Comparison of the activation kinetics of the M3 acetylcholine receptor and a constitutively active mutant receptor in living cells.

Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G(q)-coupled M(3) acetylcholine receptor (M(3)-AChR) with that of a constitutively active mutant receptor (M(3)-AChR-N514Y) using M(3)-AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M(3)-AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M(3)-AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-G-protein interaction was measured by FRET between M(3)-AChR-yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-Gγ(2). Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M(3)-AChR-N514Y reached only 12% of that of M(3)-AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged Gα(q) and CFP-tagged Gγ(2). Activation of G(q) was significantly slower than receptor activation and indistinguishable for the two agonists. However, G(q) deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agonist-stimulated coupling to G(q), agonist-stimulated G(q) activation by M(3)-AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M(3)-AChR by decreasing the rate of receptor deactivation, while having minimal effect on receptor activation.

Segmental heterogeneity of electrogenic secretions in human ascending colon and rectum.

AIMS: We have attempted to ascertain putative segmental differences in the secretory responses of the human ascending colon and rectum. METHODS: From the mucosal biopsy samples of two segments, the short-circuit current (I(sc)) and tissue resistance (R(te)) were compared under control conditions, as well as after the induction of secretion, using a modified Ussing chamber. We also performed semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to detect and quantify transport proteins. RESULTS: The spontaneous I(sc) in the ascending colon was found to be greater than that in the rectum (P<0.01), whereas isobutylmethylxanthine/forskolin and carbachol (CCh) induced a greater rise in I(sc) in the rectum than in the ascending colon (P<0.05). When coupled with indomethacin pretreatment, the increase in Delta I(sc) after the addition of CCh and forskolin was significant as compared to that observed without pretreatment (P<0.05). However, in the rectum, the secretory response to CCh and forskolin was abolished to a significant degree by indomethacin (P<0.05). Moreover, these indomethacin-induced changes were reversed by the addition of PGE2. Upon semiquantitative RT-PCR analysis, the amounts of cystic fibrosis transmembrane regulator, KCNQ1, and CLCA1 mRNAs were not found to be different between the two segments. CONCLUSION: There was a clear segmental heterogeneity with regard to electrogenic secretion in the human colon, and this difference can be explained by differences in the ascending colon and rectum.

Spatial memory in aged rats is related to PKCgamma-dependent G-protein coupling of the M1 receptor.

In the present study, individual differences in spatial memory in aged Fischer 344 (F344) rats were associated with the extent of G-protein coupling of the M1 muscarinic receptor and the dendritic-to-somal ratio of hippocampal PKCgamma (d/sPKCgamma) immunogenicity. Following testing in the eight-arm radial maze task, 7 young and 13 aged rat brains were sectioned through the dorsal hippocampal formation (HF). G-protein coupling of the M1 receptor was assessed autoradiographically using competition binding studies in the presence and absence of a G-protein uncoupler to determine high (K(H)) and low (K(L)) affinity states for agonist in the HF, neocortex, and amygdala. In aged animals, a relationship between choice accuracy in the maze and K(H), a measure of M1 receptor-G-protein coupling was seen in the dentate gyrus, CA3, CA1, and neocortex. Furthermore, choice accuracy and d/sPKCgamma immunogenicity showed a significant relationship in CA1. Lastly, a correlation was seen in the CA1 of aged animals between K(H) and d/sPKCgamma. These relationships did not hold for the amygdala. Thus, individual differences in a naturally occurring age-dependent disruption of cholinergic-PKCgamma signal transduction is associated with spatial memory dysfunction.

Reactivity of urinary bladder smooth muscle in guinea pigs to acetylcholine and carbachol--participation of acetylcholinesterase.

The authors examined the influence of acetylcholinesterase inhibitor (neostigmine) on the in vitro reactivity of urinary bladder smooth muscle (UBSM) in guinea pigs. The aim of the present study was to determine the participation of pharmacokinetic properties of acetylcholine and carbachol in different UBSM reactivity to these mediators. In vitro method of organ baths was used and reactivity of UBSM strips to cumulative doses of acetylcholine and carbachol was tested before and after the incubation with neostigmine (10(-4) mol.l(-1)). Neostigmine caused a significant increase of UBSM reactivity to acetylcholine. The UBSM reactivity to acetylcholine was significantly higher at concentrations of 10(-5) and 10(-4) mol.l(-1) compared to carbachol at the same concentrations. These findings indicate that in addition to different mediator affinity to muscarinic receptors and to their different intrinsic activity, the pharmacokinetic properties of acetylcholine and carbachol also participate in UBSM reactivity.

Decrease in GTP-sensitive high affinity agonist binding of muscarinic acetylcholine receptors in autopsied brains of dementia with Lewy bodies and Alzheimer's disease.

To determine changes in signal transduction from the muscarinic acetylcholine receptor (mAChR) to G protein in brain tissue of dementia with Lewy bodies (DLB) and Alzheimer's disease (AD), we investigated GTP-sensitive agonist high affinity binding, which is considered an index of the formation of the mAChR-G protein complex. Brain tissue was obtained at necropsy from eight patients with DLB, nine patients with Alzheimer's disease and seven patients as controls. Membrane fractions were prepared from frontal and temporal cerebral tissues. Displacement curves of [(3)H]l-quinuclidinyl benzilate (QNB) binding by carbamylcholine were analyzed by the nonlinear least-squares methods. The proportion of and affinity for the agonist in GTP-sensitive agonist high affinity binding were estimated. The percentages GTP-sensitive agonist high affinity bindings were significantly decreased in DLB (P<0.01) and Alzheimer's disease (P<0.05) only in the frontal lobe. There were no significant differences in the temporal lobe. The ratio of agonist affinity (Kd value of low affinity component/Kd value of high affinity component) did not significantly differ among groups in either the frontal lobe or temporal lobe. The concentration of mAChR-G protein complex is considered reduced in the frontal lobe of brains with DLB and Alzheimer's disease. Therefore, signal transduction from mAChR to G protein was disturbed in the frontal lobe in these diseases.

Future therapeutic strategies in autoimmune myasthenia gravis.

Antibodies against muscle acetylcholine receptor (AChR) undoubtedly play a critical role in the pathology of most myasthenia gravis (MG) cases. Selective elimination of the majority of these antibodies should result in a considerable improvement of the MG symptoms. Such a specific elimination could be achieved by AChR-based immunoadsorbents. However, sufficient quantities of native human AChR are not available while bacterially expressed recombinant domains of the AChR are unable to bind satisfactorily MG antibodies. We have undertaken the production of the extracellular domains of human AChR subunits in eukaryotic systems, in native-like conformation, for their use as potent immunoadsorbents. The N-terminal extracellular domain (amino acids 1-210; alpha(1-210)) of the alpha(1) subunit of the human muscle AChR was expressed in the yeast Pichia pastoris. The polypeptide was water-soluble, glycosylated, and in monomer form. The alpha(1-210) bound 125I-alpha-bungarotoxin (125I-alpha-BTX) with a high affinity (Kd = 5.1 +/- 2.4 nM), and this binding was blocked by unlabeled d-tubocurarine and gallamine. Several conformation-dependent anti-AChR antibodies were able to bind alpha(1-210) as did antibodies from a large proportion of MG patients. The purified protein was subsequently immobilized on Sepharose-CNBr and was used to immunoadsorb anti-AChR antibodies from 64 MG sera. It eliminated more than 50% (50-94%) of the anti-AChR antibodies in 20% of the sera, whereas from another 30% of the sera it eliminated 20-60% of their anti-AChR antibodies. Work is in progress for the expression of the extracellular domain of all other muscle AChR subunits. It is expected that their combined use may eliminate the great majority of the anti-AChR antibodies from most MG patients.

Ca(2+) channel properties in smooth muscle cells of the urinary bladder from pig and human.

Ca(2+) channel properties of pig and human bladder smooth muscle were investigated utilizing standard whole-cell patch clamp techniques. Both the amplitude obtained and the current density of Ca(2+) channel current evoked by step depolarization were larger in human than in pig myocytes. The inward currents were sensitive to an L-type Ca(2+) channel antagonist, nifedipine, the effects of which were not significantly different between species. In both species, prior application of ATP (0.1 mM) had no effect on activation of this voltage-sensitive channel current, while a muscarinic receptor agonist, carbachol (0.1 mM), significantly attenuated the amplitude of this current. Furthermore, inclusion of GDP-beta-S or Heparin in the pipette abolished or had no effect on the suppression of Ca(2+) current by carbachol, respectively. These results forward the pig as a good model for the human in detrusor Ca(2+) channel properties, especially with regard to neural modulation, although voltage-sensitive Ca(2+) channels seem to make greater contribution in human bladder physiology.

Carbachol interactions with nonsteroidal anti-inflammatory drugs.

The inhibition of cyclooxygenase enzymes by nonsteroidal anti-inflammatory drugs (NSAIDs) does not completely explain the antinociceptive efficacy of these agents. It is known that cholinergic agonists are antinociceptive, and this study evaluates the interactions between carbachol and some NSAIDs. Antinociceptive activity was evaluated in mice by the acetic acid writhing test. Dose-response curves were constructed for NSAIDs and carbachol, administered either intraperitoneally (i.p.) or intrathecally (i.t.). The interactions of carbachol with NSAIDs were evaluated by isobolographic analysis after the simultaneous administration of fixed proportions of carbachol with each NSAID. All of the drugs were more potent after spinal than after systemic administration. The combinations of NSAIDs and carbachol administered i.p. were supra-additive; however, the i.t. combinations were only additive. Isobolographic analysis of the coadministration of NSAIDs and carbachol and the fact that atropine antagonized the synergistic effect suggest that carbachol may strongly modulate the antinociceptive activity of NSAIDs; thus, central cholinergic modulation would be an additional mechanism for the antinociceptive action of NSAIDs, unrelated to prostaglandin biosynthesis inhibition.

Roles of agonist-binding sites in nicotinic acetylcholine receptor function.

Under equilibrium conditions, the nicotinic acetylcholine receptor from Torpedo electroplax carries two high affinity-binding sites for agonists. It is generally assumed that these are the only agonist sites on the receptor and that their occupancy results in rapid channel activation followed by slower conformational transitions that lead to the high affinity equilibrium state. These slow transitions are thought to reflect the physiological process of desensitization. Here we show that preequilibration of the high affinity sites with saturating concentrations of carbamylcholine does not diminish the ion flux response to subsequent exposure to higher (activating) concentrations of this agonist. This finding has profound implications with respect to receptor function: (1) occupancy of the high affinity sites per se does not desensitize the receptor and (2) these sites cannot be directly involved in receptor activation. It is thus necessary to invoke the presence of additional binding sites in channel opening.

Selective blockade of muscarinic receptor subtypes in the brain stem reticular formation in rats: effects on anesthetic requirements.

Muscarinic involvement in the modulation of general anesthesia was examined in the rat with a cannula implanted in the pontine reticular formation. Atropine microinjected into the reticular formation reversed the minimum alveolar concentration (MAC) reducing effect of carbachol on halothane anesthesia, but M(1) or M(3) antagonist had no effect. An M(2) antagonist reduced the MAC of halothane following saline and carbachol. The results suggest that any of the muscarinic receptor subtypes in this region do not independently mediate the cholinomimetic effect on halothane anesthesia.

Efficacy. II. Estimation of a newly defined efficacy related parameter.

A new method for estimation of agonist-affinity (KA) and relative efficacy was introduced. This method afforded a procedure by which relative efficacy may be estimated while the actual KA values of agonist-receptor complexes are unknown. The relative efficacy may be estimated by employing a newly defined drug parameter, namely the eES value. The eES value is related to drug efficacy and is defined in such a manner that an isolated eES is a meaningful quantity which may indicate whether or not spare receptors are present in an agonist-effector system. The estimation of eES was based on the fact that fixed agonist-competitive antagonist combinations mimic partial agonists and mediate submaximal concentration-effect curves. However, for the practical estimation of eES one may employ data acquired from agonistic concentration-effect curves determined in the absence and presence of increasing concentrations of a competitive antagonist. This procedure was illustrated by utilizing theoretical concentration-effect curves and applied practically by estimating eES and KA values acquired from sets of carbachol and salbutamol curves. The sets of carbachol and salbutamol concentration-effect curves were determined in the absence and presence of their respective competitive antagonists, namely tripitramine and pindolol.

Adenosine and carbachol are not equivalent in their effects on L-type calcium current in rabbit ventricular cells.

Adenosine is one of the most important inhibitory modulators of heart function, producing negative inotropic, chronotropic and dromotropic effects and is also a major regulator of coronary circulation. The decrease in contractility by adenosine is mediated through inhibition of adenylyl cyclase by Gi-proteins coupled to adenosine receptors. However, little is known about the developmental differences in the effect of adenosine on cardiac cells. We have now shown that there is a striking developmental difference in the inhibitory effect of adenosine on isoproterenol-stimulated ICa between adult and newborn rabbit ventricular cells. Adenosine had no significant inhibitory effect on 0.1 muM isoproterenol-stimulated ICa in adult cells, while it completely blocked the 10 muM isoproterenol-stimulated ICa in newborn cells with an inhibitory potency similar to carbachol in newborn cells. Similarly, adenosine did not decrease the isoproterenol-stimulated cAMP levels in adult cells while it inhibited isoproterenol-stimulated cAMP levels significantly and equipotently to carbachol in newborn. However, for forskolin-stimulated ICa and cAMP levels in newborn cells, adenosine had a much lower inhibitory potency than carbachol. In adult cells, forskolin-stimulated ICa and cAMP levels were not affected by adenosine. We showed previously that the Gia3 isoform of inhibitory G protein was present in newborn cell membranes, but not detectable in adult cell membranes. We have now used a synthetic decapeptide corresponding to the C-terminal sequence of Gia3 in the patch pipette and have shown a selective partial block of the inhibitory action of adenosine for isoproterenol-stimulated ICa, suggesting that the inhibitory action of adenosine on ICa is mediated primarily through the Gia3 pathway.

Evaluación clínica de la solución intraocular de carbacol al 0.01 por ciento / Clinic evaluation of the efficacy and safety of Carbacol 0.01 percent in ophthalmic solution

Se realizó un estudio prospectivo abierto para evaluar la eficacia y seguridad de la solución oftálmica de Carbacol al 0.01 por ciento, incluyendo sesenta pacientes sometidos tanto a extracción extracapsular de catarata como a facoemulsificación, enrolados en dos centros de atención oftlamológica de la Ciudad de México. Se reportó la disminución satisfactoria del diámetro popular después del procedimiento quirúrgico, así como un adecuado mantenimiento de los niveles de presión intraocular. Esto permitió que el implante intraocular permaneciera en su sitio adecuadamente y evitó la posibilidad de incrementos tensionales riesgosos. El medicamento posee mínimos efectos inflamatorios y poca interferencia con otras sustancias empleadas durante la cirugía, pudiendo modularse sus acciones según los requerimientos del cirujano y del paciente.

Regulation by thapsigargin and carbachol of the intracellular calcium concentration in rat submandibular glands.

1. Carbachol and thapsigargin both increased the intracellular calcium concentration in rat submandibular cells in the presence and in the absence of extracellular calcium. Depletion of intracellular calcium pools with thapsigargin prevented the response to carbachol. 2. The two agents also increased the influx of calcium. The muscarinic agonist stimulated the efflux of calcium outside the cell. 3. From these results it is concluded that submandibular cells possess several intracellular calcium pools sensitive to thapsigargin, among which some are sensitive to IP3. Depletion of these pools increase the uptake of extracellular calcium.

Effect of chronic central treatment with the acetylcholine analog methylcarbamylcholine on cortical nicotinic receptors: correlation between receptor changes and behavioral function.

Chronic administration of nicotine results in increased numbers of specific nicotinic acetylcholine receptors (nAChRs) within the central nervous system. The purpose of this study was to determine: 1) whether chronic administration of the nicotinic agonist, methylcarbamylcholine (MCC) or the competitive antagonist dihydro-beta-erythroidine (DHBE) in rats could alter brain nAChRs; 2) whether DHBE could inhibit the alterations produced by MCC and 3) whether such changes could be correlated with alterations in animal behavior. MCC (3-60 micrograms) or DHBE 6 or 60 micrograms) alone, or DHBE 15 min preceding MCC (30 micrograms) was injected via previously implanted i.c.v. guide cannulas, twice daily for 10 days. Chronic administration of MCC reversibly increased the apparent Bmax and Kd of cortical nAChRs in a dose- and time-dependent manner. Chronic central administration with DHBE also increased Bmax. Chronic i.c.v. pretreatment with DHBE inhibited the elevation in Bmax and Kd produced by chronic MCC injection. In freely behaving animals, MCC evoked the expression of a characteristic posture assumed in response to central nicotinic stimulation. Chronic administration of MCC resulted in tolerance to this behavioral response. DHBE pretreatment inhibited the MCC-induced behavioral changes. The time course of the development of tolerance to MCC as well as the extent of inhibition of DHBE were well correlated with the time course for receptor up-regulation and inhibition of the increase in binding parameters, respectively. Like chronic MCC treatment, chronic DHBE increased the number of [3H]cytisine binding sites but without concomitant development of behavioral tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in ligand binding and phosphoinositide turnover between M1 muscarinic receptor gene transfected cells and mouse and rat brain membranes.

The present study describes some unexpected receptor mediated effects of N-methylcarbamylcholine on mouse M1 muscarinic receptor gene transfected cell line (M1Y1) that were not evident from biochemical studies with mouse and rat brain tissue where N-methylcarbamylcholine exhibited only nicotinic properties. Although N-methylcarbamycholine was devoid of muscarinic properties in mouse and rat brain preparations, as determined by phosphoinositide turnover and inhibition of [3H]QNB binding, it exhibited significant muscarinic characteristics in the transfected M1Y1 cell line. At a concentration of 10(-6) M or greater, N-methylcarbamycholine caused a transient increase in intracellular Ca2+ of 50 s duration that was reversible by atropine or pirezepine. The Ca(2+)-transient was not elicited by other nicotinic agents such as nicotine and N,N-dimethylcarbamylcholine, a close analogue of N-methylcarbamylcholine, with comparable affinity for nicotinic receptors and devoid of muscarinic activity. N-Methylcarbamylcholine also stimulated phosphoinositide turnover in M1Y1 cells with an estimated EC50 value 10 times greater than that of carbachol, and the effect was blocked by atropine. Both carbachol and N-methylcarbamycholine inhibited [3H]QNB binding in a concentration-dependent manner; however, the IC50 for carbachol was over two orders of magnitude greater than that observed in mouse and rat brain membranes. In considering possible explanations for the differential characteristics of N-methylcarbamylcholine in mouse and rat brain as compared to the transfected M1Y1 cells, it was concluded that the difference may be attributable to differences in the receptor-transduction coupling efficiency and the microenvironment of the muscarinic receptors.

Nicotinic cholinergic receptors associated with mammalian cerebral vessels.

Current evidence suggests that the cerebral vasculature may be modulated by cholinergic nerves. We used ligand binding methods to examine the presence of nicotinic cholinergic receptors in brain vasculature. We found carbachol-displaceable [3H]acetylcholine (ACh) and [3H]nicotine (NIC) binding sites in preparations of intraparenchymal cerebral microvessels (CMV) and larger pial vessels from human and pig brains. Specific binding sites for [3H]ACh and [3H]NIC in cerebral microvessels were saturable and comparable in density to those in cerebral cortex. The Kds for the two ligands ranged 3-18 nM whereas the Bmaxs were 25-45 fmol/mg protein. In contrast, the binding of [3H]pirenzipine or [3H]quinuclidinyl benzilate, index for muscarinic receptors, was low (9-15% of cortex) in microvessels compared to the cerebral cortex. Our observations suggest the association of cholinergic nicotinic receptors with cerebral microvessels, which may be involved in the modulation of the cerebral circulation by cholinergic neurons.

Systemic pilocarpine increases deposition of and decreases responsiveness to inhaled carbachol in sheep.

The purpose of this study was to determine whether excessive airway secretions could serve as a barrier function against inhaled particulate matter. To increase airway secretions, six conscious sheep were treated with pilocarpine (0.8 mg/kg i.v.). Pilocarpine increased pulmonary resistance (RL) and total aerosol deposition within five breaths (AD5) as determined by the rebreathing of an inert monodisperse aerosol. When RL had returned to baseline, AD5 remained elevated [21 +/- 2% (SE), P < 0.05] and tracheal secretions were increased (237 +/- 77%, P < 0.05) above the values before pilocarpine administration. A carbachol aerosol dose-response curve was carried out at this time and compared with a control carbachol dose-response curve by calculating the dose of carbachol required to increase RL by 400% (PD400). Mean PD400 was increased postpilocarpine by 53 +/- 18 (P < 0.05) and 85 +/- 25% (P < 0.05) when normalized for increased aerosol deposition. Thus, pilocarpine decreased airway responsiveness to inhaled carbachol despite increasing aerosol deposition. The pilocarpine-induced airway hyporesponsiveness to inhaled carbachol is consistent with the hypothesis that excessive secretions have a protective role in the airways.

Therapeutic index of pilocarpine, carbachol, and timolol with nasolacrimal occlusion.

We assessed the effect of nasolacrimal occlusion on the therapeutic index of various antiglaucoma medications in healthy volunteers and patients with glaucoma. Nasolacrimal occlusion used with pilocarpine 2% every 12 hours gave the maximal ocular hypotensive response. Carbachol 1.5% every 12 hours with nasolacrimal occlusion gave the maximal response for this drug. For timolol, nasolacrimal occlusion collapsed the dose-response curve and extended the duration of action. A final trial of carbachol added to timolol with nasolacrimal occlusion showed that timolol 0.25% and carbachol 1.5% every 12 hours gave the maximal response for this combination. Our findings suggest that most of the commercially used ocular hypotensive agents can achieve the same maximal effect with lower concentrations and less frequent administration (never exceeding every 12 hours) than are currently recommended should nasolacrimal occlusion be performed. Furthermore, nasolacrimal occlusion should markedly decrease the systemic absorption of topical ocular drugs and lessen the chance of systemic side effects.

Muscarinic receptor coupling to inositol phospholipid metabolism in guinea-pig cerebral cortex, parotid gland and ileal smooth muscle.

Inositol phospholipid hydrolysis induced by agonist-stimulation of muscarinic receptors has been examined in slices of guinea-pig cerebral cortex, parotid gland and ileal smooth muscle. An assay measuring 3H-inositol phosphate formation from prelabelled lipids in the presence of LiCl, allowed marked stimulation by agonists to be followed. The pD2-value of carbachol differed markedly, between tissues being more than 10-fold lower in cerebral cortex than in parotid gland. The partial agonist oxotremorine showed the largest relative maximal responsiveness in parotid gland, followed by ileum and cortex. Atropine suppressed the phosphoinositide response to carbachol with an almost similar affinity in each tissue, but pirenzepine was found to have a 20-fold higher affinity in cerebral cortex, pKi = 7.7 than in parotid gland, pKi = 6.3. Carbachol, even in the presence of guanosine triphosphate (GTP), displayed complex binding against 3H-N-methylscopolamine (3H-NMS) in cortical and ileal membranes, though in membranes from the parotid gland a single homogeneous population was found. Atropine inhibition of 3H-NMS parallelled its suppression of the phosphoinositide response, the affinities in each tissue studied being similar. Pirenzepine inhibited binding from two components in cerebral cortex, the high affinity value being similar to that obtained in the phosphoinositide assay. In parotid gland, however, only low affinity pirenzepine binding sites were observed, closely resembling the affinity found for this antagonist in the functional assay. These experiments suggest (a) that there are differences between agonist occupation of muscarinic receptors and phosphoinositide hydrolysis within the different tissues, (b) that both high and low affinity pirenzepine binding sites appear to be linked to phosphoinositide metabolism, and (c) that low affinity pirenzepine sites may be more efficiently coupled to the hydrolysis of phosphoinositides.