and the permeable cyclic AMP analogue, 8-(4-chlorphenylthio)-adenosine-3'-5' monophosphate cyclic (pClpcAMP), increased basal CA secretion in a dose-dependent manner. The EC50s were 0.43 +/- 0.10 microM for forskolin and 39 +/- 9 microM for pClpcAMP. Other agonists with adenylate cyclase activity such as stimulants of adenosine receptors, beta-adrenoceptors, GABAB receptors and intestinal vasoactive peptide (VIP), also increased basal CA secretion in a highly significant manner. However, when they were added together with forskolin, CA secretion was not affected although an additive increase in cyclic AMP levels was produced. 4. Statistical analysis of the correlation between cyclic AMP levels and CA secretion evoked by these cyclic AMP increasing compounds showed that a significant direct correlation between both parameters existed only when low levels of cyclic AMP were produced by secretagogue stimulation. When the increase in intracellular cyclic AMP concentrations exceeded approximately 8 times the basal cyclic AMP levels the correlation was not significant. These results indicate a dual dose-dependent effect of cyclic AMP on basal CA secretion. 5. The stimulatory effect of low cyclic AMP on basal CA secretion was accompanied by an increase in membrane potential and in intracellular calcium concentrations ([Ca2+]j), the latter mainly being due to an increase in intracellular Ca2+ entry through L-type voltage-dependent Ca2" channels.6. The possible mechanisms involved in these cyclic AMP effects are discussed.

Ibudilast, an anti-allergic and cerebral vasodilator, modulates superoxide production in human neutrophils

We evaluated the effect of ibudilast on superoxide generation in human neutrophils by chemiluminescence development using luciferine analog, FCLA. By incubating neutrophils with ibudilast (2–200 μM) for more than 10 minutes, fMLP- or PMA-induced chemiluminescence was enhanced. However, the fMLP-induced chemiluminescence was suppressed by incubation for less than 10 minutes. This suppressed effect was missing with PMA-induced chemiluminescence. On the both fMLP- and PMA-induced chemiluminescence, the priming effect of ibudilast was further enhanced by the treatment with H-7, a protein kinase C inhibitor. In contrast, the priming effect of ibudilast on the fMLP-induced chemiluminescence was abolished by the treatment with ST-638, a selective inhibitor of tyrosine kinase. Ibudilast showed a transient stimulatory effect on cyclic AMP accumulation which continued for only a few minutes. Ibudilast showed no significant effect on phospholipase D dependent chemiluminescence, 1,4,5 triphosphate level, or protein kinase C activity. Ibudilast inhibited extracellular calcium influx. These results suggest that ibudilast acts on or through tyrosine kinase to achieved its priming effect on the fMLP-induced chemiluminescence. The early and transient increase in cyclic AMP level may explain the inhibitory effect of ibudilast on the fMLP-induced chemiluminescence after short time of incubation.

Dephosphorylation of nuclear non-histone proteins in submandibular glands of rats treated with isoproterenol.

Protein phosphatase that removed [32P]phosphate from non-histone proteins, i.e., phenol-soluble acidic proteins, more rapidly and strongly than from histone proteins was present in nuclei of rat submandibular glands, but was not associated with chromatin. Cyclic AMP (10(-4)-10(-2) mM) stimulated the dephosphorylation of non-histone proteins, but not that of histone proteins. After a single injection of isoproterenol (IPR), the dephosphorylation of non-histone proteins in rat submandibular gland nuclei increased within 15 min, reached a maximum in 30 min and returned to normal control levels within 4 h. The stimulation of dephosphorylation of non-histone proteins induced by IPR was not observed after prior treatment of the animals with dichloroisoproterenol. The dephosphorylation of histone proteins was not affected by the injection of IPR. Stimulation of beta-adrenoceptors with IPR in rat submandibular glands resulted in increase in cyclic AMP and decrease in RNA synthesis in the tissues in the first few hours after the injection. This decrease in RNA synthesis was temporary and was preceded by the increase in cyclic AMP level and in the dephosphorylation of non-histone acidic proteins in the tissues. These results suggest that protein phosphatase in nuclei plays an important part in the events controlling RNA synthesis by regulating the state of phosphorylation of non-histone acidic proteins. In addition, the phosphatase may be regulated by a function of the cytoplasmic membranes.

Suppression of lipopolysaccharide-stimulated release of tumor necrosis factor by adenosine: Evidence for A2 receptors on rat kupffer cells†

In liver grafts that will fail as a result of storage injury, reperfusion activates Kupffer cells. Overproduction of tumor necrosis factor by activated Kupffer cells may cause primary graft nonfunction, multiple organ failure and, eventually, death of graft recipients. Carolina rinse solution, adenosine, nisoldipine, pentoxifylline and prostaglandin E1 reduce graft failure from storage/reperfusion injury. To test the hypothesis that these agents act by suppressing cytokine release by activated Kupffer cells, we assessed the effect of each drug on tumor necrosis factor released from cultured rat Kupffer cells stimulated with lipopolysaccharide. Adenosine, nisoldipine and prostaglandin E1 each suppressed lipopolysaccharide-stimulated tumor necrosis factor release. The adenosine A2 receptor agonists. 5-n-ethylcarboxamidadenosine, 2-chloro-adenosine and R-phenylisopropyl adenosine also blocked tumor necrosis factor release in a potency suggestive of A2 receptor activity. Xanthine amine congener, a specific A1 receptor antagonist, failed to reverse the suppression by adenosine of tumor necrosis factor release, whereas CGS15943A, an A2 receptor antagonist, did reverse suppression by adenosine and 5-n-ethylcarboxamidadenosine. CGS15943A had no effect on suppression of lipopolysaccharide-stimulated tumor necrosis factor release by nisoldipine or prostaglandin E1. Dibutyryl-cyclicAMP also suppressed tumor necrosis factor release. Adenosine, 5-n-ethylcarboxamidadenosine, prostaglandin E1 and pentoxifylline increased cyclicAMP levels in cultured Kupffer cells, but nisoldipine did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Nicotine-induced antinociception in mice: Role of G-proteins and adenylate cyclase

The effects of pertussis toxin, forskolin, and cAMP analogues on the antinociceptive action of nicotine were examined to investigate the possible involvement of adenylate cyclase and G-proteins in nicotine's antinociceptive effect. Intrathecal injection of pertussis toxin (0.25 and 0.50 μg) in mice inhibited nicotine-induced antinociception in the tail-flick test. The effect of the toxin was dose and time dependent. Forskolin, a potent adenylate cyclase activator, and 8-(-4-chlorophenylthio) adenosine-3′:5′ monophosphate, cyclic (8-CPT-cAMP), a cAMP analogue, inhibited the antinociceptive effects of nicotine in a dose-dependent manner. EGTA reversal of 8-CPT-cAMP's inhibitory effects suggests that calcium may to be involved. These data implicate the possible involvement of a G-protein and a second messenger system (activation of a cAMP-dependent protein kinase and increase in cyclic AMP levels) in nicotine-induced analgesia in mice.

Parathyroid hormone/adenylate cyclase coupling in vascular smooth muscle cells.

Parathyroid hormone (PTH) has been implicated in hypertension, but PTH infusion results in vasodilation. PTH activates adenylate cyclase in vascular smooth muscle, but little is known about the factors that regulate PTH receptor/adenylate cyclase coupling in vascular cells. To characterize hormone-receptor signaling, we measured cyclic AMP levels in rat arterial smooth muscle cells in culture exposed to PTH (bovine 1-34). PTH yielded time- and concentration-dependent increases in cyclic AMP levels. Compared with isoproterenol, PTH was more potent, with a threshold at 2 x 10(-9) versus 5 x 10(-8) mol/L and half maximal responses at 10(-8) versus 2.4 x 10(-7) mol/L. PTH-induced increases in cyclic AMP were independent of extracellular calcium, cyclooxygenase metabolites, phospholipase C, and protein kinase C because PTH-induced increases in cyclic AMP were not prevented by variations in extracellular calcium, indomethacin, angiotensin II, vasopressin, and protein kinase C activators or inhibitors. PTH/adenylate cyclase coupling was G protein-dependent because increases in cyclic AMP were prevented by preincubation with cholera toxin but not with pertussis toxin. Prolonged exposure to PTH resulted in time- and concentration-dependent homologous desensitization of cyclic AMP responses. Desensitization occurred proximal to G protein/adenylate cyclase because after prolonged PTH, responses to forskolin and cholera toxin remained intact. Desensitization was independent of protein kinase A and receptor sequestration because cyclic AMP responses remained after prolonged exposure to forskolin and pretreatment with phenylarsine oxide, colchicine, and cytochalasin D. We conclude that in vascular smooth muscle cells, PTH is coupled to adenylate cyclase through a cholera toxin-sensitive G protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Ucb 11056, a new potential nootropic drug, amplifies induced cyclic AMP formation in rat brain tissue.

ucb 11056 [2-(4-morpholino-6-propyl-1,3,5-triazin-2-yl)aminoethanol] induced a significant (approximately 25%) increase in cyclic AMP levels in different brain areas following its intraperitoneal injection. This effect started as early as 2 min postinjection and lasted for 30 min, after which cyclic AMP levels returned to normal. In hippocampal slice preparations in vitro, ucb 11056 exerted a strong potentiation of cyclic AMP levels when it was combined with agents such as norepinephrine, forskolin, and isoproterenol. Only a slight effect on cyclic AMP levels was measured when ucb 11056 was incubated alone with hippocampal slices. The potentiating effect of ucb 11056 on norepinephrine-stimulated cyclic AMP formation was partially reduced when slices were pretreated with yohimbine and totally abolished when slices were treated with propranolol. These combined data indicate that (a) ucb 11056 rapidly increases cyclic AMP levels in the rat brain in vivo and (b) ucb 11056 potentiates stimulated cyclic AMP formation in vitro. The data also suggest that the central effect of ucb 11056 might be via the modulation of cyclic AMP generation, most probably mediated through adenylate cyclase activation mechanisms combined with a weak inhibitory activity on the cyclic nucleotide phosphodiesterase activity.

NMDA Receptor Activation Increases Cyclic AMP in Area CA1 of the Hippocampus via Calcium/Calmodulin Stimulation of Adenylyl Cyclase

We observed previously that activation of N-methyl-D-aspartate (NMDA) receptors in area CA1 of the hippocampus, through either NMDA application or long-term potentiation (LTP)-inducing high-frequency stimulation (HFS), results in an increase in cyclic AMP. In the present study, we performed experiments to determine the mechanism by which NMDA receptor activation causes this increase in cyclic AMP. As the NMDA receptor-mediated increase in cyclic AMP is dependent upon extracellular calcium, we hypothesized that NMDA receptors are coupled to adenylyl cyclase (AC) via calcium/calmodulin. In membranes prepared from area CA1, AC was stimulated by calcium in the presence of calmodulin, and the effect of calcium/calmodulin on AC in membranes was blocked by the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide (W-7) and trifluoperazine (TFP). In intact hippocampal slices, W-7 and TFP blocked the increase in cyclic AMP levels caused by both NMDA application and HFS of Schaffer collateral fibers. Exposure of hippocampal slices to elevated extracellular potassium to induce calcium influx also caused increased cyclic AMP levels; the increase in cyclic AMP caused by high potassium was also blocked by W-7 and TFP. These data support the hypothesis that NMDA receptor activation is positively coupled to AC via calcium/calmodulin and are consistent with a role for cyclic AMP metabolism in the induction of NMDA receptor-dependent LTP in area CA1 of the hippocampus.

Protein-kinase-A-dependent activator in transcription factor CREB reveals new role for CREM repressors.

Hormonally induced increases in cyclic AMP levels induce phosphorylation of the transcription factor CREB at a serine residue at position 133 by protein kinase A (ref. 1), enhancing its ability to activate transcription without affecting its intracellular location or DNA-binding activity. This effect is dependent on a 60-amino-acid region of CREB that contains Ser133 and is termed the kinase-inducible domain (KID)2, which also occurs in the CREB-related CREM-alpha and -beta proteins, although these are transcriptional repressors. Here we show that the KID domain confers a cAMP-inducible increase on the activity of the Q2 activation domain from CREB and the acidic activation domains from the yeast proteins GAL4 and GCN4. Remarkably, it retains this ability even when attached to a separate polypeptide bound to an adjacent site in the promoter. KID may therefore be the first of a new class of conditional activators that work through other promoter-bound factors to stimulate gene expression in response to hormonal stimuli.

Characteristics of serotonin-induced cyclic AMP elevation in the integument and anterior midgut of the blood-feeding bug, Rhodnius prolixus

Serotonin at 10 −6M concentration induces an average 12-fold increase in cyclic AMP levels in dorsal abdominal epidermis of fifth-instar larval Rhodnius prolixus. The epidermal serotonin receptor is inhibited by a number of mammalian 5-HT2 receptor antagonists but only α-methyl 5-HT among the 5-HT2 receptor agonists tested caused any increase in cyclic AMP levels. Isolated anterior midgut from adult male Rhodnius also shows dose-dependent cyclic AMP elevation in response to serotonin (threshold approx. 10 −7M, maximum 5 × 10 −6M) with receptor characteristics similar to the hypothesized 5-HT2-like serotonin receptor in larval epidermis. HPLC analysis showed a rapid rise in serotonin concentration in the haemolymph of adult male Rhodnius during feeding (peak 91 nM at 3 min after the onset of gorging) similar to, although less pronounced than, that previously reported for fifth-instar larvae. These findings are consistent with the hypothesis that serotonin released into the haemolymph and onto target tissues during feeding plays an important natural role in the integration of epidermal cell responses, fluid transport across the anterior midgut, diuresis, and other feeding-related functions in Rhodnius.

Reduction by arachidonic acid of prostaglandin i 2-induced cyclic amp formation : Involvement of prostaglandins E 2 and F 2α

Arachidonic acid reverses the increase in cyclic AMP levels of washed human platelets exposed to prostaglandin (PG)I 2, under conditions where the PGH 2 analogue U46619 is ineffective. This effect of arachidonic acid was inhibited by aspirin, a cyclooxygenase inhibitor, but not by the thromboxane (Tx) synthase inhibitor Ridogrel, which induces, by inhibiting the conversion of PGH 2 into TxA 2, an overproduction of PGE 2, PGD 2 and PCF 2α. Addition of PGE 2 or PGF 2α, which share a receptor with PGI 2, to washed human platelets also induced a decrease in cyclic AMP levels, but PGD 2, which interacts with a different receptor, had no effect. Thus neither PGD 2, PGG 2, PGH 2, TxA 2 nor TxB 2 formed from arachidonic acid via the cyclooxygenase pathway is involved in the decrease in cyclic AMP levels. These findings were confirmed using forskolin, a diterpene from the labdane family, which enhanced the formation of cyclic AMP synergistically with the PGs. Also, arachidonic acid, unlike U46619, is able to reverse the inhibition of platelet aggregation by PGI 2 after a lag phase of about 4 min. Our data indicate that arachidonic acid decreased cyclic AMP levels through its cyclooxygenase metabolites PGE 2 and PGF 2α probably interacting competitively with the receptor of PGI 2. In addition, intracellular cyclic AMP levels and the degree of aggregation of platelets by arachidonic acid seem to be inversely correlated.

Is vesicular exocytosis and membrane recycling a mechanism for secretin-induced choleresis in bile duct epithelium?

Intrahepatic bile duct epithelial cells, or cholangiocytes, contribute to bile secretion in response to hormones, including secretin. However, the mechanism by which secretin stimulates ductular bile flow is unknown. Since recent data in nonhepatic epithelia have suggested a role for exocytosis in fluid secretion, we tested the hypothesis that secretin stimulates exocytosis by isolated cholangiocytes. Cholangiocytes were isolated, from normal rat liver by a newly described method employing enzymatic digestion and mechanical disruption followed by immunomagnetic separation using specific monoclonal antibodies, and exocytosis was measured using a fluorescence unquenching assay employing acridine orange. Secretin caused a dose-dependent (10−12 - 10−7M) increase in acridine orange fluorescence by acridine orangeloaded cholangiocytes with a peak response at 10 min; the half-maximal concentration of secretin was 7 × 10−9M. The secretin effect was inhibited by preincubation of cholangiocytes with colchicine (30% inhibition, p<0.05) or trypsin (90% inhibition, p<0.001); no inhibition was seen with lumicolchicine and heatinactivated trypsin. Cholecystokinin, insulin, and somatostatin had no effect on fluorescence of acridine orange-loaded cholangiocytes; secretin had no effect on fluorescence of acridine orange-loaded hepatocytes or hepatic endothelial cells. Exposure of isolated cholangiocytes to secretin at doses that stimulated exocytosis caused a dose-dependent increase in cyclic AMP levels (218% maximal increase, p<0.05); moreover, an analogue of cyclic AMP stimulated exocytosis by cholangiocytes. Secretin had no effect on intracellular calcium concentration using Fura-2-loaded cholangiocytes assessed by digitized video microscopy. Our results demonstrate, for the first time, that secretin stimulates exocytosis by rat cholangiocytes. The effect is cell- and hormone-specific, dependent on intact microtubules, on a protein(s) on the external surface of cholangiocytes, and on changes in cellular levels of cyclic AMP. The results are consistent with the hypothesis that secretin-induced changes in bile flow may involve an exocytic process.

Evidence for the participation of protein kinase C and 3′,5′-cyclic AMP-dependent protein kinase in the stimulation of muscle cell proliferation by 1,25-dihydroxy-vitamin D 3

Treatment with 1,25-dihydroxy-vitamin D 3 (1,25(OH) 2D 3) (1–12 h, 10 −10 M) stimulates DNA synthesis in proliferating myoblasts, with an early response at 2–4 h of treatment followed by a maximal effect at 10 h. To investigate the mechanism involved in the mitogenic action of the hormone we studied the possible activation of intracellular messengers by 1,25(OH) 2D 3. The initial phase of stimulation of [ 3H]thymidine incorporation into DNA by the sterol was mimicked by the protein kinase C activator tetradecanoylphorbol acetate (TPA) in a manner which was dose dependent and specific as the inactive analog 4α-phorbol was without effect. Maximal responses to TPA (100 nM) were obtained at 4 h. Staurosporine, a protein kinase C inhibitor, blocked the effect of 1,25(OH) 2D 3 on myoblast proliferation at 4 h. In addition, a fast (1–5 min) elevation of diacylglycerol levels and membrane-associated protein kinase C activity was observed in response to 1,25(OH) 2D 3. The adenylate cyclase activator forskolin (20 μM) and dibutyryl-cAMP (50 μM) increased DNA synthesis reproducing the second 1,25(OH) 2D 3-dependent stimulatory phase at 10 h. Inhibitors of protein kinase A blocked the increase in muscle cell DNA synthesis induced by 1,25(OH) 2D 3 at 10 h. Significant increases in cyclic AMP levels were detected in myoblasts treated with the sterol for 1–10 h. The calcium channel antagonist nifedipine (5–10 μM) abolished both the effects of 4-h treatment with 1,25(OH) 2D 3 or TPA and 10-h treatment with 1,25(OH) 2D 3 or dibutyryl-cAMP. Similar to the calcium channel agonist Bay K8644, 1,25(OH) 2D 3 stimulated myoblast 45Ca uptake and its effects were blocked by nifedipine. Our results suggest that activation of calcium channels by phosphorylation via protein kinases C and A and is involved in the mitotic response of myoblasts to 1,25(OH) 2D 3.

P-571 - Increase of cyclic AMP levels by endothelin-1 and endothelin-3 in the isolated rat anterior, not posterior, pituitary gland.

P-571 - Increase of cyclic AMP levels by endothelin-1 and endothelin-3 in the isolated rat anterior, not posterior, pituitary gland.

Secretin stimulates exocytosis in isolated bile duct epithelial cells by a cyclic AMP-mediated mechanism.

Intrahepatic bile duct epithelial cells, or cholangiocytes, contribute to bile secretion in response to hormones, including secretin. However, the mechanism by which secretin stimulates ductular bile flow is unknown. Since recent data in nonhepatic epithelia have suggested a role for exocytosis in fluid secretion, we tested the hypothesis that secretin stimulates exocytosis by isolated cholangiocytes. Cholangiocytes were isolated from normal rat liver by a newly described method employing enzymatic digestion and mechanical disruption followed by immunomagnetic separation using specific monoclonal antibodies, and exocytosis was measured using a fluorescence unquenching assay employing acridine orange. Secretin caused a dose-dependent (10(-12)-10(-7) M) increase in acridine orange fluorescence by acridine orange-loaded cholangiocytes with a peak response at 10 min; the half-maximal concentration of secretin was 7 x 10(-9) M. The secretin effect was inhibited by preincubation of cholangiocytes with colchicine (30% inhibition, p less than 0.05) or trypsin (90% inhibition, p less than 0.001); no inhibition was seen with lumicolchicine and heat-inactivated trypsin. Cholecystokinin, insulin, and somatostatin had no effect on fluorescence of acridine orange-loaded cholangiocytes; secretin had no effect on fluorescence of acridine orange-loaded hepatocytes or hepatic endothelial cells. Exposure of isolated cholangiocytes to secretin at doses that stimulated exocytosis caused a dose-dependent increase in cyclic AMP levels (218% maximal increase, p less than 0.05); moreover, an analogue of cyclic AMP stimulated exocytosis by cholangiocytes. Secretin had no effect on intracellular calcium concentration using Fura-2-loaded cholangiocytes assessed by digitized video microscopy. Our results demonstrate, for the first time, that secretin stimulates exocytosis by rat cholangiocytes. The effect is cell- and hormone-specific, dependent on intact microtubules, on a protein(s) on the external surface of cholangiocytes, and on changes in cellular levels of cyclic AMP. The results are consistent with the hypothesis that secretin-induced changes in bile flow may involve an exocytic process.

Identification of a novel 5-HT N (nematoda) receptor from Ascaris suum muscle

The abilities of various setotonergic drugs to bind with the 5-HT receptor of Ascaris suum muscle and to affect cyclic AMP levels in muscle tissue were examined. 2. Ligands which selectively interact with either the 5-HT 1 or the 5-HT 2 receptor in mammalian systems interact with the 5-HT receptor from A. suum muscle and increase cyclic AMP levels. 3. No binding of 5-HT3 tigands to 5-HT receptors from A. suum muscle was observed. 4. The 5-HT receptor of A. suum muscle should be called the 5-HT N (for Nematoda) receptor because its pharmacological and biochemical behaviors were different from those of mammalian 5-HT receptors.

Modulation of 1,25-dihydroxyvitamin D3-dependent Ca2+ uptake in skeletal muscle by protein kinase C.

In vitro studies have shown that short exposure (1-10 min) of vitamin D-deficient chick soleus muscle to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] causes an acute stimulation of tissue 45Ca uptake through voltage-gated Ca2+ channels, with parallel increases in cyclic AMP levels, adenylate cyclase activity and membrane protein phosphorylation. We further investigated the involvement of protein kinases in the rapid effects of 1,25(OH)2D3 on skeletal muscle. The hormone was found to stimulate the protein kinase C (PKC) activity of muscle membranes. The PKC activator phorbol 12-myristate 13-acetate (PMA, 100 nM) was found to rapidly stimulate muscle 45Ca uptake, mimicking 1,25(OH)2D3. Increases of 68% and 46% were observed at 1 and 15 min of exposure to PMA respectively. The effects of PMA were dose-dependent (50-200 nM) and were specific, since the inactive analogue 4 alpha-phorbol was without effect. Analogously to the effects of the sterol, PMA-enhanced 45Ca uptake was abolished by the Ca2+ channel antagonists nifedipine (30 microM) and verapamil (50 microM). Staurosporine (10 nM), a PKC inhibitor, surprisingly potentiated 1,25(OH)2D3-dependent stimulation of 45Ca uptake. Exposure of skeletal muscle to PMA (100 nM) plus 1,25(OH)2D3 (1 nM) produced a less pronounced effect on 45Ca uptake than either agent alone. PMA also decreased muscle cyclic AMP levels. These results suggest a regulatory link between the two major transmembrane signalling systems in the mechanism of action of 1,25(OH)2D3 in skeletal muscle.

Possible Mechanisms of Action of the Antispasmodic Agent Tiropramide in the Isolated Detrusor from Rats

The effects of tiropramide hydrochloride on Ca2+-induced contraction, cytoplasmic free Ca2+ levels and tissue cyclic AMP concentrations were investigated to elucidate the mechanisms of its antispasmodic action in the isolated detrusor from rats. Tiropramide inhibited the Ca2+ (3 mM)-induced contractions of the isolated urinary bladder depolarized in a Ca2+-free medium, and the IC50 value was 3.3 × 10−6 M. When tiropramide was added during the sustained phase of the K+ (60 mM)-contracture, IC50 values of tiropramide for the contraction and the increased fluorescence were 1.9 × 10−5 M and 16.4 × 10−5M, respectively. On the other hand, the IC50 values for the K-induced contraction and fluorescence after pretreatment of the isolated urinary bladder with tiropramide were 2.1 × 10−5 M and 2.6 × 10−5 M, respectively. Tissue cyclic AMP levels at 1 min after addition of 10−5 M tiropramide were significantly increased. Papaverine, IBMX or forskolin potentiated the inhibitory effect of tiropramide on carbachol-induced contraction and its cyclic AMP-elevating effect. However, a good correlation between the degrees of potentiation of the inhibitory effect and the increase in cyclic AMP levels was not observed. The present results suggest that the smooth muscle relaxant activity of tiropramide in the isolated detrusor from rats may be intimately associated with predominant inhibition of Ca2+ influx and, to a lesser extent, an increase in intracellular cyclic AMP levels.

Possible mechanisms of action of the antispasmodic agent tiropramide in the isolated detrusor from rats.

The effects of tiropramide hydrochloride on Ca(2+)-induced contraction, cytoplasmic free Ca2+ levels and tissue cyclic AMP concentrations were investigated to elucidate the mechanisms of its antispasmodic action in the isolated detrusor from rats. Tiropramide inhibited the Ca2+ (3 mM)-induced contractions of the isolated urinary bladder depolarized in a Ca(2+)-free medium, and the IC50 value was 3.3 x 10(-6) M. When tiropramide was added during the sustained phase of the K+ (60 mM)-contracture, IC50 values of tiropramide for the contraction and the increased fluorescence were 1.9 x 10(-5) M and 16.4 x 10(-5) M, respectively. On the other hand, the IC50 values for the K(+)-induced contraction and fluorescence after pretreatment of the isolated urinary bladder with tiropramide were 2.1 x 10(-5) M and 2.6 x 10(-5) M, respectively. Tissue cyclic AMP levels at 1 min after addition of 10(-5) M tiropramide were significantly increased. Papaverine, IBMX or forskolin potentiated the inhibitory effect of tiropramide on carbachol-induced contraction and its cyclic AMP-elevating effect. However, a good correlation between the degrees of potentiation of the inhibitory effect and the increase in cyclic AMP levels was not observed. The present results suggest that the smooth muscle relaxant activity of tiropramide in the isolated detrusor from rats may be intimately associated with predominant inhibition of Ca2+ influx and, to a lesser extent, an increase in intracellular cyclic AMP levels.