Use of a modified tryptophanase promoter to direct high-level expression of foreign proteins in E. coli.

We have modified the tryptophanase promoter (PtnaA) for use as a temperature-independent promoter for the production of recombinant proteins. Although any protein will have a temperature range in which its expression is optimal, we find the tryptophanase promoter functions at all physiologically relevant temperatures (20 degrees C to 42 degrees C). Induction at temperatures below 37 degrees C avoids eliciting the heat-shock response and may favor the production of protein in the soluble state. A short segment of the E. coli tnaA promoter containing the catabolite gene activator protein (CAP) binding site but no tryptophan-responsive elements was used to direct synthesis of various proteins. Conditions for high cell density fermentation and induction control were developed. Expression was induced by depletion of glucose and was maximal when an alternative nonrepressing carbon source was supplied. Expression of certain proteins was tightly controlled; however, pre-induction expression was observed with other reporter genes. The tnaC leader portion of the tnaA promoter was found to reduce pre-induction expression in the presence of glucose, although maximal expression was observed only in the absence of this region. The effect of temperature on expression of several recombinant proteins was investigated. Although some proteins were produced only in inclusion bodies as insoluble material, the production of one protein in soluble form was clearly temperature dependent.

Magnesium transport in Salmonella typhimurium: mgtA encodes a P-type ATPase and is regulated by Mg2+ in a manner similar to that of the mgtB P-type ATPase.

Salmonella typhimurium has three distinct Mg2+ transport systems: CorA, MgtA, and MgtB, each encoded by its respective gene. corA and mgtB have been previously sequenced and characterized. This report details the sequence and properties of mgtA. Like mgtB, mgtA encodes a P-type ATPase. The mgtA gene encodes a slightly smaller protein than does mgtB, with a predicted molecular mass of about 95 kDa, running at 91 kDa on protein gels, which compares with values of 101 and 102 kDa, respectively, for the MgtB protein. The deduced amino acid sequence of MgtA is only 50% identical to that of MgtB, with a further 25% conservative amino acid substitutions, surprisingly low for such otherwise functionally similar proteins from the same organism. Codon usage for each gene is normal for S. typhimurium, however, indicating that neither gene is the result of a recent acquisition from another organism. A single open reading frame at mgtA encodes MgtA, in contrast to mgtB, which is shown to be an operon encoding (5' to 3') the 22.5-kDa MgtC and the MgtB proteins. