Cyclic AMP potentiates receptor-stimulated phosphoinositide hydrolysis in human neuroepithelioma cells.

A stimulatory role for cAMP in the regulation of receptor-activated phosphoinositide hydrolysis has been examined in human SK-N-MCIXC and SK-N-MCIIE neuroepithelioma cells. The addition of optimal concentrations of oxotremorine-M, norepinephrine, endothelin-1, and ATP enhanced the release of inositol phosphates by 2-9-fold after activation of muscarinic, alpha 1-adrenergic, endothelin, and P2 nucleotide receptors, respectively. All combinations of these agonists elicited a release of inositol phosphates that was at least additive. However, the combined presence of oxotremorine-M and norepinephrine resulted in a phosphoinositide hydrolysis that was 30% greater than additive. This potentiation of inositol lipid hydrolysis resulted from an increased activity of the muscarinic receptor after the addition or norepinephrine and persisted after alpha 1-adrenergic receptor blockade. The enhancement of muscarinic receptor-stimulated inositol phosphate release could be quantitatively mimicked by inclusion of the beta-adrenergic agonist isoproterenol (EC50 approximately 0.1 microM), but not by alpha 1- or alpha 2-adrenergic agonists. Potentiation of oxotremorine-M-stimulated inositol lipid hydrolysis observed in the presence of either norepinephrine or isoproterenol was reduced in the absence of added Ca2+. Addition of either norepinephrine or isoproterenol to SK-N-MCIXC cells also resulted in a 16-fold increase in cAMP concentration. Although the cell-permeant 8-chloro-4-phenylthio-cAMP had a small inhibitory effect on basal inositol phosphate release, its inclusion resulted in a 19-31% enhancement of muscarinic, endothelin, ATP, and alpha 1-adrenergic receptor-stimulated phosphoinositide hydrolysis. We conclude 1) that, in SK-N-MCIXC cells, the addition of beta-adrenergic agonists selectively enhances muscarinic receptor-stimulated phosphoinositide hydrolysis through a cAMP-dependent process and 2) that the ability of exogenously added cAMP to enhance the activation of all four inositol lipid-linked receptors indicates that the effects of cAMP on inositol lipid hydrolysis are compartmentalized in these cells.     

Histamine-stimulated increases in intracellular calcium in the smooth muscle cell line, DDT1MF-2

Suspensions of undifferentiated cultured vas deferens smooth muscle cells (DDT1MF-2) were loaded with the calcium-sensitive fluorescent dye fura-2. Exposure to histamine elicited a rapid and maintained increase in intracellular free calcium ([Ca2+] i) with an EC50 of 1.3 +/- 0.7 x 10(-5) M. The initial rise is a consequence of calcium release from intracellular stores, whereas the maintained or plateau phase, which is dependent upon the presence of extracellular calcium, is associated with calcium influx. Experiments in nominally Ca(2+)-free buffer attenuated the initial rise in [Ca2+]i (i.e. peak height) and virtually abolished the plateau phase. Re-addition of 2 mM Ca2+ (during experiments performed in nominally Ca(2+)-free buffer) resulted in a return of the plateau phase. Pretreatment with the H1-antagonist mepyramine (100 nM; Kd = 1.0 +/- 0.4 nM, N = 3) completely blocks the response to histamine, whereas tiotidine (2 microM; H2-antagonist) had no effect. In conclusion, the present data would suggest that functional H1-receptors found in hamster vas deferens smooth muscle cells are typical of the "classical" H1-receptor in both its control of intracellular Ca2+ and sensitivity to antagonism by mepyramine.     

Adenosine A1-receptor stimulated increases in intracellular calcium in the smooth muscle cell line, DDT1MF-2.

1. The effect of a range of adenosine receptor agonists on intracellular free calcium concentration ([Ca2+]i) has been studied in the hamster vas deferens smooth muscle cell line DDT1MF-2. 2. Adenosine receptor agonists elicited a rapid and maintained increase in [Ca2+]i in fura-2 loaded DDT1MF-2 cells. The initial rise could be maintained in the absence of extracellular calcium, whereas the maintained or plateau phase was dependent upon the presence of extracellular calcium and appeared to be associated with calcium influx. The rank order of agonist potencies was N6-cyclopentyladenosine > 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine > adenosine. 3. The response to 2-chloroadenosine was antagonized by the antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, KD 0.14 nM) and 8-phenyltheophylline (KD 112 nM). 4. Pretreatment with the 5-lipoxygenase inhibitor AA861 (20 microM) produced only a small (14 +/- 2%) inhibition of the [Ca2+]i response elicted by N6-cyclopentyladenosine (300 nM), in nominally Ca(2+)-free buffer containing 0.1 mM EGTA. The cyclo-oxygenase inhibitor, indomethacin (2 microM) was without effect. 5. The Ca(2+)-influx associated with the plateau phase required the continued presence of agonist on the receptor. The antagonist DPCPX (100 nM) attenuated the rise in [Ca2+]i observed when extracellular Ca2+ was re-applied after the cells had been stimulated with N6-cyclopentyladenosine (CPA; 300 nM) in experiments initiated in nominally Ca(2+)-free buffer. 6. Pretreatment with pertussis toxin (200 ng ml-1 for 4 h) inhibited the CPA (100 nM) stimulated intracellular Ca2+ release and Ca2+ influx but was without effect on the response to histamine (100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Bradykinin B2 receptor-mediated phosphoinositide hydrolysis in bovine cultured tracheal smooth muscle cells.

1. Bovine tracheal smooth muscle cells were established in culture to study agonist-induced phosphoinositide (PI) hydrolysis in this tissue. 2. Bradykinin (0.1 nM-10 microM) evoked a concentration-dependent increase (log EC50 (M) = -9.4 +/- 0.2; n = 8) in the accumulation of total [3H]-inositol phosphates in cultured tracheal smooth muscle cells whereas the selective B1 receptor agonist des-Arg9-bradykinin (10 microM) was significantly less effective (16% of bradykinin maximal response; relative potency = 0.2 with respect to bradykinin = 100). 3. The bradykinin-induced increase in PI hydrolysis was unaffected by the B1 receptor antagonist des-Arg9[Leu8]-bradykinin (1 nM-1 microM) but showed marked attenuation in the presence of the B2 receptor antagonists D-Arg,[Hyp3,D-Phe7]-bradykinin (10 nM-10 microM) or D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin (10 nM-10 microM). The estimated KB values obtained for these two compounds, assuming competitive antagonism, were 40 +/- 14 nM and 8.6 +/- 2.8 nM for D-Arg,[Hyp3,D-Phe7]-bradykinin and D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin respectively. 4. We conclude that bradykinin B2 receptors are expressed in cultured bovine tracheal smooth muscle cells and are coupled to PI hydrolysis mechanisms.     

Cyclic AMP-mediated regulation of vascular smooth muscle cell cyclic AMP phosphodiesterase activity

1. Rat cultured aortic vascular smooth muscle cells (VSMC) express both cyclic GMP-inhibited cyclic AMP phosphodiesterase (PDE3) and Ro 20-1724-inhibited cyclic AMP phosphodiesterase (PDE4) activities. By utilizing either cilostamide, a PDE3-selective inhibitor, or Ro 20-1724, a PDE4-selective inhibitor, PDE3 and PDE4 activities were shown to account for 15% and 55% of total VSMC cyclic AMP phosphodiesterase (PDE) activity.
2. Treatment of VSMC with either forskolin or 8-bromo-cyclic AMP caused significant concentration- and time-dependent increases in total cellular cyclic AMP PDE activity. Using cilostamide or Ro 20-1724, we demonstrated that both PDE3 and PDE4 activities were increased following forskolin or 8-bromo-cyclic AMP treatment, with a relatively larger effect observed on PDE3 activity. The increase in cyclic AMP PDE activity induced by forskolin or 8-bromo-cyclic AMP was inhibited by actinomycin D or cycloheximide, demonstrating that new mRNA synthesis and protein synthesis were required. An analogue of forskolin which does not activate adenylyl cyclase (1,9-dideoxyforskolin) or an analogue of cyclic GMP (8-bromo-cyclic GMP) did not affect total cyclic AMP PDE activity.
3. Incubation of VSMC with 8-bromo-cyclic AMP for 16 h caused a marked rightward shift in the concentration-response curves for both isoprenaline- and forskolin-mediated activation of adenylyl cyclase. A role for up-regulated cyclic AMP PDE activity in this reduced potency is supported by our observation that cyclic AMP PDE inhibitors (IBMX, cilostamide or Ro 20-1724) partially normalized the effects of isoprenaline or forskolin in treated cells to those in untreated cells.
4. We conclude that VSMC cyclic AMP PDE activity is increased following long-term elevation of cyclic AMP and that increases in PDE3 and PDE4 activities account for more than 70% of this effect. Furthermore, we conclude that increases in cyclic AMP PDE activity contribute to the reduced potency of isoprenaline or forskolin in treated VSMC. These results have implications for long-term use of cyclic AMP PDE inhibitors as therapeutic agents.

     

5-Hydroxytryptamine receptor-mediated phosphoinositide hydrolysis in canine cultured tracheal smooth muscle cells.

1. 5-Hydroxytryptamine (5-HT) has been shown to induce contraction of tracheal smooth muscle. However, the mechanisms of action of 5-HT are not known. We therefore investigated the effects of 5-HT on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and its regulation in canine cultured tracheal smooth muscle cells (TSMCs) labelled with [3H]-inositol. 5-HT-induced inositol phosphates (IPs) accumulation was time- and dose-dependent with a half-maximal response (EC50) and a maximal response at 0.38 +/- 0.05 and 10 microM, respectively. 2. Ketanserin and mianserin (10 and 100 nM), 5-HT2 receptor antagonists, were equipotent in blocking the 5-HT-induced IPs accumulation with pKB values of 8.46 and 8.21, respectively. In contrast, the dose-response curves of 5-HT-induced IPs accumulation were not shifted until the concentrations of NAN-190 and metoclopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased up to 10 microM. 3. Pretreatment of TSMCs with pertussis toxin or cholera toxin did not inhibit the 5-HT-induced IPs accumulation, but partially inhibited the AlF(4-)-induced IPs response. 4. Stimulation of IPs accumulation by 5-HT required the presence of external Ca2+ and was blocked by EGTA. The addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs directly stimulated IPs accumulation. A further Ca(2+)-dependent increase in IPs accumulation was obtained by inclusion of either guanosine 5'-O-(3-thiotriphoshate) (GTP gamma S) or 5-HT. The combination of GTP gamma S and 5-HT elicited an additive effect on IPs accumulation. 5. Treatment with phorbol 12-myristate 13-acetate (PMA, 1 microM, 30 min) abolished the 5-HT-induced IPs accumulation. The concentrations of PMA that gave a half-maximal and maximal inhibition of 5-HT-induced IPs accumulation were 2.2 +/- 0.4 nM and 1 microM, n = 3, respectively. The protein kinase C (PKC) activator, 4 alpha-phorbol 12,13-didecanoate, at 1 microM, did not influence this response. The inhibitory effect of PMA was reversed by staurosporine, a PKC inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 6. The site of this inhibition was further investigated by examining the effect of PMA on AlF(4-)-induced IPs accumulation in canine TSMCs. AlF(4-)-stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the effect of PMA is distal to the 5-HT receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serotonin-2 receptors coupled to phosphoinositide hydrolysis in a clonal cell line

A permanent line of cells has been established from the transplantable rat pituitary tumor 7315a. P11 cells have been cloned repeatedly, and after more than 60 passages their growth and characteristics are stable. Results of radioligand binding studies with 125I-lysergic acid diethylamide (125I-LSD) indicate that P11 cells express serotonin-2 (5-HT2) receptors. Analysis of the binding of 125I-LSD to membranes prepared from P11 cells revealed the presence of a single class of high affinity sites (Kd = 1.6 nM; Bmax = 211 fmol/mg of protein). The pharmacological profile of the inhibition of the binding of 125I-LSD by a panel of drugs was consistent with the expected profile of these drugs at 5-HT2 receptors. The affinity of the site for serotonin was in the low micromolar range and was decreased by GTP. Phosphoinositide hydrolysis in P11 cells, measured in the presence of lithium, was stimulated by serotonin. Increasing concentrations of the 5-HT2-selective antagonist ketanserin blocked phosphoinositide hydrolysis stimulated by serotonin, and Schild analysis was consistent with a simple competitive interaction. The Ki for ketanserin derived from Schild analysis was comparable to the Ki for ketanserin at the binding site for 125I-LSD. These results suggest that stimulation of phosphoinositide hydrolysis in P11 cells by serotonin is mediated by 5-HT2 receptors. Pretreatment of P11 cells with pertussis toxin caused ADP-ribosylation of Gi and Go, but did not affect the ability of serotonin to stimulate phosphoinositide hydrolysis. Therefore, the guaninine nucleotide-binding protein involved in the coupling of 5-HT2 receptors to phospholipase C in P11 cells is unlikely to be either Gi or Go. P11 cells expressing 5-HT2 receptors coupled to phosphoinositide hydrolysis will be a useful model system for future studies of the regulation and function of 5-HT2 receptors on cultured cells.     

Cyclic AMP and Ca-binding in microsomal fractions isolated from rabbit colon smooth muscle.

From a homogenate of rabbit colon muscle two ATP dependent Ca-accumulating microsomal fractions were isolated by differential centrifugation on a sucrose density grandient at 35% and 35-45% sucrose. Adenylate cyclase and phosphodiesterase activities were found in the fractions. The Ca-accumulation and the ATPase activity of these fractions were stimulated by cyclic AMP (10(-5)M) at an ATP concentration of 0.35 mM ATP. In the presence of higher concentrations of ATP (5 mM) cyclic AMP had no effect on the Ca-binding. The higher concentration of ATP markedly increased the cyclic AMP formation in relation to the activity found at the lower concentration of ATP. Isoprenaline (2 X 10(-6)M) stimulated the Ca-accumulation in the 35-45% fraction and increased the hydrolysis of ATP. These effects were absent in the fraction isolated at 35% sucrose. In the former fraction isoprenaline also stimulated the adenylate cyclase activity at 0.35 mM but not at 5 mM ATP. Both the effect of isoprenaline on the Ca-binding and the adenylate cyclase activity were inhibited by the adrenergic beta-receptor blocking agent sotalol. In the 35-45% fraction papaverine (1 X 10(-3)M) stimulated the Ca-accumulation and inhibited the phosphodiesterase activity. It is suggested that cyclic AMP and agents which influence the cyclic AMP metabolism in the microsomes may have a regulatory role on the Ca-binding of the microsomes.     

P2-purinoceptor-mediated membrane currents in DDT1 MF-2 smooth muscle cells

The electrophysiological response evoked by ATP was investigated in the DDT1 MF-2 smooth muscle cell line using the microelectrode technique and the whole-cell patch clamp technique. Application of ATP (10(-3) M) to the bathing solution caused a small initial depolarization of the cell membrane, followed by hyperpolarization and slow depolarization. During voltage clamping (-50 mV) a triphasic response was recorded on stimulation with ATP (10(-4)-10(-3) M). A short-lasting inward current was followed by a transient outward current and a slowly decreasing inward current. This response was not affected by the receptor antagonists, propranolol (3 X 10(-6) M), phentolamine (3 X 10(-6) M), atropine (3 X 10(-6) M) or theophylline (10(-3) M). The ATP-induced currents were not modified by the voltage-dependent channel blocking agents, tetraethyl ammonium (3 X 10(-3) M), 3,4-diaminopyridine (10(-3) M), tetrodotoxin (3 X 10(-7) M) or diltiazem (10(-5) M). The fast inward current was not detectable at a low ATP concentration (10(-5) M). The outward current showed a reversal potential near -76 mV, which equals the potassium equilibrium potential. This current was abolished after neutralization of the potassium electrochemical gradient. The outward current was suppressed under calcium-free conditions and also in the presence of tolbutamide (10(-4) M) or glipizide (5 X 10(-6) M). Guanosine triphosphate (5 X 10(-6) M) promoted the outward current, while this current was inhibited in the presence of guanosine diphosphate (5 X 10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Homologous and heterologous desensitization of histamine H1- and ATP-receptors in the smooth muscle cell line, DDT1MF-2: the role of protein kinase C.

1. The possible role of protein kinase C (PKC) in homologous and heterologous desensitization of histamine H1- and ATP-receptors has been studied in monolayers of cultured vas deferens smooth muscle cells (DDT1MF-2). Cells were loaded with the calcium-sensitive fluorescent dye fura-2 and increases in intracellular free Ca2+ concentration ([Ca2+]i) monitored in response to histamine H1- or ATP-receptor activation. 2. Histamine and ATP stimulated the release of Ca2+ from intracellular Ca2+ stores and Ca2+ influx across the plasma membrane. Activation of PKC with the phorbol ester beta-phorbol-12,13 dibutyrate (PDBu; 1 microM) attenuated histamine (100 microM) and ATP (10 microM)-induced release of intracellular Ca2+ and Ca2+ influx. 3. The selective PKC inhibitor, Ro 31-8220 (10 microM), reversed the PDBu-induced attenuation of histamine (100 microM)-stimulated Ca2+ responses. 4. Histamine H1- and ATP-receptors are readily susceptible to homologous desensitization since short-term exposure to histamine or ATP (450 s) attenuated the Ca2+ responses elicited by a second application of the same agonist. Furthermore, H1-receptor activation-induced heterologous desensitization of ATP stimulated Ca2+ responses and vice versa. 5. Homologous and heterologous desensitization of histamine and ATP Ca2+ responses still occurred in the presence of the PKC inhibitor, Ro 31-8220 (10 microM). 6. These data suggest that PKC activation can attenuate histamine H1- and ATP-receptor mediated Ca2+ responses. However, based on our experimental data, PKC-independent mechanisms appear to be involved in the homologous and heterologous desensitization of histamine H1- and ATP-receptor mediated Ca2+ responses in DDT1MF-2 cells.     

Regulation of histamine H1 receptor-mediated phosphoinositide hydrolysis by histamine and phorbol esters in DDT1 MF-2 cells

The regulation of histamine-stimulated phosphoinositide turnover by histamine and phorbol esters was examined in intact DDT1 MF-2 cells grown in suspension culture. Histamine increased the incorporation of 32P into phosphatidylinositol (PI) in these cells, and this stimulation was inhibited by the H1 antagonist diphenhydramine but not by the H2 antagonist cimetidine. Pretreatment of cells with histamine or with phorbol 12-myristate 13-acetate (PMA) or other activators of protein kinase C induced a marked decrease in the subsequent stimulation by histamine. PMA, but not histamine, also decreased the ability of epinephrine to stimulate PI labelling through alpha 1-adrenoceptors. Thus, histamine appears to induce homologous desensitization of histamine H1 receptor-mediated PI turnover, whereas direct activation of protein kinase C in the absence of receptor occupancy by agonist induces nonspecific heterologous desensitization of both histamine H1- and alpha 1-adrenoceptor-mediated responses.     

Proteome analysis reveals a distinct molecular profile of cellular stress following incubation of DDT1-MF2 smooth muscle cells in the presence of a high concentration of hyperforin

The acylphloroglucinol derivative hyperforin is a major constituent of St. John's wort extracts ( Hypericum perforatum L.), which has been demonstrated to contribute to the antidepressant action of this herbal drug. In previous investigations we observed that hyperforin causes a rapid stimulation of intracellular calcium mobilization and enhances extracellular acidification in the hamster vas deferens smooth muscle cell line DDT (1)-MF2. To obtain further insight into its mode of action, we have now examined if these effects are accompanied by changes in protein expression. Cells were incubated with hyperforin for 15 min at a concentration of 1 microg/mL. Proteome analysis in cell lysates was accomplished by two-dimensional gel electrophoresis (2D-PAGE) and proteins were visualized by silver staining. Differences in the expression pattern between hyperforin- and vehicle-treated cells were displayed by computer-assisted differential display and identification of selected protein spots was performed by peptide mass fingerprinting after digestion with trypsin. Following incubation with hyperforin marked changes in the expression of several proteins were evident. A particularly strong change was observed for 6 proteins, which were identified as tubulin-beta, enolase 3, SYNCRIP, endoplasmin, elongation factor 2 and HSP84. As these proteins are known to be involved in cellular responses to stress by regulating energy metabolism as well as synthesis, intracellular transport and folding of proteins, our results suggest that the effects observed are not components of the normal pharmacological activity profile of hyperforin but are rather indicative of cellular stress promoting activity at higher concentrations.     

Characterization of adenosine A1 receptors in intact DDT1 MF-2 smooth muscle cells

Adenosine receptors in the smooth muscle cell line DDT1 MF-2 were studied by radioligand binding using the A1 receptor-selective antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine [( 3H]DPCPX) as the ligand. Binding characteristics were similar in intact cells and in membranes (KD value of approximately 1 nM). The maximum binding amounted to 183 fmol/10(6) intact cells or 344 fmol/mg of membranes. To characterize the receptor, competition experiments were performed by inhibiting [3H]DPCPX binding with several adenosine agonists and antagonists. Adenosine receptor antagonists appeared to bind to a single class of binding site, both in membranes and intact cells. The order of potency was DPCPX = CGS 15943A greater than 8-cyclopentyl-1,3-dimethylxanthine greater than 8-(p-sulfophenyl)-theophylline greater than 3-isobutyl-1-methylxanthine greater than theophylline. Competition curves with adenosine agonists in membranes were best described by a two-site rather than a one-site model. At equilibrium in intact cells, only a single site was detected at both 4 degrees and 25 degrees. However, short term incubations (1-4 min) at 25 degrees showed biphasic binding curves in intact cells. The equilibrium KD values for intact cells were similar to the low affinity KD values in membranes (KL). The order of potency was N6-cyclopentyladenosine greater than or equal to (-)-(R)-N6-phenylisopropyladenosine[(R)-PIA] greater than or equal to N6-cyclohexyl adenosine greater than 5'-N-ethylcarboxamidoadenosine (NECA) greater than 2-chloroadenosine greater than adenosine (intact cells only) greater than 2-phenylaminoadenosine (CV 1808). Treatment of cells with pertussis toxin ADP-ribosylated GTP-binding proteins and eliminated the high affinity agonist binding in membranes but did not affect binding to intact cells. The addition of GTP (100 microM) also shifted the competition curves from bi- to monophasic curves in membranes. Adenosine receptor agonists inhibited the formation of cAMP induced by isoprenaline (IC50 for (R)-PIA, 0.4 nM). This inhibition could be prevented with adenosine receptor antagonists. Pretreatment with pertussis toxin also reversed these effects and actually revealed functional A2 receptors, as shown by the formation of cAMP induced by NECA. In conclusion, the equilibrium binding of A1 receptor agonists to intact smooth muscle cells is similar to the low affinity binding observed in membranes. In addition, it is suggested that agonists may transiently convert the A1 receptor from a "resting" low affinity state to a high affinity state coupled to a GTP-binding protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Endothelin-1-induced contractions of isolated pig detrusor and vesical arterial smooth muscle: calcium dependence and phosphoinositide hydrolysis.

1. In isolated pig detrusor and vesical arterial smooth muscle preparations, endothelin-1 (ET-1) caused concentration-dependent contractions. Nifedipine (10(-6) M) did not significantly affect the action of ET-1 in the vessels, but almost abolished its effect in the detrusor. Incubation for 30 min in Ca(2+)-free solution markedly reduced the ET-1-induced contractions in both detrusor and vesical arteries. 2. The protein kinase C inhibitor H-7 (3 x 10(-5) M), reduced the response to ET-1 in detrusor muscle as well as in vessels, and abolished the contractions evoked by ET-1 in Ca(2+)-free solution. 3. ET-1 caused an increase in the accumulation of inositol phosphates (IPs) in preparations prelabelled with myo-[3H]inositol. After exposure to ET-1 (10(-7) M) for 60 min, an approx. 4-fold increase in IPs levels were demonstrated, compared to untreated controls, in both detrusor and vessel preparations. Pretreatment with nifedipine (10(-6) M) did not reduce IPs formation. In contrast, no increase in IPs formation was demonstrated in Ca(2+)-free medium. 4. The increase in accumulation of IPs was slow in onset in both detrusor and vesical arteries, with no significant accumulation demonstrable during the first 30 min. Time-course studies of tension development for ET-1 revealed that maximum tension was reached before significant levels of IPs could be detected.     

Cyclic AMP dependent down regulation in the relaxation of smooth muscle cells of cat esophagitis

We investigated whether the signal mechanism for relaxation may be affected by inflammation of the cat esophagus. Acute esophagitis was induced by perfusion with 0.1N HCI at a rate of 1 mL/min for 45 min over three consecutive days. We then isolated esophageal smooth muscle cells by enzymatic digestion with collagenase. We pre-contracted the isolated smooth cells with acetylcholine (ACh) (10(-5) M) and compared the agonist-induced relaxation of pre-con tracted normal cells with those of esophagitic cells. Vasoactive intestinal polypeptide (VIP) caused a dose-dependent relaxation in normal cells, and this curve was down shifted in esophagitic cells. Sodium nitroprusside (SNP) or SIN-1 (NO donor) produced dose-dependent relaxation in normal cells, which was not affected by esophagitis. 8-Br-cGMP (a cGMP ana log) also induced dose-dependent relaxation to a similar extent in both normal and esoph agitic cells. Forskolin (a cAMP activator) or db-cAMP (a cAMP analog) produced dose-dependent relaxation in normal cells, and this relaxation curve was down shifted in esoph agitic cells. Western blotting was used to determine what subtype of adenylyl cyclase was involved in the cAMP pathway. Western blot analysis of homogenates derived from esophageal smooth muscle using antibodies against adenylyl cyclase types II, III, IV and V/VI revealed the presence of type V and/or type VI only. This result suggests that relaxation via a cAMP-dependent pathway rather than a cGMP dependent-pathway is down regulated in cat acute esophagitis. This subsensitivity of the cAMP related pathway may be related to the activ ity of adenylyl cyclase V/VI.     

Dibutyryl cyclic AMP and forskolin inhibit phosphatidylinositol hydrolysis, Ca2+ influx and contraction in vascular smooth muscle.

Dibutyryl cyclic AMP and forskolin inhibited the contraction induced by norepinephrine (NE) more strongly than the high K(+)-induced contraction in isolated rat aorta. These inhibitors inhibited the 45Ca2+ influx stimulated by NE but not that by high K+, and they inhibited NE-induced inositol monophosphate accumulation. These results suggest that cAMP inhibits NE-induced contraction, at least partly, by inhibiting the alpha-adrenoceptor-mediated signal transduction and high K(+)-induced contraction by decreasing Ca2+ sensitivity but not Ca2+ influx.     

Bradykinin stimulation of phosphoinositide hydrolysis in guinea-pig ileum longitudinal muscle.

1. Bradykinin (BK)-induced contraction of ileal smooth muscle is assumed to be due to phosphoinositide hydrolysis but this has never been reported. We have investigated whether BK receptors are linked to this transduction mechanism in guinea-pig ileum longitudinal muscle and determined whether these receptors are equivalent to those labelled in [3H]-BK binding assays. 2. In membranes prepared from longitudinal muscle, [3H]-BK bound to a single class of sites with high affinity. Characterization of the binding with BK analogues indicated that the radioligand selectivity labelled a B2 type receptor. 3. BK significantly elevated tissue levels of [3H]-inositol phosphates in longitudinal muscle slices preincubated with [3H]-myo-inositol. The agonists potencies of BK, Lys-BK, Met-Lys-BK, Tyr5-BK and Tyr8-BK were in agreement with their relative potencies in the binding assay. The B1 receptor agonist des-Arg9-BK, did not stimulate inositol phosphate production. The response to BK was blocked by known B2 receptor antagonists but not by the B1 antagonist des-Arg9, Leu8-BK. 4. BK-induced phosphoinositide hydrolysis was unaffected by exposure of muscle slices to either atropine or indomethacin. 5. The results indicate that the B2 receptors linked to phosphoinositide turnover in ileal longitudinal muscle exhibit properties similar to those involved in contractile responses. Also, the receptor mediating the phosphoinositide response is likely to be that labelled in the [3H]-BK binding studies.     

Demonstration of both A1 and A2 adenosine receptors in DDT1 MF-2 smooth muscle cells

Adenosine receptors of the A1 and A2 subtypes were characterized in membranes from DDT1 MF-2 smooth muscle cells. These cells possess a high density of A1 adenosine receptors (Bmax = 0.8-0.9 pmol/mg of protein), as measured by both agonist and antagonist radioligands. Agonists compete for [125I]N6-[2-(4-amino-3-iodophenyl)ethyl]-adenosine (A1 receptor-selective radioligand) binding with the following potency series: (R)-phenylisopropyladenosine [(R)-PIA] greater than 5'-N-ethylcarboxamide adenosine (NECA) greater than (S)-PIA, indicative of their interaction with A1 adenosine receptors. Agonist competition for [3H]8-(4-[[[(2-aminoethyl)amino]carbonyl)methyl)oxy]phenyl)-1, 3-dipropylxanthine [( 3H]XAC) (an antagonist radioligand for the A1 adenosine receptor) was described by a two-state model of 1.3 nM (high affinity state, KK) and 370 nM (low affinity state, KL), with 70% of the receptors in the high affinity state (RH). Addition of guanosine 5'-[beta, alpha-imido]triphosphate (100 microM) shifted the (R)-PIA competition curves to the right to lower affinities. Photoaffinity labeling with the agonist photoprobe [125I]N6-[2-(4-amino-3-iodophenyl) ethyl]adenosine indicates that the A1 adenosine receptor binding subunit is a Mr 38,000 protein. Adenosine receptor agonists [(R)-PIA, NECA, and (S)-PIA] inhibited isoproterenol-stimulated adenylate cyclase activity in DDT1 MF-2 cell membranes with IC50 values of 62, 538, and 750 nM, respectively. Inhibition of adenylate cyclase by (R)-PIA was attenuated by the A1 receptor antagonist XAC and following inactivation of Gi with pertussis toxin (100 ng/ml). Using a recently developed A2 adenosine receptor agonist radioligand 2-[4-(2-[( 4-aminophenyl]methylcarbonyl)ethyl) phenyl]ethylamino-5'-N-ethylcarboxamido adenosine (125I-PAPA-APEC), we have demonstrated the presence of A2 adenosine receptors in this cell line. Saturation curves with 125I-PAPA-APEC indicated the Bmax and Kd values to be 0.21 pmol/mg of protein and 4.0 nM, respectively. In competition experiments, NECA was more potent at inhibiting 125I-PAPA-APEC binding than (R)-PIA, with their respective IC50 values being 5.6 and 351 nM. The photolabeled A2 adenosine receptor migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an Mr of 42,000. Finally, adenosine receptor agonists stimulated adenylate cyclase activity by approximately 2-3 fold with the following potency series: PAPA-APEC greater than or equal to NECA greater than (R)-PIA, indicative of their interaction at A2 receptors. These data represent the first demonstration of the presence of both A1 and A2 receptors in a single cell line, DDT1 MF-2 smooth muscle cells.     

Thrombin induces DNA synthesis and phosphoinositide hydrolysis in airway smooth muscle by activation of distinct receptors

Chronic airway inflammation induces numerous structural changes of the airways involving hypertrophy and hyperplasia of airway smooth muscle (ASM). Thrombin has been identified in the bronchoalveolar lavage fluid of asthmatic subjects and displays potent bronchoconstrictor and mitogenic activity towards ASM. This study has addressed which proteinase-activated receptors (PARs) and signalling pathways are involved in mediating distinct effects of thrombin. Using cultured bovine tracheal smooth muscle (BTSM) cells as a model system, thrombin stimulated a marked increase in [³H]inositol phosphate ([³H]InsPs) accumulation, which was fully mimicked by a selective PAR1 activating peptide. In contrast, PAR1, PAR2, PAR3 and PAR4 activating peptides were unable to replicate the ability of thrombin to stimulate DNA synthesis as assessed by [³H]thymidine incorporation. Further investigation demonstrated that the mitogenic effect of thrombin did not involve stimulation of PDGF secretion but did involve activation of PDGF or EGF receptors and a G_i/o-dependent activation of phosphoinositide 3-kinase. Thrombin, but not the PAR1, PAR2, PAR3 or PAR4 activating peptides was able to stimulate PtdIns(3,4,5)P₃ mass accumulation. PAR3 antisense oligonucleotides substantially inhibit thrombin-stimulated [³H]thymidine incorporation and PtdIns(3,4,5)P₃ generation but had no effect on thrombin-induced phosphoinositide hydrolysis. These data indicate that while PI hydrolysis and Ca²⁺ mobilisation induced by thrombin operates via PAR1-dependent activation of phospholipase C, phosphoinositide 3-kinase activation and DNA synthesis occurs via a distinct proteinase-activated receptor pathway, possibly involving PAR3.