Effects Of Muscarinic Receptor Stimulation Research Articles

Dual Mode of Action of Acetylcholine on Cytosolic Calcium Oscillations in Pancreatic Beta and Acinar Cells In Situ.

Cholinergic innervation in the pancreas controls both the release of digestive enzymes to support the intestinal digestion and absorption, as well as insulin release to promote nutrient use in the cells of the body. The effects of muscarinic receptor stimulation are described in detail for endocrine beta cells and exocrine acinar cells separately. Here we describe morphological and functional criteria to separate these two cell types in situ in tissue slices and simultaneously measure their response to ACh stimulation on cytosolic Ca2+ oscillations [Ca2+]c in stimulatory glucose conditions. Our results show that both cell types respond to glucose directly in the concentration range compatible with the glucose transporters they express. The physiological ACh concentration increases the frequency of glucose stimulated [Ca2+]c oscillations in both cell types and synchronizes [Ca2+]c oscillations in acinar cells. The supraphysiological ACh concentration further increases the oscillation frequency on the level of individual beta cells, inhibits the synchronization between these cells, and abolishes oscillatory activity in acinar cells. We discuss possible mechanisms leading to the observed phenomena.

Effects of muscarinic receptor stimulation on Ca2+ transient, cAMP production and pacemaker frequency of rabbit sinoatrial node cells

We investigated the contribution of the intracellular calcium (Cai2+) transient to acetylcholine (ACh)-mediated reduction of pacemaker frequency and cAMP content in rabbit sinoatrial nodal (SAN) cells. Action potentials (whole cell perforated patch clamp) and Cai2+ transients (Indo-1 fluorescence) were recorded from single isolated rabbit SAN cells, whereas intracellular cAMP content was measured in SAN cell suspensions using a cAMP assay (LANCE®). Our data show that the Cai2+ transient, like the hyperpolarization-activated “funny current” (I f) and the ACh-sensitive potassium current (I K,ACh), is an important determinant of ACh-mediated pacemaker slowing. When I f and I K,ACh were both inhibited, by cesium (2 mM) and tertiapin (100 nM), respectively, 1 μM ACh was still able to reduce pacemaker frequency by 72%. In these I f and I K,ACh-inhibited SAN cells, good correlations were found between the ACh-mediated change in interbeat interval and the ACh-mediated change in Cai2+ transient decay (r 2 = 0.98) and slow diastolic Cai2+ rise (r 2 = 0.73). Inhibition of the Cai2+ transient by ryanodine (3 μM) or BAPTA-AM (5 μM) facilitated ACh-mediated pacemaker slowing. Furthermore, ACh depressed the Cai2+ transient and reduced the sarcoplasmic reticulum (SR) Ca2+ content, all in a concentration-dependent fashion. At 1 μM ACh, the spontaneous activity and Cai2+ transient were abolished, but completely recovered when cAMP production was stimulated by forskolin (10 μM) and I K,ACh was inhibited by tertiapin (100 nM). Also, inhibition of the Cai2+ transient by ryanodine (3 μM) or BAPTA-AM (25 μM) exaggerated the ACh-mediated inhibition of cAMP content, indicating that Cai2+ affects cAMP production in SAN cells. In conclusion, muscarinic receptor stimulation inhibits the Cai2+ transient via a cAMP-dependent signaling pathway. Inhibition of the Cai2+ transient contributes to pacemaker slowing and inhibits Cai2+-stimulated cAMP production. Thus, we provide functional evidence for the contribution of the Cai2+ transient to ACh-induced inhibition of pacemaker activity and cAMP content in rabbit SAN cells.

