Effect of M2 and M3 muscarinic receptors on airway responsiveness to carbachol in bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs.

The expression balance of M2 and M3 muscarinic receptor subtypes on the pathogenesis of airway hyperresponsiveness was investigated by using two congenitally related strains of guinea pigs, bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR). CCh-induced airway responses in vivo and in vitro were investigated by comparing the effects of muscarinic receptor subtype antagonists, and the relative amounts of M2 and M3 muscarinic receptor mRNA in tracheal smooth muscle and lung tissue were investigated. After treatment with muscarinic receptor subtype antagonists, the ventilatory mechanics (VT, Raw, and Cdyn) of response to CCh aerosol inhalation were measured by the bodyplethysmograph method. The effects of these antagonists on CCh-induced tracheal smooth muscle contraction were also investigated. The effects of M2 muscarinic receptor blockade were less but the effects of M3 muscarinic receptors blockade on the airway contractile responses were greater in BHS than in BHR. In M3 muscarinic receptor blockades, CCh-induced tracheal contractions in BHS were significantly greater than those in BHR. In tracheal smooth muscle from BHS, the relative amount of M2 muscarinic receptors mRNA was less but that of M3 muscarinic receptor mRNA was more than those in BHR. These results suggest that the high ACh level as a consequence of dysfunction of M2 muscarinic autoreceptors and the excessive effect of M3 muscarinic receptors on the airway smooth muscle may play an important role in the pathogenesis of airway hyperresponsiveness.     

Caveolae facilitate muscarinic receptor-mediated intracellular Ca2+ mobilization and contraction in airway smooth muscle

Contractile responses of airway smooth muscle (ASM) determine airway resistance in health and disease. Caveolae microdomains in the plasma membrane are marked by caveolin proteins and are abundant in contractile smooth muscle in association with nanospaces involved in Ca(2+) homeostasis. Caveolin-1 can modulate localization and activity of signaling proteins, including trimeric G proteins, via a scaffolding domain. We investigated the role of caveolae in contraction and intracellular Ca(2+) ([Ca(2+)](i)) mobilization of ASM induced by the physiological muscarinic receptor agonist, acetylcholine (ACh). Human and canine ASM tissues and cells predominantly express caveolin-1. Muscarinic M(3) receptors (M(3)R) and Galpha(q/11) cofractionate with caveolin-1-rich membranes of ASM tissue. Caveolae disruption with beta-cyclodextrin in canine tracheal strips reduced sensitivity but not maximum isometric force induced by ACh. In fura-2-loaded canine and human ASM cells, exposure to methyl-beta-cyclodextrin (mbetaCD) reduced sensitivity but not maximum [Ca(2+)](i) induced by ACh. In contrast, both parameters were reduced for the partial muscarinic agonist, pilocarpine. Fluorescence microscopy revealed that mbetaCD disrupted the colocalization of caveolae-1 and M(3)R, but [N-methyl-(3)H]scopolamine receptor-binding assay revealed no effect on muscarinic receptor availability or affinity. To dissect the role of caveolin-1 in ACh-induced [Ca(2+)](i) flux, we disrupted its binding to signaling proteins using either a cell-permeable caveolin-1 scaffolding domain peptide mimetic or by small interfering RNA knockdown. Similar to the effects of mbetaCD, direct targeting of caveolin-1 reduced sensitivity to ACh, but maximum [Ca(2+)](i) mobilization was unaffected. These results indicate caveolae and caveolin-1 facilitate [Ca(2+)](i) mobilization leading to ASM contraction induced by submaximal concentrations of ACh.     

Efficacy of muscarinic stimulation and mode of excitation-contraction coupling in bovine trachealis muscle

We have compared the efficacy of cromakalim and nifedipine to inhibit acetylcholine (ACh) and pilocarpine-induced tonic contractions in control preparations and in tissues where a fraction of the muscarinic receptor population had been removed by alkylation with phenoxybenzamine (PBZ). Both agonists induced contractions by stimulating pharmacologically similar receptors, probably of the M3 muscarinic subtype. The receptor reserve was larger, and the coupling between stimulation and contraction (E-C coupling) more efficient when ACh was the stimulating agonist. For stimulations that produced equal levels of muscle response, cromakalim was more efficacious in inhibiting contractions induced by pilocarpine. The efficacy of cromakalim in relaxing contractions induced by ACh increased when the number of functional receptors decreased. Cromakalim and nifedipine decreased the efficiency of E-C coupling for ACh and pilocarpine. Cromakalim efficacy decreased in a sigmoid manner when stimulating concentrations of ACh (and receptor occupancy) increased, and there was an inverse relationship between receptor occupancy by ACh and cromakalim efficacy. In the presence of TEA, a K+ channel blocker, nifedipine almost completely inhibited contractions induced by the M3 muscarinic agonist bethanechol. These data indicate that in bovine tracheal smooth muscle, electro-mechanical coupling is an inherent part of muscarinic E-C coupling, but its functional expression is dependent upon the efficacy of stimulation. The data also suggest that the M3 receptor is coupled to a cellular pathway linked with the activation of K+ channels that exerts a potent functional antagonism against activation of voltage-dependent Ca2+ entry.     

Critical intracellular Ca2+ concentration for all-or-none Ca2+ spiking in single smooth muscle cells.

Neurotransmitters induce contractions of smooth muscle cells initially by mobilizing Ca2+ from intracellular Ca2+ stores through inositol 1,4,5-trisphosphate (InsP3) receptors. Here we studied roles of the molecules involved in Ca2+ mobilization in single smooth muscle cells. A slow rise in cytoplasmic Ca2+ ([Ca2+]i) in agonist-stimulated smooth muscle cells was followed by a wave of rapid regenerative Ca2+ release as the local [Ca2+]i reached a critical concentration of approximately 160 nM. Neither feedback regulation of phospholipase C nor caffeine-sensitive Ca(2+)-induced Ca2+ release was found to be required in the regenerative Ca2+ release. These results indicate that Ca(2+)-dependent feedback control of InsP3-induced Ca2+ release plays a dominant role in the generation of the regenerative Ca2+ release. The resulting Ca2+ release in a whole cell was an all-or-none event, i.e. constant peak [Ca2+]i was attained with agonist concentrations above the threshold value. This finding suggests a possible digital mode involved in the neural control of smooth muscle contraction.     

Differential regulation of intracellular Ca2+ signalling induced by high K+ and endothelin-1 in single smooth muscle cells of intact canine basilar artery: detection by means of confocal laser microscopy

Changes in intracellular calcium concentration ([Ca2+]i) in smooth muscle cells play the key role in regulation of vascular smooth muscle tone and pathogenesis of cerebral vasospasm. In this study, we adopted the confocal laser microscopy to detect the fluorescence signals arising from the individual smooth muscle cells of canine basilar artery. Ring preparations were made, loaded with fluo-3 and changes in fluorescence induced by high K+ and endothelin-1 (ET-1) were measured by confocal laser microscopy. In some unstimulated smooth muscle cells Ca2+ waves arising from discrete region of the cell propagated to the whole cell with a velocity of approximately 10 microm/s. High K+ (80 mmol/L) induced a rapid rise in [Ca2+]i, the peak level being consistently reached approximately 10 s after stimulation. In contrast, the time to peak level of [Ca2+]i induced by ET-1 (0.3 micromol/L) varied widely between 13 and 26 s among individual cells, an indication that the extent of nonuniform coordination of increases in [Ca2+]i in individual cells may be partly responsible for the different time courses of tension development of vascular smooth muscle in response to the vasoactive stimulants. The increase in [Ca2+]i induced by ET-1 was transient but a pronounced and sustained contraction developed further in response to ET-1. Thus ET-1 has a biological property as a potential candidate to elicit cerebral vasospasm. Confocal laser microscopy could be a useful tool to measure the changes in [Ca2+]i in individual smooth muscle cells of cerebral artery.     

Relationship between asynchronous Ca2+ waves and force development in intact smooth muscle bundles of the porcine trachea

Fluctuations in intracellular calcium concentration ([Ca2+]i) constitute the main link in excitation-contraction coupling (E-C coupling) in airway smooth muscle cells (ASMC). It has recently been reported that ACh induces asynchronous recurring Ca2+ waves in intact ASMC of murine bronchioles. With the use of a novel technique allowing us to simultaneously measure subcellular [Ca2+]i and force generation in ASMC located within an intact tracheal muscle bundle, we examined a similar pattern of Ca2+ signaling in the trachea. We found that application of ACh resulted in the generation of recurring intracellular Ca2+ waves progressing along the longitudinal axis of the ribbon-shaped intact ASMC. These Ca2+ waves were not synchronized between neighboring cells, and induction of wave-like [Ca2+]i oscillations was temporally associated with development of force by the tracheal muscle bundle. By comparing the concentration dependence of force generation and the parameters characterizing the [Ca2+]i oscillations, we found that the concentration-dependent increase in ACh-induced force development by the tracheal smooth muscle bundle is achieved by differential recruitment of intact ASMC to initiate Ca2+ waves and by enhancement in the frequency of [Ca2+]i oscillations and elevation of interspike [Ca2+]i once the cells are recruited. Our findings demonstrate that asynchronous recurring Ca2+ waves underlie E-C coupling in ACh-induced contraction of the intact tracheal smooth muscle bundle. Furthermore, in contrast to what was reported in enzymatically dissociated ASMC, Ca2+ influx through the L-type voltage-gated Ca2+ channel was not an obligatory requirement for the generation of [Ca2+]i oscillations and development of force in ACh-stimulated intact ASMC.     

Age differences in cholinergic airway responsiveness in relation with muscarinic receptor subtypes

Children seem more susceptible to increased airway reactivity than adults. Such an age-dependent discrepancy in airway reactivity may involve different airway smooth muscle functions. Therefore, we compared the in vivo and in vitro responsiveness of airway smooth muscles between two age groups of animals. Rats of 6 and 21 weeks old were challenged in vivo with acetylcholine (ACh) infused intravenously and airway resistance (R(aw)) was measured. Tracheal muscle was also isolated and the isometric force developed to ACh or KCl was measured. Furthermore, the level of genes encoding muscarinic receptor subtypes (M(1-3)) and acetylcholinesterase (AChE) expressed in the tracheal muscle was determined by RT-PCR. In results, the basal R(aw) was similar in the two age groups. The R(aw) at each ACh dose was significantly greater in young rats than older rats (p<0.05, n=22-27). Tracheal muscles from young rats were more sensitive to ACh than older rats (p<0.05, n=20-21), while receptor-independent muscle contraction to KCl was greater in older rats (p<0.05, n=10-19). Genes encoding AChE, M(2) and M(3) muscarinic receptors were more highly expressed in the tracheal muscles from young than older rats (p<0.05, n=4-6). In conclusion, airway smooth muscle in young rat is more sensitive to cholinergic stimulation in vivo and in vitro compared to older rats, which may be due to a higher expression of M(2) and M(3) muscarinic receptors in airway smooth muscle.     

Endothelin-1 inhibits and enhances contraction of porcine coronary arterial strips with an intact endothelium.

Using front-surface fluorometry and fura-2-loaded porcine coronary arterial strips with an intact endothelium, changes in cytosolic Ca2+ concentrations ([Ca2+]i) and tension of smooth muscle were simultaneously monitored in an attempt to determine the vasoactive properties of endothelin-1 (ET-1). ET-1 in low concentrations (0.1-1nM) caused a significant transient decrease in [Ca2+]i and tension of the strips precontracted with 10(-7) M U-46619. The maximal decreases in [Ca2+]i and tension were obtained with 0.6nM ET-1. In higher concentrations (1nM-100nM), there was no reduction in [Ca2+]i or tension; the contraction induced by U-46619 was potentiated. The decreases in [Ca2+]i and tension induced by ET-1 were inhibited by the mechanical removal of the endothelium or by pretreatment with NG-nitro-L-arginine and were slightly attenuated by indomethacin. Thus, ET-1 in low concentrations can induce endothelium-dependent transient relaxations accompanied by transient reductions of [Ca2+]i in isolated porcine coronary arteries. This effect is mainly mediated by the release of endothelium-derived relaxing factor.     

Cell calcium and its regulation in smooth muscle

Two novel methods used to study smooth muscles-electron probe X-ray microanalysis and Ca2+-sensitive indicators (which are used for resolving, respectively, the spatial distribution and temporal distribution of calcium)-are briefly reviewed and the major findings obtained are summarized. In smooth muscle the sarcoplasmic reticulum is the major intracellular source of Ca2+; mitochondria do not play a significant role in the physiological regulation of [Ca2+]i. Under pathological conditions mitochondria can reversibly accumulate large amounts of calcium. Resting [Ca2+]i generally ranges from 80 to 200 nM, and is lower in phasic than in tonic smooth muscles. Removal of extracellular Ca2+ and Ca2+ entry blockers can reduce [Ca2+]i, but the effects of beta-adrenergic agents are variable. Increases in [Ca2+]i are triggered by electrical stimulation, depolarization with high K+, and excitatory agonists. Stretch, after a delay of several seconds, can cause an increase in [Ca2+]i in some smooth muscles. There is also a delay of approximately 200-400 ms between the initiation of the rise of Ca2+ and contraction that follows spontaneous action potentials or electrical stimulation. Agonist-induced Ca2+ release, a major mechanism of pharmacomechanical coupling, has been demonstrated in smooth muscles depolarized with high K; evidence suggests that it is mediated by G proteins that couple receptors to phospholipase C. Ca2+ release can be triggered directly in permeabilized smooth muscle with inositol 1,4,5-trisphosphate. Even though Ca2+ is the major physiological regulator of contraction, Ca2+ sensitivity of the regulatory-contractile apparatus differs in different (phasic and tonic) smooth muscles, and can be modulated in a given smooth muscle. The force [Ca2+]i ratio is higher during agonist-stimulated than during high K+-induced contractions, owing to agonist-induced increases in Ca2+ sensitivity mediated by G proteins. In some phasic smooth muscles (guinea pig ileum), the time course of the initial myosin light chain phosphorylation is extremely rapid and returns to basal levels while force remains elevated. In these smooth muscles there is also a marked decrease in the Ca2+ sensitivity of the regulatory-contractile apparatus during maintained depolarization in Ca2+-free or low Ca2+ solutions. It has been suggested that regulation of myosin light chain phosphatase plays a major role in the modulation of the Ca2+ sensitivity manifested as either potentiation or desensitization to [Ca2+]i.     

Expression and coupling of neurokinin receptor subtypes to inositol phosphate and calcium signaling pathways in human airway smooth muscle cells

Neuropeptide tachykinins (substance P, neurokinin A, and neurokinin B) are present in peripheral terminals of sensory nerve fibers within the respiratory tract and cause airway contractile responses and hyperresponsiveness in humans and most mammalian species. Three subtypes of neurokinin receptors (NK1R, NK2R, and NK3R) classically couple to Gq protein-mediated inositol 1,4,5-trisphosphate (IP3) synthesis and liberation of intracellular Ca2+, which initiates contraction, but their expression and calcium signaling mechanisms are incompletely understood in airway smooth muscle. All three subtypes were identified in native and cultured human airway smooth muscle (HASM) and were subsequently overexpressed in HASM cells using a human immunodeficiency virus-1-based lentivirus transduction system. Specific NKR agonists {NK1R, [Sar9,Met(O2)11]-substance P; NK2R, [beta-Ala8]-neurokinin A(4-10); NK3R, senktide} stimulated inositol phosphate synthesis and increased intracellular Ca2+ concentration ([Ca2+]i) in native HASM cells and in HASM cells transfected with each NKR subtype. These effects were blocked by NKR-selective antagonists (NK1R, L-732138; NK2R, GR-159897; NK3R, SB-222200). The initial transient and sustained phases of increased [Ca2+]i were predominantly inhibited by the IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) or the store-operated Ca2+ channel antagonist SKF-96365, respectively. These results show that all three subtypes of NKRs are expressed in native HASM cells and that IP3 levels are the primary mediators of NKR-stimulated initial [Ca2+]i increases, whereas store-operated Ca2+ channels mediate the sustained phase of the [Ca2+]i increase.     

Impaired endothelin-induced vasoconstriction in coronary arteries of Zucker obese rats is associated with uncoupling of [Ca2+]i signaling

Although insulin resistance (IR) is a major risk factor for coronary artery disease, little is known about the regulation of coronary vascular tone in IR by endothelin-1 (ET-1). We examined ET-1 and PGF(2alpha)-induced vasoconstriction in isolated small coronary arteries (SCAs; approximately 250 microM) of Zucker obese (ZO) rats and control Zucker lean (ZL) rats. ET-1 response was assessed in the absence and presence of endothelin type A (ET(A); BQ-123), type B (ET(B); BQ-788), or both receptor inhibitors. ZO arteries displayed reduced contraction to ET-1 compared with ZL arteries. In contrast, PGF(2alpha) elicited similar vasoconstriction in both groups. ET(A) inhibition diminished the ET-1 response in both groups. ET(B) inhibition alone or in combination with ET(A) blockade, however, restored the ET-1 response in ZO arteries to the level of ZL arteries. Similarly, inhibition of endothelial nitric oxide (NO) synthase with N(omega)-nitro-l-arginine methyl ester (l-NAME) enhanced the contraction to ET-1 and abolished the difference between ZO and ZL arteries. In vascular smooth muscle cells from ZO, ET-1-induced elevation of myoplasmic intracellular free calcium concentration ([Ca2+]i) (measured by fluo-4 AM fluorescence), and maximal contractions were diminished compared with ZL, both in the presence and absence of l-NAME. However, increases in [Ca2+]i elicited similar contractions of the vascular smooth muscle cells in both groups. Analysis of protein and total RNA from SCA of ZO and ZL revealed equal expression of ET-1 and the ET(A) and ET(B) receptors. Thus coronary arteries from ZO rats exhibit reduced ET-1-induced vasoconstriction resulting from increased ET(B)-mediated generation of NO and diminished elevation of myoplasmic [Ca2+]i.     

Role of capacitative Ca2+ entry in bronchial contraction and remodeling.

Asthma is characterized by airway inflammation, bronchial hyperresponsiveness, and airway obstruction by bronchospasm and bronchial wall thickening due to smooth muscle hypertrophy. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) may serve as a shared signal transduction element that causes bronchial constriction and bronchial wall thickening in asthma. In this study, we examined whether capacitative Ca2+ entry (CCE) induced by depletion of intracellular Ca2+ stores was involved in agonist-mediated bronchial constriction and bronchial smooth muscle cell (BSMC) proliferation. In isolated bronchial rings, acetylcholine (ACh) induced a transient contraction in the absence of extracellular Ca2+ because of Ca2+ release from intracellular Ca2+ stores. Restoration of extracellular Ca2+ in the presence of atropine, an M-receptor blocker, induced a further contraction that was apparently caused by a rise in [Ca2+]cyt due to CCE. In single BSMC, amplitudes of the store depletion-activated currents (I(SOC)) and CCE were both enhanced when the cells proliferate, whereas chelation of extracellular Ca2+ with EGTA significantly inhibited the cell growth in the presence of serum. Furthermore, the mRNA expression of TRPC1, a transient receptor potential channel gene, was much greater in proliferating BSMC than in growth-arrested cells. Blockade of the store-operated Ca2+ channels by Ni2+ decreased I(SOC) and CCE and markedly attenuated BSMC proliferation. These results suggest that upregulated TRPC1 expression, increased I(SOC), enhanced CCE, and elevated [Ca2+]cyt may play important roles in mediating bronchial constriction and BSMC proliferation.     

Nicotonic and muscarinic components in acetylcholine stimulation of porcine adrenal medullary cells

Adrenal medullary chromaffin cells secrete catecholamines (CA) in response to cholinergic receptor activation by acetylcholine (ACh) released from splacnic nerve terminals. In cultured bovine chromaffin cells nicotinic receptors play a preponderant (> 90%) role in the control of CA release. By contrast, we found and report here that up to 40% of the ACh-evoked CA secretion from cultured porcine chromaffin cells can be associated with muscarinic receptor activation. The following results support our belief that in porcine adrenal medullary cells ACh (100 M) evoked CA secretion is mediated by both nicotinic and muscarinic cholinergic receptors. 1) Hexamethonium (100 M), a nicotinic receptor antagonist, inhibited ACh-induced CA secretion to ca. 40% of the control release and atropine (1 M), a muscarinic receptor antagonist, inhibited to ca. 60% of the control value. 2) We also found that ACh (100 M) evoked intracellular Ca2+ concentration ([Ca2+]i) rise was inhibited by these receptor antagonists to a different extent, and reversibly reduced by lowering the concentration of Ca2+ in the external medium ([Ca2+]o). This last maneuver ([Ca2+]o < 0.1 M) per se caused a marked reduction in the peak phase of the [Ca2+]i rise evoked by ACh (40% of the control response). Switching the external medium back to physiologic [Ca2+]o in the continued presence of ACh caused a partial recovery of the elevated [Ca2+]i. This [Ca2+]o-dependent [Ca2+]i rise was blocked by hexamethonium (100 M) but not by atropine (1 M). Conversely, the ACh-evoked [Ca2+]i rise in low external [Ca2+]o was blocked by atropine but not by hexamethonium. From these data we conclude that in porcine adrenal medullary cells an important fraction (ca. 0.4) of both ACh-induced CA secretion and peak [Ca2+]i rise is due to muscarinic receptor activation.     

Agonist activation of cytosolic Ca2+ in subfornical organ cells projecting to the supraoptic nucleus

The subfornical organ (SFO) is sensitive to both ANG II and ACh, and local application of these agents produces dipsogenic responses and vasopressin release. The present study examined the effects of cholinergic drugs, ANG II, and increased extracellular osmolarity on dissociated, cultured cells of the SFO that were retrogradely labeled from the supraoptic nucleus. The effects were measured as changes in cytosolic calcium in fura 2-loaded cells by using a calcium imaging system. Both ACh and carbachol increased intracellular ionic calcium concentration ([Ca2+]i). However, in contrast to the effects of muscarinic receptor agonists on SFO neurons, manipulation of the extracellular osmolality produced no effects, and application of ANG II produced only moderate effects on [Ca2+]i in a few retrogradely labeled cells. The cholinergic effects on [Ca2+]i could be blocked with the muscarinic receptor antagonist atropine and with the more selective muscarinic receptor antagonists pirenzepine and 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP). In addition, the calcium in the extracellular fluid was required for the cholinergic-induced increase in [Ca2+]i. These findings indicate that ACh acts to induce a functional cellular response in SFO neurons through action on a muscarinic receptor, probably of the M1 subtype and that the increase of [Ca2+]i, at least initially, requires the entry of extracellular Ca2+. Also, consistent with a functional role of M1 receptors in the SFO are the results of immunohistochemical preparations demonstrating M1 muscarinic receptor-like protein present within this forebrain circumventricular organ.     

Production of nitric oxide from endothelial cells by 31-amino-acid-length endothelin-1, a novel vasoconstrictive product by human chymase

Human chymase selectively converts big endothelin (ET)-1 to 31-amino-acid-length ET-1 [ET-1(1-31)]. In this study we examined effect of ET-1(1-31) on endothelial function. ET-1(1-31) evoked contraction in a concentration-dependent manner at > 10(-8) M, which was about 10 times weaker than that of conventional ET-1 [ET-1(1-21)]. BQ485, an ETA receptor antagonist, completely abolished ET-1(1-31)-induced contraction, but BQ788, an ETB receptor antagonist, slightly enhanced it, suggesting that ET-1(1-31) relaxes artery via endothelium. On endothelial cells, ET-1(1-21) and ET-1(1-31) increased [Ca2+]i and produced NO, both of which were significantly inhibited by BQ788 and not by BQ485. These results indicate that ET-1(1-31) increased [Ca2+]i and produced NO in endothelial cells through ETB receptor similarly with ET-1(1-21), although slight difference in effect on smooth muscle cells.     

