Contribution of adrenergic and "purinergic" neurotransmission to contraction in rabbit detrusor.

In the presence of propranolol, norepinephrine produced an alpha adrenoceptor mediated contraction in isolated rabbit detrusor. Phenoxybenzamine (3.3 x 10(-8) M) antagonized this response but failed to affect the contraction produced by field stimulation either in normal or in hemicholinium-3-treated tissue. Higher concentrations of phenoxybenzamine were antagonistic to carbachol. Electrically induced contractions were also unaffected by guanethidine (1 x 10(-4) M) in vitro. Reserpine pretreatment produced no change in the contractile response although the tissue was depleted of catecholamine fluorescence on histology. It is concluded that adrenergic neurotransmission does not account for noncholinergic excitatory neurotransmission in rabbit detrusor. In rabbit detrusor adenosine 5'-triphosphate (ATP) produced a transient contraction which was not antagonized by tetrodotoxin (1 x 10(-7) M), atropine (4 x 10(-7) M) or phenoxybenzamine (3.3 x 10(-7) M). Adenosine, adenine phosphate and adenosine 5'-monophosphate had little or no effect, while sodium tripolyphosphate and adenosine 5'-diphosphate produced a smaller response than ATP. Dipyridamole (1 x 10(-8)-1 x 10(-5) M) did not unmask a response to adenosine and did not potentiate the response to ATP or field stimulation. Theophylline (5 x 10(-5) M) and 2, 2'-pyridylisatogen (PIT) (1 X 10(-5) M) depressed responses to ATP without antagonizing those to carbachol. At these doses, theophylline and 2, 2'-pyridylisatogen also antagonized the electrically induced contraction. Desensitization with ATP (1.5 X 10(-3) M for 30 min) selectively depressed responses to ATP but not to carbachol, and also depressed the response to field stimulation, particularly at frequencies of 10 Hz and lower. It is at these frequences that the noncholinergic component of the contractile response is most significant. Combination of the desensitization procedure with atropine produced an additive effect, suggesting that the two mechanisms affected are independent. Combination of the desensitization procedure with hemicholinium-3 produced less than an additive effect, suggesting an interference between the two treatments. It is concluded that ATP plays a role in the noncholinergic component of excitatory neurotransmission in rabbit detrusor.     

Pharmacological characterization of the M1 muscarinic receptors expressed in murine fibroblast B82 cells

The muscarinic receptors in a B82 cell line which were transfected with the rat m1 muscarinic receptor gene (cTB10 cells) were studied by using radioligand binding assays. Their possible coupling to the hydrolysis of inositol lipids and cyclic AMP formation were also investigated. [(-)-[3H]Quinuclidinyl benzilate [(-)-[3H]QNB] binding to the intact cTB10 cells was saturable and displaceable by 1 microM atropine sulfate. The Kd and maximum binding values of (-)-[3H]QNB from saturation studies were 12 pM and 17 fmol/10(6) cells, respectively. Inhibition studies of (-)-[3H]QNB binding to intact cTB10 cells suggested that these muscarinic receptors are of the M1 type defined by their high affinity for pirenzepine and low affinity for AF-DX 116 [11-[2-diethylamino methyl-1-piperidinylacetyl]-5,11-dihydro-6H-pyrido(2,3-b) (1,4)benzodiazepine-6-one]. The muscarinic agonist carbachol stimulated [3H]inositol monophosphate accumulation in the cTB10 cells, which could be reversed by the muscarinic antagonists atropine, pirenzepine or AF-DX 116. The rank order of potency of the muscarinic antagonists in inhibiting carbachol-stimulated [3H]inositol monophosphate accumulation was atropine greater than pirenzepine greater than AF-DX 116, in agreement with that from ligand/(-)-[3H]QNB competition experiments. Pertussis toxin and 4 beta-phorbol, 12-beta-myristate, 13-alpha-acetate reduced carbachol-stimulated [3H]inositol monophosphate accumulation. Prostaglandin E1 stimulated cyclic AMP formation in the cTB10 cells. Carbachol at the concentration of 10 mM exhibited no stimulatory or inhibitor effect on the basal or prostaglandin E1-stimulated cyclic AMP formation. These results suggest that the muscarinic receptors encoded by the transfected m1 gene in the cTB10 cells are of the M1 type and are coupled to the hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein.     

Muscarinic receptors: relationships among phosphoinositide breakdown, adenylate cyclase inhibition, in vitro detrusor muscle contractions and in vivo cystometrogram studies in guinea pig bladder

The relationships between activation of muscarinic receptors in guinea pig bladder measured as carbachol-stimulated inositol phosphate (IP) accumulation, oxotremorine-induced adenylate cyclase (AC) inhibition and bladder detrusor smooth muscle contraction determined in vitro as well as in vivo in the slow filling cystometrogram (CMG), were analyzed from the potencies of a number of muscarinic antagonists to block these responses. Significant positive linear correlations were found among the inhibitory potencies of 10 muscarinic antagonists to inhibit phosphoinositide (PI) turnover and both detrusor muscle contraction in vitro, as well as peak intravesical bladder pressure in vivo in the CMG (r = 0.8, P less than .01). In contrast, there was no significant correlation between the potency of antagonists to block the AC inhibitory response and either in vitro or in vivo guinea pig bladder contractions (P greater than .05). Muscarinic agonists inhibited basal AC activity to a maximum of 20% in a GTP-dependent, Na+-sensitive manner and dose dependently stimulated both PI breakdown (3- to 4-fold) and isolated detrusor contractions. Again, a significant correlation (r = 0.9, P less than .01) was calculated among the potencies of seven muscarinic agonists to elicit PI turnover and in vitro muscle contraction, whereas no significant correlation was observed between their potencies to inhibit AC activity and contractile responses in vitro. Collectively, the data suggest that IP accumulation and presumably IP-induced Ca++ release may function as the transducing mechanism for cholinergic contraction of the urinary bladder.(ABSTRACT TRUNCATED AT 250 WORDS)     

The relationship between agonist states of the M1 muscarinic receptor and the hydrolysis of inositol lipids in transfected murine fibroblast cells (B82) expressing different receptor densities

We studied the relationship between the M1 muscarinic receptor density and the receptor-mediated hydrolysis of inositol lipids in cloned murine fibroblast B82 cells which were transfected with the m1 muscarinic receptor gene. Of the seven clones examined, the M1 muscarinic receptor densities in these cells characterized by (-)[3H]methyl-3-quinuclidinyl benzilate ([-)-[3H]MQNB binding ranged from 12 fmol/10(6) cells in LK3-1 cells to 260 fmol/10(6) cells in the LK3-8 cells. Carbachol/(-)[3H]MQNB competition curves for the LK3-1 cells (with low receptor density) had a Hill coefficient close to unity. The competition curves for carbachol in the clones with higher receptor densities had Hill coefficients less than 1 and were best fitted by a computerized nonlinear least-squares regression program for the two-site model. The percentage of the M1 muscarinic receptors which had high affinity for carbachol decreased as the receptor density increased, suggesting that the presence of endogenous factors in these cells may be important for the agonist affinity state of the receptor. Concentration-response curves for carbachol-stimulated [3H]inositol monophosphate [( 3H]IP1) accumulation were also obtained. A significant correlation was observed between the density of M1 muscarinic receptor with high affinity for carbachol and the maximum [3H]IP1 accumulation in these cells. There is no significant difference among the EC50 values and the dissociation constant of high-affinity state values of the carbachol/(-)[3H] MQNB competition curves. These results suggest that the high-affinity state for carbachol may be the functional state of the M1 muscarinic receptors in these transfected B82 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological evidence for distinct muscarinic receptor subtypes coupled to the inhibition of adenylate cyclase and to the increased generation of inositol phosphates in the guinea pig myometrium

In the guinea pig myometrium, muscarinic receptor activation leads to contraction and elicits two biochemical responses viz. an increased formation of inositol phosphates (via a guanine nucleotide regulatory protein, distinct from the stimulatory and inhibitory G proteins of the adenylate cyclase system and a decreased synthesis of cyclic AMP involving inhibitory G protein activation. We now describe two major differences in the effects of muscarinic agonists. First, the greater potency of carbachol in inhibiting cyclic AMP formation (EC50 = 8 nM) than in stimulating the accumulation of inositol phosphates and tension (EC50 = 15 and 2 microM, respectively). Second, carbachol, oxotremorine and pilocarpine were equally effective in eliciting cyclic AMP inhibition but the order of potency for inositol phosphate formation was carbachol greater than oxotremorine and pilocarpine was without effect. The partial agonists, pilocarpine and oxotremorine, inhibited carbachol-mediated inositol phosphate formation. Pirenzepine, selective for muscarinic M1 receptor subtype, displayed a low affinity for antagonizing cyclic AMP inhibition, inositol phosphate generation and tension due to carbachol (Ki = 286, 92 and 110 nM, respectively). AF-DX116 (11-[( 2-[(diethylamino)methyl]-1- piperidinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine- 6-one), selective for cardiac M2 receptors blocked cyclic AMP inhibition with high affinity (Ki = 1.14 nM) while it antagonized inositol phosphate formation with low affinity (Ki = 346 nM). Both high (Ki = 1 nM) and low (Ki = 100 nM) affinities were displayed by AF-DX116 in antagonizing contractions due to carbachol (24 and 76% inhibition, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

A transfected m5 muscarinic acetylcholine receptor stimulates phospholipase A2 by inducing both calcium influx and activation of protein kinase C

Receptor-mediated arachidonic acid release and its relationship to phospholipase A2 and phospholipase C activation were investigated in Chinese hamster ovary cells transfected with and expressing the m5 muscarinic receptor. Carbachol, a muscarinic receptor agonist, stimulated the release of arachidonic acid and inositol phosphates with similar potencies. In addition, carbachol and the phorbol ester, phorbol-12-myristate, 13-acetate (PMA), stimulated protein kinase C (PKC) activity. PMA potentiated the carbachol-stimulated release of arachidonic acid, but had no effect on release of inositol phosphates. Long-term preincubation with PMA or carbachol inhibited PKC activity and prevented carbachol-stimulated release of arachidonic acid, but not inositol phosphates, suggesting that release of arachidonic acid, but not release of inositol phosphates, required activation of PKC. Carbachol stimulated the release of [3H]lysophosphatidylcholine from [3H]choline prelabeled cells, suggesting that phospholipase A2 was involved in the release of arachidonic acid. The role of calcium in carbachol-stimulated release of arachidonic acid was also investigated. Carbachol stimulated a transient followed by a sustained increase in intracellular calcium. In the absence of extracellular calcium, the transient rise in intracellular calcium was maintained but the sustained increase in intracellular calcium and the release of arachidonic acid were abolished. Carbachol stimulated a sustained influx of 45Ca++. We conclude that the combined effect of PKC activation and sustained elevation of intracellular calcium, from an extracellular source, is essential for m5 muscarinic receptor activation of phospholipase A2.     

Regional characterization of agonist and depolarization-induced phosphoinositide hydrolysis in rat brain

The ability of various receptor agonists and elevated extracellular potassium to initiate inositol phospholipid hydrolysis in various regions of rat brain was examined by using a direct assay which involves prelabeling slices with [3H]inositol and assaying [3H]inositol phosphates ([3H]IPs) in the presence of lithium. Both carbachol and noradrenaline evoked an increase in [3H]IP accumulation in all cerebral regions, although there were marked topographical differences in maximal responsiveness. These marked differences do not seem to be due to regional differences in coupling as similar apparent affinities of full agonists and the relative intrinsic activities of partial agonists were obtained. Both carbachol and noradrenaline responses were antagonized equipotently in all the brain regions tested by the muscarinic and alpha-1 antagonists atropine and prazosin, respectively. However, the putatively selective muscarinic antagonist pirenzepine did show an (approximately 10-fold) apparent selectivity against the carbachol responses elicited in the forebrain regions from those in the pons-medulla. Evaluation of extracellular potassium to 18 mM resulted in an increased production of [3H]IPs in all brain regions except the cerebellum. Incubation of slices with the cholinesterase inhibitor physostigmine (50 microM) and the dihydropyridine Ca++ channel activator BAY-K-8644 (1 microM) greatly enhanced the responses produced by elevated K+ in the forebrain regions but had markedly weaker effects in the hindbrain regions. The elevated K+ response alone and the enhanced response in the presence of BAY-K-8644 were both antagonized significantly by the dihydropyridine antagonist (+)-PN-205-033 in all brain regions, by 70 to 80 and 70 to 95%, respectively.     

Inhibitory effects of substance P and carbachol on the saturable sodium-dependent uptake process of myo-inositol in rat parotid gland.

myo-Inositol uptake in prisms of rat parotid glands was investigated by measuring both the accumulation of free myo-[3H] inositol into the cytosol and its incorporation into phospholipids. Total myo-[3H]inositol uptake involved two distinct processes, a prominent one which is saturable and sodium-dependent (Km, 95 microM; Vmax, 8 pmol/mg of protein per min) and a minor one, nonsaturable and sodium-independent. Phloretin and cytochalasin B, two inhibitors of hexose transport, and D-glucose, but only at high concentrations (greater than 10 mM), inhibited myo-[3H]inositol uptake. Dixon plots of the data indicated that D-glucose inhibition was noncompetitive suggesting that myo-inositol and D-glucose are transported by different carriers. Electrogenic cotransport of sodium and myo-inositol, rather than energy derived from mitochondrial oxidative metabolism, seems to be involved in the transport process. Thus, ouabain, monensin or veratridine, all of which increase intracellular sodium concentrations, reduced myo-[3H]inositol uptake, whereas dinitrophenol, potassium cyanide and carbonyl cyanide m-chlorophenyl hydrazone were without effect. Substance P affected only the sodium-dependent uptake process of myo-[3H]inositol, this inhibitory effect requiring extracellular calcium. Similar observations were made with the muscarinic agonist carbachol. From these results, an increase in intracellular sodium concentration linked to the activation of calcium-sensitive cation-permeant channels appears to be responsible for the inhibitory effects of substance P and carbachol on myo-[3H]inositol uptake, these effects being mediated respectively by NK1 and muscarinic receptors coupled to a phospholipase C.     

The contribution of cholinergic postganglionic neurotransmission to contractions of rabbit detrusor.

Field stimulation was used to elicit a contractile response in muscle strips from rabbit detrusor. The blockade of this response by tetrodotoxin (1 X 10(-7) M) ranged from 100% at 1 Hz to 86% at 40 Hz. At concentrations which produced strictly muscarinic antagonism (up to 4 X 10(-7) M) atropine depressed the frequency-response curve by about 42% at maximum but was much less effective at frequencies below 10 Hz. Similarly, treatment of the strips with hemicholinium-3 (5.2 X 10(-4) M) for 90 minutes in the presence of field stimulation at 60 Hz, depressed the frequency-response curve by 52% at maximum but produced less depression below 10 Hz. The hemicholinium-3-resistant response was neither depressed further by atropine (4 X 10(-7) M) nor potentiated by physostigmine (2 X 10(-6) M). Although hemicholinium-3 has antimuscarinic and anticholinesterase properties, these were found not to interfere with the tests for residual cholinergic transmission. Therefore, it was concluded that only part of the motor neurotransmission in rabbit detrusor is cholinergic. The remaining portion is predominant at frequencies below 10 Hz and is postulated to involve a chemical mediator other than acetylcholine.     

Effects of osmolality on phosphoinositide hydrolysis in renal medulla

We have reported previously that carbachol stimulates hydrolysis of phosphoinositides (PIs) in the renomedullary slices when incubated in a buffer of 300 mOsm/kg of H2O. However, the mammalian renal medulla has a hypertonic environment that changes with the state of hydration of the animal. In order to determine if the change in renal osmolality produces a change in the response of the inner medulla to hormones and neurotransmitters, we determined the effects of osmolality on carbachol-stimulated hydrolysis of PIs in the inner medullary slices of the rabbit kidney. The hydrolysis was determined by incorporation of [3H]inositol into PIs and the release of [3H]inositol phosphates in the presence and absence of 1 mM carbachol. The osmolality of the incubation media was increased from 300 to 1200 mOsm/kg of H2O in increments of 300 mOsm/kg of H2O by addition of either urea, NaCl, mannitol or an equiosmolar mixture of urea and NaCl. Increasing the osmolality of the incubation media by any one of these solutes decreased carbachol-stimulated release of inositol phosphates in the inner medullary slices of the rabbit kidney. Our results suggest that the effect of carbachol on PI messenger system in the renal medulla in vivo will depend on the tissue osmolality that itself depends on the state of hydration of the animal.     

Pharmacological characterization of the muscarinic receptors mediating phosphoinositide hydrolysis in rat myometrium

Activation of rat uterine myometrial muscarinic receptors with a variety of agonists results in increased phosphatidylinositol metabolism. Activation with carbachol is concentration- and time-dependent and is most apparent by following the accumulation of inositol monophosphate although there are small but significant increases of inositol bisphosphate and inositol trisphosphate. Carbachol stimulation of phospholipid turnover is greatest in the upper third of the uterus. The carbachol-induced increase of inositol monophosphate is antagonized by atropine and by the selective M-3 muscarinic receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methobromide. Pirenzepine, a selective M-1 receptor antagonist is less active, whereas gallamine and 11-2[[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one, selective M-2 receptor antagonists, are minimally effective suggesting that muscarinic M-3 receptors modulate phospholipid turnover in the rat myometrium. Displacement of tritium-quinuclidinyl benzilate binding by muscarinic antagonists also supports the presence of M-3 receptors in the uterus. Incubation with phorbol 12, 13-dibutyrate significantly reduced the accumulation of inositol monophosphate induced by carbachol implying that protein kinase C might modulate the responsiveness of the M-3 receptors in the rat uterus. Our results suggest that the intracellular concentration of calcium required for the contraction of the rat myometrium may be modulated, in part, through M-3 muscarinic receptors coupled to phospholipase C-activated turnover of phosphoinositides.     

Carbachol- and norepinephrine-stimulated phosphoinositide metabolism in rat brain: effect of chronic cholinesterase inhibition

Activation of cholinergic muscarinic receptors leads to several biochemical events including an increased turnover of phosphoinositides. In this study we have investigated whether repeated administration of the organophosphorus insecticide disulfoton, known to cause the development of tolerance to this compound, would affect phosphoinositide metabolism in rat brain. Basal and carbachol-stimulated phosphoinositide metabolism were measured in cerebral cortex slices, by measuring the accumulation of inositol phosphates (InsPs) in the presence of lithium. In control animals carbachol caused a 600% increase in InsPs accumulation with an EC50 of 100 microM. Maximal effect occurred with a LiCl concentration of 7.5 mM and required the presence of calcium. Administration of disulfoton for 10 days (2 mg/kg/day by gavage), decreased the number of muscarinic receptors in cortex from 1.1 to 0.7 pmol/mg of protein without changing the affinity of the receptors (both measured by binding of [3H]quinuclidinyl benzilate). Acetylcholinesterase was inhibited by 85%. Basal InsPs accumulation was unchanged in disulfoton-treated rats, whereas carbachol-stimulated InsPs accumulation decreased by 18%. No changes of norepinephrine-stimulated InsPs formation and of alpha-1 adrenoceptors were present in cortices from disulfoton-treated rats. Recovery of muscarinic receptor binding and carbachol-stimulated InsPs accumulation occurred at a similar rate and was completed 2 to 3 weeks after the end of the treatment, whereas acetylcholinesterase activity was still 38% inhibited 3 weeks later. These results support the hypothesis that a functional adaptation of muscarinic receptors is involved in the development of tolerance to organophosphates.     

Glucose and carbachol generate 1,2-diacylglycerols by different mechanisms in pancreatic islets.

Diacylglycerols (DAG) modulate secretory responses by the activation of protein kinase C. Early changes in DAG formation induced by the muscarinic receptor agonist carbachol were compared to those caused by the nutrient secretagogue glucose in pancreatic islets. Turnover rates of DAG were investigated in radiolabeling experiments, whereas changes in total mass and fatty acid composition of DAG were assessed by gas-liquid chromatography. When islet lipids were labeled to steady state in tissue culture with [3H]glycerol, carbachol induced a rapid (10 s) and sustained increase of [3H]DAG generation. In contrast, glucose stimulation failed to increase [3H]glycerol containing DAG, and this was probably due to the isotopic dilution of the label secondary to enhanced glycolysis. This was substantiated by following the transfer of 14C from glucose into DAG. Within 1 min of acute exposure of islets to D-[U-14C]-glucose at stimulatory concentrations, DAG labeling increased fivefold representing up to 2% of total glucose usage. Similar stimulation of 14C incorporation into other neutral lipids and inositol phospholipids was observed, suggesting the enhanced de novo synthesis of phosphatidic acid, the common precursor for DAG, and inositol phospholipids from glycolytic intermediates. Transfer of 14C from glucose was not stimulated by agents such as carbachol and exogenous phospholipase C that act primarily on inositol phospholipid breakdown. The total mass of islet DAG was increased by 60% after both carbachol and glucose stimulation. However, analysis of the fatty acid composition of carbachol-generated DAG revealed at the early time point (10 s) a prevalent stearoyl-arachidonoyl configuration similar to that reported for inositol phospholipids. This pattern shifted to a DAG enriched in palmitic acid at a later time point. Glucose-stimulated islets displayed a predominance of palmitic acid containing DAG, indicating increased de novo synthesis of the putative second messenger rather than its formation by inositol phospholipid hydrolysis. Indeed, steady-state labeling of these phospholipids with [3H]inositol confirmed this idea since only carbachol caused detectable inositol phospholipid hydrolysis. Thus, although protein kinase C may be activated by both carbachol and glucose, the two secretagogues generate diacylglycerols through different mechanisms.     

Prejunctional inhibitory effects of prostanoids on sympathetic neurotransmission in the rabbit iris-ciliary body

Both naturally occurring and synthetic prostaglandins (PGs) caused concentration-dependent inhibition of electrically evoked [3H]norepinephrine (NE) overflow from the isolated, superfused rabbit iris-ciliary body without affecting basal tritium efflux. The rank order of potencies of the agonists was: sulprostone greater than 16, 16-dimethyl-PGE2 greater than PGE2 greater than 11-deoxy-PGE1 greater than iloprost (stable PGl2 analog) greater than PGF2 alpha greater than or equal to PGD2. However, the Tx-mimetic, U-46619, was without effect on transmitter release at concentrations up to 1 microM. The selective EP1-receptor antagonists, AH 6809 (30 microM) or SC-19220 (10 microM) had no effect on basal or field-stimulated [3H]NE secretion, nor did they antagonize the PGE2-mediated reduction of evoked [3H]NE overflow. Indomethacin (3 microM) and the 5-lipoxygenase inhibitor, BW A4C (1 microM) were without effect on basal or evoked [3H]NE release, suggesting that endogenously formed arachidonic acid metabolites have no significant modulatory role in this in vitro system. Inhibitory effects of submaximal or maximal concentrations of PGE2 combined with corresponding concentrations of clonidine or carbachol were not additive, suggesting that prejunctional PGE2 receptors coexist with alpha-2 adrenergic and muscarinic receptors at neurotransmitter release sites. In the presence of yohimbine (100 nM) and/or atropine (100 nM), however, the inhibition produced by PGE2 was enhanced markedly, suggesting that tonic activation of prejunctional alpha-2 adrenergic or muscarinic receptors by endogenously released transmitters may impair the response to exogenous PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacologic comparison of selected agonists for the M1 muscarinic receptor in transfected murine fibroblast cells (B82).

The radioligand binding and functional properties of 10 muscarinic agonists for the M1 muscarinic receptors were characterized on the murine fibroblast B82 cells, which have been transfected with the m1 gene. All of the muscarinic agonists completely inhibited [3H](-)methyl-3-quinuclidinyl benzilate binding to the M1 muscarinic receptor in the transfected B82 cells. Their apparent inhibition constant values for agonist/[3H](-)methyl-3-quinuclidinyl benzilate inhibition experiments correlate well with their EC50 values in stimulating phosphatidylinositide hydrolysis. Based on the maximal functional effects: (+)-cismethyl-dioxolane, oxotremorine-M, acetylcholine, carbachol and methacholine are most efficacious, McN-A-343 and arecoline are least efficacious, whereas the efficacies of oxotremorine and pilocarpine are intermediate. In addition, McN-A-343 inhibited carbachol-stimulated phosphatidylinositide hydrolysis. Spare receptors were detected for oxotremorine-M, methacholine and carbachol, but not the rest of the agonists, by comparing the receptor-occupancy curves with the concentration-response curves. These results suggest that the presence of a quaternary nitrogen (trimethylammonium group) within the structure of the agonist may be important for the expression of full agonist activity.     

Relationship between agonist-induced muscarinic receptor loss and desensitization of stimulated phosphoinositide turnover in two neuroblastomas: methodological considerations

The addition of both full and partial muscarinic agonists to human SK-N-SH neuroblastoma cells resulted in a sustained phosphoinositide (PPI) hydrolysis for up to 2 hr, as monitored either by the accumulation of [3H]inositol phosphates (IP) in the presence of Li+, or alternatively, by an increased labeling of phosphatidate and phosphatidylinositol (PI) from 32Pi. This enhanced PPI hydrolysis was maintained even though 40-50% of cell-surface muscarinic cholinergic receptors (mAChRs) became sequestered. Desensitization of carbachol-stimulated PPI hydrolysis was only detected after a 2-4 hr prior exposure of the cells to the agonist and was accompanied by a comparable reduction in total mAChR number. Prolonged incubation of SK-N-SH cells with the partial agonist bethanechol also resulted in a desensitization of stimulated PPI turnover but at a slower rate than that observed for carbachol and with a loss of fewer mAChR sites. Desensitization of mAChR-stimulated [3H]IP formation appeared to occur more rapidly in mouse NIE-115 neuroblastoma. However, Li+ could not fully prevent the degradation of accumulated [3H]IP in these cells. When an increase in [32P]phosphatidylinositol labeling was used to assess PPI turnover in NIE-115 cells, no desensitization was evident for up to 2 hr. We conclude that 1) in SK-N-SH cells, desensitization more closely parallels the down-regulation than the sequestration of mAChRs, 2) in both neuroblastomas, stimulated PPI hydrolysis desensitizes relatively slowly and 3) the appearance of desensitization can vary as a function of the assay method employed.     

Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent [3H]acetylcholine release from rabbit retina labeled in vitro with [3H]choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of [3H]acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of [3H]acetylcholine with the following order of potency: apomorphine greater than or equal to SKF(R)82526 greater than SKF 85174 greater than SKF(R)38393 greater than or equal to pergolide greater than or equal to dopamine (EC50 = 4.5 microM) greater than SKF(S)82526 greater than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of [3H]acetylcholine: SCH 23390 (IC50 = 1 nM) greater than (+)-butaclamol greater than or equal to cis-flupenthixol greater than fluphenazine greater than perphenazine greater than trans-flupenthixol greater than R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating [3H]acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by [3H]SCH 23390, or as determined by adenylate cyclase activity. [3H]SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of [3H]SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate [3H]acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at [3H]SCH 23390 binding sites (r = 0.755, P greater than .05, n = 8). The potencies of antagonists to inhibit dopamine-evoked [3H]acetylcholine release were correlated with their potencies to inhibit the dopamine-stimulated adenylate cyclase (r = 0.759, P less than .05, n = 5) and with their affinities at [3H]SCH 23390 binding sites (r = 0.998, P less than .01, n = 7). We conclude that in rabbit retina dopamine evokes calcium-dependent [3H]acetylcholine release through activation of a site with the pharmacological characteristics of a D-1 dopamine receptor.     

Catecholamines relax detrusor through beta 2-adrenoceptors in mouse and beta 3-adrenoceptors in man

(-)-Isoproterenol [4-[1-hydroxy-2-[(1-methylethyl)amino]ethyl]-1,2-benzene diol hydrochloride] relaxes murine detrusor through beta-adrenoceptors (ARs); however, the beta-AR subtypes involved are unknown. beta(2)-ARs have been associated with caveolae, plasma-lemmal scaffolding domains that are absent in caveolin-1 (cav-1) knockout (KO) mice. Here, we studied detrusor responses in the absence and presence of beta-AR subtype-selective antagonists in wild-type (WT) and cav-1 KO mice. To inquire whether the murine detrusor model is relevant to man, beta-AR subtypes that mediate (-)-isoproterenol-evoked human detrusor relaxation were investigated. In WT mice, (-)-isoproterenol concentration-dependently relaxed the KCl (40 mM)-precontracted detrusor (-logEC(50)M = 8.04, E(max) = 62%). The effects of (-)-isoproterenol were surmountably antagonized by the beta(2)-AR-selective antagonist ICI 118,551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol] (pK(B) = 9.28) but not affected by the beta(1)-AR-selective antagonist CGP 20712 [1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol] and beta(3)-AR-selective L-748,337 [(S)-M-[4-[2-[3-[3-[acetamidomethyl)phenoxy)-2-hydroxypropyl]-amino]-ethyl]-phenylbenzsulfonamide)], suggesting involvement of beta(2)-AR only. The cav-1 KO detrusor displayed significant contractile dysfunction. (-)-Isoproterenol was less potent and efficient in relaxing detrusor from cav-1 KO (-logEC(50)M, 7.76; E(max) = 44%), but ICI 118,551 caused similar antagonism (pK(B) = 9.15), suggesting that beta(2)-AR function persisted in cav-1 KO. The beta(3)-AR-selective antagonist L-748,337 in the presence of ICI 118,551 and CGP 20712 caused additional blockade of (-)-isoproterenol effects in cav-1 KO, consistent with a beta(3)-AR involvement during relaxation and suppression of this effect in WT. (-)-Isoproterenol relaxed human detrusor muscle precontracted with carbachol (-logEC(50)M = 6.39, E(max) = 52%). However, the effects of (-)-isoproterenol in human detrusor were not blocked by CGP 20712 or ICI 118,551 but antagonized by L-748,337 (pK(B) = 7.65). We conclude that murine detrusor relaxation occurs via beta(2)-AR, and loss of caveolae does not perturb beta(2)-AR function but unmasks an additional activation of beta(3)-AR. In contrast, detrusor relaxation in man is mediated exclusively via beta(3)-AR.     

Characterization of high affinity [3H]pirenzepine and (-)-[3H] quinuclidinyl benzilate binding to muscarinic cholinergic receptors in rabbit peripheral lung

We have characterized the binding of the selective muscarinic antagonist [3H]pirenzepine ([3H])PZ) and the classical muscarinic antagonist (-)-[3H]quinuclidinyl benzilate ((-)-[3H]QNB) to muscarinic cholinergic sites in rabbit peripheral lung membranes. For both radioligands, high affinity binding with pharmacologic specificity was demonstrated. The high affinity Kd for [3H]PZ binding determined from saturation isotherms was 4.5 nM and the Kd for (-)-[3H]QNB binding was 6.2 pM. Comparison of the total binding capacity values determined by saturation experiments with [3H] PZ and (-)-[3H]QNB demonstrates that approximately 78% of the total muscarinic binding sites in rabbit peripheral lung bind [3H]PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of (-)-[3H]QNB binding by the muscarinic agonist carbachol in peripheral lung membranes. If the pulmonary muscarinic receptor with high affinity for PZ proves to have an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.