Light Vesicles Research Articles

Regulation of cardiac beta-adrenergic receptors by captopril. Implications for congestive heart failure.

The interaction of the renin-angiotensin system and the sympathetic nervous system in patients with congestive heart failure is not well understood. We tested the hypothesis that angiotensin-converting enzyme inhibitors can resensitize the beta-adrenergic receptor system. Guinea pigs were given captopril, isoproterenol, or both for 2 weeks. At death, cardiac sarcolemmal and light vesicle fractions and intact mononuclear leukocytes were prepared. Captopril treatment led to an up-regulation of cardiac beta 1- but not mononuclear leukocyte beta 2-adrenergic receptors and an increase in isoproterenol-stimulated adenylate cyclase activity in the heart. Animals treated with isoproterenol developed cardiac hypertrophy, had increased plasma norepinephrine levels, and had a decreased number and responsiveness of both cardiac and mononuclear leukocyte beta-adrenergic receptors. Concomitant treatment with captopril attenuated alterations of heart weight, plasma norepinephrine levels, and cardiac beta-receptor density and function. In contrast to its cardiac effects, captopril treatment did not diminish the down-regulation of mononuclear leukocyte beta 2-adrenergic receptors by isoproterenol. Our data suggest that captopril may resensitize the cardiac but not the mononuclear leukocyte beta-adrenergic receptor-adenylate cyclase system after long-term catecholamine exposure.

Regulation of Steady State Filling in Sarcoplasmic Reticulum: Roles of back-inhibition, leakage, and slippage of the calcium pump

Abstract Calcium filling of sarcoplasmic reticulum vesicles in the steady state is greatly increased by precipitation of lumenal calcium with oxalate. We find that low concentrations (1 mM) of Pi also allow greater loading by forming a soluble complex with lumenal calcium, an effect that is likely to be of physiological relevance. Furthermore, ADP scavenging by ATP regenerating systems favors calcium loading by preventing reversal of the pump. We also find that uncoupling of ATPase and transport activities is another factor limiting calcium loading. In fact, calcium uptake and ATP utilization occur with a molar ratio of 2:1 in the transient state following addition of ATP but decrease to much lower values in the steady state. Even in the absence of the highly conductive channel which is present only in heavy vesicles, light vesicles display calcium leakage which is inhibited by medium Ca2+ in the concentration range of ATPase activation and is likely related to an ATPase channel which is involved in calcium transport. It is apparent that, under conditions of ATPase turnover and in the presence of high lumenal Ca2+ and ADP, slippage of calcium through this channel produces true uncoupling of catalytic and transport activities. Coupling is improved by complexation of lumenal Ca2+ and by ATP regeneration and is influenced by the solvent characteristics of the reaction medium. The synergistic effects of lumenal Ca2+ and ADP, and the role of alternate pathways for phosphoenzyme cleavage, are clarified by steady state analysis of a multiple step reaction mechanism. It is concluded that the ideal (2:1) stoichiometric coupling of transport and ATPase activities is not insured by an obligatory pathway of catalysis (as predicted by all reaction schemes published so far); rather, coupling is influenced by the concentrations of ligands and their effects on second order reactions and the consequent distribution of intermediate states.

An Acute Exercise-Induced Translocation of β-Adrenergic Receptors in Rat Myocardium1

The effect of acute exercise (treadmill running) on rat myocardium beta-adrenergic receptors (beta-AR) was studied. beta-AR was identified in purified sarcolemmal membrane fractions and light vesicle fractions. In control hearts, the number of beta-AR was 21.25 +/- 2.25 and 20.89 +/- 2.89 fmol/mg protein (mean +/- SE) in sarcolemmal membranes and light vesicles, respectively. Immediately after a single bout of dynamic exercise, about 35% of beta-AR was transferred from light vesicles to sarcolemmal membranes (p less than 0.05); concomitantly, isoproterenol-stimulated adenylate cyclase activity also significantly increased in sarcolemmal membranes (p less than 0.05). These results suggest that acute exercise provokes the translocation of beta-AR from a presumably intracellular site (light vesicles) to functional membrane fractions (sarcolemmal membranes) in rat myocardium.

Biochemical evidence for the presence of small and large noradrenergic storage vesicles isolated from cat ovary in isoosmotic conditions. Distribution of dopamine-β-hydroxylase activity

The cat ovary presents unusually high levels of noradrenaline that change according to the endocrine status of the animal. Their functional meaning remains unknown. The cat ovary innervation, unlike that of other organs receiving noradrenergic innervation, has been poorly characterized on biochemical grounds. We present here a biochemical characterization of the neurotransmitter storage. By using hyperosmotic and isoosmotic gradients evidence is presented that noradrenaline is associated to two different populations of vesicles. In hyperosmomolarity conditions (sucrose gradients) "light" vesicles (density 1.12 g/ml) and "heavy" vesicles (density 1.17 g/ml) appeared. In both vesicles, noradrenaline and dopamine-?-hydroxylase were found. In isoosmotic Percoll gradients distribution of the markers also suggested the presence of two vesicle populations. Light vesicles (density 1.033 g/ml) with high dopamine-?-hydroxylase activity but very low levels of noradrenaline and adenosine triphosphate; [(3)H]noradrenaline, used as a specific exogenous vesicle marker, was feebly incorporated in this fraction. Heavy vesicles (density 1.041 g/ml) containing high levels of noradrenaline, adenosine triphosphate, low levels of dopamine-?-hydroxylase activity are able to incorporate high amounts of [(3)H]noradrenaline. In these gradients, Mg(2+) activated ATPase activity was present in both vesicle fractions. Sedimentation analysis by analytical differential centrifugation also disclosed two types of vesicles: large vesicles with a sedimentation coefficient between 348 and 308 S and small vesicles with a sedimentation coefficient of 96 S . Large vesicles were associated with noradrenaline-?-hydroxylase activity, while small vesicles were associated only with noradrenaline. In isoosmotic conditions the use of other microsomal markers allowed us to define the degree of contamination of the vesicle fractions. It was found that the noradrenergic heavy vesicles fraction presented under 11% of 5?-nucleotidase activity of the total activity present in the gradient and less than 5% of acid phosphatase, NADH-cytochrome c reductase and monoaminooxidase of the total activities in the gradients. In isoosmotic conditions the physical properties of presumed vesicles were apparently undisturbed supporting the current morphometric observations. Our results then suggest prevailing roles for each type of vesicle: synthesis for light vesicles, and storage and/or release for heavy ones.

Vasopressin‐induced transfer via light vesicles of receptors and water channels from basolateral to apical membrane of toad bladder

Toad bladder epithelial cells were homogenized and fractionated by Percoll density-gradient centrifugation. Binding of tritium-labeled vasopressin ([3H]AVP) was measured in surface membranes (SM), microsomes (M), and a 100,000 g (60-min) microsomal supernatant fraction (S). More than two-thirds of the total receptors were in S. Receptors in SM--but not in S--were tightly coupled to G-protein as suggested by inhibition of [3H]AVP binding by GTP. GTP-sensitivity of [3H]AVP binding was not altered by vasotocin (AVT) stimulation, although the distribution of receptors shifted from SM to S. Intact bladders, exposed on the serosal side to 1-desamino, 7-lysine-(4-azidobenzoyl), 8-arginine vasotocin (d7-N3-AVT) in the presence of UV light, exhibited a persistent hydroosmotic response compared to controls stimulated with photoaffinity analog in the dark. Cell fractions from the irradiated bladders showed a reduction in [3H]AVP binding in SM and S. Intact bladders, exposed on the mucosal side to d7-N3-AVT in the presence of UV light (while stimulated from the serosal side with AVP) exhibited a decrease in [3H]AVP binding in SM compared to controls without d7-N3-AVT.(ABSTRACT TRUNCATED AT 250 WORDS)

.BETA.-Adrenergic receptor adaptation after an acute exercise in rat myocardium.

An acute dynamic exercise provokes the translocation of beta-adrenergic receptors (beta-AR) from light vesicle fractions to sarcolemmal membranes in rat myocardium. However, 15 min after an acute exercise the density of beta-AR in both fractions returned to the pre-exercise level. The mean maximal activity of adenylate cyclase in response to isoproterenol roughly paralleled the redistribution of beta-AR. The dose-response curves, however, were substantially shifted to the right with increase in EC50 for isoproterenol stimulation of adenylate cyclase. Thus, the sensitivity of sarcolemmal beta-AR was found to be blunted 15 min afterwards.

Vasopressin-induced transfer via light vesicles of receptors and water channels from basolateral to apical membrane of toad bladder

Vasopressin-induced transfer via light vesicles of receptors and water channels from basolateral to apical membrane of toad bladder

The subcellular location in rat kidney of the peripheral benzodiazepine acceptor.

In rat kidney high-affinity binding sites for [3H]Ro-5-4864 and [3H]PK-11195 with the properties of the peripheral-type acceptor were found enriched in mitochondrial (M) and light-mitochondrial-lysosomal (L) fractions on differential centrifugation. When the combined M and L fractions were subjected to sucrose density gradient centrifugation, these binding sites were found enriched at a density of 1.155 g/ml coincident with a population of light mitochondria, whereas a population of heavier mitochondria (rho = 1.175 g/ml) had few or no binding sites. Transmission electron microscopy showed that whereas the heavier mitochondria appeared highly pure and intact, the lighter mitochondria appeared less intact and to be contaminated with vesicular structures. After fractionation of the light mitochondria and vesicles by centrifugation, both fractions showed the same ratio of [3H]Ro-4864 binding sites to monoamine oxidase activity consistent with the vesicles being of mitochondrial outer-membrane origin. Digitonin pre-treatment had no effect on the density of acceptor-rich fractions on sucrose density gradient centrifugation. However, pretreatment with succinate/iodophenylnitrophenylphenyltetrazolium (INT) perturbed equally the density of acceptor-rich fractions and mitochondrial marker enzymes. When mitochondrial fractions were subjected to sonication prior to density gradient centrifugation the binding sites were now found highly enriched in a much lighter fraction coincident with the monoamine oxidase activity and thus consistent with being outer-membrane vesicles. When a mitochondrial fraction was subjected to hypotonic treatment before assay no evidence for activation/unmasking of binding sites was found. The hypotonic treatment did not release any inhibitor of the binding sites. These results are consistent with the peripheral benzodiazepine acceptor having an outer-membrane location on a sub-population of rat kidney mitochondria. Those mitochondria showing high levels of the acceptor are either light mitochondria or appear more susceptible to osmotic damage than those mitochondria in which the acceptor is absent or at low levels.

Identification of the Ca2+-release activity and ryanodine receptor in sarcoplasmic-reticulum membranes during cardiac myogenesis

Ca2+-induced Ca2+ release and pH-induced Ca2+ release activities were identified in sarcoplasmic-reticulum (SR) vesicles isolated from adult- and fetal-sheep hearts. Ca2+-induced Ca2+ release and pH-induced Ca2+ release appear to proceed via the same channels, since both phenomena are similarly inhibited by Ruthenium Red. Ca2+ release from fetal SR vesicles is inhibited by higher concentrations of Ruthenium Red than is that from adult membranes. Both fetal and adult SR vesicles bind ryanodine. Fetal SR shows higher ryanodine-binding capacity than adult SR vesicles. Scatchard analysis of ryanodine binding revealed only one high-affinity binding site (Kd 6.7 nM) in fetal SR vesicles compared with two distinct binding sites (Kd 6.6 and 81.5 nM) in the adult SR vesicles. SR vesicles isolated from fetal and adult hearts were separated on discontinuous sucrose gradients into light (free) and heavy (junctional) SR vesicles. Heavy SR vesicles isolated from adult hearts exhibited most of the Ca2+ release activities. In contrast, Ca2+-induced Ca2+ release, pH-induced Ca2+ release and ryanodine receptors were detected in both light and heavy fetal SR. These results suggest that fetal SR may not be morphologically and functionally as well differentiated as that of adult cardiac muscle and that it may contain a greater number of Ca2+-release channels than that present in adult SR membranes.

Calcium binding protein changes of sarcoplasmic reticulum from rat denervated skeletal muscle.

Two Ca2+ sequestering proteins were studied in fast-twitch (EDL) and slow-twitch (soleus) muscle sarcoplasmic reticulum (SR) as a function of denervation time. Ca2+-ATPase activity measured in SR fractions of normal soleus represented 5% of that measure in SR fractions of normal EDL. Denervation caused a severe decrease in activity only in fast-twitch muscle. Ca2+-ATPase and calsequestrin contents were affected differently by denervation. In EDL SR, Ca2+-ATPase content decreased progressively, whereas in soleus SR, no variation was observed. Calsequestrin showed a slight increase in both muscles as a function of denervation time correlated with increased 45Ca-binding. These results indicate first that Ca2+-ATPase activity in EDL was under neural control, and that because of low Ca2+-ATPase activity and content in slow-twitch muscle no variation could be detected, and secondly that greater calsequestrin content might represent a relative increasing of heavy vesicles or decreasing of light vesicles as a function of denervation time in the whole SR fraction isolated in both types of muscles.

Effect of ammonium chloride on subcellular distribution of lysosomal enzymes in human fibroblasts

Three subcellular fractions enriched in lysosomal enzyme activities have been isolated recently from human cultured fibroblasts with Percoll gradients: the dense lysosomes (DL), light lysosomes (LL), and light membranous vesicles (LM). They were shown to have different morphological, cytochemical, biochemical, and immunological properties. We now report on the dramatic but different effects of a primary amine, NH 4Cl, on these subfractions. The lysosomes, as detected with a specific ultrastructural cytochemical stain for the lysosomal enzyme, arylsulfatase A, were swollen significantly in all these fractions, increasing their volumes by 64% (DL), 53% (LL), and 95% (LM), respectively. When arylsulfatase A enzyme activity was monitored, about half of the DL content was diverted to the LL. However, when newly synthesized arylsulfatase A enzyme protein was monitored with metabolic labeling and immunoprecipitation, about 80% of the enzyme protein was depleted from both the DL and LL. In contrast, neither the enzyme activity nor the newly synthesized enzyme protein of arylsulfatase A was greatly altered in the LM fraction by the treatment. Since primary amines caused newly synthesized lysosomal enzymes to diverge from the lysosomal route to a secretory pathway, it was deduced that (i) the LM fraction corresponded to a prelysosomal compartment whose lysosomal enzyme content was not affected by the amine and was thus proximal to the point of diversion between the secretory and lysosomal pathways; (ii) the LL and DL fractions were distal to the point of diversion since both fractions were depleted of their newly synthesized lysosomal enzyme; and (iii) the sorting of newly synthesized lysosomal enzyme may be different from that of the preexisting pool of the same enzyme since the LL fraction was depleted of its newly synthesized enzyme protein while accumulating excessive enzyme activity.

Cathepsin D is membrane-associated in macrophage endosomes.

Previously we identified an acid protease activity which was located in the endosomes of rabbit alveolar macrophages (Diment, S., and Stahl, P.D. (1985) J. Biol. Chem. 260, 15311-15317). In this study, the endosomal protease is identified as cathepsin D by immunoprecipitation with polyclonal antibodies raised against rabbit cathepsin D and by NH2-terminal sequence. In order to elucidate the mechanism for targeting of cathepsin D to endosomes, we first examined the membrane association of cathepsin D with light (rho = 1.05 g/ml) and heavy density (rho = 1.1 g/ml) vesicles from Percoll density gradients. After sequential washes, 8.4 and 21.9% of cathepsin D activity remained associated with heavy and light density vesicles, respectively. This membrane-associated cathepsin D could not be solubilized in either buffer at pH 5.0 containing mannose 6-phosphate and EDTA or in buffer at pH 10.6. Solubilization required the detergent Triton X-100. To determine whether membrane-associated cathepsin D was found in endosomes, the enzyme was radioiodinated within endosomes and lysosomes with internalized lactoperoxidase. The membrane-associated form was detected in endosomes, but much less in lysosomes. Biosynthetic studies combined with the same extraction procedure revealed that macrophage cathepsin D is first synthesized as an inactive membrane-associated precursor. The precursor is processed to an active, membrane-associated form and then to the active soluble form found in lysosomes. Our studies provide evidence that 1) cathepsin D is in endosomes of macrophages; 2) cathepsin D is transported to endosomes as a membrane-associated form; and 3) the membrane-associated form is a biosynthetic precursor for the soluble form found in endosomes and lysosomes.

Properties of light and heavy vesicles simultaneously prepared from hog gastric mucosae.

We obtained two kinds of vesicle preparations which were of different density from the same gastric mucosae of hogs stimulated with food before slaughter. Both kinds contained H+,K+-ATPase. The light vesicle preparation differed from the heavy vesicle preparation as follows: the KCl permeability across the membrane of heavy vesicles was larger than that of light vesicles, the actin (46-kDa peptide on SDS-polyacrylamide gel) content of heavy vesicles was much higher than that of light vesicles, and the H+,K+-ATPase activity of heavy vesicles was less sensitive to a monoclonal antibody raised against light vesicles (HK2032) than that of light vesicles. Furthermore, there was a drastic difference in reactivity to SCH 28080, which is an H+,K+-ATPase-specific inhibitor and reacts competitively with the K+-high affinity site. SCH 28080 is more potent in light vesicles than in heavy vesicles. These results suggest that the conformation of H+,K+-ATPase changed during the translocation from tubulovesicles to the apical plasma membrane. On the other hand, H+,K+-ATPase activities in both vesicles had similar pH and [K+] dependences.

Monoclonal Antibody Characterization of the 1,4‐Dihydropyridine Receptor of Rabbit Skeletal Muscle

The 1,4-dihydropyridine receptor (DHPR) of the voltage-dependent Ca channel has been purified from transverse tubular membranes of skeletal muscle. Curtis and Catterall have shown that it consists of three polypeptides of 160,000 daltons, 50,000 Da, and 32,000 Da and that under reducing conditions the apparent molecular weight of the 160,000 Da subunit shifted to 130,000. Borsotto et al. have identified three polypeptides of 142,000 Da, 33,000 Da, and 32,000 Da in their preparation of the dihydropyridine receptor. Furthermore, they have shown by immunoblotting with polyclonal antibodies that the 142,000 Da and 32,000 Da subunits are produced by the reduction of a 170,000 Da polypeptide. We report here the identification and characterization with monoclonal antibodies (MAb) of an additional high molecular weight subunit of the DHPR which is distinct from that described by Curtis and Catterrall and Borsotto et al. Monoclonal antibodies capable of specifically immunoprecipitating the [H]PN200-110-labeled DHPR of rabbit skeletal muscle were produced by immunizing BALB/c mice initially with skeletal muscle triad vesicles followed by booster immunizations with purified DHPR from skeletal muscle triads. Hybridoma supernatants were screened for the production of anti-DHPR antibodies with an immunodot assay. Supernatants that reacted positively against the partially purified DHPR were then tested for their ability to immunoprecipitate the [H]PN200-110-labeled receptor from digitonin-solubilized triads (see TABLE 1). All three MAbs (IIC12, IIF7, IIID5) that were capable of immunoprecipitating the DHPR recognized a protein with an Mr of 170,000 on nitrocellulose transfers of skeletal muscle triads and transverse tubular membranes separated on sodium dodecyi sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions (FIGURE la). Wheat-germ agglutinin (WGA) peroxidase stained a 175,000 Da protein on similar nitrocellulose transfers. Neither the 170,000 Da polypeptide nor the 175,000 Da polypeptide was detected in light sarcoplasmic reticulum vesicles, a preparation devoid of DHPR. Under reducing conditions, the Mr of the 170,000 Da

Ischemia- and agonist-induced changes in alpha- and beta-adrenergic receptor traffic in guinea pig hearts.

We have used radioligand binding techniques and subcellular fractionation to assess whether changes in expression of myocardial alpha 1- and beta-adrenergic receptors are mediated by a redistribution of receptors between various membrane fractions. Three fractions were prepared from the left ventricles of guinea pigs that underwent either 1 h of ischemia or injection of epinephrine (0.25 mg/kg ip): a crude membrane, a purified sarcolemma, and a light vesicle fraction. In control animals alpha 1-adrenergic receptors ([3H]prazosin binding) in light vesicles was only 25% of the total alpha 1-receptor density found in sarcolemmal and light vesicle fractions as compared with 50% for beta-adrenergic receptors ([125I]iodocyanopindolol binding sites). Although ischemia was associated with a 53% decrease in the number of light vesicle beta-adrenergic receptors and a 42% increase in the number of sarcolemma beta-receptors (P less than 0.05), there was no change in the number of light vesicle alpha 1-receptors, even though the number of sarcolemmal alpha 1-receptors increased 34%. Epinephrine treatment promoted internalization of beta-adrenergic receptors; sarcolemma beta-receptors decreased 37% and light vesicle beta-receptors increased 28% (P less than 0.025). For alpha 1-receptors, epinephrine treatment decreased the number of sarcolemmal receptors 41% (P less than 0.025) but failed to increase the number of receptors in the light vesicle fraction. The changes in receptor binding to beta-adrenergic receptors in sarcolemmal fractions were mirrored by parallel changes in isoproterenol-stimulated adenylate cyclase activity. These results indicate that alpha 1- and beta-adrenergic receptors may undergo a different cellular itinerary in guinea pig myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of local anesthetics on the passive permeability of sarcoplasmic reticulum vesicles to Ca 2+ and Mg 2+

Sarcoplasmic reticulum vesicles are used here as model membrane system to question the hypothesis of enhancement of permeability of cations by anesthetics, particularly that of Ca 2+ and of Mg 2+. The effects of dibucaine (up to 800 μM), tetracaine (up to 2 mM), lidocaine (up to 10 mM) and procaine (up to 10 mM) on the permeability of these membranes to Ca 2+ and Mg 2+ have been measured. We have used an experimental approach based on the light scattering method (Kometani, T. and Kasai, M. (1978) J. Membrane Biol. 41, 295–308). It has been found that all the local anesthetics cited above markedly increase the permeability of sarcoplasmic reticulum vesicles to Mg 2+ and, in the concentration range tested herein, only dibucaine and tetracaine increase the permeability to Ca 2+. The kinetic analysis of the time dependence of the light-scattering data after the osmotic shock shows that, in the absence of local anesthetics, the Mg 2+ influx can be described as proceeding through a unique type of channel. However, Ca 2+ influx appears to involve two channel of different kinetic properties. Because the relative fraction of both types of Ca 2+ channel is similar to the average ratio between light and heavy vesicles in unfractionated sarcoplasmic reticulum, we suggest that each type of channel can be preferentially located in one of these fractions. The determined rate constants for Ca 2+ permeability through both types of channel are 0.77 ± 0.08 min −1 (fast channels) and 0.025 ± 0.005 min −1 (slow channels) and that for Mg 2+ is 0.08 ± 0.02 min −1. These results agree with data obtained by other groups using different experimental approaches. Dibucaine and tetracaine significantly alter the rate of Mg 2+ and Ca 2+ influx through the slow channels. In addition, these two local anesthetics also produce the effect that the Mg 2+ influx cannot be described with only one exponential process, thus suggesting a differential effect on vesicles of different density. The increase of Ca 2+ and Mg 2+ permeability by dibucaine and by tetracaine is found at concentrations of these drugs that do not produce a noticeable inhibition of the (Ca 2+ + Mg 2+)-ATPase activity of sarcoplasmic reticulum vesicles.

Identification and characterization of the high affinity [3H]ryanodine receptor of the junctional sarcoplasmic reticulum Ca2+ release channel.

The high affinity ryanodine receptor of the Ca2+ release channel from junctional sarcoplasmic reticulum of rabbit skeletal muscle has been identified and characterized using monoclonal antibodies. Anti-ryanodine receptor monoclonal antibody XA7 specifically immunoprecipitated [3H]ryanodine-labeled receptor from digitonin-solubilized triads in a dose-dependent manner. [3H]Ryanodine binding to the immunoprecipitated receptor from unlabeled digitonin-solubilized triads was specific, Ca2+-dependent, stimulated by millimolar ATP, and inhibited by micromolar ruthenium red. Indirect immunoperoxidase staining of nitrocellulose blots of various skeletal muscle membrane fractions has demonstrated that anti-ryanodine receptor monoclonal antibody XA7 recognizes a high molecular weight protein (approximately 350,000 Da) which is enriched in isolated triads but absent from light sarcoplasmic reticulum vesicles and transverse tubular membrane vesicles. Thus, our results demonstrate that monoclonal antibodies to the approximately 350,000-Da junctional sarcoplasmic reticulum protein immunoprecipitated the ryanodine receptor with properties identical to those expected for the ryanodine receptor of the Ca2+ release channel.

A vesicular intermediate in the transport of hepatoma secretory proteins from the rough endoplasmic reticulum to the Golgi complex.

We have identified a vesicle fraction that contains alpha 1-antitrypsin and other human HepG2 hepatoma secretory proteins en route from the rough endoplasmic reticulum (RER) to the cis face of the Golgi complex. [35S]Methionine pulse-labeled cells were chased for various periods of time, and then a postnuclear supernatant fraction was resolved on a shallow sucrose-D2O gradient. This intermediate fraction has a density lighter than RER or Golgi vesicles. Most alpha 1-antitrypsin in this fraction (P1) bears N-linked oligosaccharides of composition similar to that of alpha 1-antitrypsin within the RER; mainly Man8GlcNac2 with lesser amounts of Man7GlcNac2 and Man9GlcNac2; this suggests that the protein has not yet reacted with alpha-mannosidase-I on the cis face of the Golgi complex. This light vesicle species is the first post-ER fraction to be filled by labeled alpha 1-antitrypsin after a short chase, and newly made secretory proteins enter this compartment in proportion to their rate of exit from the RER and their rate of secretion from the cells: alpha 1-antitrypsin and albumin faster than preC3 and alpha 1-antichymotrypsin, faster, in turn, then transferrin. Deoxynojirimycin, a drug that blocks removal of glucose residues from alpha 1-antitrypsin in the RER and blocks its intracellular maturation, also blocks its appearance in this intermediate compartment. Upon further chase of the cells, we detect sequential maturation of alpha 1-antitrypsin to two other intracellular forms: first, P2, a form that has the same gel mobility as P1 but that bears an endoglycosidase H-resistant oligosaccharide and is found in a compartment--probably the medial Golgi complex--of density higher than that of the intermediate that contains P1; and second, the mature sialylated form of alpha 1-antitrypsin.

Propranolol treatment externalizes beta-adrenergic receptors in guinea pig myocardium and prevents further externalization by ischemia.

Purified sarcolemmal and light vesicle (intracellular) fractions of beta-adrenergic receptors were used to examine the effects of propranolol on receptor translocation in guinea pig heart. Guinea pigs were given propranolol (0.15 mg/kg/hr) via minipumps for 7 days and either killed or made ischemic for 1 hour via a coronary ligature. Propranolol treatment led to an externalization of beta-receptors from light vesicle to sarcolemmal fractions. This externalization increased the number of surface beta-adrenergic receptors that were functional, as assessed by isoproterenol-stimulated adenylate cyclase activity. After chronic propranolol treatment, ischemia did not further alter receptor distribution. These results suggest that externalization of beta-adrenergic receptors from a light vesicle fraction to the sarcolemma contributes to up-regulation of beta-receptors that occur in response to both propranolol treatment and ischemia. Because propranolol-treated animals show blunting in externalization after myocardial ischemia, propranolol treatment and myocardial ischemia appear to access the same pool of intracellular beta-adrenergic receptors. Depletion of this pool of receptors along with receptor blockade may thus contribute to the mechanism by which the drug is efficacious in preventing some adverse effects of ischemia.

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.