Muscarinic receptor reserve of airway smooth muscle and the response to isoproterenol

It has been hypothesized that the muscarinic receptor reserve for contraction of airway smooth muscle is an important determinant of the potency with which isoproterenol relaxes submaximal muscarinic contractions. The goals of this study were to inactivate, with phenoxybenzamine, a fraction of the muscarinic receptors present in canine tracheal smooth muscle, and then to determine whether this decrease in muscarinic receptor reserve altered the potency with which isoproterenol relaxed submaximal muscarinic contractions. Strips of smooth muscle were suspended from force transducers in vitro and preincubated with either vehicle (untreated) or phenoxybenzamine (10(-5) M) for 30 min. For muscarinic contractions induced by carbachol that were approximately 70-80% of maximum, the half-maximally effective concentration of isoproterenol was 2.4 +/- 0.8 x 10(-7) M for untreated strips but 5.8 +/- 1.3 x 10(-9) M for strips treated with phenoxybenzamine (n = 6, P less than 0.05). We concluded that treatment with phenoxybenzamine increased the sensitivity of a submaximal muscarinic contraction to isoproterenol. The results support the hypothesis that the muscarinic receptor reserve for contraction is an important determinant of the potency with which isoproterenol relaxes submaximal muscarinic contractions.     

Contractile role of M2 and M3 muscarinic receptors in gastrointestinal, airway and urinary bladder smooth muscle

Both M(2) and M(3) muscarinic receptors are expressed in smooth muscle and influence contraction through distinct signaling pathways. M(3) receptors interact with G(q) to trigger phosphoinositide hydrolysis, Ca(2+) mobilization and a direct contractile response. In contrast, M(2) receptors interact with G(i) and G(o) to inhibit adenylyl cyclase and Ca(2+)-activated K(+) channels and to potentiate a Ca(2+)-dependent, nonselective cation conductance. Ultimately, these mechanisms lead to the prediction that the influence of the M(2) receptor on contraction should be conditional upon mobilization of Ca(2+) by another receptor such as the M(3). Mathematical modeling studies of these mechanisms show that the competitive antagonism of a muscarinic response mediated through activation of both M(2) and M(3) receptors should resemble the profile of the directly acting receptor (i.e., the M(3)) and not that of the conditionally acting receptor (i.e., the M(2)). Using a combination of pharmacological and genetic approaches, we have identified two mechanisms for the M(2) receptor in contraction: 1) a high potency inhibition of the relaxation elicited by agents that increase cytosolic cAMP and 2) a low potency potentiation of contractions elicited by the M(3) receptor. The latter mechanism may be involved in muscarinic agonist-mediated heterologous desensitization of smooth muscle, which requires activation of both M(2) and M(3) receptors.     

Muscarinic receptor subtypes modulating smooth muscle contractility in the urinary bladder

Normal physiological voiding as well as generation of abnormal bladder contractions in diseased states is critically dependent on acetylcholine-induced stimulation of contractile muscarinic receptors on the smooth muscle (detrusor) of the urinary bladder. Muscarinic receptor antagonists are efficacious in treating the symptoms of bladder hyperactivity, such as urge incontinence, although the usefulness of available drugs is limited by undesirable side-effects. Detrusor smooth muscle is endowed principally with M2 and M3 muscarinic receptors with the former predominating in number. M3 muscarinic receptors, coupled to stimulation of phosphoinositide turnover, mediate the direct contractile effects of acetylcholine in the detrusor. Emerging evidence suggests that M2 muscarinic receptors, via inhibition of adenylyl cyclase, cause smooth muscle contraction indirectly by inhibiting sympathetically (beta-adrenoceptor)-mediated relaxation. In certain diseased states, M2 receptors may also contribute to direct smooth muscle contraction. Other contractile mechanisms involving M2 muscarinic receptors, such as activation of a non-specific cationic channel and inactivation of potassium channels, may also be operative in the bladder and requires further investigation. From a therapeutic standpoint, combined blockade of M2 and M3 muscarinic receptors would seem to be ideal since this approach would evoke complete inhibition of cholinergically-evoked smooth muscle contractions. However, if either the M2 or M3 receptor assumes a greater pathophysiological role in disease states, then selective antagonism of only one of the two receptors may be the more rational approach. The ultimate therapeutic strategy is also influenced by the extent to which pre-junctional M1 facilitatory and M2 inhibitory muscarinic receptors regulate acetylcholine release and also which subtypes mediate the undesirable effects of muscarinic receptor blockade such as dry mouth. Finally, the consequence of muscarinic receptor blockade in the central nervous system on the micturition reflex, an issue which is poorly studied and seldom taken into consideration, should not be ignored.     

Lan-1: A Human Neuroblastoma Cell Line With M1 and M3 Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization

The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin.