Effector Coupling Research Articles

Interaction of Anesthetics and Catecholamines on Conduction in the Canine His--Purkinje System

The findings in papillary muscles that epinephrine facilitates conduction at Purkinje fiber-muscle junctions and in the endocardium are consistent with older observations that activation of myocardial beta-adrenergic receptors speeds conduction and activation in the heart and thereby increases the synergy of contraction (46,47). The cellular mechanism underlying this action is probably increased cell-to-cell coupling between muscle fibers secondary to elevation of cyclic AMP (19,48). However, the findings that epinephrine alone or with halothane transiently slows conduction in the Purkinje layer while simultaneously improving conduction across Purkinje-muscle junctions and in the endocardium may represent proarrhythmic actions. These actions could facilitate arrhythmogenesis by transiently increasing regional differences of activation and repolarization times in the conduction system and myocardium and thereby increasing vulnerability to induction of reentry by premature impulses. Such a proarrhythmic effect could explain an older observation that low-dose norepinephrine infusions decrease the threshold for induction of fibrillation by two premature beats in pentobarbital-anesthetized animals (49). The cellular basis underlying the different responses of Purkinje fibers and the endocardial muscle layer to catecholamines, in which velocity decreased and increased, respectively, is not known. Our working hypothesis to explain this action in canine Purkinje fibers is a mechanism involving activation of WB4101-sensitive alpha 1-adrenoceptor, G-protein coupling to phospholipase C and the generation of DAG and IP3 leading to modulation of cell-to-cell coupling, which is potentiated in the presence of partial uncoupling by halothane. The different responses of Purkinje and myocardial fibers are speculated to result from differences in the relative density of this subtype of alpha 1-adrenoceptor, differences in the subcellular effector coupling mechanisms, or differences in the specific connexin proteins forming gap junctions between Purkinje and myocardial fibers (50).

2] Specific peptide probes for G-protein interaction with effectors

This chapter describes regions on G-protein α subunits involved in interactions with receptors. The main model system to be employed is the light receptor rhodopsin and the rod G protein transducin (G t ). The visual signaling pathway is similar to that of other transduction cascades involving receptor–G-protein–effector coupling. Light activation of rhodopsin leads to the formation of metarhodopsin II (Meta II), which displays sites on the cytoplasmic surface for interaction with heterotrimeric G t . Meta II catalyzes a conformational change in the G t α subunit ( α t ) which opens its nucleotide binding pocket and decreases the affinity of guanosine-diphosphate (GDP). “Empty-pocket” G t displays transient high affinity for rhodopsin which normally is terminated when guanosine-5'-triphosphate (GTP) binds and causes a second conformational change, leading to decreased affinity of α t for both rhodopsin and βγ . In the absence of GTP, the molecular basis for the high-affinity interaction between receptor and G protein is examined in the chapter in great detail.

Permeable Cell Systems as Models for Studying Disruption, by Site-Specific Synthetic Peptides, of Receptor–G Protein–Effector Coupling

Synthetic peptides corresponding to the carboxyl terminal of Gsα have been demonstrated to block activation of Gsα by the β-adrenergic receptor and the subsequent activation of adenylyl cyclase. These same peptides appear to mimic Gsα in promoting high-affinity agonist binding to that receptor. Curiously, attempts to achieve these responses in membranes have met with limited success. Thus, it appears that the cellular milieu surrounding the plasma membrane is best left intact. To disrupt the coupling between receptors and Gsα, a mechanism for making cells permeable has been devised to allow an effective examination of both adenylyl cyclase and radioligand binding in an environment that resembles an intact cell. Described herein is a method for making cells permeable in order utilize synthetic peptides to probe structure–function relationships of G protein α subunits in their ability to interface with both receptors and effector molecules. Specific methods for assaying adenylyl cyclase and the number and affinity of β-adrenergic receptors are described.

Distribution and second messenger coupling of four somatostatin receptor subtypes expressed in brain

The mRNA distribution in the brain and the coupling to cellular effector systems of four somatostatin receptors (SSTR1-4) was studied. All four SRIF receptor subtypes were expressed in cortex and hippocampus. In addition, SSTR1 mRNA was relatively abundant in the spinal cord whereas SSTR2 mRNA was also present in the striatum. The SSTR3 gene was predominantly expressed in the olfactory bulb and in the cerebellum. Conflicting results about the effector coupling of SSTR1-3 have been published previously. We have stably expressed human SSTR1-4 in HEK 293 human embryonal kidney cells. Agonist binding to the receptor subtypes, including the recently cloned SSTR4, inhibited the formation of forskolin-induced cAMP. Is is concluded that, in an appropriate cellular environment, all four receptor subtypes can functionally couple to the inhibition of adenylyl cyclase.

Resistance of β2-adrenoceptor-mediated responses of lung strips to desensitization by long-term agonist exposure - comparison with atrial β1-adrenoceptor-mediated responses

In vitro desensitization of β 2-adrenoceptor-mediated relaxation of guinea-pig isolated parenchymal strips was examined. Concentration-response curves for isoprenaline were obtained and after long-term incubation with isoprenaline, followed by washout, a second curve was obtained. Correction for time-dependent loss of sensitivity was made from time-matched controls. After incubation with 10 −5 M isoprenaline for 0.5, 1, 2, 4 and 8 h, loss responsiveness of carbachol-contracted lung strips was observed after 4h as a reduced post-incubation maximum response. When the concentration was reduced to 10 −6 M, a 4 h incubation with 1 h washout no longer induced a shift of the post-incubation curve in carbachol-contracted lung strips. In contrast, lung strips with intrinsic tone displayed reduced responsiveness to isoprenaline after 4 h incubation with 10 −6 M isoprenaline. Incubation of the tissue for 4 h with lanthanum (1.4 × 10 −6 M), a relaxant not operating through β 2-adrenoceptors or their effector coupling, had the same effect upon isoprenaline concentration-response curves as incubation with isoprenaline. This was irrespective of whether intrinsic tone (10 −6 M isoprenaline) or carbachol-contracted (10 −5 M isoprenaline) lung strips were used. It was concluded that the loss of β 2-adrenoceptor responsiveness after incubation with 10 −6 M isoprenaline was due to the prolonged maximal relaxation of the tissue for 4 h rather than desensitization of the β 2-adrenoceptor. Indeed, after correction for maximal relaxation and for time, no significant change in β 2-adrenoceptor sensitivity of the lung occurred after incubation with 10 −6 M isoprenaline for 4 h. This contrasts with significant rightwards shifts of the concentration-response curves for the β 1-adrenoceptor-mediated increases in rate and tension of guinea-pig right and left atria after identical incubation conditions. Thus whereas β 1-adrenoceptor-mediated responses displayed desensitization after long-term in vitro agonist exposure, the β 2-adrenoceptor-mediated responses were resistant.

Recombinant Gq alpha. Mutational activation and coupling to receptors and phospholipase C.

Gq mediates hormonal stimulation of phosphoinositide-specific phospholipase C (PI-PLC). We mutated the alpha subunit of Gq (alpha q) to replace arginine 183 with cysteine. Mutations that substitute cysteine for the corresponding arginine residues of alpha s and alpha i2 constitutively activate their respective effector pathways, creating the gsp and gip2 oncogenes. Transient expression of alpha q-R183C in COS-7 and HEK-293 cells constitutively activates PI-PLC, but wild type (WT) alpha q does not. This suggests that the mutated arginines in alpha s, alpha i2, and alpha q share a common function in regulating the active state of these proteins and that the alpha q gene may serve as a target for oncogenic mutations in human tumors. In an attempt to develop an assay for receptor stimulation of recombinant alpha q, we co-expressed receptors with alpha q-WT. We found that the alpha 2-adrenoceptor stimulates PI-PLC activation in HEK-293 cells in a fashion that depends completely on co-expression of alpha q-WT. These findings create an experimental model, similar to that provided for alpha s by S49 cyc- cells, that should make it possible to analyze receptor and effector coupling by mutant alpha q against a null background.

Multiple second messenger pathways of alpha-adrenergic receptor subtypes expressed in eukaryotic cells.

The alpha-adrenergic receptors mediate the effects of epinephrine and norepinephrine on cellular signaling systems via guanine nucleotide binding regulatory proteins (G-proteins). Three alpha-adrenergic receptor subtypes have been cloned: the alpha 1, the alpha 2-C10, and the alpha 2-C4 adrenergic receptors. To investigate functional differences between the different subtypes, we assessed the ability of each to interact with adenylyl cyclase and polyphosphoinositide metabolism by permanently and transiently expressing the DNAs encoding the alpha 1, the alpha 2-C10, and the alpha 2-C4 adrenergic receptors in cells lacking endogenous alpha-adrenergic receptors. Both alpha 2-C10 and alpha 2-C4 couple primarily to inhibition of adenylyl cyclase and to a lesser extent to stimulation of polyphosphoinositide hydrolysis. alpha 2-C10 inhibits adenylyl cyclase more efficiently than alpha 2-C4. Effects of the alpha 2-adrenergic receptors on adenylyl cyclase inhibition and on polyphosphoinositide hydrolysis are both mediated by pertussis toxin-sensitive G-proteins. The major coupling system of the alpha 1-adrenergic receptor is activation of phospholipase C via a pertussis toxin-insensitive G-protein. alpha 1-Adrenergic receptor stimulation can also increase intracellular cAMP by a mechanism that does not involve direct activation of adenylyl cyclase. As with the muscarinic cholinergic receptor family our results show that each of the alpha-adrenergic receptor subtypes can couple to multiple signal transduction pathways and suggest several generalities about the effector coupling mechanisms of G-protein-coupled receptors.

Location of a region of the muscarinic acetylcholine receptor involved in selective effector coupling

Chimaeric muscarinic acetylcholine receptors (mAChR) in which corresponding portions of mAChR I and mAChR II are replaced with each other have been produced in Xenopus oocytes by expression of cDNA constructs encoding them. Functional analysis of the chimaeric mAChRs indicates that a region mostly comprising the putative cytoplasmic portion between the proposed transmembrane segments V and VI is involved in selective coupling of mAChR I and mAChR II with different effector systems. In contrast, the exchange of this region between mAChR I and mAChR II does not significantly affect the antagonist binding properties of the two mAChR subtypes.

Neurotransmitter receptor interactions with G-proteins: A critical link in receptor response mechanisms

The interaction of membrane bound receptors with guanine nucleotide binding regulatory proteins is necessary for the transduction of many extracellular signals across the cell membrane. Age-related impairments of this interaction illustrate that the efficiency of receptor effector coupling is complex, and is an additional factor for consideration in the future study of receptor mediated functions.

Heterogenous properties of alpha2 adrenoceptors in particulate and soluble preparations of human platelet and rat and rabbit kidney

Alpha2 adrenoceptors of the human platelet and rat and rabbit renal cortex were compared in binding studies using the selective antagonist ligand [3H]rauwolscine. Significant differences in the pharmacological characteristics of the alpha2 adrenoceptor were observed between the tissues with reference to both absolute drug affinities as well as rank order of drug potency. Two subsets of the alpha2 adrenoceptor sites could be described: one exhibiting equal affinity for the alpha2 selective diastereoisomers, yohimbine and rauwolscine, and low affinity for the alpha1 antagonist prazosin (human platelet); the other displaying significantly higher affinity for rauwolscine than yohimbine but also relatively high affinity for prazosin (rat and rabbit kidney). Digitonin solubilised alpha2 adrenoceptors from these tissues identified by [3H]rauwolscine generally displayed reduced drug affinities. This was most apparent for agonists (10–50-fold lower) indicating separation of the soluble receptors from the guanine nucleotide binding proteins. However, the solubilised alpha2 adrenoceptors retained the overall pharmacological properties of their respective membrane receptors, including rank order of drug potency, reflecting similar inter-tissue differences. These results suggest that the pharmacological differences in alpha2 adrenoceptors observed are not species specific and are not related to variation in receptor effector coupling mechanisms or problems of ligand access due to membrane constraints. This may reflect true intrinsic differences in the molecular structure of these receptors.

Purification of the muscarinic acetylcholine receptor from rat forebrain.

The muscarinic acetylcholine receptor (mAChR) is a member of a class of neurotransmitter and hormone receptors which can activate more than one type of transmembrane signalling system. In the case of the mAChR, established mechanisms include (a) GTP-binding-proteinmediated inhibition of adenylate cyclase (Jakobs et al., 1979), (b) stimulation of the breakdown of triphosphoinositides (Jacobson et al., 1985), and (c) the opening of a certain category of K+ channels (Soejima & Noma, 1984). In each of these cases, there is evidence for the activation of distinct membrane-bound effector species by the agonistreceptor complex. The mAChR thus provides an example of a divergence of biochemical action at the level of effectuation of the response. The converse situation is also common, an example being the convergence of mAChR, crl -adrenoceptors and substance P receptors on the same pool of mobilizable intracellular Ca2+ in parotid gland cells (Putney , 1979). A likely implication of such functional diversity is that receptors such as the mAChR are, essentially, modular constructs, in which ligand-binding domains of different specificities exposed on the extracellular surface are linked to distinct effector recognition domains, or catalytic sites exposed to the intracellular milieu. Appropriate structural analogies may be provided by tyrosine kinases such as the epidermal growth factor and insulin receptors (Ullrich et al., 1984, 1985), by proteins of the major histocompatibility complex (Hood et al., 1982) or by immunoglobulins (Tonegawa, 1983). Conformational differences in the ligand-binding site induced by linkage to a variety of effector recognition sites might provide an explanation for the origin of the 'subtypes' detectable by selective ligands, in the case of the mAChR, by the selective antagonist pirenzepine (Pz) (Hammer et al., 1980; Birdsall & Hulme, 1983). However, conformational effects on the ligandbinding site could equally result from differences in effector coupling, or tissue-specific post-translational modifications, thus leading to the appearance of pharmacological subtypes without recourse to differences in the primary structure of the receptor. Convincing differentiation between these possibilities requires structure determination, and in particular the primary amino acid sequence of the receptor. This provides a major impetus for the purification, partial sequencing and cloning of receptors such as the mAChR. Further stimulus is provided by the prospect of obtaining monoclonal antibodies directed against different parts of the receptor sequence which may be used to detect structural homologies between different receptors and their subtypes (Andre et al., 1984; Venter et al., 1984), and which hold out the prospect of detailed immunohistochemical studies of receptor localization. To this end, we have purified the mAChR from rat forebrain by a straightforward procedure which exploits the properties of the o-atninobenzhydry-

Functional and biochemical basis for multiple muscarinic acetylcholine receptors

1. The novel antimuscarinic compound pirenzepine (PZ) has generated considerable interest in the basis and the implications of muscarinic acetylcholine receptor (mAChR) heterogeneity. 2. [ 3H]PZ has been used extensively to identify and characterize the putative M 1 (high affinity for PZ) mAChR subtype, which predominates in central nervous system (CNS) and ganglia. 3. The heterogeneity sensed by PZ is not identical to the heterogeneity sensed by agonists. 4. Differences in effector coupling do not necessarily provide a simple explanation for the molecular basis of these putative M 1 and M 2 subtypes. 5. Therapeutic and untoward effects of muscarinic drugs may be mediated by independent mAChR subpopulations which may be pharmacologically exploited to produce more highly selective as well as efficacious new drugs.

Activation, desensitization, and recycling of frog erythrocyte beta-adrenergic receptors. Differential perturbation by in situ trypsinization.

We have utilized limited in situ trypsinization of the adenylate cyclase-coupled beta-adrenergic receptor of frog erythrocytes to probe the processes of receptor activation, desensitization, and recycling. Treatment of intact erythrocytes with trypsin (1 mg/ml) for 1 h at 20 degrees C converts all the receptor peptides (identified by photoaffinity labeling with p-azido-125I-benzylcarazolol) from a Mr approximately 58,000 to a Mr approximately 40,000 species. Nonetheless, the trypsinized beta-adrenergic receptors bind agonists and antagonists with unaltered affinity and with no change in the number of binding sites. Moreover, the ability of the proteolyzed receptors to interact with the nucleotide regulatory protein to form a high affinity guanine nucleotide-sensitive state and to activate adenylate cyclase were also unaltered. However, upon exposure of intact cells to the agonist isoproterenol, trypsinized beta-adrenergic receptors were more rapidly and more completely cleared from the plasma membranes ("down-regulated") than untrypsinized receptors. Whereas down-regulated receptors from nontrypsinized cells appear to recycle to the cell surface after removal of the agonist, internalized trypsinized beta-adrenergic receptors do not recycle to the plasma membrane and appear to be degraded within the cell. Moreover, when internalized receptors, recovered in a light vesicle fraction, were fused with a heterologous adenylate cyclase system, untreated but not trypsinized receptors reconstituted catecholamine stimulation of the enzyme. These data suggest that the beta-adrenergic receptor contains a trypsin-sensitive site which is exposed on the outer surface of the plasma membrane. Proteolysis at this site releases a fragment which though not critically involved in either ligand binding or "effector coupling" might be important for anchoring the receptors in the plasma membrane. These data also suggest that in situ proteolysis of the receptors might serve as a physiological trigger for their internalization and degradation.

Receptor to effector coupling in the receptor-dependent adenylate cyclase system.

The mode of coupling between the components of hormone-regulated adenylate cyclases is discussed. In view of the structural knowledge and kinetic experiments, an attempt is made to analyze critically the current molecular models suggested for the mode of hormone stimulation and hormone inhibition of adenylate cyclase.

In vivo binding of 3H-etorphine in morphine-dependent rats

The opiate agonist 3H-etorphine was used to search for potential changes in in vivo opiate receptor binding in rats following chronic exposure to morphine sulfate. A rapid filtration method was employed to allow assessment of in vivo binding; receptor dissociation in vitro following in vivo labeling was also measured. No significant differences in total binding were seen with addicted animals, naive controls and naive animals pre-injected with morphine, at two different 3H-etorphine doses. In vitro dissociation under several conditions also yielded no differences. However, the rate of in vitro dissociation in the presence of both Na + and a guanine nucleotide showed a small but significant decrease in dependent animals, suggesting a possible impairment of receptor effector coupling with morphine addiction.