Regulation of ciliary beat frequency by autonomic mechanisms: in vitro

The ciliated epithelium of the mammalian trachea separates the neurohumoral milieu of the tissue from that of the environment of the airway lumen. To determine whether specific autonomic receptors regulating ciliary beat frequency (CBF) were located on mucosal or serosal sides, we measured CBF by heterodyne mode correlation analysis laser light scattering in bovine tracheal tissues mounted in a two-sided chamber. A beta 2-adrenergic agonist, fenoterol, at 10(-7) M, stimulated serosal CBF from 7.9 +/- 1.3 to 20.2 +/- 5.8 Hz (P less than 0.01) and mucosal CBF from 6.6 +/- 0.9 to 14.7 +/- 4.6 Hz (P less than 0.01). A muscarinic cholinergic agonist, methacholine, at 10(-7) M, increased mucosal CBF from 8.4 +/- 1.0 to 19.5 +/- 5.5 Hz (P less than 0.01) and serosal CBF from 8.0 +/- 0.9 to 15.4 +/- 5.0 Hz (P less than 0.01). The differences in stimulation of CBF on the mucosal and serosal sides between fenoterol and methacholine were significant (P less than 0.01). Studies in which these autonomic agonist stimulating effects were inhibited by their respective antagonists, propranolol and atropine sulfate, demonstrated that CBF can be regulated independently by mediators both in the submucosa and within the mucus lining.     

Pathways of substance P stimulation of canine tracheal ciliary beat frequency.

Substance P (SP), an inflammatory neuropeptide, may be released by intraepithelial nerves in response to an irritant or inflammatory stimulus. To investigate the neural and humoral pathways mediating the response of tracheal ciliary beat frequency (CBF) to topically applied SP, CBF was measured on the ventral midtracheal surface of anesthetized beagles by using heterodyne-mode correlation analysis laser light scattering. In the first study, aerosolized SP, delivered to the lungs of eight beagle dogs, stimulated CBF in a dose-dependent manner from a baseline of 4.9 +/- 0.4 Hz to a maximum of 14.9 +/- 1.5 Hz at dose of 10(-7) M. In the second study, the tracheal lumen was isolated from the bronchial airways by inflating the cuff of an endotracheal tube near the carina. Intravenous hexamethonium bromide (2 mg/kg), ipratropium bromide (0.5 micrograms/kg), and indomethacin (2 mg/kg) were used as blocking agents to inhibit the nicotinic, muscarinic, and cyclooxygenase pathways, respectively. Aerosolized 10(-9), 10(-8), or 10(-7) M SP was delivered sequentially to the tracheal lumen for 3 min at 30-min intervals. SP caused two distinct CBF stimulatory episodes at 4 min (mean time of the maximal response) and at 18 min (mean time of the maximal response) after onset of delivery and returned to baseline after 25 min. SP stimulated CBF from the baseline of 5.1 +/- 0.4 Hz to a maximum of 14.2 +/- 2.5 Hz during the first episode (P less than 0.01) and to 10.4 +/- 0.6 Hz during the second episode (P less than 0.01) at dose of 10(-8) M. These responses were inhibited by all the blocking agents. These data suggest that SP stimulates CBF via a cyclooxygenase-dependent parasympathetic reflex.     

Effects of albuterol enantiomers on ciliary beat frequency in ovine tracheal epithelial cells.

beta(2)-Adrenergic agonists stimulate ciliary beat frequency (CBF), an integral part of mucociliary clearance. To evaluate the differential effects of albuterol enantiomers and their racemic mixture on ciliary function, CBF and intracellular calcium were measured at room temperature from single ovine airway epithelial cells with use of digital videomicroscopy. Baseline CBF was 7.2 +/- 0.2 (SE) Hz (n = 80 measurements). R-albuterol (10 microM to 1 mM) stimulated CBF in a dose-dependent manner to maximally 24.4 +/- 5.4% above baseline. Racemic albuterol stimulated CBF to maximally 12.8 +/- 3.6% above baseline, a significantly lower increase compared with R-albuterol alone, despite identical R-enantiomer amounts in both groups. Simultaneous recordings of intracellular calcium concentration and CBF from single cells indicated that the CBF increase in response to R-albuterol was mediated through beta-receptors and stimulation of protein kinase A, in a calcium-dependent and -independent fashion. S-albuterol had a negligible effect on CBF and did not change intracellular calcium. Together, these results suggest that R-albuterol is more efficacious than racemic albuterol in stimulating CBF. Thus S-albuterol may interfere with the ability of R-albuterol to increase CBF.     

