Transition probability cell cycle model. Part I--Balanced growth

In this study, we have identified and characterized functional alpha2-adrenergic receptor (alpha2-AR) subtypes in human corpus cavernosum and in cultured human corpus cavernosum smooth muscle cells. Analysis of total RNA, isolated from whole corpus cavernosum tissue and smooth muscle cells, by RNase protection assays, demonstrated expression of mRNA for alpha2A, alpha2B, and alpha2C adrenergic receptor subtypes in whole tissue and alpha2A and alpha2C subtypes in cultured smooth muscle cells. Binding studies with [3H]RX821002 (a highly selective and specific ligand for alpha2-adrenergic receptor) in isolated membrane fractions of human corpus cavernosum smooth muscle cells, demonstrated specific alpha2-AR binding sites with high affinity (Kd = 0.63 nM) and limited capacity (25-30 fmol/mg protein). Binding of [3H]RX821002 was displaced with the nonselective alpha-AR antagonist, phentolamine, and with the alpha-AR agonist, norepinephrine, in a dose-dependent manner, but not by the selective alpha1-AR agonist, phenylephrine. Binding of [3H]rauwolscine was also displaced by phentolamine. UK 14,304, a selective alpha2-AR agonist, inhibited forskolin-induced cyclic adenosine monophosphate (cAMP) synthesis in cultured human corpus cavernosum smooth muscle cells and induced dose-dependent contractions of tissue strips in organ bath chambers. UK 14,304-induced contractions were inhibited with alpha2-AR selective antagonists, rauwolscine and delquamine (RS 15385-197). These observations suggest that in human corpus cavernosum, norepinephrine (NE) and epinephrine may activate postsynaptic alpha2-AR subtypes, in addition to activating alpha1-AR subtypes, on smooth muscle cells, contributing to local control of human corpus cavernosum smooth muscle tone, in vivo.     

Physiological consequences of beta-adrenergic receptor disruption

Activation of beta-adrenergic receptors (beta-ARs) in vivo is an important means by which animals regulate cardiac performance, vascular tone, lipid and carbohydrate metabolism, and behavior. The advent of targeted gene disruption in mice has led to significant advances in our understanding of the role that beta-AR subtypes play in these processes, and this technique has become an important tool for the study of G protein coupled receptors in general. To date, targeted disruption of both beta1- and beta3-ARs in mice has been reported. Mice lacking beta1-ARs are unresponsive to cardiac beta-AR stimulation, suggesting that neither beta2- nor beta3-ARs couple to inotropic or chronotropic responses in the mouse. Conversely, mice lacking beta3-ARs retain at least some adipose beta-AR responsiveness through remaining beta1- and beta2-ARs, suggesting that all three beta-AR subtypes mediate similar functions in this tissue. While these knockout models have been extremely valuable tools for revealing the roles that individual beta-ARs play in whole animal physiology, it is also useful to integrate the results of experiments derived from either transgenic overexpression of beta-ARs or purely pharmacological approaches to the study of beta-AR function in order to create a comprehensive model of beta-AR function in vivo.     

Targeted disruption of the mouse beta1-adrenergic receptor gene: developmental and cardiovascular effects

At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes.     

Norepinephrine-induced contraction of isolated rabbit bronchial artery: role of alpha 1- and alpha 2-adrenoceptor activation

The contractile effect of norepinephrine (NE) on isolated rabbit bronchial artery rings (150-300 microns in diameter) and the role of alpha 1- and alpha 2-adrenoceptors (AR) on smooth muscle and endothelium were studied. In intact arteries, NE increased tension in a dose-dependent manner, and the sensitivity for NE was further increased in the absence of endothelium. In intact but not in endothelium-denuded arteries, the response to NE was increased in the presence of both indomethacin (Indo; cyclooxygenase inhibitor) and NG-nitro-L-arginine methyl ester [L-NAME; nitric oxide (NO) synthase inhibitor], indicating that two endothelium-derived factors, NO and a prostanoid, modulate the NE-induced contraction. The alpha 1-AR antagonist prazosin shifted the NE dose-response curve to the right, and phenylephrine (alpha 1-AR agonist) induced a dose-dependent contraction that was potentiated by L-NAME or removal of the endothelium. The sensitivity to NE was increased slightly by the alpha 2-AR antagonists yohimbine and idazoxan, and this effect was abolished by Indo or removal of the endothelium. Similarly, contractions induced by UK-14304 (alpha 2-AR agonist) were potentiated by Indo or removal of the endothelium. These results suggest that NE-induced contraction is mediated through activation of alpha 1- and alpha 2-ARs on both smooth muscle and endothelium. Activation of the alpha 1- and alpha 2-ARs on the smooth muscle causes contraction, whereas activation of the endothelial alpha 1- and alpha 2-ARs induces relaxation through release of NO (alpha 1-ARs) and a prostanoid (alpha 2-ARs).     

Atypical beta-adrenoceptors in the rat isolated common carotid artery

1. The possible existence of atypical beta-adrenoceptors in vascular smooth muscle of the rat common carotid artery was examined in this study. 2. Isoprenaline produced concentration-dependent relaxation of noradrenaline (10(-7) M) precontracted ring segments of the carotid artery. The relaxation was not affected by endothelial denudation. 3. Propranolol (10(-8) M-3 x 10(-7) M) shifted the isoprenaline curve to the right without suppressing the maximum response. However, the slope (0.74) of the Schild plot was significantly (P < 0.05) less than 1. 4. Salbutamol (beta 2), CGP 12177 and BRL 37344 (beta 3) also concentration-dependently relaxed noradrenaline precontracted artery segments. These relaxations were not affected by propranolol (10(-7) M). Pretreatment of the artery segments with BRL 37344 did not desensitize the tissue to the relaxant effect of isoprenaline, CGP 12177 and salbutamol. 5. It is concluded that atypical beta-adrenoceptors exist in vascular smooth muscle of the common carotid artery.     

Diffuse bile duct papillomatosis: high rate of recurrence and risk of malignant transformation

Magnesium (Mg2+) is an important cofactor in many intracellular biochemical reactions; however, its role in the signal transduction pathways of pulmonary vascular smooth muscle is poorly defined. The purpose of this study was to examine the following mechanisms of pulmonary vascular smooth relaxation in the presence and in the absence of Mg2+: (1) Endothelium-dependent cGMP-mediated relaxation (response to acetylcholine, ACh), (2) Endothelium-independent cGMP-mediated relaxation (response to sodium nitroprusside, SNP), and (3) beta2-adrenergic cAMP-mediated relaxation (response to isoproterenol, ISO). Dose response curves were generated in isolated rat pulmonary artery rings preconstricted with phenylephrine. With Mg2+, ACh 10(-6) M produced complete ring relaxation but in the absence of Mg2+, only 66% relaxation was produced in response to ACh 10(-6) M (P < 0.05). On the other hand, endothelium-independent cGMP-mediated relaxation (response SNP) was not impaired without Mg2+. Beta2-adrenergic cAMP-mediated relaxation was also impaired in the absence of Mg2+. In the presence of Mg2+, ISO 10(-6) M produced complete relaxation but without Mg2+, only 30% relaxation was produced (P < 0.05). We conclude that Mg2+ is essential for cGMP- and cAMP-mediated mechanisms of pulmonary vasorelaxation. Hypomagnesemia should be avoided to prevent pulmonary vasomotor dysfunction.     

Agonist interactions with chimeric and mutant beta1- and beta3-adrenergic receptors: involvement of the seventh transmembrane region in conferring subtype specificity

beta1- and beta3-adrenergic receptors (AR) are the predominant beta-AR subtypes in adipocytes, and analysis of native and recombinant beta-AR has revealed several pharmacological and biochemical differences between these subtypes. This study used chimeric and mutated rat beta-AR expressed in Chinese hamster ovary cells to examine the basis of certain characteristic differences in the agonist properties of catecholamines and prototypic beta3-AR agonists. The exchange of sequence beyond transmembrane (TM) region 6 between the beta-AR subtypes had dramatic and reciprocal effects on the affinity and efficacy of the prototypic beta3-AR agonists BRL 37,344 and CL 316,243, without affecting the interactions with catecholamines. Mutation of Phe350 and Phe351 in TM7 of the beta1-AR to Ala and Leu found in the beta3-AR was sufficient to allow activation by prototypic beta3-AR agonists. Interestingly, this mutation did not affect catecholamine action and it did not impair the ability of propranolol to block the actions of isoproterenol or the selective beta3-AR agonists. beta1-AR containing beta3-AR sequence from predicted TM5 through TM6 exhibited reduced affinity for catecholamines without altering agonist potency, suggesting enhanced coupling efficiency. Inclusion of the homologous beta1-AR sequence in the beta3-AR, however, did not produce reciprocal effects. These results are the first to define a major determinant of beta3-AR subtype-selective agonism in TM7 and demonstrate that the determinants of selective phenethanolamines, catecholamines, and propranolol action are distinct.     

Mechanism of rat uterine smooth muscle contraction induced by endothelin-1

1. Endothelin (ET)-1 has been demonstrated to cause contraction of uterine smooth muscle. We investigated the role of ET receptor subtypes (ETA and ETB receptors) in ET-1-induced contraction of rat uterine smooth muscle by using the ETA receptor antagonist BQ-123 and the ETB receptor agonist BQ-3020. 2. ET-1 caused a contraction with superimposed oscillations of the rat isolated uterus suspended in Krebs-Ringer solution; both the amplitude of contraction as well as the oscillation frequency increased in a dose-dependent manner (10(-11)-10(-7)M). 3. BQ-123 (10(-6)M) markedly shifted the dose-response curve of ET-1 for both contractile effects and oscillation frequency to the right. 4. BQ-3020 (10(-11)-3 x 10(-7) M) did not cause uterine contraction; neither did it affect the dose-response curve of ET-1 for either the contractile effect or the increase in oscillation frequency. Thus, stimulation of ETB receptors is not involved in these responses. 5. The present findings suggest that ET-1-induced contractile responses and the increase in oscillation frequency in rat uterine smooth muscle is mediated through ETA receptors, and that ETB receptors play no role in these responses.     

Inhibitory effects of diazepam and midazolam on Ca2+ and K+ channels in canine tracheal smooth muscle cells

BACKGROUND: Benzodiazepines have a direct bronchodilator action in airway smooth muscle, but the mechanisms by which these agents produce muscle relaxation are not fully understood. The current study was performed to identify the effects of the benzodiazepines diazepam and midazolam on Ca2+ and K+ channels in canine tracheal smooth muscle cells. METHODS: Whole-cell patch-clamp recording techniques were used to evaluate the effects of the benzodiazepines diazepam (10(-8) to 10(-3) M) and midazolam (10(-8) to 10(-3) M) on inward Ca2+ and outward K+ channel currents in dispersed canine tracheal smooth muscle cells. The effects of the antagonists flumazenil (10(-5) M) and PK11195 (10(-5) M) on these channels were also studied. RESULTS: Each benzodiazepine tested significantly inhibited Ca2+ currents in a dose-dependent manner, with 10(-6) M diazepam and 10(-5) M midazolam each causing approximately 50% depression of peak voltage-dependent Ca2+ currents. Both benzodiazepines promoted the inactivated state of the channel at more-negative potentials. The Ca2+-activated and voltage-dependent K+ currents were inhibited by diazepam and midazolam (> 10(-5) M and > 10(-4) M, respectively). Flumazenil and PK11195 had no effect on these channel currents or on the inhibitory effects of the benzodiazepines. CONCLUSIONS: Diazepam and midazolam had inhibitory effects on voltage-dependent Ca2+ channels, which lead to muscle relaxation. However, high concentrations of these agents were necessary to inhibit the K+ channels. The lack of antagonized effects of their antagonists is related to the non-gamma-aminobutyric acid-mediated electrophysiologic effects of benzodiazepines on airway smooth muscle contractility.     

Mechanism of prostaglandin E2-, F2alpha- and latanoprost acid-induced relaxation of submental veins

The mechanism of prostaglandin E2-, prostaglandin F2alpha- and latanoprost acid (13,14-dihydro-17-phenyl-18,19,20-trinor-prostaglandin F2alpha)-induced relaxation of the rabbit submental vein was studied. Prostaglandin E2 caused maximum relaxation of endothelin-1 precontracted vessels (EC50: 1.8 x 10(-8) M). Much of the relaxation could be abolished by denuding the endothelium with the nitric oxide synthase inhibitor, L-NAME (N(G)-Nitro-L-arginine methylester). CGRP-(8-37) (calcitonin gene-related peptide fragment (8-37)), a calcitonin gene-related peptide receptor antagonist, exhibited a partial blocking effect, whereas the tachykinin NK1 receptor blocker, GR 82334 ([D-Pro9[Spiro-gamma-Lactam]Leu10,Trp11]physalaemin (1-11)), markedly attenuated the response. Both prostaglandin F2alpha and the relatively selective FP receptor agonist, latanoprost acid, caused relaxation of the veins to about 50% of the precontracted state in the presence of GR 32191B ([1R-[1alpha(Z),2beta,3beta,5alpha]]-(+)-7-[5-([1,1'-b iphenyl]-4-ylmethoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-he ptenoic acid), a thromboxane receptor antagonist (EC50: for prostaglandin F2alpha 7.9 x 10(-9) M, and for latanoprost acid 4.9 x 10(-9) M). L-NAME, as well as denuding the endothelium, completely abolished the effect. In addition, most or at least a large part of the relaxation was also blocked by CGRP-(8-37) as well as GR 82334. These results indicate that the FP receptor-mediated relaxation of veins is based on release of nitric oxide in addition to involvement of calcitonin gene-related peptide and substance P, or some other tachykinin, probably released from perivascular sensory nerves. The more pronounced relaxation induced by prostaglandin E2 could be due to vasodilator EP receptors in the smooth muscle layer of the veins.     

A novel vascular relaxing agent, N-(6--aminohexyl)-5-chloro-1-naphthalensulfonamide which affects vascular smooth muscle actomyosin

The relaxing effect and possible mechanism of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on isolated rabbit artery were investigated. The addition of W-7 in concentrations ranging from 1 X 10(-6) to 3 X 10(-4) M caused a significant relaxation of isolated rabbit vascular strips contracted by KCl, prostaglandin F2alpha, norepinephrine, histamine, CaCl2, serotonin or angiotensin II. W-7 also caused a shift to the right of the dose-response curves for all agonists tested. Propranolol and atropine did not affect W-7 induced relaxation, suggesting that this drug does not act through beta adrenergic or cholinergic receptors. Superprecipitation of actomyosin from bovine aorta smooth muscle was inhibited by the addition of W-7 in a dose-dependent fashion. The concentration of W-7 which inhibited superprecipitation of bovine aorta smooth muscle actomyosin was in good agreement with the dose producing relaxation of isolated vascular strips. These facts suggest that W-7 produces relaxation of isolated vascular strips by inhibiting actin and myosin interaction.     

Mosaicism in trisomy 8: phenotype differences according to tissular repartition of normal and trisomic clones

The functional role of non-adrenergic non-cholinergic (NANC) nerves in the autonomic control of the male mini-pig bladder neck was investigated in the present study. Electrical stimulation of muscle strips from male mini-pig bladder neck showed biphasic response with initial phasic contraction followed by post-contractile relaxation. Electrical stimulation in the presence of four autonomic blockers (atropine 10(-6) M, propanolol 10(-6) M, phentolamine 10(-6) M) showed suppression of 68 +/- 15% of the contractile response (P < 0.05, n = 8) but no significant change in the relaxation response. Alpha-chymotrypsin 2 U/ml, L-NG-monomethyl-L-arginine acetate (a nitric oxide synthetase inhibitor) 10(-4) M, 8-phenylthlophylline (a P1-purinoceptor antagonist) 10(-6) M, and pyridoxal-phosphate-6-azophenyl-2', 4'-disulphoric acid tetrasodium salt (a P2Y-purinoceptor antagonist) 3 x 10(-5) M did not alter the NANC response significantly. On the other hand, reactive blue-2 (a P2Y-purinoceptor antagonist) 3 x 10(-5) significantly reduced the relaxation by 79 +/- 9%. The result suggested that the P2Y-purinoceptor is involved in the electrically induced NANC post-contractile relaxation of the mini-pig bladder neck smooth muscle.     

Prostaglandin E receptor subtypes in cultured rat microglia and their role in reducing lipopolysaccharide-induced interleukin-1beta production

Prostaglandins (PGs) are potent modulators of brain function under normal and pathological conditions. The diverse effects of PGs are due to the various actions of specific receptor subtypes for these prostanoids. Recent work has shown that PGE2, while generally considered a proinflammatory molecule, reduces microglial activation and thus has an antiinflammatory effect on these cells. To gain further insight to the mechanisms by which PGE2 influences the activation of microglia, we investigated PGE receptor subtype, i.e., EP1, EP2, EP3, and EP4, expression and function in cultured rat microglia. RT-PCR showed the presence of the EP1 and EP2 but not EP3 and EP4 receptor subtypes. Sequencing confirmed their identity with previously published receptor subtypes. PGE2 and the EP1 agonist 17-phenyl trinor PGE2 but not the EP3 agonist sulprostone elicited reversible intracellular [Ca2+] increases in microglia as measured by fura-2. PGE2 and the EP2/EP4-specific agonists 11-deoxy-PGE1 and 19-hydroxy-PGE2 but not the EP4-selective agonist 1-hydroxy-PGE1 induced dose-dependent production of cyclic AMP (cAMP). Interleukin (IL)-1beta production, a marker of activated microglia, was also measured following lipopolysaccharide exposure in the presence or absence of the receptor subtype agonists. PGE2 and the EP2 agonists reduced IL-1beta production. IL-1beta production was unchanged by EP1, EP3, and EP4 agonists. The adenylyl cyclase activator forskolin and the cAMP analogue dibutyryl cAMP also reduced IL-1beta production. Thus, the inhibitory effects of PGE2 on microglia are mediated by the EP2 receptor subtype, and the signaling mechanism of this effect is likely via cAMP. These results show that the effects of PGE2 on microglia are receptor subtype-specific. Furthermore, they suggest that specific and selective manipulation of the effects of PGs on microglia and, as a result, brain function may be possible.     

Distribution of prostaglandin IP and EP receptor subtypes and isoforms in platelets and human umbilical artery smooth muscle cells

Prostaglandins act through specific receptors to stimulate cyclic AMP formation which inhibits platelet activation and relaxes vascular smooth muscle. We have used RT-PCR combined with Southern blot analysis to determine the subtypes of prostaglandin receptor on platelets. Platelets expressed the EP4 rather than the EP2 prostaglandin EP receptor subtype, whereas vascular smooth muscle cells predominantly expressed the EP2 receptor. The IP receptor, which binds prostacyclin and couples to stimulation of adenylyl cyclase, and three isoforms of the inhibitory EP3 receptor were equally expressed in platelets, HEL cells and umbilical artery smooth muscle cells. The EP3-II isoform showed variation in level of expression among the three cell types. As a positive control for the presence of platelet RNA, PCR was performed using primers specific for the alpha chain of the platelet membrane glycoprotein Ib. As a negative control for the absence of T and B cell contamination in the platelet RNA, PCR was performed using primers specific for the cell specific cluster determinants CD2 (a T-cell marker) and CD20 (a B-cell marker). The finding that platelets express both stimulatory and inhibitory prostaglandin receptors provides confirmation of a homeostatic model of regulation of platelet adenylyl cyclase previously proposed.     

Heart rate variability: significance and clinic application

In vitro effects of two bioactive forms of pituitary adenylate cyclase activating polypeptide (PACAP): PACAP-38 and PACAP-27 were studied on rabbit vascular and non-vascular smooth muscle. Segments of the ovarian artery and muscle strips from the fallopian tube were used. Two series of experiments were performed on vessels: the dose-response relationship of PACAP-38 (10(-10)-10(-7) M) was established on noradrenaline- (NA, 10(-6) M) contracted vessels. In the other set of experiments the contractile effect of 10(-8)-10(-4) M NA added cumulatively, was studied on arterial segments incubated with PACAP-38 (10(-7) M), PACAP-27 (10(-7) M) or VIP (10(-7) M). The effect of PACAP-38, PACAP-27 and VIP (10(-10)-10(-6) M) was investigated on spontaneously contracting smooth muscle of the fallopian tube. Longitudinally as well as transversally cut specimens were investigated. PACAP-38 produced a significant dose-related relaxation on the NA-precontracted vessels. However, pre-incubation of the vessels with 10(-7) M PACAP-38, PACAP-27 and vaso active intestinal polypeptide (VIP) did not induce a general rightward shift of the NA concentration-response curves, although a tendency to inhibition in the low-dose interval was observed. The peptides caused a significant, dose-dependent inhibition of both frequency and amplitude on the fallopian tube smooth muscle activity. The effects of the three peptides on longitudinally as well as transversally cut specimens were alike.     

Tuberculosis control in health care workers: an algorithmic approach

Smooth muscle cells isolated from cecal circular smooth muscle of the guinea pig were used to determine whether thyrotropin-releasing hormone (TRH) can inhibit the contractile response produced by 10(-6) M carbachol by exerting a direct action on muscle cells. In addition, the inhibitory effect of 2',5'-dideoxyadenosine (an inhibitor of adenylate cyclase), phorbol 12-myristate 13-acetate (an inhibitor of particulate guanylate cyclase), 6-anilinoquinoline-5,8-quinone (an inhibitor of nitric oxide synthase) on the TRH-induced relaxation of cecal circular smooth muscle cells was examined. TRH inhibited the contractile response produced by 10(-6) M carbachol in a concentration-dependent manner, with an IC50 value of 4 nM, 2',5'-Dideoxyadenosine and phorbol 12-myristate 13-acetate did not have any significant effect on the TRH-induced relaxation. On the other hand, 6-anilinoquinoline-5,8-quinone and N omega-nitro-L-arginine methyl ester significantly inhibited the relaxation produced by TRH. Our findings show that TRH has a direct inhibitory effect on the isolated cecal circular smooth muscle cells via activation of nitric oxide synthase and soluble guanylate cyclase.     

Mechanism of nitric oxide-induced contraction in the rat isolated small intestine

1. The contractile response to nitric oxide (NO) in ral ileal myenteric plexus-longitudinal muscle strips was pharmacologically analysed. 2. NO (10(-7) M) induced only contraction while 10(-6) M NO induced contraction followed by relaxation. Methylene blue (up to 10(-4) M) did not affect the NO-induced contractions but significantly reduced the relaxation evoked by 10(-6) M NO. Administration of 8-bromo-cyclic GMP (10(-6)-10(-4) M) only induced relaxation. 3. Sodium nitroprusside (SNP; 10(-7)-10(-5) M) induced concentration-dependent contractions per se; the contractile response to NO, administered within 10 min after SNP, was concentration-dependently reduced. The guanosine 3':5'-cyclic monophosphate (cyclic GMP) content of the tissues was not increased during contractions with 10(-8) M NO and 10(-6) M SNP; it was increased by a factor of 2 during contraction with 10(-7) M NO, and by a factor of 12 during relaxation with 3 x 10(-6) M NO. 4. The NO-induced contractions were not affected by ryanodine (3 x 10(-5) M) but were concentration-dependently reduced by nifedipine (10(-8)-10(-7) M) and apamin (3 x 10(-9)-3 x 10(-8) M). 5. These results suggest that cyclic GMP is not involved in the NO-induced contraction in the rat small intestine. The NO-induced contraction is related to extracellular Ca2+ influx through L-type Ca2+ channels, that might be activated in response to the closure of Ca(2+)-dependent K+ channels.     

Functional role of M2 and M3 muscarinic receptors in the urinary bladder of rats in vitro and in vivo

1. Urinary bladder smooth muscle is enriched with muscarinic receptors, the majority of which are of the M2 subtype whereas the remaining minority belong to the M3 subtype. The objective of the present study was to assess the functional role of M2 and M3 receptors in the urinary bladder of rat in vitro and in vivo by use of key discriminatory antagonists. 2. In the isolated bladder of rat, (+)-cis-dioxolane produced concentration-dependent contractions (pEC50 = 6.3) which were unaffected by tetrodotoxin (0.1 microM). These contractions were antagonized by muscarinic antagonists with the following rank order of affinity (pA2) estimates: atropine (9.1) > 4-diphenyl acetoxy-methyl piperidine methiodide (4-DAMP) (8.9) > darifenacin (8.5) > para fluoro hexahydrosiladifenidol (p-F-HHSiD) (7.4) > pirenzepine (6.8) > methoctramine (5.9). These pA2 estimates correlated most favourably (r = 0.99, P < 0.001) with the binding affinity (pKi) estimates of these compounds at human recombinant muscarinic m3 receptors expressed in Chinese hamster ovary cells, suggesting that the receptor mediating the direct contractile responses to (+)-cis-dioxolane equates with the pharmacologically defined M3 receptor. 3. As M2 receptors in smooth muscle are negatively coupled to adenylyl cyclase, we sought to determine whether a functional role of M2 receptors could be unmasked under conditions of elevated adenylyl cyclase activity (i.e., isoprenaline-induced relaxation of KCl pre-contracted tissues). Muscarinic M3 receptors were preferentially alkylated by exposing tissues to 4-DAMP mustard (40 nM, 1 h) in the presence of methoctramine (0.3 microM) to protect M2 receptors. Under these conditions, (+)-cis-dioxolane produced concentration-dependent reversal (re-contraction) of isoprenaline-induced relaxation (pEC50 = 5.8) but had marginal effects on pinacidil-induced, adenosine 3':5'-cyclic monophosphate (cyclic AMP)-independent, relaxation. The re-contractions were antagonized by methoctramine and darifenacin, yielding pA2 estimates of 6.8 and 7.6, respectively. These values are intermediate between those expected for these compounds at M2 and M3 receptors and were consistent with the involvement of both of these subtypes. 4. In urethane-anaesthetized rats, the cholinergic component (approximately 55%) of volume-induced bladder contractions was inhibited by muscarinic antagonists with the following rank order of potency (ID35%inh, nmol kg-1, i.v.): 4-DAMP (8.1) > atropine (20.7) > methoctramine (119.9) > darifenacin (283.3) > pirenzepine (369.1) > p-F-HHSiD (1053.8). These potency estimates correlated most favourably (r = 0.89, P = 0.04) with the pKi estimates of these compounds at human recombinant muscarinic m2 receptors. This is consistent with a major contribution of M2 receptors in the generation of volume-induced bladder contractions, although the modest potency of darifenacin does not exclude a role of M3 receptors. Pretreatment with propranolol (1 mg kg-1, i.v.) increased the ID35%inh of methoctramine significantly from 95.9 to 404.5 nmol kg-1 but had no significant effects on the inhibitory responses to darifenacin. These data suggest an obligatory role of beta-adrenoceptors in M2 receptor-mediated bladder contractions in vivo. 5. The findings of the present study suggest that both M2 and M3 receptors can cause contraction of the rat bladder in vitro and may also mediate reflex bladder contractions in vivo. It is proposed that muscarinic M3 receptor activation primarily causes direct contraction of the detrusor whereas M2 receptor activation can contract the bladder indirectly by reversing sympathetically (i.e. beta-adrenoceptor)-mediated relaxation. This dual mechanism may allow the parasympathetic nervous system, which is activated during voiding, to cause more efficient and complete emptying of the bladder.     

Antagonist properties of a phosphono isoxazole amino acid at glutamate R1-4 (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid receptor subtypes

All three subtypes of beta-adrenoceptors are coupled to stimulation of adenylyl cyclase activity via the stimulatory guanine-nucleotide-binding protein. Nevertheless, the beta3 adrenoceptor (beta3-AR) differs significantly from the other subtypes in terms of pharmacology. Most strikingly, it recognizes as agonists several compounds acting as potent beta1-AR and beta2-AR antagonists. Furthermore, the human beta3-AR is quite different from the animal beta3-AR. Molecular modelling studies followed by site-directed mutagenesis was used here to identify some of the amino acid residues which may be implicated in ligand binding and signal transduction of the beta3-AR. Three contiguous residues, valine-leucine-alanine, which are present in the first transmembrane domain at positions 48-50 of the human receptor but are absent in all known rodent sequences, were thought to be important for species specificity. When these three residues were deleted from the human receptor, no 'rodent-like' pharmacological profile was obtained in terms of either binding or adenylyl cyclase activation. Glycine at position 53, also in the first transmembrane domain in the human beta3-AR, has been suggested to participate in beta2-/beta3-AR subtype selectivity. Replacement of this glycine residue by phenylalanine, which is the residue present at the homologous position in the human beta2-AR, left the beta3-AR pharmacological profile unaltered in terms of specificity and selectivity. Aspartate residue 117, in the third transmembrane domain, has been found to be essential for ligand binding and consequently adenylyl cyclase activation in several bioamine receptors. When this residue was replaced by a leucine residue in the beta3-AR, ligand binding and signal transduction were suppressed. Finally, replacement of asparagine at position 312 in the sixth transmembrane domain by an alanine residue, led to alterations in the signal-transduction pathway.