Deletion of TRPC4 and TRPC6 in Mice Impairs Smooth Muscle Contraction and Intestinal Motility In Vivo

Downstream effects of muscarinic receptor stimulation in intestinal smooth muscle include contraction and intestinal transit. We thought to determine whether classic transient receptor potential (TRPC) channels integrate the intracellular signaling cascades evoked by the stimulated receptors and thereby contribute to the control of the membrane potential, Ca-influx, and cell responses. We created trpc4-, trpc6-, and trpc4/trpc6-gene-deficient mice and analyzed them for intestinal smooth muscle function in vitro and in vivo. In intestinal smooth muscle cells TRPC4 forms a 55 pS cation channel and underlies more than 80% of the muscarinic receptor-induced cation current (mI(CAT)). The residual mI(CAT) depends on the expression of TRPC6, indicating that TRPC6 and TRPC4 determine mI(CAT) channel activity independent of other channel subunits. In TRPC4-deficient ileal myocytes the carbachol-induced membrane depolarizations are diminished greatly and the atropine-sensitive contraction elicited by acetylcholine release from excitatory motor neurons is reduced greatly. Additional deletion of TRPC6 aggravates these effects. Intestinal transit is slowed down in mice lacking TRPC4 and TRPC6. In intestinal smooth muscle cells TRPC4 and TRPC6 channels are gated by muscarinic receptors and are responsible for mI(CAT). They couple muscarinic receptors to depolarization of intestinal smooth muscle cells and voltage-activated Ca(2+)-influx and contraction, and thereby accelerate small intestinal motility in vivo.

Effects of muscarinic receptor stimulation and nitric oxide synthase inhibition on gastric tone and gastric myoelectrical activity in canines

This study was designed to assess whether the muscarinic receptor stimulation and nitric oxide synthase inhibition were equally effective on gastric fundic tone or gastric myoelectrical activity (GMA) in canines, and the correlation between gastric fundic tone and GMA. Gastric fundic tone and GMA were recorded on seven dogs implanted with serosal electrodes and a gastric cannula. Bethanechol and L-nitro-N-arginine (L-NNA) significantly increased gastric fundic tone; gastric volume was decreased with bethanechol or L-NNA (P < 0.05). Increased spike activities were observed after both bethanechol and L-NNA. The percentage of slow waves superimposed with spikes was increased with bethanechol (P < 0.001) and L-NNA (P < 0.05). There was a significant reduction in dominant frequency (DF) (P < 0.05), dominant power (DP) (P < 0.05) and percentage of normal slow waves (%N) (P < 0.05) with bethanechol, while no significant change was observed with L-NNA. The variation of gastric tone was not correlated with parameters of GMA. Muscarinic receptor stimulation and nitric oxide synthase inhibition have similar effects on gastric tone and gastric spike activities, but different effects on gastric slow waves. Gastric fundic tone does not correlate with gastric slow waves.

Muscarinic M3 receptor stimulation increases cigarette smoke-induced IL-8 secretion by human airway smooth muscle cells

Acetylcholine is the primary parasympathetic neurotransmitter in the airways and is known to cause bronchoconstriction and mucus secretion. Recent findings suggest that acetylcholine also regulates aspects of remodelling and inflammation through its action on muscarinic receptors. In the present study, we aimed to determine the effects of muscarinic receptor stimulation on cytokine production by human airway smooth muscle cells (primary and immortalised cell lines). The muscarinic receptor agonists carbachol and methacholine both induced modest effects on basal interleukin (IL)-8 and -6 secretion, whereas the secretion of RANTES, eotaxin, vascular endothelial growth factor-A and monocyte chemoattractant protein-1 was not affected. Secretion of IL-8 and -6 was only observed in immortalised airway smooth muscle cells that express muscarinic M3 receptors. In these cells, methacholine also significantly augmented IL-8 secretion in combination with cigarette smoke extract in a synergistic manner, whereas synergistic effects on IL-6 secretion were not significant. Muscarinic M3 receptors were the primary subtype involved in augmenting cigarette smoke extract-induced IL-8 secretion, as only tiotropium bromide and muscarinic M3 receptor subtype selective antagonists abrogated the effects of methacholine. Collectively, these results indicate that muscarinic M3 receptor stimulation augments cigarette smoke extract-induced cytokine production by airway smooth muscle. This interaction could be of importance in patients with chronic obstructive pulmonary disease.

Homeostatic Regulation of Sleep in a Genetic Model of Depression in the Mouse: Effects of Muscarinic and 5-HT1A Receptor Activation

In depressed patients, sleep undergoes marked alterations, especially sleep onset insomnia, sleep fragmentation, and disturbances of the Rapid Eye Movement (REM) sleep. Abnormalities of rest-activity rhythms and of hypothalamic-pituitary-adrenocortical function have also been described in these patients. In the present study, we examined the presence of such abnormalities in a recently developed line of mice (Helpless mice-H) that exhibit depression-like behaviors in validated tests, compared to the nonhelpless (NH) line derived from the same colony. Experiments were essentially carried out in females for which previous studies showed marked differences between H and NH lines. Compared to NH mice, the H line exhibited (i) lower basal locomotor activity, (ii) sleep fragmentation, shift towards lighter sleep stages, and facilitation of REM sleep reflected by increased amounts and decreased latency, (iii) larger response to the REM sleep promoting effect of muscarinic receptor stimulation (by arecoline). In contrast, H and NH mice were equally responsive to the REM sleep inhibitory effect of 5-HT1A receptor stimulation (by 8-OH-DPAT). In addition, a deficiency in delta power enhancement after sleep deprivation was observed in the H group, and acute immobilization stress in this group failed to elicit a REM sleep rebound and was associated with a long-lasting raise in serum corticosterone levels. These results further validate H mice as a depression model and suggest they might be of particular interest for investigating the neurobiological mechanisms and possibly genetic substrates underlying sleep alterations associated with depression.

Muscarinic M3 receptor‐dependent regulation of airway smooth muscle contractile phenotype

1. Airway smooth muscle (ASM) cells are known to switch from a contractile to a proliferative and synthetic phenotype in culture in response to serum and growth factors. Phenotype switching in response to contractile agonists, however, is poorly characterised, despite the possible relationship between ASM phenotype and airway remodelling in asthma. 2. To investigate the effects of muscarinic receptor stimulation on ASM phenotype, we used organ-cultured bovine tracheal smooth muscle (BTSM) strips, in which contractile responsiveness, contractile protein expression and proliferation were measured after pretreatment with methacholine. 3. Long-term methacholine pretreatment (8 days) decreased maximal contraction and sensitivity to methacholine as well as to histamine and KCl. This decrease was dose-dependent (pEC(50)=5.2+/-0.1). Pretreatment with the highest concentration of methacholine applied (100 microm) could suppress maximal histamine-induced contraction to 8+/-1% of control. In addition, contractile protein expression (myosin, actin) was downregulated two-fold. No concomitant increase in proliferative capacity was observed. 4. The M(3)/M(2) muscarinic receptor antagonist DAU 5884 (0.1 microm) completely inhibited the observed decrease in contractility. In contrast, the M(2)/M(3) muscarinic receptor antagonist gallamine (10 microm) was ineffective, demonstrating that M(2) receptors were not involved. 5. Pretreatment (8 days) with 60 mm KCl could mimick the strong decreases in contractility. This was completely prevented by pretreatment with verapamil (1 microm). 6. Regulation of contractility was not affected by protein kinase C inhibition, whereas inhibitors of phosphatidyl inositol 3-kinase and p42/p44 mitogen activated protein kinase were partially effective. 7. These results show that long-term methacholine pretreatment (8 days) induces an M(3) receptor-dependent decrease in BTSM contractility without increased proliferative capacity.

Cl(-)/HCO(3)(-) exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca(2+)](i) increase in salivary acinar cells.

Large volumes of saliva are generated by transepithelial Cl(-) movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl(-) uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na(+) independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an approximately 170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive Cl(-)/HCO(3)(-) exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the Cl(-)/HCO(3)(-) exchanger activity in acinar cells from both glands. Intracellular Ca(2+) chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca(2+) concentration with the Ca(2+)-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating Cl(-)/HCO(3)(-) exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca(2+)-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.

Nuclear calcium signaling evoked by cholinergic stimulation in hippocampal CA1 pyramidal neurons.

The cholinergic system is thought to play an important role in hippocampal-dependent learning and memory. However, the mechanism of action of the cholinergic system in these actions in not well understood. Here we examined the effect of muscarinic receptor stimulation in hippocampal CA1 pyramidal neurons using whole-cell recordings in acute brain slices coupled with high-speed imaging of intracellular calcium. Activation of muscarinic acetylcholine receptors by synaptic stimulation of cholinergic afferents or application of muscarinic agonist in CA1 pyramidal neurons evoked a focal rise in free calcium in the apical dendrite that propagated as a wave into the soma and invaded the nucleus. The calcium rise to a single action potential was reduced during muscarinic stimulation. Conversely, the calcium rise during trains of action potentials was enhanced during muscarinic stimulation. The enhancement of free intracellular calcium was most pronounced in the soma and nuclear regions. In many cases, the calcium rise was distinguished by a clear inflection in the rising phase of the calcium transient, indicative of a regenerative response. Both calcium waves and the amplification of action potential-induced calcium transients were blocked the emptying of intracellular calcium stores or by antagonism of inositol 1,4,5-trisphosphate receptors with heparin or caffeine. Ryanodine receptors were not essential for the calcium waves or enhancement of calcium responses. Because rises in nuclear calcium are known to initiate the transcription of novel genes, we suggest that these actions of cholinergic stimulation may underlie its effects on learning and memory.

Nuclear Calcium Signaling Evoked by Cholinergic Stimulation in Hippocampal CA1 Pyramidal Neurons

The cholinergic system is thought to play an important role in hippocampal-dependent learning and memory. However, the mechanism of action of the cholinergic system in these actions in not well understood. Here we examined the effect of muscarinic receptor stimulation in hippocampal CA1 pyramidal neurons using whole-cell recordings in acute brain slices coupled with high-speed imaging of intracellular calcium. Activation of muscarinic acetylcholine receptors by synaptic stimulation of cholinergic afferents or application of muscarinic agonist in CA1 pyramidal neurons evoked a focal rise in free calcium in the apical dendrite that propagated as a wave into the soma and invaded the nucleus. The calcium rise to a single action potential was reduced during muscarinic stimulation. Conversely, the calcium rise during trains of action potentials was enhanced during muscarinic stimulation. The enhancement of free intracellular calcium was most pronounced in the soma and nuclear regions. In many cases, the calcium rise was distinguished by a clear inflection in the rising phase of the calcium transient, indicative of a regenerative response. Both calcium waves and the amplification of action potential-induced calcium transients were blocked the emptying of intracellular calcium stores or by antagonism of inositol 1,4,5-trisphosphate receptors with heparin or caffeine. Ryanodine receptors were not essential for the calcium waves or enhancement of calcium responses. Because rises in nuclear calcium are known to initiate the transcription of novel genes, we suggest that these actions of cholinergic stimulation may underlie its effects on learning and memory.

Muscarinic mediation of the urethro genital reflex in spinal cord-transected rats

The effects of muscarinic receptor stimulation were tested on the urethro genital reflex (UGR) in anesthetized and spinal cord-transected rats. Drugs were applied directly to the spinal cord. The electromyographic activity (EMG) of the bulbospongiosus (BS) muscle was used for recording UGR. In six animals BS as well as soleus, posterior biceps or peroneus tertius muscle EMG was recorded simultaneously. Muscarine (5, 10, 20, 50 and 100 μg) was applied in 22 animals after cutting L6–S1 dorsal roots. Some observations were made on another six animals, to which an extensive bilateral dorsal rhizotomy (L3–S2) was performed. Rhythmic bursts of similar frequency and size to those seen during UGR were found in BS muscle a few minutes after muscarine application. No rhythmic bursting was found on the hindlimb muscles, but exclusively on BS muscles. The effects of homatropine (25, 50, 100 and 200 μg), an acetylcholine muscarinic receptor antagonist, were tested in 21 rats after UGR was elicited three times at low stimulation intensity (7 mm Hg). Homatropine produced two effects: (i) A significant increase in the latency of UGR. (ii) A facilitation of UGR inhibition. In view of these results it can be speculated that muscarinic receptor stimulation is involved in the elicitation of UGR.

Positive and negative inotropic effects of muscarinic receptor stimulation in mouse left atria

In isolated mouse left atria, acetylcholine (ACh) produced a biphasic inotropic response; a transient decrease in developed tension was followed by an increase. Both negative and positive responses were concentration dependent and were inhibited by atropine. The negative and positive inotropic responses were also observed with a nonselective muscarinic stimulant, oxotremorine-M, but not with an M1-receptor selective stimulant, McN-A343. Pirenzepine, an M1-receptor antagonist, inhibited both negative and positive inotropic responses at high concentrations. Gallamine, an M2-receptor antagonist, inhibited the negative response. Hexahydro-siladifenidol hydrochloride, p-fluoro analog (p-F-HHSiD), an M3-receptor antagonist, inhibited the positive response with no effect on the negative phase. In pertussis toxin (PTX) treated preparations, negative inotropic response to ACh was not observed. These results suggest that the negative and positive inotropic responses to acetylcholine in mouse atria are mediated by M2 and M3 receptors, respectively. The negative phase, but not the positive phase, was mediated by a PTX-sensitive G protein.

P-596 - Negative and positive inotropic effect of muscarinic receptor stimulation in mouse left atria

P-596 - Negative and positive inotropic effect of muscarinic receptor stimulation in mouse left atria

Mutual inhibitory effects between dopamine and carbachol on the excitatory synaptic transmission in the rat neostriatum.

The interactions between dopamine and carbachol on the excitatory synaptic transmission were studied in rat neostriatal slices using an intracellular recording method. Excitatory postsynaptic potentials (EPSPs) were evoked by cortical stimulation. Application of dopamine (DA; 0.1 microM) or carbachol (0.1 microM) produced a dramatic and reversible inhibition of the EPSP amplitude. The inhibitory effect induced by carbachol was markedly attenuated in the presence of either DA (0.1 microM) or the selective D2 dopaminergic receptor agonist (+/-)-2-(N-phenylethyl-N-propyl) amino-5-hydroxytertralin (PPHT; 0.1 microM), but not by the D1 dopaminergic receptor agonist (+/-)-7, 8-dihydroxy-3-allyl-1-phenyl-2, 3, 4, 5-tetrahydro-1H-3-benzazepine (SKF-38393; 0.1 microM) or the D3 dopaminergic receptor agonist R(-)-(4aS, 10bS)-3, 4, 4a, 10b-tetrahydro-4-propyl-2H, 5H-[1] benzogyrano-[4,3-b]-1, 4-oxazin-9-ol (PD-128,907; 0.1 microM). Conversely, muscarinic receptor activation with carbachol (0.1 microM) also completely abolished the DA-induced depression of the EPSP amplitude. In addition, the inhibitory effect of DA on the carbachol-induced depression of the EPSP amplitude was antagonized by sulpiride (1 microM), a selective D2 dopaminergic receptor antagonist. However, D1 dopaminergic receptor antagonist (+/-)-7-bromo-8-hydroxy-3-methyl-1-phenyl-2, 3, 4, 5-tetrahydro-3-benzazepine (SKF-83566; 1 microM) did not affect DA's inhibition. Rp-adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS; 25 microM), a potent inhibitor of cAMP-dependent protein kinase A (PKA), alone decreased the amplitude of EPSP below baseline values and mimicked the inhibitory effect of DA on the carbachol-induced depression of the EPSP amplitude. Based on these findings, we conclude that the inhibitory effects of D2 dopaminergic receptor and muscarinic receptor activation on the excitatory synaptic transmission in the neostriatum are non-additive and therefore are antagonistic interactions. furthermore, the effect of muscarinic receptor stimulation will depend on the extent of D2 dopaminergic receptor activation and the modulation of the cellular PKA-dependent messenger system seems to contribute to their interactions.

Inhibitory effect of muscarinic receptor activation on Ca2+ channel current in smooth muscle cells of guinea‐pig ileum.

1. The effect of muscarinic receptor stimulation on voltage-gated calcium channel currents was examined in whole-cell voltage-clamped smooth muscle cells of the guinea-pig ileum. 2. In cells voltage clamped at -60 mV and in which calcium channel currents (ICa) were elicited repeatedly by depolarizing pulses (25 ms duration, 0.25 Hz frequency) to 0 mV, carbachol (CCh, 10 microM) induced an inward current (ICCh) and were suppressed ICa, in a biphasic manner; an initial transient component was followed by a more sustained one. 3. A calcium channel current (IBa), when Ba2+ was used as a charge carrier, was also suppressed by CCh in a biphasic manner, as with ICa. The sustained phase of the IBa suppression was significantly smaller than that of the ICa suppression, suggesting that Ca2+ entry exerts a potentiating effect on the current suppression. 4. CCh had little or no effect on calcium channel currents (ICa and IBa) in cells dialysed with a pipette solution containing EGTA (20 mM). 5. Inclusion of GDP-beta-S (1 mM) in the pipette solution abolished ICCh and the suppression of IBa. With GTP-gamma-S (10 microM) in the pipette, the sustained phase of the IBa suppression remained almost unchanged even after removal of CCh. 6. Pretreatment with 2 micrograms ml-1 pertussis toxin (PTX), which abolished ICCh, did not change noticeably the initial transient and sustained phases of IBa suppression. 7. Neomycin (100 microM) or heparin (5 mg ml-1) in the pipette each abolished the initial transient component of ICCh as well as the initial transient phase of IBa suppression. 8. The biphasic effect of CCh on IBa was observed in the presence of either staurosporine (1 microM) or 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (100 microM). Phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate (up to 10 microM) had no inhibitory effect on ICa and IBa. 9. The results suggest that stimulation of the muscarinic receptor causes a biphasic suppression of the voltage-gated calcium channel currents through a PTX-insensitive G protein in guinea-pig ileal smooth muscle cells. The initial transient phase may be brought about by the release of Ca2+ from internal storage sites, and the sustained phase by a Ca(2+)-dependent mechanism which is independent of the phosphatidylinositol pathway.

Effects of muscarinic receptor stimulation of sympathetic preganglionic neurones of neonatal rat spinal cord in vitro

Whole cell current-clamp recordings were made from 85 sympathetic preganglionic neurones (SPN) of the neonatal spinal cord in vitro. Superfusion of up to 500 μM acetylcholine (2–30 sec) gave weak responses. Carbachol (CChol; 5–50 μM) superfused for 1–20 sec, hyperpolarized SPN. This response was associated with a mean reduction in input resistance of 22%. Ion substitution studies suggested that potassium is the likely carrier of at least part of the current underlying the CChol-induced hyperpolarization. Some SPN display spontaneous rhythmic oscillations in their membrane potentials which may be due to action potential discharge in electrically-coupled neurones. CChol also acts to inhibit these oscillations concomitant with the hyperpolarization. Responses to CChol were unaffected by addition of 500 nM TTX to the bathing medium suggesting that CChol acts directly upon SPN. Carbachol-induced hyperpolarizing responses were totally abolished by the non-specific muscarinic receptor antagonist atropine (20–50 μM). Pirenzepine, at concentrations over 5 μM reversibly reduced the responses to CChol. Gallamine, an M2 receptor antagonist applied at 25 μM also reversibly abolished the CChol responses. These results suggest that CChol-mediated hyperpolarizations may be due to M2 receptor activation.

Parasympathetic muscarinic stimulation limits noradrenaline induced myocardial creatine kinase release: a study in the isolated perfused working rat heart.

It has long been known that high concentrations of catecholamines may induce myocardial damage, and aggravate ischaemic injury. It has also been shown that beta-blockade may protect the myocardium from ischaemic damage. Stimulation of muscarinic receptors modulates beta-adrenergic receptor affinity for isoproterenol and attenuates isoproterenol induced adenylyl cyclase activation. Effects of muscarinic receptor stimulation were therefore investigated in isolated anterogradely perfused rat hearts under different experimental conditions. One group of hearts was perfused with noradrenaline, 10(-6) mol l-1 for 45 min, and another group was perfused with different carbachol concentrations (3 x 10(-7)-10(-5) mol l-1) with or without noradrenaline 10(-6) mol l-1, for 45 min. Release of creatine kinase to the perfusion buffer was taken as a sign of cell damage. Heart rate, left ventricular maxdP/dt and left ventricular pressure were measured throughout the perfusion time by insertion of a 20 gauge cannula through the left ventricular wall near the base. Carbachol (3 x 10(-7) mol l-1) alone induced a decrease of heart rate by 25% and maxdP/dt by 13%. Noradrenaline produced a 20% increase in heart rate, whereas the combination of noradrenaline plus carbachol induced a minor decrease in heart rate. Muscarinic receptor stimulation alone decreased myocardial contractility. However, when combined with noradrenaline no decrease in contractility was seen. Also, the release of creatine kinase to the perfusion buffer containing the combination of carbachol plus noradrenaline was reduced. Thus, muscarinic receptor stimulation protected the myocardium from catecholamine induced damage at concentrations where no change in contractility was seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of the effects of acetylcholine on the contractile properties and Ca2+ transients in ferret ventricular muscles.

1. We investigated the mechanism of signal transduction during the effect of muscarinic receptor stimulation on Ca2+ transients, tension, Ca2+ sensitivity and the cross-bridge cycling rate (CCR). 2. Membrane-permeable derivatives of cyclic GMP (8-bromo-cyclic GMP and dibutyryl cyclic GMP) did not cause any significant changes in the peaks of Ca2+ transients and tension and the time courses of either signal modulated by isoprenaline (Iso) (0.1 microM). 3. Nitroprusside (0.1-1 mM) likewise did not change the peaks or the time courses of Ca2+ transients and tension in the Iso-treated preparations. 4. In papillary muscles excised from ferrets treated with pertussis toxin (islet-activating protein, IAP), which is known to abolish the function of GTP-binding proteins (Gi, Go and Gt), similar changes in Ca2+ transients and tension produced by treatment with Iso (0.1 microM) were noted as in non-IAP-treated preparations. However, no effects of acetylcholine (ACh; 1 microM) on either signal were observed. 5. The relation between [Ca2+]i and tension measured during the steady state of tetanic contraction was shifted to the right by Iso (0.1 microM), and cyclic GMP derivatives (1 mM) did not change the altered relation. In the IAP-treated preparations, ACh (1 microM) did not influence the relation altered by Iso (0.1 microM). 6. Cyclic GMP derivatives (1 mM) did not alter the Iso (0.1 microM)-increased CCR measured by perturbation analysis. ACh (1 microM) did not restore the Iso-increased CCR in the IAP-treated preparations. 7. These results suggest that signal transduction in muscarinic receptor stimulation is primarily mediated by inhibition of adenylate cyclase via IAP-sensitive GTP-binding proteins, and that cyclic GMP does not play an important role in the effect of muscarinic receptor stimulation on Ca2+ transients, tension, Ca2+ sensitivity or CCR.

Depolarization and Agonist‐Stimulated Changes in Inositol 1,4,5‐Trisphosphate and Inositol 1,3,4,5‐Tetrakisphosphate Mass Accumulation in Rat Cerebral Cortex

Muscarinic receptor stimulation or depolarization with elevated extracellular K+ induced rapid and sustained increases in mass accumulations of myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] in cerebral cortex slices. Synergistic but transient responses of both inositol polyphosphate second messengers were observed when slices were stimulated with carbachol under depolarizing conditions; this synergy was observed as an increase in the maximal responsiveness, with no significant change in EC50 values for carbachol. Omission of buffer Ca2+ ([Ca2+]e 10-20 microM) reduced basal Ins(1,4,5)P3 and Ins(1,3,4,5)P4 concentrations; the relative stimulatory effects of muscarinic receptor stimulation were maintained, but the effects of depolarization were markedly attenuated under these conditions. A component of the response to depolarization appeared to be indirectly mediated by the release of acetylcholine, because the K(+)-evoked increase in Ins(1,3,4,5)P4 was enhanced by the cholinesterase inhibitor physostigmine, and was partially attenuated by atropine. An additive suppression by nitrendipine suggests that entry of Ca2+ through L-type Ca2+ channels may serve to accelerate phosphorylation of Ins(1,4,5)P3 by 3-kinase. Norepinephrine did not significantly increase Ins(1,4,5)P3 or Ins(1,3,4,5)P4 accumulation; however, in the presence of depolarizing K+, norepinephrine caused a dramatic increase in Ins(1,3,4,5)P4 mass accumulation. In contrast, the excitatory amino acid quisqualate caused significant increases in the mass accumulations of both inositol polyphosphates measured, with no further increase being observed under depolarizing conditions. The results are discussed with respect to the interactive effects of agonist and depolarization stimuli on inositol polyphosphate accumulation which might more accurately reflect the conditions pertaining in vivo.

Effect of muscarinic receptor stimulation on release of cysteinyl-leukotrienes and thromboxane B2 from anaphylactic guinea-pig hearts.

The effects of muscarinic receptor stimulation by infusions of methacholine (6.25 x 10(-8) mol/min or 1.9 x 10(-7) mol/min) into isolated perfused, spontaneously beating sensitized guinea-pig hearts on the anaphylactic release of cysteinyl-leukotrienes (LT) and thromboxane (TX) B2 were investigated. Methacholine increased coronary flow and decreased heart rate under basal conditions. Furthermore, infusions of methacholine (1.9 x 10(-7) mol/min) significantly increased the anaphylactic release of TXB2 as well as of immunoreactive cysteinyl-LT, which were demonstrated by reversed phase high pressure liquid chromatography to consist of a mixture of LTC4, LTD4 and LTE4. Infusions of atropine (1.3 x 10(-7) mol/min) alone did not significantly affect coronary flow and heart rate prior to ovalbumin injection nor anaphylactic release of cysteinyl-LT. The anaphylactic release of TXB2 was, however, significantly decreased in the presence of atropine. Atropine (1.3 x 10(-7) mol/min) infused in addition to methacholine (1.9 x 10(-7) mol/min) abolished the effects of the muscarinic receptor agonist on spontaneous heart rate and significantly antagonized the increase in coronary flow prior to ovalbumin injection. Similarly, the simultaneous infusion of atropine abolished the effects of methacholine on the anaphylactic release of TXB2 and cysteinyl-LT. After antigen challenge hearts infused with methacholine, atropine or the combination of both drugs did not exhibit any differences with respect to anaphylactic changes of heart rate or the time course of anaphylactic coronary flow reduction. Thus, in the isolated perfused anaphylactic guinea-pig heart, muscarinic receptor stimulation significantly enhanced the release of the arachidonic acid-derived mediators TXB2 and cysteinyl-LT.(ABSTRACT TRUNCATED AT 250 WORDS)