Stimulation of tracheal ciliary beat frequency by capsaicin

To determine the possible involvement of neural and cyclooxygenase pathways whereby irritants might affect cilia activity in vivo, the temporal response of canine tracheal ciliary beat frequency (CBF) to the inhaled surrogate irritant capsaicin was studied. CBF was measured on the ventral midtracheal surface of barbiturate-anesthetized eucapnically ventilated beagle dogs by heterodyne-mode laser light scattering. After base-line CBF was established, hexamethonium bromide (2 mg/kg iv), ipratropium bromide (0.5 microgram/kg iv), indomethacin (2 mg/kg iv), or intravenous 0.9% saline was administered. Aerosolized 3 Z 10(-9) M capsaicin in 0.9% saline was delivered for 2 min, and CBF was measured for the following 60 min. Control experiments used 0.9% saline sham aerosol with a 0.9% saline sham block. Aerosolized capsaicin stimulated CBF from a base line of 6.2 +/- 1.4 (SD) Hz (n = 230) to a mean maximum of 17.7 +/- 7.3 Hz (n = 16) 23 min after aerosol delivery, and CBF returned to base line within 60 min. Neither hexamethonium bromide, ipratropium bromide, nor indomethacin changed CBF from base-line values. The episodic CBF stimulatory response to capsaicin after commencement of aerosol was completely inhibited by hexamethonium bromide. Ipratropium bromide partially inhibited the first 15 min and totally inhibited the following 45 min of stimulatory response. Indomethacin inhibited the initial 15 min but had less effect on the following 45 min of stimulatory response. These data indicate that multiple stimulatory mechanisms function over a prolonged period of time to affect the removal of irritants from the airways and that these mechanisms differ from those involved in the maintenance of basal CBF.     

Ciliary activity in the oviduct of cycling, pregnant, and muscarinic receptor knockout mice

The transport of the oocyte and the embryo in the oviduct is managed by ciliary beating and muscular contractions. Because nonneuronally produced acetylcholine influences ciliary beating in the trachea via the muscarinic receptors M2 and M3, we supposed that components of the cholinergic system may also modulate ciliary activity in the oviduct. To address this issue, we analyzed the expression profile of muscarinic receptors (CHRMs) in the murine oviduct by RT-PCR and assessed ciliary beat frequency (CBF) and cilia-driven particle transport speed (PTS) on the mucosal surface of opened oviductal segments in correlation with histomorphological investigations. RT-PCR of laser-assisted microdissected epithelium revealed expression of Chrm subtypes Chrm1 and Chrm3. In opened isthmic segments, particle transport was barely seen, correlating with a significantly lower number of ciliated cells compared to the ampulla. In the ampulla, basal PTS and CBF were high (71 μm/sec and 21 Hz, respectively) both in cycling and pregnant wild-type mice and in mice with targeted deletion of the Chrm genes Chrm1, Chrm3, Chrm4, and Chrm5. In contrast to the trachea, where basal ciliary activity was low and largely enhanced by muscarinic stimulation, muscarinic agonists and antagonists did not affect the high ampullar PTS. Our results imply that this high oviductal autonomous ciliary activity is independent from the intrinsic cholinergic system and serves to maintain optimal clearance of the tube throughout all stages of the estrous cycle and early pregnancy.     

Time course of isolated rat fundus response to muscarinic agonists: a measure of intrinsic efficacy.

The establishment of a dose-response relationship and its quantification is the usual procedure for analysing drug action on an isolated organ. However, the time course of the effect seems to be an inherent characteristic of the agonist which produces it. In our study, we have analyzed the time-response curves of four cholinergic agonists (acetylcholine, methacholine, carbachol and bethanechol) which produce tonic contractions of the isolated rat gastric fundus. The order of affinity of agonists to muscarinic receptors on the rat fundus were carbachol > bethanechol > methacholine > acetylcholine (K(A) values: 46 +/- 12, 84 +/- 21, 380 +/- 110 and 730 +/- 120 nM, respectively). The effective concentrations which produced 60% of the maximal response (EC60) were used for establishing the time-response curves. The time-response curves were also recorded after partial alkylation of muscarinic receptors with phenoxybenzamine, after exposure of the isolated rat fundus to physostigmine and after addition of supramaximal concentrations of the agonists. The experimental time-response curve for acetylcholine was on the extreme left, followed by curves for methacholine, bethanechol and carbachol, respectively. Phenoxybenzamine and supramaximal doses of the agonists did not change the order of response development in time, but supramaximal doses shifted all curves to the left and phenoxybenzamine shifted all time-response curves to the right. Only physostigmine shifted the time-response curve for methacholine to the right. The results of our study suggest that the response rate of the isolated rat gastric fundus to cholinergic agonists depends on the intrinsic activity of these agents, but not on their affinity for muscarinic receptors.     

Mechanisms of airway hypercontractility in basenji-greyhound dogs

The comparative effects of contractile agonists and physiological stimulation of the tracheal and bronchial smooth muscle (BSM) response were studied isometrically in situ in five Basenji-greyhound (BG) and six mongrel dogs. Frequency-response curves generated by bilateral stimulation of the vagus nerves (0-20 Hz, 15-20 V, 2-ms duration) elicited greater maximal contraction in mongrel trachea (36.8 +/- 8.1 vs. 26.9 +/- 4.0 g/cm; P less than 0.02) and exhibited greater responsiveness in mongrel BSM (half-maximal response to electrical stimulation 3.0 +/- 1.1 vs. 7.0 +/- 0.5 Hz; P less than 0.05) compared with BG dogs. However, muscarinic sensitivity to intravenous methacholine (MCh) was substantially greater in BG dogs; MCh caused contraction greater than 1.5 g/cm at a mean dose of 3.0 X 10(-10) mol/kg for BG dogs compared with 5.1 X 10(-9) mol/kg for mongrel controls (P less than 0.03, Mann-Whitney rank-sum test). In contrast to the muscarinic response, the contractile response elicited by intravenous norepinephrine after beta-adrenergic blockade was similar in trachea and bronchus for both mongrel and BG dogs. Our data confirm previous in vitro demonstration of tracheal hyporesponsiveness in BG dogs and demonstrate that the contraction resulting from efferent parasympathetic stimulation is less in the BG than mongrel dogs. However, postsynaptic muscarinic responsiveness of BG BSM is substantially increased. We conclude that a component of airway responsiveness in BG dogs depends directly on contractile forces generated postsynaptically that are nongeometry dependent, postjunctional, and agonist specific.     

Ciliogenesis in cryopreserved mammalian tracheal epithelial cells cultured at the air–liquid interface

To determine air–liquid interface (ALI) culture derived from cryopreserved mammalian tracheal ciliated cells is a viable ciliated cell model for the investigations of regulatory mechanisms of ciliary beat frequency (CBF), two studies were performed using ovine and porcine tracheae obtained from local slaughterhouses. The protease-digested tracheal ciliated cells were harvested and cultured at the ALI using collagen-coated, porous membrane inserts. In study 1, the ALI culturing protocols were established using non-cryopreserved ovine tracheal ciliated cells. Ciliogenesis was documented with immuno-histology and electron micrographs. Vigorous beating cilia were video-recorded. CBF was measured by laser light scattering. The functional integrity of the autonomic receptors of the ciliated cells was confirmed with the stimulatory responses of CBF using luminal methacholine and basolateral terbutaline. In study 2, porcine tracheal ciliated cells stored in liquid nitrogen for a minimum of 4 weeks were used. The cryopreserved cells were thawed and cultured using the ALI protocol established in study 1. After two months, cilia outgrowths were confirmed using video microscopy and scanning electron micrograph (SEM). The trans-epithelial resistances were 28.5 kΩ (n = 4). Luminal applications of 1 μM and 10 μM methacholine stimulated CBF from a baseline of 7.4 ± 0.2 Hz to 8.4 ± 0.8 Hz and 7.7 ± 0.4 Hz, respectively (n = 5). Basolateral applications of 1 μM and 10 μM terbutaline stimulated CBF from a baseline of 7.5 ± 0.3 Hz to 8.2 ± 0.4 Hz and 8.0 ± 0.4 Hz, respectively (n = 5). These data demonstrated that a ciliated cell bank can be established using cryopreserved ciliated cells for pulmonary drug discovery and toxicological screening.     

Stimulation of tracheal ciliary beat frequency by localized tissue incision

Since measurements of basal ciliary beat frequency (CBF) were significantly lower in our intact canine experiments than reports of ciliary activity in rabbits involving surgical intervention, we hypothesized that local tissue trauma stimulates CBF. The effects of minor neck surgery on tracheal CBF in eight barbiturate-anesthetized eucapnically ventilated beagles were investigated. Each dog underwent two studies. Measurements of CBF were made at 1-min intervals on the right lateral midtracheal surface by means of heterodyne mode correlation analysis laser light scattering. In the control study, CBF was measured in each dog for at least 160 min. In the incision study, base-line CBF was measured for at least 40 min. The overlying sternohyoidus muscles were then separated, and a longitudinal 2- to 3-cm incision was made in the trachea caudally from the fourth to the fifth cartilage ring. CBF was measured at least 5 cm distally from the site of tracheal injury for an additional 120 min. Electrocardiogram, rectal temperature, tracheal pressure, exhaled CO2, and arterial blood pressure, PO2, PCO2, and pH remained stable throughout both studies. The mean base-line CBF was 4.7 +/- 0.4 Hz. It increased to 19.5 +/- 2.9 Hz (P less than 0.0001) 100 min after the incision and remained elevated until the end of the study period (P less than 0.0001). The mechanism(s) causing this stimulation may also be responsible for the high "basal" CBF observed in other studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine activation of A(2B) receptor(s) is essential for stimulated epithelial ciliary motility and clearance

Mucociliary clearance, vital to lung clearance, is dependent on cilia beat frequency (CBF), coordination of cilia, and the maintenance of periciliary fluid. Adenosine, the metabolic breakdown product of ATP, is an important modulator of ciliary motility. However, the contributions of specific adenosine receptors to key airway ciliary motility processes are unclear. We hypothesized that adenosine modulates ciliary motility via activation of its cell surface receptors (A(1), A(2A), A(2B), or A(3)). To test this hypothesis, mouse tracheal rings (MTRs) excised from wild-type and adenosine receptor knockout mice (A(1), A(2A), A(2B), or A(3), respectively), and bovine ciliated bronchial epithelial cells (BBECs) were stimulated with known cilia activators, isoproterenol (ISO; 10 μM) and/or procaterol (10 μM), in the presence or absence of 5'-(N-ethylcarboxamido) adenosine (NECA), a nonselective adenosine receptor agonist [100 nM (A(1), A(2A), A(3)); 10 μM (A(2B))], and CBF was measured. Cells and MTRs were also stimulated with NECA (100 nM or 10 μM) in the presence and absence of adenosine deaminase inhibitor, erythro-9- (2-hydroxy-3-nonyl) adenine hydrochloride (10 μM). Both ISO and procaterol stimulated CBF in untreated cells and/or MTRs from both wild-type and adenosine knockout mice by ~3 Hz. Likewise, CBF significantly increased ~2-3 Hz in BBECs and wild-type MTRs stimulated with NECA. MTRs from A(1), A(2A), and A(3) knockout mice stimulated with NECA also demonstrated an increase in CBF. However, NECA failed to stimulate CBF in MTRs from A(2B) knockout mice. To confirm the mechanism by which adenosine modulates CBF, protein kinase activity assays were conducted. The data revealed that NECA-stimulated CBF is mediated by the activation of cAMP-dependent PKA. Collectively, these data indicate that purinergic stimulation of CBF requires A(2B) adenosine receptor activation, likely via a PKA-dependent pathway.     

Agonist-induced changes in the modulation of K+ permeability and beating rate by muscarinic agonists in cultured heart cells

The correlation between number of muscarinic cholinergic receptor sites as measured by binding of the muscarinic antagonist [3H]methylscopolamine ([3H]MS) and the ability of muscarinic agonists to mediate a physiologic response was determined in intact heart cells cultured from chick embryos 10 d in ovo. The increase in K+ permeability and the decrease in beating rate mediated by the muscarinic agonist carbamylcholine were the responses studied. Exposure to 10(-3) M carbamylcholine caused a 15% decrease in beating rate and a 33% increase in the rate of 42K+ efflux from cells labeled to equilibrium. An assay for binding of [3H]MS to intact cells was developed. [3H]MS bound specifically to intact heart cells (185 fmol/mg protein) with a Kd of 0.48 nM. Exposure of cells for various times to 10(-3) M carbamylcholine followed by binding of [3H]MS to intact cells demonstrated that a gradual loss of 70% of [3H]MS binding sites took place over the next 6 h with a T 1/2 of 30 min. A decrease in the ability of carbamylcholine to stimulate K+ efflux and to decrease beating rate was observed after pre-exposure of cells to muscarinic agonists. A close correlation was found between the loss of the subclass of muscarinic receptors subject to agonist control and the loss of physiologic responsiveness after agonist exposure. The data suggest the absence of significant numbers of "spare" receptors within this group.     

Thromboxane A2 mimetic U46619 stimulates ciliary motility of rabbit tracheal epithelial cells.

To elucidate whether thromboxane A2 (TxA2), one of the important arachidonic acid metabolites that may play a role in the development of airway inflammation, affects respiratory ciliary motility and, if so, what the mechanism of action is, we measured ciliary beat frequency (CBF) of rabbit cultured tracheal epithelium in response to U46619, a TxA2 mimetic agonist, by a photoelectric method. Addition of U46619 (10(-5) M) increased CBF from 17.7 +/- 0.7 to 22.8 +/- 1.4 Hz (mean +/- SE, p less than 0.01) within 5 min, which was followed by a decline to the baseline value by 10 min. This effect was concentration-dependent, the maximal increase from the baseline value and the drug concentration required to produce a half-maximal effect (EC50) being 26.9 +/- 4.6% (p less than 0.01) and 3 x 10(-7) M, respectively. The U46619-induced increase in CBF was abolished by SQ29548, and TxA2 receptor antagonist, and inhibited by verapamil, a Ca(2+)-entry blocker, and H-7, a protein kinase C inhibitor. These results suggest that TxA2 stimulates ciliary motility through the activation of airway epithelial TxA2 receptors, and that this effect may be exerted from Ca(2+)-influx and protein kinase C.     

Electrically evoked release of [(3)H]noradrenaline from mouse cultured sympathetic neurons: release-modulating heteroreceptors

Cultured neurons from the thoracolumbar sympathetic chain of newborn mice are known to possess release-inhibiting alpha(2)-autoreceptors. The present study was carried out in a search for release-modulating heteroreceptors on these neurons. Primary cultures were preincubated with [(3)H]noradrenaline and then superfused and stimulated by single pulses, trains of 8 pulses at 100 Hz, or trains of 36 pulses at 3 Hz. The cholinergic agonist carbachol reduced the evoked overflow of tritium. Experiments with antagonists indicated that the inhibition was mediated by M(2) muscarinic receptors. The cannabinoid agonist WIN 55,212-2 reduced the evoked overflow of tritium through CB(1) receptors. Prostaglandin E(2), sulprostone, and somatostatin also caused presynaptic inhibition. The inhibitory effects of carbachol, WIN 55,212-2, prostaglandin E(2), and somatostatin were abolished (at the highest concentration of WIN 55, 212-2 almost abolished) by pretreatment of the cultures with pertussis toxin (250 ng/ml). Several drugs, including the beta(2)-adrenoceptor agonist salbutamol, opioid receptor agonists, neuropeptide Y, angiotensin II, and bradykinin, failed to change the evoked overflow of tritium. These results demonstrate a distinct pattern of presynaptic inhibitory heteroreceptors, all coupled to pertussis toxin-sensitive G proteins. The lack of operation of several presynaptic receptors known to exist in adult mice in situ may be due to the age of the (newborn) donor animals or to the culture conditions.     

Tracheal mucosal blood flow responses to autonomic agonists

In lightly anesthetized adult sheep, we determined tracheal mucosal blood flow (Qtr) by measuring the steady-state uptake of dimethyl ether from a tracheal chamber created by an endotracheal tube provided with two cuffs. Qtr normalized for carotid arterial pressure [Qtr(n)] was determined before and after the exposure of the tracheal mucosa to aerosolized phenylephrine (0.25-2.0 mg), isoproterenol (0.05-0.8 mg), and methacholine (2.5-20 mg). The same doses of methacholine were also administered during the intravenous infusion of vasopressin. The measurements were repeated after intravenous pretreatment with the respective antagonists phentolamine, propranolol, and atropine. Mean +/- SE base-line Qtr(n) was 1.2 +/- 0.1 ml.min-1.mmHg-1.10(2). The autonomic antagonists had no effect on mean Qtr(n). Phenylephrine produced a dose-dependent decrease in mean Qtr(n) (-70% at the highest dose), which was blunted by phentolamine, and isoproterenol produced a dose-dependent increase in mean Qtr(n) (40% at the highest dose), which was blocked by propranolol. Methacholine failed to alter mean Qtr(n) even when Qtr was first decreased by vasopressin. We conclude that in lightly anesthetized adult sheep 1) base-line Qtr(n) is not under adrenergic or cholinergic control, 2) a locally administered alpha-adrenergic agonist decreases and beta-adrenergic agonist increases Qtr(n) via specific receptor activation, and 3) a locally administered cholinergic muscarinic agonist has no effect on Qtr(n).     

Cholinergic modulation of progesterone-induced maturation of Xenopus oocytes in vitro

Mixed and muscarinic cholinergic agonists (acetylcholine, carbamylcholine, methacholine, oxotremorine, and pilocarpine) accelerated in a dose-dependent manner the progesterone-induced maturation of Xenopus laevis oocytes. None of these agonists induced oocyte maturation in the absence of progesterone. The accelerating effect of cholinergic agonists was blocked in a dose-dependent manner by specific muscarinic antagonists (atropine and scopolamine) but not by specific nicotinic antagonists (d-tubocurarine and hexamethonium). The specific nicotinic agonist, dimethylphenylpiperazine, alone induced maturation in the absence of progesterone. The optimal promoting effect of acetylcholine was observed when oocytes were exposed to acetylcholine for 30 min, 5 min after the addition of progesterone, and was markedly better than when oocytes were exposed to acetylcholine throughout their incubation with progesterone. The effect of acetylcholine was observed in both follicle-enclosed and in defolliculated oocytes, indicating that follicular cells were not the target of the cholinergic drugs.     

Comparative effect of atropine on the adrenergic and muscarinic stimulation of phospholipid 32P labelling in isolated parotid cells: atropine, a possible blocker of alpha-adrenergic receptors

The inhibitory effect of atropine on phospholipid 32P labelling stimulated by muscarinic or alpha-adrenergic agonists was studied in isolated parotid cells. Atropine (10(-11) to 10(-4) M) had no effect on phospholipid 32P labelling in unstimulated cells. In contrast, 10(-8) to 10(-7) M atropine provoked a competitive inhibition of the cholinergic stimulation (i.e. this effect was completely wiped out at high agonist concentration). The atropine app. KD for the muscarinic receptor was 5 X 10(-9) M. Moreover, atropine inhibits the adrenergic stimulation of phospholipid 32P labelling by decreasing the efficacity and potency of the adrenergic agonists. The atropine app. KD for the alpha-adrenergic receptor can be estimated at 10(-5) M. This inhibition of alpha-adrenergic stimulation appears to be specific since atropine was without effect on the substance P or beta-adrenergic stimulation. At very low concentration (10(-10) - 10(-9) M) atropine seems to be a modulator (activator) of the muscarinic or adrenergic agonist-receptor complex. From the present data, it is suggested that atropine, besides its classical blocker effect at the muscarinic receptor, at high concentration is a specific alpha-adrenergic antagonist.     

Comparative effect of atropine on the adrenergic and muscarinic stimulation of phospholipid 32P labelling in isolated parotid cells: atropine,a possible blocker of alpha-adrenergic receptors

The inhibitory effect of atropine on phospholipid 32P labelling stimulated by muscarinic or alpha-adrenergic agonists was studied in isolated parotid cells. Atropine (10(-11) to 10(-4) M) had no effect on phospholipid 32P labelling in unstimulated cells. In contrast, 10(-8) to 10(-7) M atropine provoked a competitive inhibition of the cholinergic stimulation (i.e. this effect was completely wiped out at high agonist concentration). The atropine app. KD for the muscarinic receptor was 5 × 10(-9) M. Moreover, atropine inhibits the adrenergic stimulation of phospholipid 32P labelling by decreasing the efficacity and potency of the adrenergic agonists. The atropine app. KD for the alpha-adrenergic receptor can be estimated at 10(-5) M. This inhibition of alpha-adrenergic stimulation appears to be specific since atropine was without effect on the substance P or beta-adrenergic stimulation. At very low concentration (10(-10) — 10(-9) M) atropine seems to be a modulator (activator) of the muscarinic or adrenergic agonist-receptor complex. From the present data, it is suggested that atropine, besides its classical blocker effect at the muscarinic receptor, at high concentration is a specific alpha-adrenergic antagonist.     

Arachidonic acid release in cell lines transfected with muscarinic receptors: a simple functional assay to determine response of agonists.

Muscarinic agonists stimulated arachidonic acid release from 10- to 32-fold in Chinese hamster ovary (CHO) cells transfected with muscarinic M1, M3 and M5 receptor subtypes. Muscarinic agonists liberated arachidonic acid from the cAMP-coupled M2 and M4 cells only in the presence of ATP. Partial agonists were less efficacious at liberating arachidonic acid than full agonists. The ability of muscarinic agonists to liberate arachidonic acid and stimulate phosphoinositide hydrolysis in the same CHO M1, M3 and M5 cells was well correlated; however, partial agonists were more efficacious at stimulating phosphoinositide hydrolysis than arachidonic acid release. The efficacy and potency of 13 muscarinic agonists to liberate arachidonic acid was characterised. Influx of external calcium was required for arachidonic acid release even after initiation of agonist-induced release. It is concluded that arachidonic acid release is a simple assay suitable for evaluation of muscarinic agonists, antagonists and the flux of external calcium into cells.     

Regulation of atrial natriuretic peptide secretion by cholinergic and PACAP neurons of the gastric antrum

Atrial natriuretic peptide (ANP) released from enterochromaffin cells helps regulate antral somatostatin secretion, but the mechanisms regulating ANP secretion are not known. We superfused rat antral segments with selective neural agonists/antagonists to identify the neural pathways regulating ANP secretion. The nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) stimulated ANP secretion; the effect was abolished by hexamethonium but doubled by atropine. Atropine's effect implied that DMPP activated concomitantly cholinergic neurons that inhibit and noncholinergic neurons that stimulate ANP secretion, the latter effect predominating. Methacholine inhibited ANP secretion. Neither bombesin nor vasoactive intestinal polypeptide stimulated ANP secretion, whereas pituitary adenylate cyclase-activating polypeptide (PACAP)-27, PACAP-38, and maxadilan [PACAP type 1 (PAC1) agonist] each stimulated ANP secretion. The PAC1 antagonist M65 1) abolished PACAP-27/38-stimulated ANP secretion; 2) inhibited basal ANP secretion by 28 +/- 5%, implying that endogenous PACAP stimulates ANP secretion; and 3) converted the ANP response to DMPP from 109 +/- 21% above to 40 +/- 5% below basal, unmasking the cholinergic component and indicating that DMPP activated PACAP neurons that stimulate ANP secretion. Combined atropine and M65 restored DMPP-stimulated ANP secretion to basal levels. ANP secretion in the antrum is thus regulated by intramural cholinergic and PACAP neurons; cholinergic neurons inhibit and PACAP neurons stimulate ANP secretion.     

Thermic response of selective muscarinic agonists and antagonists in rat

Effect of some selective agonists and antagonists of cholinergic M receptor subtypes on rectal temperature was investigated in rats at an ambient temperature of 25 degrees +/- 2 degrees C. Centrally administered acetylcholine (ACh) induced transient hypothermia, whereas the muscarinic M1 receptor agonists, arecholine (ip) and McN-A-343 (McN) (icv), induced sustained and dose-related hypothermia. However, the nonspecific muscarinic receptor agonist, oxotremorine, and physostigmine, induced hypothermia at a lower dose and hyperthermia, accompanied by tremors, at higher doses. The muscarinic M2 receptor agonist, carbachol (icv) also produced a dose-related dual effect, hyperthermia and hypothermia being induced by the lower and higher doses, respectively. The M1 receptor antagonists, scopolamine (ip) and pirenzepine (icv), induced hyperthermia, whereas the M2 receptor antagonists, gallamine (icv) and AF-DX 116 (AFDX) (ip), produced hypothermia. The hypothermic effects of ACh. arecholine, McN, physostigmine, oxotremorine and carbachol were attenuated by scopolamine and pirenzepine. However, although scopolamine also inhibited the hyperthermic and tremorogenic effects of the higher dose of oxotremorine, it had a synergistic effect with the hyperthermia-inducing higher dose of physostigmine. AFDX attenuated the hyperthermic effect of the lower dose of carbachol, indicating that it was M2 receptor-mediated. Hemicholinium, an ACh synthesis inhibitor, had a transient hypothermic effect followed by slight hyperthermia. However, it markedly antagonized the hypothermic effects of gallamine and AFDX, indicating that their effects were dependent upon the availability of neuronal ACh. The results indicate that cholinergic hypothermia is a function of central muscarinic M1 receptors, with the M2 receptors serving as automodulators.(ABSTRACT TRUNCATED AT 250 WORDS)