Mitogen-activated protein kinase inhibitors suppress prostaglandin F(2alpha)-induced myosin-light chain phosphorylation and contraction in iris sphincter smooth muscle

The purpose of this study was to investigate the potential role of mitogen-activated protein (MAP) kinase in contraction by monitoring MAP kinase phosphorylation (activation) and contraction during agonist stimulation of cat iris sphincter smooth muscle. Changes in tension in response to prostaglandin F(2alpha), latanoprost, a prostaglandin F(2alpha) analog used as an anti-glaucoma drug, and carbachol were recorded isometrically, and MAP kinase activation was monitored by Western blot using a phosphospecific p42/p44 MAP kinase antibody. We found that treatment of the muscle with 2'-Amino-3'-methoxyflavone (PD98059) (10 microM), a specific inhibitor of MAP kinase kinase (MEK), inhibited significantly prostaglandin F(2alpha)- and latanoprost-induced phosphorylation and contraction, but had little effect on those evoked by carbachol. Prostaglandin F(2alpha) increased MAP kinase phosphorylation in a concentration-dependent manner with EC(50) value of 1.1 x 10(-8) M and increased contraction with EC(50) of 0.92 x 10(-9) M. The MAP kinase inhibitors PD98059, Apigenin and 1,4-Diamino-2,3-dicyano-1, 4bis(2-aminophenylthio)butadiene (UO126) inhibited prostaglandin F(2alpha)-induced contraction in a concentration-dependent manner with IC(50) values of 2.4, 3.0 and 4.8 microM, respectively. PD98059 had no effect on prostaglandin F(2alpha)- or on carbachol-stimulated inositol-1,4,5-trisphosphate (IP(3)) production. In contrast, the MAP kinase inhibitor inhibited prostaglandin F(2alpha)-induced myosin-light chain (MLC) phosphorylation, but had no effect on that of carbachol. N-[2-(N-(4-Chloro-cinnamyl)-N-methylaminomethyl)phenyl]-N-[2- hydroxyethyl]-4-methoxybenzenesulfonamide (KN-93) (10 microM), a Ca(2+)-calmodulin-dependent protein kinase inhibitor, and Wortmannin (10 microM), an MLC kinase inhibitor, inhibited significantly (by 80%) prostaglandin F(2alpha)- and carbachol-induced contraction. It can be concluded that in this smooth muscle p42/p44 MAP kinases are involved in the mechanism of prostaglandin F(2alpha)-, but not in that of carbachol, induced contraction. In addition, these data clearly indicate that the stimulation of the iris sphincter with prostaglandin F(2alpha) and carbachol activate two distinct pathways, the MAP kinase pathway and the Ca(2+) mobilization pathway.     

The effect of M & B 22948 on carbachol-induced inositol trisphosphate accumulation and contraction in iris sphincter smooth muscle.

The effect of a cyclic GMP phosphodiesterase inhibitor, M & B 22948, on carbachol-induced phosphatidylinositol 4,5-bis-phosphate (PIP2) breakdown and phosphatidic acid labeling, 1,4,5-inositol trisphosphate (IP3) accumulation and muscle contraction was studied in bovine iris sphincter smooth muscle. Addition of carbachol (10 microM) to 32P-labeled tissue resulted in increased labeling of phosphatidic acid and hydrolysis of PIP2. In myo[3H]inositol labeled tissue, carbachol caused rapid accumulation of IP3 which reached its maximum at about 2 min. Under identical experimental conditions, carbachol initiated a rapid increase in muscle contraction (phasic component) which was followed by a slightly lower contractile response (tonic component) that lasted for several minutes. Pretreatment of the iris sphincter with M & B 22948 did not alter carbachol-stimulated PIP2 breakdown and phosphatidic acid labeling, IP3 accumulation, or phasic component of the contractile response. However, the tonic component of the contractile response was increasingly attenuated by increasing concentrations of the drug. In conclusion, the data presented demonstrate a close correlation between carbachol-induced IP3 accumulation and muscle contraction, and that M & B 22948 does not inhibit carbachol-induced responses in the iris sphincter.     

Contribution of Ca2+ influx to carbachol-induced detrusor contraction is different in human urinary bladder compared to pig and mouse

Carbachol-induced detrusor contractions are mainly mediated via M3 receptor subtype and depend not only on Ca2+ release from the intracellular calcium stores but also on Ca2+ influx via L-type Ca2+ channels. The purpose of this study was to examine the different contributions of Ca2+ influx and Ca2+ release underlying muscarinic receptor-mediated contractions in human, porcine and murine urinary bladder. Detrusor contractions were measured in urothelium-denuded detrusor strips as responses to cumulatively increasing carbachol concentrations, release of intracellular Ca2+ was determined in Chinese hamster ovary cells stably transfected with human muscarinic M3 (hM3) receptors. In human tissue, 1 microM of the L-type Ca2+-channel blocker nifedipine reduced carbachol contractions to 74%, in pig to 18% and in mouse to 27% of pre-drug controls. 2-aminoethoxyphenyl borate (2-APB, 300 microM), which impairs inositol trisphosphate (IP3)-induced release of Ca2+, reduced carbachol responses in human detrusor to 60%, in pig to 35% and in mouse to 20%, whereas block of the Ca2+-induced Ca2+ release with ryanodine had no significant effect on carbachol contractions in all three species. Carbachol-induced release of intracellular Ca2+ in Chinese hamster ovary cells expressing muscarinic hM3 receptors was completely prevented by 100 microM 2-APB. The direct intracellular IP3 receptor antagonist xestospongin C (10 microM) reduced carbachol-stimulated intracellular Ca2+ to 41% of the control value. Blockade of ATP-dependent Ca2+ uptake into intracellular stores with thapsigargin was associated with a concentration-dependent increase of detrusor contraction, but limited on-top contractions with carbachol. In conclusion, carbachol-induced contractions in human, porcine and mouse detrusor depend differently on Ca2+ influx, since potency of nifedipine reducing muscarinic receptor-mediated detrusor contraction is lower in human bladder. On the other hand, slight species differences are also found when inhibiting IP3-induced Ca2+ release and Ca2+ reuptake into intracellular stores. Taken together, our data show considerable species differences between human, porcine and murine detrusor regarding the relative contributions of Ca2+ influx and maybe also carbachol-induced Ca2+ release that could be of relevance when using different animal models.     

The M3 muscarinic receptor links to three different transduction mechanisms with different efficacies in circular muscle of guinea-pig stomach.

1. In a previous publication, we showed that 10 microM carbachol induced contraction by activating three independent transduction mechanisms in circular smooth muscle of guinea-pig gastric fundus (Parekh & Brading, 1991). These were: inositol trisphosphate-mediated intracellular Ca2+ release, Ca2+ influx through a nifedipine-sensitive route and Ca2+ influx through a receptor operated nifedipine-insensitive pathway. The former two processes contribute to the phasic contraction and the latter two to the tonic contraction. In this paper, we have studied the effects of muscarinic receptor antagonists with known selectivity for different muscarinic receptor subtypes, on the contraction evoked by 10 microM carbachol. 2. Low concentrations of pirenzepine (M1 selective) had little effect on the contraction initiated by carbachol. Higher concentrations (greater than 1 microM) reduced only the phasic component. This concentration of pirenzepine greatly reduced the contraction evoked by 10 microM carbachol in Ca(2+)-free solution, indicating inhibition of intracellular Ca2+ release. 3. In the presence of 10 microM nifedipine, the tonic contraction evoked by 10 microM carbachol (reflecting the receptor-operated nifedipine-insensitive route) was abolished by 10 microM pirenzepine. In the absence of nifedipine pretreatment, however, 10 microM pirenzepine did not abolish the contraction to 10 microM carbachol. This contraction was subsequently abolished by nifedipine. 4. Only high concentrations (greater than 10 microM) of the M2-selective antagonist, gallamine, inhibited the contraction to 10 microM carbachol. Like pirenzepine, gallamine preferentially inhibited the phasic component of the contraction, indicating an effect on intracellular Ca2+ release. 5. The non-selective muscarinic receptor antagonist, atropine, abolished all components of the contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential coupling of muscarinic receptors to Ca2+ mobilization and cyclic AMP in SH-SY5Y and IMR 32 neuroblastoma cells.

Muscarinic receptor-linked Ca2+ mobilization and changes in cyclic AMP were studied in SH-SY5Y and IMR 32 human neuroblastoma cell lines. Muscarinic agonists acetylcholine, carbachol, methacholine and muscarine induced an increase in cytosolic free Ca2+ in a pertussis toxin (100 ng/ml)-insensitive manner in both cell lines. The ED50 values in IMR 32 cells (8-98 microM) were one order of magnitude higher than in SH-SY5Y cells (0.3-1.6 microM). Oxotremorine and pilocarpine failed to mobilize Ca2+ in IMR 32 cells. Pirenzepine antagonized carbachol-induced Ca2+ mobilization in SH-SY5Y cells with a Ki value in the range of 150-189 nM whereas the corresponding values in IMR 32 cells were 24-28 nM. Atropine inhibited a carbachol-stimulated increase in cytosolic Ca2+ with an equal potency in both cell lines (Ki 2-3 nM). Carbachol stimulated cyclic AMP (cAMP) accumulation in SH-SY5Y cells in a pertussis toxin-insensitive manner. In IMR 32 cells carbachol inhibited prostaglandin E1-stimulated cAMP accumulation. Treatment of IMR 32 cells with pertussis toxin abolished the inhibition of stimulated cAMP accumulation. These results suggest that in SH-SY5Y cells the M3 muscarinic receptor couples to both Ca2+ mobilization and stimulation of cAMP accumulation. In IMR 32 cells the M1 receptor seems to couple to Ca2+ mobilization whereas the inhibition of stimulated cAMP accumulation is coupled to a non-M1 subtype by an inhibitory G-protein.     

Contractile and relaxant effects of phorbol ester in the intestinal smooth muscle of guinea-pig taenia caeci.

1. Effects of phorbol esters on the cytosolic Ca2+ level ([Ca2+]i) and muscle tension in the intestinal smooth muscle of guinea-pig taenia caeci were examined. 2. 12-Deoxyphorbol 13-isobutyrate (DPB, 1 microM) did not change the [Ca2+]i and tension in resting muscle. 3. In high K(+)-stimulated muscle, 1 microM DPB transiently augmented the contraction and decreased [Ca2+]i. 12-Deoxyphorbol 13-isobutyrate 20-acetate (1 microM) and phorbol 12, 13-dibutyrate (1 microM) showed similar effects to DPB whereas phorbol 12-myristate 13-acetate (1 microM) and phorbol 12, 13-didecanoate (1 microM) were ineffective. 4. DPB (1 microM) inhibited both [Ca2+]i and tension stimulated by 300 nM carbachol or 3 microM histamine. In the presence of a higher concentration of carbachol (1 microM), DPB decreased [Ca2+]i and transiently increased muscle tension. 5. In the muscle strips permeabilized with bacterial alpha-toxin, 1 microM DPB shifted the Ca(2+)-tension curve to the left. An inhibitor of protein kinase C, H-7 (30 microM), inhibited the effect of DPB. 6. DPB did not change the high K(+)-induced contraction in the muscle strips pretreated with 3 microM phorbol 12-myristate 13-acetate for 24 h. 7. These results suggest that activation of protein kinase C has dual effects; it augments contraction by increasing the Ca2+ sensitivity of the contractile elements and it inhibits contraction by decreasing [Ca2+]i.     

Intracellular calcium in canine cultured tracheal smooth muscle cells is regulated by M3 muscarinic receptors.

1. The regulation of cytosolic Ca2+ concentrations ([Ca2+]i) during exposure to carbachol was measured directly in canine cultured tracheal smooth muscle cells (TSMCs) loaded with fura-2. Stimulation of muscarinic cholinoceptors (muscarinic AChRs) by carbachol produced a dose-dependent rise in [Ca2+]i which was followed by a stable plateau phase. The EC50 values of carbachol for the peak and sustained plateau responses were 0.34 and 0.33 microM, respectively. 2. Atropine (10 microM) prevented all the responses to carbachol, and when added during a response to carbachol, significantly, but not completely decreased [Ca2+]i within 5 s. Therefore, the changes in [Ca2+]i by carbachol were mediated through the muscarinic AChRs. 3. AF-DX 116 (a selective M2 antagonist) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, a selective M3 antagonist) inhibited the carbachol-stimulated increase in [Ca2+]i with pKB values of 6.4 and 9.4, respectively, corresponding to low affinity for AF-DX 119 and high affinity for 4-DAMP in antagonizing this response. 4. The plateau elevation of [Ca2+]i was dependent on the presence of external Ca2+. Removal of Ca2+ by the addition of 2 mM EGTA caused the [Ca2+]i to decline rapidly to the resting level. In the absence of external Ca2+, only an initial transient peak of [Ca2+]i was seen which then declined to the resting level; the sustained elevation of [Ca2+]i could then be evoked by the addition of Ca2+ (1.8 mM) in the continued presence of carbachol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of relaxing action of the antiasthmatic drug, azelastine, in isolated porcine tracheal smooth muscle.

Azelastine (1-300 microM) inhibited contractions of isolated porcine trachea induced by high K+, carbachol and endothelin-1 (ET-1) with a decrease in [Ca2+]cyt (as measured by fura-2-fluorescence). Verapamil (0.1-10 microM) also inhibited the high K(+)-induced increases in [Ca2+]cyt and contraction, although it only partially inhibited the responses evoked by carbachol or ET-1. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), carbachol induced a transient increase in [Ca2+]cyt and force by releasing Ca2+ from cellular stores. Azelastine (100 microns) completely inhibited these contransient changes. In the absence of extracellular Ca2+, carbachol and 12-deoxyphorbol 13-isobutyrate (DPB) induced small sustained contractions without increasing [Ca2+]cyt. Azelastine inhibited these contractions. In muscle permeabilized with alpha-toxin, Ca2+ (0.3-3 microM) induced contraction in a concentration-dependent manner. DPB (without GTP) and carbachol or ET-1 (with GTP) enhanced the Ca(2+)-induced contraction. Azelastine partially inhibited the contraction induced by 0.3 microM Ca2+ but not the contraction induced by 3 microM Ca2+, and strongly inhibited the potentiating effects of DPB, carbachol and ET-1. Azelastine had no effect on the content of cyclic AMP or cyclic GMP. These results suggest that azelastine inhibits smooth muscle contraction by (i) decreasing [Ca2+]cyt, by inhibition of Ca2+ channels, (ii) decreasing agonist-induced Ca2+ release, and (iii) direct inhibition of contractile elements.     

Muscarinic receptors in canine colonic circular smooth muscle. II. Signal transduction pathways.

We have, in the accompanying work, demonstrated the coexistence of M2 and M3 muscarinic receptors in the circular smooth muscle of canine colon. In the present study, the effects of muscarinic receptor stimulation on phosphoinositide turnover and adenylate cyclase activity were examined. In myo-[3H]inositol-labeled circular smooth muscle strips, carbachol caused a concentration-dependent (EC50 = 5 microM) increase in [3H]inositol phosphate production. The more M3 receptor-selective muscarinic antagonist pirenzepine (KB = 53 nM) was approximately 60 times more potent than the more M2-selective agent AF-DX 116 (KB = 3 microM) in blocking carbachol-elicited accumulation of [3H]inositol phosphates. The carbachol-stimulated increase in [3H]inositol phosphate accumulation was not affected by pretreatment of the tissue with pertussis toxin (200 ng/ml, 3 hr). Within the first minute, carbachol (100 microM) caused a rapid and transient increase of [3H]inositol 1,4,5-trisphosphate production that oscillated continuously in the presence of agonist (120 min). The accumulation of [3H]inositol 1,3,4-trisphosphate was also extremely rapid, reaching a peak at 15 sec. The accumulation of [3H]inositol monophosphate was delayed and progressively increased over 30 min. [3H]inositol 1,3,4,5-tetrakisphosphate, although not detectable in the first minute, accumulated to significant levels over 30 min in the presence of agonist. Addition of carbachol in the adenylate cyclase assay caused inhibition of forskolin-stimulated [32P]cAMP production and blocked forskolin-stimulated cAMP accumulation in the intact tissue. The inhibitory effects of carbachol on adenylate cyclase were blocked by atropine, AF-DX 116, and 4-diphenylacetoxy-N-methylpiperidine methobromide but were unaffected by the more M3-selective agent pirenzepine (1 microM). Pretreatment of tissues with pertussis toxin completely eliminated M2 receptor-mediated inhibition of adenylate cyclase activity, without altering inositol 1,4,5-trisphosphate accumulation. We conclude that muscarinic receptor stimulation of inositol trisphosphate production is mediated by the M3 receptor coupled to a pertussis toxin-insensitive GTP-binding protein and results in the rapid formation of inositol tetrakisphosphate, whereas inhibition of adenylate cyclase activity is mediated by the M2 subtype of muscarinic receptor coupled to the pertussis toxin-sensitive GTP-binding protein Gi.     

Inhibitory effect of diazepam on muscarinic receptor-stimulated inositol 1,4,5-trisphosphate production in rat parotid acinar cells

1. This study examined the effect of diazepam (DZP) on phosphoinositide turnover, which plays an important role in the regulation of salivary secretion, in rat parotid acinar cells. 2. DZP (10(-9) M to 10(-5) M), a potent agonist of both central- and peripheral-type benzodiazepine receptors, dose-dependently decreased inositol 1,4,5-trisphosphate IP3 production stimulated by carbachol, a muscarinic receptor agonist, in the cells. 3. DZP produced a maximum inhibitory response at a concentration of 10(-5) M, with IP3 production decreased to 63% of maximal levels. The concentration inducing half maximal inhibition of IP3 production was approximately 3.5 x 10 (-8) M. 4. An inhibitory response to DZP was produced by a short-term pretreatment (<3 min) of the cells and prevented by antagonist and competing ligand for the central- and peripheral-type benzodiazepine receptors, flumazenil and PK 11195, respectively. 5. DZP showed a non-competitive inhibition of carbachol-stimulated IP3 production. It did not directly inhibit the activities of GTP-binding regulatory proteins and phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) in the parotid gland membranes, though choline chloride inhibited PLC activity. 6. DZP (10(-5) M) attenuated the increase in the intracellular Ca2+ concentration ([Ca(2+)](i)) in the cells following stimulation of the muscarinic and alpha(1)-adrenoceptors. 7. These results suggest that in the parotid acinar cells, DZP inhibits muscarinic receptor-stimulated IP3 production through benzodiazepine receptors and that PLC activity which produces IP3 is inhibited by chloride. The decreases in IP3 and [Ca(2+)](i) in the cells may be connected with the suppression of salivary secretion induced by DZP.     

Mechanism of smooth muscle relaxation by tiropramide

Experiments on rabbit isolated colon show that a carbachol-induced contraction is accompanied by a decrease in cAMP content of the smooth muscle. (+/-)-alpha-(Benzoylamino)-4-[2-(diethylamino)-ethoxy]-NN-di-propylbenzen-propanamid (tiropramide, CR 605), a new tyrosine derivative with antispastic properties, increases cAMP concentrations within the same dose range that produces smooth muscle relaxation with or without carbachol (0.1 microM). These effects are potentiated by 1 mM theophylline, a phosphodiesterase inhibitor. In a purified microsomal preparation from rabbit colon smooth muscle, corresponding to sarcoplasmic reticulum, tiropramide induced a dose-dependent increase Ca2+ binding in the presence of ATP and Mg2+. Tiropramide inhibited phosphodiesterase activity in rabbit colon homogenates in a range of doses about ten times that producing relaxation, cAMP content enhancement and increase Ca2+ binding to sarcoplasmic reticulum. The effects of tiropramide on the carbachol-stimulated rabbit colon in the presence of theophylline, indomethacin of PGE1 are more in agreement with an action of tiropramide as inhibitor of cAMP catabolism than as a result of a prostaglandin-mediated effect. These observations suggest that the smooth muscle relaxant activity of tiropramide arises from drug-induced increase of cAMP concentrations possibly because of inhibition of cAMP catabolism. This effect is accompanied by the binding to the sarcoplasmic reticulum of Ca2+, preventing its interaction with the contractile proteins of the smooth muscle. A direct effect of tiropramide-enhanced cAMP content on contractile proteins in the smooth muscle cannot be excluded.     

Effect of extracellular Ca2+ on cholinergic, KCl and phorbol ester-mediated phosphoinositide turnover and guinea pig urinary bladder contraction.

The effect of extracellular Ca2+ ([Ca2+]o) on cholinergic, KCl and phorbol ester-mediated detrusor contractions was related to phosphoinositide (PI) breakdown in guinea pig urinary bladder. Carbachol (1.0 mM) elicited a 20-fold increase in inositol phosphate (IP) accumulation both in presence and absence of [Ca2+]o yielding the same EC50 value (approximately 12 microM). In contrast, carbachol-induced detrusor contractions were reduced by 35% without [Ca2+]o, but maximal efficacy was restored with Ca2+ replenishment. In absence of [Ca2+]o, repeated cholinergic stimulation yielded contractions only if tissues were intermittently equilibrated in [Ca2+]o. High K+ and PDBu evoked [Ca2+]o-dependent contractions. Ca2+ channel antagonists and divalent metal cations inhibited high K+ more potently than carbachol-mediated contractions. Together, these findings suggest multiple sources of Ca2+ for urinary bladder contraction, where voltage-sensitive responses depend primarily on [Ca2+]o and PI-linked muscarinic responses involved Ca2+ mobilization from intracellular stores as well. Clinical agents used for the treatment of urinary incontinence inhibited both carbachol-induced PI turnover and muscle contraction with the same rank order of potency both in presence and absence of [Ca2+]o. These findings suggest that the cholinergic mechanism of action of these agents involves the PI-Ca2+ effector system.     

Role of Ca2+ mobilization in muscarinic receptor-mediated membrane depolarization in guinea pig ileal smooth muscle cells

In single smooth muscle cells dispersed from guinea pig ileum, the muscarinic agonist carbachol (CCh) at 2 microM produced an oscillatory or sustained type of depolarization and at 100 microM, the latter type depolarization. Depletion of internal Ca2+ stores blocked the oscillatory response, but not the sustained responses to 2 microM and 100 microM CCh, although their decay after reaching the peak became faster. Blocking voltage-dependent Ca2+ channels (VDCCs) blocked both types of response to 2 microM CCh, but only slowed the initial rising phase of 100 microM CCh responses. Combination of Ca2+ store depletion and VDCC blockade abolished the responses to 2 microM CCh again and decreased those to 100 microM CCh in peak amplitude and persistency. Combination of Ca2+ store depletion with removal of extracellular Ca2+ markedly reduced or abolished the 100 microM CCh responses. The results suggest that muscarinic depolarization of the ileal cells requires Ca2+ mobilization for its generation and persistence; at weak muscarinic stimulation, both Ca2+ entry via VDCCs and Ca2+ release from internal stores may contribute to the Ca2+ mobilization; and under strong muscarinic stimulation, Ca2+ entry pathways resistant to VDCC blockers may also contribute to it.     

Effects of cooling on the contractile response of the rat tracheal muscle to pilocarpine in vitro

Experiments were designed to determine the effect of cooling on the contractile response of rat tracheal strip-chain preparation to the partial agonist pilocarpine. The preparation was suspended in an organ bath containing Krebs bicarbonate solution for isometric tension recording. A decrease of the bath temperature from 37 to 30, 20 or 10 degrees C (cooling) did not affect or inhibited the contractile response of the trachea caused by pilocarpine (1-100 microM), while it augmented the carbachol (0.1-1 microM)-induced response. At 37 degrees C, the pilocarpine (3-100 microM)-induced contraction was relatively resistant to removal of extracellular Ca2+ or to the Ca2+ entry blocker, verapamil (1 microM). Similar results were also obtained for the carbachol (0.3-10 microM)-induced response. On the other hand, the affinity of pilocarpine for the tracheal muscarinic receptors, determined from its dissociation constant (KA), was significantly decreased at a lower temperature. It is concluded from these observations that cooling-induced subsensitivity of the rat tracheal muscle to pilocarpine is mainly associated with a decrease in the affinity of the agonist for muscarinic receptors.     

Effect of membrane depolarization by high K+ on carbachol-stimulated phosphoinositides hydrolysis in guinea pig cerebral cortical slices

Stimulation of phosphoinositide hydrolysis by carbachol was studied in slices of guinea pig cerebral cortex under normal conditions (4.7 mM K+) and depolarization conditions with high K+ (42 mM K+). Slices were labeled with [myo-3H]-inositol, and the effects of carbachol and high K+ on the formation of inositol-bisphosphates (IP2) and inositol-trisphosphates (IP3) were determined. Carbachol (10 mM) caused only 140% stimulation of the formations of IP2 and IP3 over the control value in normal Krebs Ringer Buffer (KRB), but about 200% stimulation in high K+ medium. Dose-response curves for the effect of carbachol on the formations of IP2 and IP3 showed that high K+ medium selectively decreased the ED50 value of carbachol for IP2 formation about 3-fold. A Ca++ channel blocker, verapamil, inhibited the synergistic effect of carbachol and high K+ on IP2 formation, and a decrease in extracellular Ca++ also inhibited IP2 formation induced by high K+, but these treatments had little, if any, effect on IP3 formation. The possibility that IP2 may be directly generated by hydrolysis of phosphatidylinositol 4-monophosphate (PIP) as well as from hydrolysis of IP3 was discussed.     

cGMP formation and phosphoinositide turnover in rat brain slices are mediated by pharmacologically distinct muscarinic acetylcholine receptors.

The cGMP response and the accumulation of inositol monophosphate (IP) induced by carbachol were compared in slices of different rat brain structures. Basal cGMP and the responses of cGMP to carbachol appeared dependent on the concentration of added Ca2+, suggesting that distinct Ca(2+)-mediated and Ca(2+)-sensitive muscarinic receptor-mediated mechanisms stimulate guanylate cyclase. Regional responses of cGMP to carbachol or to direct stimulation of guanylate cyclase with sodium nitroprusside were markedly distinct, indicating that a major proportion of guanylate cyclase in the cortex, an intermediate proportion in other forebrain regions, and only a minor proportion in the brainstem is sensitive to muscarinic receptor stimulation. The regional patterns of IP and cGMP responses to carbachol were different in the forebrain. Maximal IP accumulation was found in the cortex, whereas cGMP responses were highest in the hippocampus. Moreover, IP and cGMP formation in the hippocampus were differently antagonized by atropine, 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP), the M2-receptor subtype-preferring antagonist AF-DX 116 and the M1-selective antagonist pirenzepine. These data support the notion that the IP formation induced by carbachol in the forebrain predominantly is mediated by muscarinic receptors of the M1 subtype, and indicate the involvement of muscarinic receptors of the M3 subtype in the carbachol-induced cGMP formation.     

Studies on the mechanism of [3H]-noradrenaline release from SH-SY5Y cells: the role of Ca2+ and cyclic AMP.

1. The roles of both Ca2+ and adenosine 3':5'-cyclic monophosphate (cyclic AMP) in carbachol and K(+)-stimulated [3H]-noradrenaline release from SH-SY5Y human neuroblastoma cells were examined. 2. Both carbachol and K+ caused a time- and dose-related stimulation of [3H]-noradrenaline release. The release event in perfused cells was monophasic. Half-maximum stimulation measured in statically incubated (3 min) cells was 38 +/- 4 microM and 63 +/- 4 mM respectively. K+ (100 mM, added)-evoked release was greater than that produced by carbachol (1 mM). 3. Both carbachol and K+ caused a time- and dose (measured at 3 min)-related stimulation of cyclic AMP formation with half-maximum stimulation occurring at 5 +/- 1 microM and 49 +/- 2 mM respectively. In contrast to its effects on release, carbachol produced a greater stimulation of cyclic AMP formation than K+. 4. K(+)-stimulated [3H]-noradrenaline release was entirely dependent on Ca2+ entry as 2.5 mM Ni2+ abolished release. However, carbachol-evoked (1 mM) release appeared to be unaffected by Ni2+ pretreatment. 5. These data suggest that in SH-SY5Y cells, elevated cyclic AMP levels are not directly involved in [3H]-noradrenaline release. In addition, carbachol-stimulated release is largely independent of extracellular Ca2+ possibly implying a role for intracellular stored Ca2+ in the release process.     

Tachykinin receptors mediating responses to sensory nerve stimulation and exogenous tachykinins and analogues in the rabbit isolated iris sphincter.

1. We have used selective tachykinin receptor agonists and antagonists to investigate the nature of the receptors mediating responses to endogenous and exogenous tachykinins in the rabbit iris sphincter preparation in vitro. 2. The NK1-selective agonist, substance P methyl ester, induced contraction with a pD2 of 9.16 indicating the presence of NK1 receptors. In confirmation, the NK1-selective antagonist, GR82334, competitively antagonized responses to substance P methyl ester with high affinity (pKB 7.46). 3. NK3 receptors also mediate contraction since NK3-selective agonists exhibited high potency, e.g. the pD2 of [Me-Phe7]-neurokinin B was 9.67, and their responses were not inhibited by GR82334 (10 microM). 4. NK2 receptor activation does not seem to contribute to contraction since the NK2-selective agonist [beta-Ala8]-neurokinin A(4-10) had relatively low potency (pD2 6.43), and the NK2-selective antagonists MEN10207 (1 microM) and L-659,877 (10 microM) were inactive or had low affinity, respectively. 5. GR82334 (1 microM) significantly inhibited responses to electrical field-stimulation of non-adrenergic non-cholinergic sensory nerves (3, 10 and 30 Hz), and caused a rightward shift of the log concentration-response curve to bradykinin (lateral shift ca. 1000 fold). Higher concentrations of GR82334 (10 microM) significantly attenuated responses to capsaicin (1-60 microM) whilst completely abolishing responses to field-stimulation (3, 10 and 30 Hz) and bradykinin (1 nM- 3 microM). 6. In conclusion, NK1 and NK3 receptor activation results in contraction of the rabbit iris sphincter. The contractile response following sensory nerve stimulation by bradykinin, capsaicin and electrical field stimulation results from NK1 receptor activation.     

Muscarinic receptor subtypes coupled to generation of different second messengers in isolated tracheal smooth muscle cells.

1. Activation of muscarinic receptor subtypes leads to contraction, an increase in the accumulation of inositol phosphates (IPs) and a decrease in adenosine 3': 5'-cyclic monophosphate (cyclic AMP) synthesis in tracheal smooth muscle. The concentrations of carbachol that produced a half-maximal effect (EC50) in inhibition of cyclic AMP generation, stimulation of IPs formation and contraction were 15 nM, 2.0 microM and 0.17 microM, respectively. 2. Pirenzepine, a selective M1 antagonist, displayed a low affinity for antagonizing cyclic AMP inhibition, IPs formation and contraction induced by carbachol (pKB = 6.8, 7.0, and 7.1, respectively). 3. Methoctramine, a cardioselective M2 antagonist, blocked cyclic AMP inhibition with a high affinity (pKB = 7.5), while it antagonized IPs formation and contraction with a low affinity (pKB = 6.2 and 6.1, respectively). 4. 4-Diphenylacetoxy-N-methylpiperidine (4-DAMP), a selective smooth muscle M3 antagonist, possessed a high affinity in blocking IPs formation (pKB = 8.8) and contraction (pKB = 9.2) as well as a low affinity for antagonism of cyclic AMP inhibition (pKB = 8.1). 5. In conclusion, we have demonstrated that M2 and M3 receptor subtypes are coupled to different effector systems in tracheal smooth muscle. An M1 receptor subtype is not involved in the generation of the second messengers examined. Inhibition of cyclic AMP formation may be coupled to the M2 receptor subtype. The accumulation of IPs and presumably IP-induced Ca2+ release may function as the transducing mechanism for cholinergic contraction of tracheal smooth muscle through the activation of M3 receptors.     

Muscarinic regulation of cytosolic free calcium in canine tracheal smooth muscle cells: Ca2+ requirement for phospholipase C activation.

1. The relationship between muscarinic receptor-mediated phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown and the increase of intracellular Ca2+ ([Ca2+])i has been examined in canine cultured tracheal smooth muscle cells (TSMCs). 2. Addition of acetylcholine (ACh) and carbachol led to a 2-3 fold increase in [Ca2+]i over the resting level as determined by fura-2, with half-maximal stimulation (EC50) obtained at concentrations of 97 and 340 nM, respectively. Addition of the partial agonist, bethanechol, showed a smaller increase in PIP2 turnover and [Ca2+]i than did ACh or carbachol. 3. Addition of ACh or carbachol to TSMCs that had been prelabelled with [3H]-inositol led to the rapid (5-15 s) release of inositol mono, bis and trisphosphates IP1, IP2 and IP3. The time course of IP3 accumulation is correlated with the time course of the peak rise in [Ca2+]i. 4. Inclusion of EGTA lowered the resting [Ca2+]i and markedly reduced the extent of the agonist-induced rise in [Ca2+]i. When assayed under conditions similar to those used for the [Ca2+]i measurements, EGTA reduced the muscarinic agonist-stimulated inositol phosphates (IPs) accumulation. Conversely, ionomycin could stimulate IPs accumulation and elevate [Ca2+]i. The addition of Ca2+ (2.7-617 nM) to digitonin-permeabilized TSMCs directly stimulated IPs accumulation. 5. Both Ca2+ and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) stimulated the formation of IPs in digitonin-permeabilized TSMCs prelabelled with [3H]-inositol. A further calcium-dependent increase in IPs accumulation was obtained by inclusion of either GTP gamma S or carbachol.(ABSTRACT TRUNCATED AT 250 WORDS)