Biomedical subjects

R H Strasser

Publications and source records attributed to R H Strasser.

At least 55 records · Page 3Linked to original sources

PubMed1992

Alpha 1-receptor-independent activation of protein kinase C in acute myocardial ischemia. Mechanisms for sensitization of the adenylyl cyclase system.

The activity of the adrenergic system plays an important role in the genesis of malignant arrhythmias and the spreading of the infarcted zone in acute myocardial ischemia. Acute myocardial ischemia induces an increased activity of adenylyl cyclase. This sensitization at the enzyme level as shown in the isolated perfused rat heart occurs rapidly after the onset of ischemia (5-15 minutes) and is rapidly reversible on reperfusion. With prolonged ischemia, it is only transient and is followed by a gradual loss of the adenylyl cyclase activity. The increased activity of adenylyl cyclase is even retained after partial purification, suggesting a covalent modification of the enzyme. Blockade of alpha 1-adrenergic receptors does not prevent this sensitization, demonstrating that it occurs independently of alpha 1-adrenergic receptor activation. Only blockade of protein kinase C by various inhibitors, such as polymyxin B or staurosporine, is able to completely prevent this sensitization process. Moreover, in acute myocardial ischemia an activation of protein kinase C could be identified using its translocation from the cytosol to the particulate fraction as an indicator. Blockade of alpha 1-adrenergic receptors using prazosin fails to prevent the activation of protein kinase C and consequently the sensitization of the adenylyl cyclase system, indicating that the ischemia-induced translocation of protein kinase C occurs independently of alpha 1-adrenergic receptors. These data characterize for the first time an important interaction of two effector enzymes of two distinct signal transduction pathways, i.e., the adenylyl cyclase system and the protein kinase C system in acute myocardial ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗

PubMed1991

Sensitization of the beta-adrenergic system in acute myocardial ischaemia by a protein kinase C-dependent mechanism.

In acute myocardial infarction the occurrence of malignant arrhythmias and the spreading of the infarcted zone followed by the development of heart failure determine the clinical outcome of the disease. The activity of the adrenergic system plays an important role in both. At various levels acute myocardial ischaemia induces an inadequate activation of the adrenergic system. The increased presynaptic release of endogenous catecholamines does not promote the expected desensitization at the postsynaptic level. In contrast, acute ischaemia leads to a rapid and persistent increase of functionally coupled beta-adrenergic receptors, which in the early phase of acute ischaemia, induce an increased responsiveness of the adenylyl cyclase system to beta-adrenergic stimulation. This sensitization at the receptor level is superimposed by a receptor-independent sensitization of the adenylyl cyclase and a loss of tonic inhibition due to the functional impairment of the inhibitory G protein. At the enzyme level a transient sensitization of adenylyl cyclase in acute myocardial ischaemia is due to a modification of the enzyme, which is tightly associated with the purified enzyme. Only inhibition of protein kinase C is able to block completely the ischaemia-induced sensitization of adenylyl cyclase. Based on these data, it could be demonstrated that acute myocardial ischaemia leads to a rapid activation of protein kinase C by an as yet undefined mechanism. Beyond the sensitization of adenylyl cyclase, activation of protein kinase C may directly activate ion channels or the N+/H+ echanger, and it may induce the increased expression of oncogenes and thus crucially influence the clinical outcome of an acute myocardial infarction.

Adenylyl Cyclases↗

PubMed1990

Supersensitivity of the adenylyl cyclase system in acute myocardial ischemia: evaluation of three independent mechanisms.

Malignant arrhythmias and the spreading of the infarcted zone in acute myocardial ischemia may be influenced by the sympathetic system. It has been known for quite some time that acute ischemia leads to an increased release of endogenous catecholamines. Adaptive mechanisms at the postsynaptic level such as receptor desensitization, which are operative under normoxic conditions, are abolished in acute myocardial ischemia. On the contrary, three newly characterized, distinct mechanisms lead to a transiently increased activity of the beta-adrenergic system in the early phase of acute ischemia: 1) Functionally coupled beta-adrenergic receptors are rapidly and persistently increased at the cell surface due to the impairment of beta-agonist-promoted uncoupling and internalization. 2) Despite the reversible increase of inhibitory, muscarinic M2 receptors, the inhibitory pathway of the adenylyl cyclase systems becomes ineffective since the coupling protein, Gi, is rapidly impaired. Both the Gi-linked GTPase-activity and the binding of [gamma-35S]GTP are reduced by 25-30% without any loss of the total protein. Stimulatory effects prevail at the G-protein level since in the early period of ischemia the stimulatory G-protein, Gs, remains intact. 3) The adenylyl cyclase is transiently sensitized by about 30%. This increased activity is closely associated with the partially purified enzyme and may be due to a rapidly reversible covalent modification. Prolonged ischemia, in contrast, results in a general decrease of the cyclase activity notwithstanding any changes at the receptor or G-protein level. The individual mechanisms may play distinct and/or complimentary roles in the early sensitization of the adenylyl cyclase system in acute myocardial ischemia.

Adenylyl Cyclase Inhibitors↗

PubMed1990

Dual sensitization of the adrenergic system in early myocardial ischemia: independent regulation of the beta-adrenergic receptors and the adenylyl cyclase.

Acute myocardial ischemia provokes sensitization of the adenylyl cyclase system. This sensitization can be differentiated in a receptor-specific and an enzyme-specific sensitization. The receptor-linked sensitization is characterized by an increase of beta-adrenergic receptors in the plasma membranes after 15 mins of global ischemia (49.8 +/- 3.6 to 67 +/- 6 fmol/mg protein) followed by a further increase (89 +/- 4 fmol/mg protein) after 50 min of ischemia in isolated perfused hearts. Concomitantly functionally coupled receptors which are able to bind the beta-agonist with high affinity, increased by 32% after 15 min and by 57% after 50 min of ischemia. The affinities of the receptors for their agonists or their antagonists remain unchanged. Maximally isoproterenol-stimulated adenylyl cyclase activity rose from 66 +/- 7 to 101 +/- 10 pmol cAMP/min/mg protein after 15 min of global ischemia indicating the beta-receptor-specific sensitization of the beta-adrenergic system. This sensitization was followed by a gradual decline of the adenylyl cyclase activity after 30 and 50 min of global ischemia. Additionally, 15 min of myocardial ischemia induced an enzyme-linked sensitization of the adenylyl cyclase activity as indicated by an increase of the forskolin-stimulated activity by about 25% (300 +/- 20 vs 378 +/- 25 pmol cAMP/min/mg protein). In contrast after 50 min of ischemia the total adenylyl cyclase activity declined (232 +/- 24 pmol cAMP/min/mg protein) despite the persistent increase of beta-adrenergic receptors in the plasma membranes. These data demonstrate that the enzyme-specific sensitization is only transient. The early sensitization and late inactivation of the adenylyl cyclase activity occurred independently of receptor activation and could not be prevented by beta-blockade (10(-6) M alprenolol). Cyanide perfusion (1 mM), used to block energy metabolism, lead to energy depletion similar to acute myocardial ischemia. This resulted in an increase of functionally coupled receptors with a time course comparable to that of global ischemia. Additional perfusion with desensitizing concentrations of the beta-agonist isoproterenol did not induce uncoupling or internalization of beta-adrenergic receptors in cyanide treated hearts, suggesting that the rise in functionally coupled receptors is due to a redistribution in part caused by the abolition of continuous receptor internalization. In contrast, the enzyme-linked sensitization is independent of cellular localization of the beta-adrenergic receptors. The increased activity was carried by the enzyme even after partial purification with solubilization and wheat germ affinity chromatography. These data suggest an ischemia-induced, covalent modification of the adenylyl cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclases↗

PubMed1990

Independent sensitization of beta-adrenoceptors and adenylate cyclase in acute myocardial ischaemia.

1. Acute myocardial ischaemia provokes sensitization of the adenylate cyclase system. This sensitization could be differentiated in a receptor-linked and an enzyme-linked sensitization. The increase in the number of beta-adrenoceptors in the plasma membranes was observed already after 15 min of global ischaemia (50 +/- 2 to 67 +/- 6 fmol mg-1 protein) and persisted after 50 min of ischaemia. The maximally isoprenaline-stimulated adenylate cyclase activity rose from 66 +/- 7 to 100 +/- 10 pmol cAMP min-1 mg-1 protein after 15 min of global ischaemia indicating the receptor-mediated sensitization of the beta-adrenergic system. However, after 50 min of ischaemia the isoprenaline-stimulated adenylate cyclase was reduced by about 50% despite the continuous increase of beta-adrenoceptors in the plasma membranes. 2. Additionally direct stimulation of the adenylate cyclase by forskolin revealed an increased enzyme activity after 15 min of global ischaemia (300 +/- 20 vs 378 +/- 25 pmol cAMP min-1 mg-1). Prolonged periods of ischaemia, however, caused a decline of the total adenylate cyclase activity (232 +/- 24 pmol cAMP min-1 mg-1 protein). This demonstrates an enzyme-specific sensitization of the adenylate cyclase, which in contrast to the rise in beta-adrenoceptors is only transient. This enzyme-specific sensitization or the late inactivation of the enzyme occur independently of receptor activation and cannot be prevented by beta-adrenoceptor blockade (10(-6) M alprenolol) prior to the ischaemic insult.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗

PubMed1990

Adrenergic receptors and sensitization of adenylyl cyclase in acute myocardial ischemia.

Acute myocardial ischemia leads to a gradual increase in beta-adrenergic receptors at the cell surface. This increase occurs rapidly after onset of global ischemia (15 minutes) and persists even after prolonged periods of global ischemia. This alteration can be observed both in vivo and in vitro in isolated perfused hearts. Several groups have previously shown that ischemia induces a local release of endogenous catecholamines. Here, we show that these endogenous catecholamines are sufficiently high to induce receptor desensitization with internalization of beta-adrenergic receptors in normal hearts. In acute myocardial ischemia, however, agonist-promoted internalization and functional uncoupling of beta-adrenergic receptors is abolished. Consequently, the balance of internalization and externalization of receptors is shifted toward an increase in functionally coupled receptors at the cell surface. Similarly, but inconsistently, the density of alpha 1-adrenergic receptors in the plasma membrane is increased in acute myocardial ischemia. In regard to function, the increase of coupled beta-adrenergic receptors leads to an augmented responsiveness of the adenylyl cyclase system to beta-adrenergic stimulation. This receptor-specific sensitization is superimposed by a transient increase of total adenylyl cyclase activity in the very early phase of global ischemia (0-20 minutes). The enhanced activity of adenylyl cyclase to direct stimulation is tightly associated with the partially purified enzyme, suggesting a covalent modification of the enzyme molecule. However, after prolonged periods (greater than 30 minutes) of global ischemia, the ischemia-induced enzyme-specific sensitization is displaced by a general reduction in enzyme activity, both in vivo and in vitro. The persistent sensitization at the receptor level then meets an unresponsive adenylyl cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗

PubMed1988

The beta-adrenergic receptor kinase: role in homologous desensitization in S49 lymphoma cells.

Phosphorylation of the beta-adrenergic receptor (beta AR) is closely associated with homologous desensitization of the beta-adrenergic receptor-coupled adenylate cyclase system. Homologous desensitization and receptor phosphorylation also occur in cell mutants which are deficient in their cAMP-dependent protein kinase (kin- mutant of S49 lymphoma cells). beta AR phosphorylation is mediated by a cAMP-independent protein kinase which phosphorylates the receptor only when it is occupied by a beta-agonist. During the time course of desensitization the beta AR kinase (beta ARK) activity is translocated from a cytoplasmic to a plasma membrane location. beta ARK translocation can also be effected by prostaglandin E1 (PGE1) suggesting that this beta ARK may represent a more general enzyme capable of phosphorylating other adenylate cyclase-coupled receptors. Thus, beta ARK may play a key role in the process of homologous desensitization of adenylate cyclase coupled receptors. Extracellular hormones interact with specific receptors at the outer surface of the plasma membrane and thus initiate a cellular response. One of the best studied transmembrane signalling systems known to be coupled to the occupancy of cell surface receptors is adenylate cyclase. The adenylate cyclase system is composed of various components all of which have been purified to homogeneity (Shorr et al., 1982; Homcy et al., 1983; Benovic et al., 1984; Codina et al., 1984; Northup et al., 1980; Sternweis et al., 1981; Bokoch et al., 1984; Pfeuffer et al., 1985). Initially, agonist binding to the receptor promotes coupling of the occupied receptor to one of the guanine nucleotide binding regulatory proteins. These proteins are members of a family of heterotrimeric proteins consisting of alpha, beta and gamma subunits. Stimulatory receptors like the beta-adrenergic (Cerione et al., 1984) or glucagon (Iyengar et al., 1979) receptors couple to the stimulatory regulatory protein Ns (or Gs) whereas inhibitory receptors like the alpha 2-adrenergic (Jacobs et al., 1976) or M2-muscarinic (Harden et al., 1982) receptors couple to the inhibitory regulatory protein Ni (or Gi). Prolonged exposure to agonist hormones, either stimulatory or inhibitory, results in an attenuation of the response to the hormonal activation, a phenomenon called tachyphylaxis or desensitization (Harden, 1983; Sibley and Lefkowitz, 1985; Sharma et al., 1975). One of the best studied models for desensitization is the beta-adrenergic receptor-coupled adenylate cyclase system. In this system two different forms of desensitization have been characterized.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Regulation of beta-adrenergic receptors: impaired desensitization in myocardial ischemia.

The dynamic regulation of the adrenergic system plays an important role in the adaptation of the cardiovascular system in health and in disease. In myocardial infarction, large quantities of catecholamines are presynaptically released. The mechanisms of adaptive regulation of the beta-adrenergic receptors to beta-agonist stimulation involves functional uncoupling of the beta-adrenergic receptors from stimulatory guanine nucleotide binding protein Gs by local sequestration with the domain of the plasma membranes and by internalization of the beta-adrenergic receptors. During this course of adaptive regulation, also called desensitization, the beta-adrenergic receptors become phosphorylated by the newly discovered beta-adrenergic receptorkinase in a cAMP-independent process. In myocardial infarction, however, large quantities of beta-adrenergic agonists are contrasted by an increased number of functionally coupled beta-adrenergic receptors. The increase of functionally coupled beta-adrenergic receptors is dependent on the loss of high energy phosphates like ATP. This loss occurring in myocardial ischemia can be mimicked by the perfusion of isolated hearts with cyanide. The perfusion with cyanide results in an increase of functionally coupled receptors, additionally demonstrating that the loss of high energy phosphates is responsible for the increase of the beta-adrenergic receptors in acute myocardial ischemia. For the first time it could be demonstrated that in myocardial ischemia the processes of beta-agonist-induced receptor uncoupling and internalization are abolished. They lead to the functional sensitization of the beta-adrenergic system and are at least in part responsible for the occurrence of malignant arrhythmias in myocardial infarction.

Animals↗

PubMed1986

A novel catecholamine-activated adenosine cyclic 3',5'-phosphate independent pathway for beta-adrenergic receptor phosphorylation in wild-type and mutant S49 lymphoma cells: mechanism of homologous desensitization of adenylate cyclase.

Virtually all known biological actions stimulated by beta-adrenergic and other adenylate cyclase coupled receptors are mediated by cAMP-dependent protein kinase. Nonetheless, "homologous" or beta-adrenergic agonist-specific desensitization does not require cAMP. Since beta-adrenergic receptor phosphorylation may be involved in desensitization, we studied agonist-promoted receptor phosphorylation during homologous desensitization in wild-type S49 lymphoma cells (WT) and two mutants defective in the cAMP-dependent pathway of beta-agonist-stimulated protein phosphorylation (cyc- cannot generate cAMP in response to beta-adrenergic agonists; kin- lacks cAMP-dependent kinase). All three cell types demonstrate rapid, beta-adrenergic agonist-promoted, stoichiometric phosphorylation of the receptor which is clearly not cAMP mediated. The amino acid residue phosphorylated is solely serine. These data demonstrate, for the first time, that catecholamines can promote phosphorylation of a cellular protein (the beta-adrenergic receptor) via a cAMP-independent pathway. Moreover, the ability of cells with mutations in the adenylate cyclase-cAMP-dependent protein kinase pathway to both homologously desensitize and phosphorylate the beta-adrenergic receptors provides very strong support for the notion that receptor phosphorylation may indeed be central to the molecular mechanism of desensitization.

Adenosine Triphosphate↗

PubMed1986

Beta-agonist- and prostaglandin E1-induced translocation of the beta-adrenergic receptor kinase: evidence that the kinase may act on multiple adenylate cyclase-coupled receptors.

beta-Adrenergic receptor kinase (beta-AR kinase) is a cytosolic enzyme that phosphorylates the beta-adrenergic receptor only when it is occupied by an agonist [Benovic, J. Strasser, R. H., Caron, M. G. & Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. USA 83, 2797-2801.] It may be crucially involved in the processes that lead to homologous or agonist-specific desensitization of the receptor. Stimulation of DDT1MF-2 hamster smooth muscle cells or S49 mouse lymphoma cells with a beta-agonist leads to translocation of 80-90% of the beta-AR kinase activity from the cytosol to the plasma membrane. The translocation process is quite rapid, is concurrent with receptor phosphorylation, and precedes receptor desensitization and sequestration. It is also transient, since much of the activity returns to the cytosol as the receptors become sequestered. Stimulation of beta-AR kinase translocation is a receptor-mediated event, since the beta-antagonist propranolol blocks the effect of agonist. In the kin- mutant of the S49 cells (lacks cAMP-dependent protein kinase), prostaglandin E1, which provokes homologous desensitization of its own receptor, is at least as effective as isoproterenol in promoting beta-AR kinase translocation to the plasma membrane. However, in the DDT1MF-2 cells, which contain alpha 1-adrenergic receptors coupled to phosphatidylinositol turnover, the alpha 1-agonist phenylephrine is ineffective. These results suggest that the first step in homologous desensitization of the beta-adrenergic receptor may be an agonist-promoted translocation of beta-AR kinase from cytosol to plasma membrane and that beta-AR kinase may represent a more general adenylate cyclase-coupled receptor kinase that participates in regulating the function of many such receptors.

Alprostadil↗

PubMed1986

Phosphorylation/dephosphorylation of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase and subcellular distribution.

Prolonged exposure of cells or tissues to drugs or hormones such as catecholamines leads to a state of refractoriness to further stimulation by that agent, known as homologous desensitization. In the case of the beta-adrenergic receptor coupled to adenylate cyclase, this process has been shown to be intimately associated with the sequestration of the receptors from the cell surface through a cAMP-independent process. Recently, we have shown that homologous desensitization in the frog erythrocyte model system is also associated with increased phosphorylation of the beta-adrenergic receptor. We now provide evidence that the phosphorylation state of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase, subcellular translocation, and recycling to the cell surface during the process of agonist-induced homologous desensitization. Moreover, we show that the receptor phosphorylation is reversed by a phosphatase specifically associated with the sequestered subcellular compartment. At 23 degrees C, the time courses of beta-adrenergic receptor phosphorylation, sequestration, and adenylate cyclase desensitization are identical, occurring without a lag, exhibiting a t1/2 of 30 min, and reaching a maximum at approximately 3 hr. Upon cell lysis, the sequestered beta-adrenergic receptors can be partially recovered in a light membrane vesicle fraction that is separable from the plasma membranes by differential centrifugation. The increased beta-adrenergic receptor phosphorylation is apparently reversed in the sequestered vesicle fraction as the sequestered receptors exhibit a phosphate/receptor stoichiometry that is similar to that observed under basal conditions. High levels of a beta-adrenergic receptor phosphatase activity appear to be associated with the sequestered vesicle membranes. The functional activity of the phosphorylated beta-adrenergic receptor was examined by reconstituting purified receptor with its biochemical effector the guanine nucleotide regulatory protein (Ns) in phospholipid vesicles and assessing the receptor-stimulated GTPase activity of Ns. Compared to controls, phosphorylated beta-adrenergic receptors, purified from desensitized cells, were less efficacious in activating the Ns GTPase activity. These results suggest that phosphorylation of the beta-adrenergic receptor leads to its functional uncoupling and physical translocation away from the cell surface into a sequestered membrane domain. In the sequestered compartment, the phosphorylation is reversed thus enabling the receptor to recycle back to the cell surface and recouple with adenylate cyclase.

Adenylyl Cyclases↗

PubMed1986

Beta-adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor.

Agonist-promoted desensitization of adenylate cyclase is intimately associated with phosphorylation of the beta-adrenergic receptor in mammalian, avian, and amphibian cells. However, the nature of the protein kinase(s) involved in receptor phosphorylation remains largely unknown. We report here the identification and partial purification of a protein kinase capable of phosphorylating the agonist-occupied form of the purified beta-adrenergic receptor. The enzyme is prepared from a supernatant fraction from high-speed centrifugation of lysed kin- cells, a mutant of S49 lymphoma cells that lacks a functional cAMP-dependent protein kinase. The beta-agonist isoproterenol induces a 5- to 10-fold increase in receptor phosphorylation by this kinase, which is blocked by the antagonist alprenolol. Fractionation of the kin- supernatant on molecular-sieve HPLC and DEAE-Sephacel results in a 50- to 100-fold purified beta-adrenergic receptor kinase preparation that is largely devoid of other protein kinase activities. The kinase activity is insensitive to cAMP, cGMP, cAMP-dependent kinase inhibitor, Ca2+-calmodulin, Ca2+-phospholipid, and phorbol esters and does not phosphorylate general kinase substrates such as casein and histones. Phosphate appears to be incorporated solely into serine residues. The existence of this novel cAMP-independent kinase, which preferentially phosphorylates the agonist-occupied form of the beta-adrenergic receptor, suggests a mechanism that may explain the homologous or agonist-specific form of adenylate cyclase desensitization. It also suggests a general mechanism for regulation of receptor function in which only the agonist-occupied or "active" form of the receptor is a substrate for enzymes inducing covalent modification.

Adrenergic beta-Agonists↗

PubMed1986

Identification of the subunit structure of rat pineal adrenergic receptors by photoaffinity labeling.

The adrenergic receptors of rat pineal gland were investigated using radiolabeled ligand binding and photoaffinity labeling techniques. 125I-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone (125I-HEAT) and 125I-cyanopindolol (125I-CYP) labeled specific sites on rat pineal gland membranes with equilibrium dissociation constants (KD) of 48 (+/- 5) pM and 30 (+/- 5) pM, respectively. Binding site maxima were 481 (+/- 63) and 1,020 (+/- 85) fmol/mg protein. The sites labeled by 125I-HEAT had the pharmacological characteristics of alpha 1-adrenergic receptors. 125I-CYP-labeled beta-adrenergic receptors were characterized as a homogeneous population of beta 1-adrenergic receptors. The alpha 1- and beta 1-adrenergic receptors were covalently labeled with the specific photoaffinity probes 4-amino-6,7-dimethoxy-2-(4-[5-(4-azido-3-[125I]iodophenyl) pentanoyl]-1-piperazinyl) quinazoline (125I-APDQ) and 125I-p-azidobenzylcarazolol (125I-pABC). 125I-APDQ labeled an alpha 1-adrenergic receptor peptide of Mr = 74,000 (+/- 4,000), which was similar to peptides labeled in rat cerebral cortex, liver, and spleen. 125I-pABC labeled a single beta 1-adrenergic receptor peptide with a Mr = 42,000 (+/- 1,500), which differed from the 60-65,000 peptide commonly seen in mammalian tissues. Possible reasons for these differences are discussed.

Affinity Labels↗

PubMed1985

Homologous desensitization of beta-adrenergic receptor coupled adenylate cyclase. Resensitization by polyethylene glycol treatment.

Brief (approximately 20-min) exposure of S49 lymphoma cells to beta-agonists such as isoproterenol leads to a homologous form of desensitization in which beta-agonist but not prostaglandin E1-sensitive or NaF-sensitive adenylate cyclase is reduced. The desensitized receptors (R) appear to be sequestered away from the effector system (guanine nucleotide regulatory protein (Ns) and adenylate cyclase (C)). Membrane perturbants such as polyethylene glycol are known to reorient membrane proteins and lipids. Thus, we fused agonist-desensitized S49 lymphoma cells to each other, using polyethylene glycol as fusogen, in an attempt to functionally reunite the R, N, and C components which might have become sequestered in microdomains of the plasma membrane during desensitization. Such treatment completely restored isoproterenol-stimulated adenylate cyclase to normal and re-established the ability of R and N to functionally couple as assessed by the ability to form a high affinity, guanine nucleotide-sensitive state of the receptor. These results support the concept that agonist-promoted sequestration plays a functionally significant role in the homologous desensitization of the beta-adrenergic receptor.

Adenylyl Cyclases↗

PubMed1985

Homologous desensitization of adenylate cyclase is associated with phosphorylation of the beta-adrenergic receptor.

We recently demonstrated that heterologous desensitization of adenylate cyclase in turkey erythrocytes is highly correlated with phosphorylation of the beta-adrenergic receptor. In contrast, little is known of the biochemical mechanisms underlying the homologous form of beta-adrenergic receptor desensitization, which is agonist-specific and not cAMP-mediated. Accordingly, the present studies were undertaken to examine if phosphorylation of the beta-adrenergic receptor is also associated with this form of desensitization in a well studied model system, the frog erythrocyte. Preincubation of these cells with the beta-adrenergic agonist isoproterenol leads to a 45% decline in isoproterenol-stimulated adenylate cyclase activity without significant changes in basal, prostaglandin E1-, NaF-, guanyl-5'-yl-imidodiphosphate-, forskolin-, or MnCl2-stimulated enzyme activities. There is also a 48% decline in [125I]iodocyanopindolol membrane binding sites. Conversely, preincubation of the cells with prostaglandin E1 attenuates only the prostaglandin E1-stimulated enzyme activity and does not affect [125I]iodocyanopindolol binding. Phosphorylation of the beta-adrenergic receptor was assessed by preincubating the cells with 32Pi and desensitizing them, and subsequently purifying the receptors by affinity chromatography. Under basal conditions there is about 0.62 mol of phosphate/mol of receptor whereas after desensitization with isoproterenol this increases to 1.9 mol/mol. This isoproterenol-induced receptor phosphorylation exhibits stereospecificity and is blocked by the beta-adrenergic antagonist propranolol. In addition, preincubation with prostaglandin E1 does not promote beta-adrenergic receptor phosphorylation. These data suggest that receptor phosphorylation is involved in homologous as well as heterologous forms of desensitization and may provide a unifying mechanism for desensitization of adenylate cyclase-coupled hormone receptors.

Adenylyl Cyclases↗

PubMed1985

Homologous desensitization of the beta-adrenergic receptor. Functional integrity of the desensitized receptor from mammalian lung.

Previous work has demonstrated that injection of rats with isoproterenol is rapidly (10 min) followed by the development of a homologous form of desensitization of the beta-agonist-coupled adenylate cyclase in lung membranes. Half the receptor pool becomes sequestered in a light membrane fraction while the other half remains in the plasma membranes but becomes functionally uncoupled. In the present work we sought to assess whether "local sequestration" of the functionally intact receptor away from the effector adenylate cyclase in the plasma membrane contributes to the uncoupling of the beta-adrenergic receptor observed in the plasma membranes. We tested the functionality of the desensitized beta-adrenergic receptor in three different ways. We reconstituted the affinity chromatography purified control and "desensitized" receptors with pure Ns from human erythrocytes and assessed the ability to induce GTPase activity in Ns. Both control and desensitized beta-adrenergic receptors stimulate similar levels of GTPase activity in Ns (852 +/- 38 versus 738 +/- 49 fmol of Pi released/30 min (p greater than 0.05, n = 4). To further assess the relative ability of control and desensitized beta-adrenergic receptors to couple to another source of Ns we fused reconstituted beta-adrenergic receptors to Xenopus laevis erythrocytes, which contain Ns and adenylate cyclase but essentially no beta-adrenergic receptors. The functional interactions of control and desensitized beta-adrenergic receptor with the adenylate cyclase system of the acceptor cells was assessed by measuring the beta-agonist-stimulated adenylate cyclase activity and the agonist-induced formation of the high affinity state of the beta-adrenergic receptor (RH). Again both control and desensitized beta-adrenergic receptors appeared to interact with Ns to the same extent. To test if a local sequestration of the beta-adrenergic receptor away from Ns within the plasma membrane might contribute to the uncoupling of the beta-adrenergic receptors during desensitization, plasma membranes from control and desensitized lungs were treated with the fusogen polyethylene glycol to disrupt any compartmentalization of protein components within the plasma membrane. After polyethylene glycol treatment the previously uncoupled beta-adrenergic receptors could be recoupled to Ns as assessed by the formation of RH in agonist competition curves. These data suggest that in marked contrast to the heterologous type of desensitization, homologous desensitization may involve a local sequestration of a functionally intact beta-adrenergic receptor away from the adenylate cyclase effector system.

Adenylyl Cyclases↗

PubMed1984

Endogenous proteinases modulate the function of the beta-adrenergic receptor-adenylate cyclase system.

Photoaffinity labeling techniques have recently demonstrated that mammalian beta 1- and beta 2-adrenergic receptors reside on peptides of Mr 62 000-64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of Mr 30 000-55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the beta 2-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of Mr 40 000-55 000 are fully functional with respect to their ability to bind beta-adrenergic antagonist radioligands such as [3H]dihydroalprenolol and beta-adrenergic antagonist photoaffinity reagents such as p-azido-m-[125I]iodobenzylcarazolol. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc- variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of beta-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.

Adenylyl Cyclases↗

PubMed1984

Translocation and uncoupling of the beta-adrenergic receptor in rat lung after catecholamine promoted desensitization in vivo.

Beta-adrenergic agonists and antagonists are widely used in many clinical situations to regulate beta-adrenergic stimulation. However, responsiveness to beta-stimulation may be reduced by the process of desensitization. We have established an in vivo mammalian model system, the rat lung, to study the cellular and biochemical basis for beta-agonist induced desensitization. After in vivo administration of a beta-agonist [(-)isoproterenol] the adenylate cyclase becomes rapidly insensitive to further stimulation by beta-agonists with no change in basal or NaF-stimulated activity. The in vivo desensitization can be blocked by the simultaneous administration of a beta-antagonist [(+/-)propranolol] and the process displays the pharmacological characteristics typifying the beta 2 receptor of rat lung. This indicates that the in vivo desensitization is itself a receptor-mediated event. The processes of de- and resensitization are very rapid with onset within 5 min, maximal effect at 10 min, and complete reversal by 2-3 h. The change of adenylate cyclase sensitivity is paralleled by a translocation of approximately 40% of the beta-receptors from the plasma membrane fraction to a light membrane fraction, which has very low activities of plasma membrane marker enzymes. The receptors translocated to the light membrane fraction as well as those remaining in the plasma membranes are uncoupled with loss of their ability to form the high affinity, nucleotide sensitive, physiologically active state of the receptor. During resensitization the receptors in the plasma membrane fraction are recoupled before all the translocated receptors have returned. This suggests that translocation and uncoupling of the receptors are two distinct, probably independent processes. During the entire process of de- and resensitization no structural change of the receptor protein residing in the plasma membranes or light membrane fraction can be demonstrated as visualized by photoaffinity labeling.

5'-Nucleotidase↗