Role of sympathetic nervous system in rat model of chronic visceral pain.

BACKGROUND: Changes in central pain modulation have been implicated in generalized pain syndromes such as irritable bowel syndrome (IBS). We have previously demonstrated that reduced descending inhibition unveils a role of sympathoneuronal outflow in decreasing peripheral sensory thresholds, resulting in stress-induced hyperalgesia. We investigated whether sympathetic nervous system (SNS) exacerbation of pain sensation when central pain inhibition is reduced is relevant to chronic pain disorders using a rat colon irritation (CI) model of chronic visceral hypersensitivity with hallmarks of IBS. METHODS: Rats were treated to a series of colorectal balloon distensions (CRD) as neonates resulting in visceral and somatic hypersensitivity and altered stool function that persists into adulthood. The visceral sensitivity was assessed by recording electromyographic (EMG) responses to CRD. Somatic sensitivity was assessed by paw withdrawal thresholds to radiant heat. The effects on the hypersensitivity of (i) inhibiting sympathoneuronal outflow with pharmacological and surgical interventions and (ii) enhancing the outflow with water avoidance stress (WAS) were tested. KEY RESULTS: The alpha2-adrenergic agonist, clonidine, and the alpha1-adrenergic antagonist, prazosin, reduced the visceral hypersensitivity and WAS enhanced the pain. Chemical sympathectomy with guanethidine and surgical sympathectomy resulted in a loss of the chronic visceral hypersensitivity. CONCLUSIONS & INFERENCES: The results support a role of the SNS in driving the chronic visceral and somatic hypersensitivity seen in CI rats. The findings further suggest that treatments that decrease sympathetic outflow or block activation of adrenergic receptors on sensory nerves could be beneficial in the treatment of generalized pain syndromes.

Transient allodynia pain models in mice for early assessment of analgesic activity.

BACKGROUND AND PURPOSE: The most common preclinical models of neuropathic pain involve surgical ligation of sensory nerves, which is especially difficult in mice. Transient models of chemically sensitized allodynia are potentially useful for rapidly characterizing the analgesic profile of compounds and conducting mechanistic studies. EXPERIMENTAL APPROACH: Increasing doses of NMDA, sulprostone (an EP1/EP3 prostaglandin receptor agonist) or phenylephrine (an alpha (1) adrenoceptor agonist) were injected intrathecally (i.t.) or i.p., and animals were subsequently assessed for allodynia. The effects of receptor antagonists and analgesic compounds on allodynia were also assessed. KEY RESULTS: A comparison of total body doses that cause allodynia following spinal or systemic administration indicated that NMDA induces allodynia in the spinal cord while sulprostone and phenylephrine act through a peripheral mechanism. Inhibition of the allodynia with receptor antagonists indicated that each agent induces allodynia by a distinct mechanism. The three models were benchmarked using compounds known to be active in neuropathic pain patients and nerve injury animal models, including gabapentin, amitriptyline and clonidine. CONCLUSIONS AND IMPLICATIONS: These transient allodynia models are a useful addition to the toolbox of preclinical pain models. They are simple, rapid and reproducible, and will be especially useful for characterizing the pain phenotype of knockout mice.

Pharmacological characterization of a novel antiglaucoma agent, Bimatoprost (AGN 192024).

Replacement of the carboxylic acid group of prostaglandin (PG) F(2alpha) with a nonacidic moiety, such as hydroxyl, methoxy, or amido, results in compounds with unique pharmacology. Bimatoprost (AGN 192024) is also a pharmacologically novel PGF(2alpha) analog, where the carboxylic acid is replaced by a neutral ethylamide substituent. Bimatoprost potently contracted the feline lung parenchymal preparation (EC(50) value of 35-55 nM) but exhibited no meaningful activity in a variety of PG-sensitive tissue and cell preparations. Its activity seemed unrelated to FP receptor stimulation according to the following evidence. 1) Bimatoprost exhibited no meaningful activity in tissues and cells containing functional FP receptors. 2) Bimatoprost activity in the cat lung parenchyma is not species-specific because its potent activity in this preparation could not be reproduced in cells stably expressing the feline FP receptor. 3) Radioligand binding studies using feline and human recombinant FP receptors exhibited minimal competition versus [(3)H]17-phenyl PGF(2a) for Bimatoprost. 4) Bimatoprost pretreatment did not attenuate PGF(2alpha)-induced Ca(2+) signals in Swiss 3T3 cells. 5) Regional differences were apparent for Bimatoprost but not FP agonist effects in the cat lung. Bimatoprost reduced intraocular pressure in ocular normotensive and hypertensive monkeys over a 0.001 to 0.1% dose range. A single-dose and multiple-dose ocular distribution/metabolism studies using [(3)H]Bimatoprost (0.1%) were performed. Within the globe, bimatoprost concentrations were 10- to 100-fold higher in anterior segment tissues compared with the aqueous humor. Bimatoprost was overwhelmingly the predominant molecular species identified at all time points in ocular tissues, indicating that the intact molecule reduces intraocular pressure.

The pharmacology of bimatoprost (Lumigan).

Bimatoprost (Lumigan) is a pharmacologically unique and highly efficacious ocular hypotensive agent. It appears to mimic the activity of a newly discovered family of fatty acid amides, termed prostamides. One biosynthetic route to the prostamides involves anandamide as the precursor. Bimatoprost pharmacology has been extensively characterized by binding and functional studies at more than 100 drug targets, which comprise a diverse variety of receptors, ion channels, and transporters. Bimatoprost exhibited no meaningful activity at receptors known to include antiglaucoma drug targets as follows: adenosine (A(1-3)), adrenergic (alpha(1), alpha(2), beta(1), beta(2)), cannabinoid (CB(1), CB(2)), dopamine (D(1-5)), muscarinic (M(1-5)), prostanoid (DP, EP(1-4), FP, IP, TP), and serotonin (5HT(1-7)). Bimatoprost does, however, exhibit potent inherent pharmacological activity in the feline iris sphincter preparation, which is prostamide-sensitive. Bimatoprost also resembles the prostamides in that it is a potent and highly efficacious ocular hypotensive agent. A single dose of bimatoprost markedly reduces intraocular pressure in dogs and laser-induced ocular hypertensive monkeys. Decreases in intraocular pressure are well maintained for at least 24 hr post-dose. Human studies have demonstrated that systemic exposure to bimatoprost is low and that accumulation does not occur. The sclera is the preferred route of accession to the eye. The high scleral permeability coefficient Papp is a likely contributing factor to the rapid onset and long-acting ocular hypotensive profile of bimatoprost.

Role of alpha-2 adrenergic receptors in neuroprotection and glaucoma.

The loss of retinal ganglion cells (RGCs) in glaucoma occurs progressively over many years. A neuroprotective drug should enhance survival of RGCs in the presence of chronic stress/injury. Four criteria are proposed for assessing the likely therapeutic utility in human glaucoma of drugs that have demonstrated neuroprotective activity in animal models: 1) A specific receptor target must be in the retina/optic nerve; 2) Activation of the target must trigger pathways that enhance a neuron's resistance to stress/injury and/or suppresses toxic insults; 3) The drug must reach the retina/vitreous at pharmacologic doses; and 4) The neuroprotective activity should be demonstrated in clinical trials. Data are presented that illustrate how the specific and potent alpha-2 agonist, brimonidine, meets these criteria. The alpha-2A receptor was localized in the inner rat retina by immunohistochemistry. Brimonidine reduced the rate of RGC loss in the calibrated rat optic nerve injury model even when dosed 12 and 24 hours before injury, consistent with a long-term enhancement of RGC resistance to stress. Brimonidine was also neuroprotective in the lasered chronic hypertensive rat model, reducing RGC loss over three weeks from 33% to 15%. A clinical trial has been initiated to determine brimonidine's neuroprotective activity in patients with non-arteritic ischemic optic neuropathy.

alpha(2)-Adrenoceptor agonists inhibit vitreal glutamate and aspartate accumulation and preserve retinal function after transient ischemia.

Recent studies have suggested that alpha(2)-adrenergic agonists prevent neuronal cell death in a number of animal models, although the mechanism of alpha(2)-neuroprotection remains unclear. In a retinal ischemia model, the alpha(2)-specific agonist brimonidine (1 mg/kg i.p.) preserves approximately 80% of the electroretinogram (ERG) b-wave. The protective effect of brimonidine is completely blocked by coadministration of the alpha(2)- antagonist rauwolscine. Brimonidine treatment preserves the ERG b-wave if animals are treated 1 or 3 h before ischemia, but has no effect if it is injected during ischemia. The 3-h pretreatment effect is blocked by i.v. injection of rauwolscine 2 h later (1 h before ischemia). A comparison of vitreous humor glutamate levels between untreated and brimonidine-treated eyes shows that 1) after ischemia, glutamate levels rise 2- to 3-fold in the untreated animals, and 2) glutamate levels in the brimonidine-treated animals are comparable to the nonischemic controls. Hence, the mechanism for brimonidine-mediated protection in the retinal ischemia model requires activation of the alpha(2)-adrenergic receptors immediately before and during ischemia. These data suggest that activation of the alpha(2)-adrenergic receptor may reduce ischemic retinal injury by preventing the accumulation of extracellular glutamate and aspartate.

Synthetic modification of prostaglandin f(2alpha) indicates different structural determinants for binding to the prostaglandin F receptor versus the prostaglandin transporter.

Several principles governing the binding of E series prostaglandins to EP receptors have emerged in recent years. The C-1 carboxyl group binds electrostatically to a conserved arginine residue in the seventh transmembrane segment of the receptor. Prostaglandin E analogs involving bioisosteric replacements of the carboxyl group, such as acylsulfonamide, are also active. In addition, structurally similar esters may also exhibit similar affinity, presumably by virtue of hydrogen bonding. Other regions of the substrate molecule appear to bind to other domains of EP receptors, either via hydrophobic interactions or by hydrogen bonding. Less information is available about the structural requirements for substrate binding to FP receptors. Prostanoids also bind to the prostaglandin transporter PGT. In this case, a conserved C-1 carboxyl group is critically important, since C-1 esters exhibit little affinity. Here we examined the binding of chemically diverse PGF(2alpha) structural analogs to the FP receptor and compared these with binding by the PG transporter. PGT recognized a wide range of anionic substituents. In contrast, the carboxylic acid group was essential for optimal binding to the FP receptor, since replacement by larger moieties with a similar pK(a), such as acylsulfonamide and tetrazole, substantially decreased binding affinity. Interestingly, insertion of cyclic substituents in the omega chain increased binding to the FP receptor but reduced affinity for PGT, and substitution for the 15-hydroxyl group produced only a modest reduction in FP receptor binding, but eliminated binding by PGT. Because extracellular PGF(2alpha) may compete for binding between FP receptors and PGT, these findings have implications for designing PGF(2alpha) analogs for treating disease states.

Replacement of the carboxylic acid group of prostaglandin f(2alpha) with a hydroxyl or methoxy substituent provides biologically unique compounds.

Replacement of the carboxylic acid group of PGF(2alpha) with the non-acidic substituents hydroxyl (-OH) or methoxy (-OCH(3)) resulted in an unexpected activity profile. Although PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) exhibited potent contractile effects similar to 17-phenyl PGF(2alpha) in the cat lung parenchymal preparation, they were approximately 1000 times less potent than 17-phenyl PGF(2alpha) in stimulating recombinant feline and human FP receptors. In human dermal fibroblasts and Swiss 3T3 cells PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) produced no Ca(2+) signal until a 1 microM concentration was exceeded. Pretreatment of Swiss 3T3 cells with either 1 microM PGF(2alpha) 1-OH or PGF(2alpha) 1-OCH(3) did not attenuate Ca(2+) signal responses produced by PGF(2alpha) or fluprostenol. In the rat uterus, PGF(2alpha) 1-OH was about two orders of magnitude less potent than 17-phenyl PGF(2alpha) whereas PGF(2alpha) 1-OCH(3) produced only a minimal effect. Radioligand binding studies on cat lung parenchymal plasma membrane preparations suggested that the cat lung parenchyma does not contain a homogeneous population of receptors that equally respond to PGF(2alpha)1-OH, PGF(2alpha)1-OCH(3), and classical FP receptor agonists. Studies on smooth muscle preparations and cells containing DP, EP(1), EP(2), EP(3), EP(4), IP, and TP receptors indicated that the activity of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) could not be ascribed to interaction with these receptors. The potent effects of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) on the cat lung parenchyma are difficult to describe in terms of interaction with the FP or any other known prostanoid receptor.

A single amino-acid substitution in the EP2 prostaglandin receptor confers responsiveness to prostacyclin analogs.

A high degree of homology between the four Gs-coupled prostaglandin (PG) receptors [EP2, EP4, prostacyclin (IP), PGD2 (DP)] and the four Gq/Gi-coupled receptors [EP1, EP3, PGF2alpha (FP), thromboxane A2 (TP)] suggests that prostaglandin receptors evolved functionally from an ancestral EP receptor before the development of distinct binding epitopes. If so, ligand selectivity should be determined by a limited number of amino acids. EP2 receptor transmembrane domain residues that are similar to those in the EP4 receptor but differ from those in the IP receptor were mutated to the corresponding IP receptor residue. Activation of the mutant receptors by PGE2 (EP2 ligand), iloprost (stable prostacyclin analog), and PGE1 (EP2/IP ligand) was determined using a cAMP-dependent reporter gene assay. A Leu304-to-tyrosine substitution in the seventh transmembrane domain enhanced iloprost potency approximately 100-fold. A glycine substitution at Ser120 in the third transmembrane domain had no effect on drug potency but improved the response of the Tyr304 mutant. The potency of the natural prostaglandins PGF2alpha and PGD2 was not enhanced by the mutations. In contrast, the potency of all prostaglandins was reduced 10- to 100-fold when arginine 302, which is thought to be a counterion for the prostaglandin carboxylic acid, was mutated. Thus, a single amino acid change resulted in a selective gain of function for iloprost, which is consistent with the proposed phylogeny of the prostaglandin receptors.

Muscarinic receptor subtypes in human iris-ciliary body measured by immunoprecipitation.

PURPOSE: To determine the relative levels of the five muscarinic receptor subtypes in the anterior segment of the human eye. METHODS: Antisera selective for each of the five muscarinic receptor proteins were incubated with [3H]-QNB bound receptors solubilized from human iris sphincter, ciliary muscle, and ciliary processes. Precipitation of the radiolabeled receptor-antibody complexes and scintillation counting enabled quantitation of the subtypes in the various tissues. Reverse transcription-polymerase chain reaction was performed on the tissues and cultured smooth muscle cells derived from them. RESULTS: Approximately 60% to 75% of the muscarinic receptors in the human iris sphincter and ciliary body are the m3 subtype. Lower levels (5% to 10%) of the m2 and m4 receptors are present in these tissues. The m1 receptor (7%) was detected in the ciliary processes and iris sphincter and the m5 receptor (5%), which is usually found only in the central nervous system, was present in the iris sphincter. CONCLUSIONS: The m3 subtype is the predominant muscarinic receptor in the anterior segment of the human eye. The extensive heterogeneity of muscarinic receptors makes it difficult to predict whether subtype-selective drugs will have an improved efficacy and side-effect profile.

Cloning of a carboxyl-terminal isoform of the prostanoid FP receptor.

An FP prostanoid receptor isoform, which appears to arise from alternative mRNA splicing, has been cloned from a mid-cycle ovine large cell corpus luteum library. The isoform, named the FP(B) receptor, is identical to the original isoform, the FP(A), throughout the seven transmembrane domains, but diverges nine amino acids into the carboxyl terminus. In contrast to FP(A), whose carboxyl terminus continues for another 46 amino acids beyond the nine shared residues, the FP(B) terminates after only one amino acid. The FP(A) isoform appears to arise by the failure to utilize a potential splice site, while a 3.2-kilobase pair intron is spliced out from the FP gene to generate the FP(B) isoform mRNA. The two isoforms have indistinguishable radioligand binding properties, but seem to differ in functional coupling to phosphatidylinositol hydrolysis. Thus, in COS-7 cells transiently transfected with either the FP(A) or the FP(B) receptor cDNAs, prostaglandin F(2alpha) stimulates inositol phosphate accumulation to the same absolute maximum, but the basal level of inositol phosphate accumulation is approximately 1.3-fold higher in cells transfected with the FP(B) as compared with cells transfected with the FP(A) isoform. Using the polymerase chain reaction, mRNA encoding the FP(B) isoform was identified in the ovine corpus luteum.

Localization of alpha 2-adrenergic receptor subtypes in the anterior segment of the human eye with selective antibodies.

PURPOSE: To develop antibodies that selectively recognize each of the alpha 2-adrenergic receptor (AR) subtypes and to determine the expression and localization of these subtypes in the anterior segment of the human eye. METHODS: Recent studies have shown that there are three subtypes of the alpha 2-ARs, termed alpha 2-C10 (alpha 2A), alpha 2-C2 (alpha 2B), and alpha 2-C4 (alpha 2C). Polymerase chain reaction was used to amplify portions of these receptors fused (in-frame) to a cDNA encoding glutathione-S-transferase (GST). The expressed fusion proteins were used to immunize chickens, and antibodies were generated. Immunofluorescence microscopy was used to localize the alpha 2-AR subtypes in sections of human and rabbit ciliary body. Polymerase chain reaction and dot blot hybridization were used to determine which subtypes were present in RNA from primary cultures of human nonpigmented epithelium (NPE) and rabbit iris-ciliary body (ICB). RESULTS: Immunofluorescence microscopy of COS cells transfected with the alpha 2-AR subtypes showed that the antibodies raised against the GST-receptor fusion proteins specifically recognized their respective receptor subtypes. In the human ciliary body, alpha 2 B and alpha 2C immunoreactivity were present in the NPE and ciliary muscle. In the rabbit ciliary body, alpha 2A immunoreactivity also was present. Polymerase chain reaction and dot blot hybridization indicated that RNA encoding the alpha 2B and alpha 2C subtypes was present in human NPE and that RNA encoding all three subtypes was present in the rabbit ICB. CONCLUSIONS: Multiple alpha 2-adrenergic subtypes are expressed in the ciliary body. In the human, alpha 2B and alpha 2C predominate, whereas all three are present in the rabbit. This could be important with respect to animal models of glaucoma and to the development of drugs for lowering intraocular pressure.

Cloning of a receptor for prostaglandin F2 alpha from the ovine corpus luteum.

A complementary DNA clone encoding a functional receptor for prostaglandin F2 alpha (PGF2 alpha) has been isolated from an ovine large luteal cell complementary DNA library (prepared from day 10 mid-luteal phase RNA). This receptor, which has been designated FP, consists of 362 amino acids (M(r) = 40,982) and is a member of the family of G protein-coupled receptors. Radioligand binding studies with membranes prepared from transfected COS cells demonstrated specific 17-[3H]phenyl-trinor-PGF2 alpha binding that was displaced by prostanoids in the order of 17-phenyl-trinor-PGF2 alpha > PGF2 alpha > fluprostenol > PGD2 > PGE2 >> 8-epi PGF2 alpha. Xenopus laevis oocytes injected with RNA encoding the ovine FP receptor responded to 17-phenyl-trinor-PGF2 alpha with increased membrane chloride conductance in calcium-free medium. Northern blot analysis with RNA from day 10 corpus luteum showed a major band of approximately 6.1 kilobases. On day 14, when luteolysis usually starts, the abundance of this 6.1-kilobase band was variable between individual ewes, and on day 16, when luteolysis is underway, the message was uniformly less abundant. This variability appeared to correlate with circulating progesterone. Thus, when the progesterone level was high (days 10 and 14 depending on whether luteolysis had started), the amount of FP receptor message was high, whereas when the progesterone level was low or falling (day 16), the amount of FP receptor message decreased. We have cloned an ovine FP receptor whose expression confers appropriate functional activity in COS cells and Xenopus oocytes. Furthermore, the level of messenger RNA encoding the FP receptor is high in the midluteal phase ovine corpus luteum and decreases during luteolysis.

Cytogenetic and molecular localization of tipE: a gene affecting sodium channels in Drosophila melanogaster.

Voltage-sensitive sodium channels play a key role in nerve cells where they are responsible for the increase in sodium permeability during the rising phase of action potentials. In Drosophila melanogaster a subset of temperature-sensitive paralytic mutations affect sodium channel function. One such mutation is temperature-induced paralysis locus E (tipE), which has been shown by electrophysiology and ligand binding studies to reduce sodium channel numbers. Three new gamma-ray-induced tipE alleles associated with either visible deletions in 64AB or a translocation breakpoint within 64B2 provide landmarks for positional cloning of tipE. Beginning with the flanking cloned gene Ras2, a 140-kb walk across the translocation breakpoint was completed. Germline transformation using a 42-kb cosmid clone and successively smaller subclones localized the tipE gene within a 7.4-kb genomic DNA segment. Although this chromosome region is rich in transcripts, only three overlapping mRNAs (5.4, 4.4, and 1.7 kb) lie completely within the smallest rescuing construct. The small sizes of the rescuing construct and transcripts suggest that tipE does not encode a standard sodium channel alpha-subunit with four homologous repeats. Sequencing these transcripts will elucidate the role of the tipE gene product in sodium channel functional regulation.

Molecular characterization and ocular hypotensive properties of the prostanoid EP2 receptor.

The cloning of the genes that encode for prostaglandin (PG) receptors has resolved much of the complexity and controversy in this area by confirming the classification proposed by Coleman, et al. Two issues that remained unresolved were (1) the inability of the EP2 agonist butaprost to interact with the cloned putative EP2 receptor and (2) molecular biological confirmation of a fourth PGE2-sensitive receptor, which was pharmacologically designated EP4. In order to provide clarification, we attempted to clone further PGE2-sensitive receptors. By using a cDNA probe that encodes for the human EP3A receptor, a cDNA clone that encoded for a novel PGE2-sensitive receptor was obtained by screening a human placenta library. This cDNA clone was transfected into COS-7 cells for pharmacological studies. The cDNA clone obtained from human placenta had only about 30% amino acid identity with cDNAs for other PG receptors, including those that encode for the previously proposed murine and human EP2 receptors. Radioligand binding studies on the novel EP receptor expressed in COS-7 cells revealed that selective EP2 agonists such as butaprost, AH 13205, AY 23626 and 19(R)-OH PGE2 all competed with 3H-PGE2 for its binding sites, whereas selective agonists for other PG receptor subtypes had minimal or no effect. This receptor was coupled to adenylate cyclase and EP2 agonists caused dose-related increases in cAMP. It appears that the cDNA described herein encodes for the pharmacologically defined EP2 receptor. Ocular studies revealed that AH 13205 decreased intraocular pressure in normal and ocular hypertensive monkeys by a mechanism that does not appear to involve inhibition of aqueous humor secretion.

Cloning of a novel human prostaglandin receptor with characteristics of the pharmacologically defined EP2 subtype.

A cDNA that when expressed has the binding and functional characteristics of the pharmacologically defined EP2 prostaglandin (PG) receptor [Cardiovasc. Drug Rev. 11:165-179 (1993)] has been cloned from a human placenta library. This clone, known as Hup-4, encodes a protein of 358 amino acids that has only approximately 30% overall identity with other PG receptors, including mouse and human clones that have been designated as EP2 receptors [J. Biol. Chem. 268:7759-7762 (1993); Biochem. Biophys. Res. Commun. 197:263-270 (1993)]. In COS-7 cells transfected with Hup-4, PGE2 stimulated the formation of cAMP with an EC50 of approximately 50 nM. The EP2-selective agonists AH13205 and butaprost were also active, with EC50 values in the range of 2-6 microM. The order of potency of PGs for competition with binding of [3H]PGE2 to membranes prepared from COS-7 cells transfected with Hup-4 was PGE2 > or = PGE1 > 16,16-dimethyl-PGE2 > or = 11-deoxy-PGE1 > butaprost > AH13205 > 19(R)-OH-PGE2. Natural PGs and analogues that are selective for the FP (PGF2a), DP (PGD2), EP1 (sulprostone), EP3 (MB 28767), and EP4 (1-OH-PGE1) receptors were inactive or competed poorly with the binding of [3H]PGE2 (< 50% displacement of specific binding at 10 microM). Northern blot analysis showed the presence of a Hup-4 message of approximately 3.1 kilobases in mRNA from human lung and placenta. Reverse transcription-polymerase chain reaction studies also indicated that Hup-4 is probably expressed in human uterus and in HL-60 (human promyelocytic leukemia) cells. Our findings suggest that Hup-4 encodes the pharmacologically defined EP2 receptor, whereas the mouse and human cDNAs previously classified as EP2 may represent another EP receptor subtype or the recently defined EP4 subtype [Prostaglandins 47:151-168 (1994)].

Muscarinic cholinergic receptor binding sites differentiated by their affinity for pirenzepine do not interconvert.

Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M1 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4 degrees C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands [3H]quinuclidinyl benzilate or [3H]PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of [3H]quinuclidinyl benzilate in a biphasic manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Pirenzepine distinguishes between muscarinic receptor-mediated phosphoinositide breakdown and inhibition of adenylate cyclase.

Subtypes of muscarinic cholinergic receptors have been proposed to exist, but the biochemical responses mediated by the putative subtypes are unknown. In the present study, muscarinic receptor-mediated phosphoinositide breakdown and inhibition of adenylate cyclase activity were characterized in rat brain as well as rat parotid and heart. To study whether these responses are mediated by separate subtypes of muscarinic receptors, the potencies of agonists and antagonists were determined in both assays. Antagonist potencies were calculated by Schild analysis. In the brain, the putatively selective muscarinic receptor antagonist, pirenzepine, exhibited Ki values of 21 nM in the assay of phosphoinositide breakdown and 310 nM in the assay of adenylate cyclase activity. Similarly, using radioligand binding techniques, it distinguished two binding sites with Kd values of 12 and 168 nM. The antagonist, atropine, on the other hand, was equipotent in the two biochemical assays and the radioligand binding assay with Ki values of approximately 1 to 2 nM. In peripheral tissues with robust muscarinic receptor-mediated phosphoinositide (parotid) and adenylate cyclase (heart) responses, pirenzepine exhibited a similar selectivity (19-fold) for the phosphoinositide assay that was seen in the brain, but it was 6- to 7-fold less potent in both peripheral tissues than in the central nervous system. In addition, the potencies of pirenzepine in binding and functional studies in each tissue were not as well correlated as in the brain. Atropine and other antagonists were 4- to 9-fold selective for inhibiting oxotremorine-stimulated phosphoinositide breakdown in the peripheral tissues.(ABSTRACT TRUNCATED AT 250 WORDS)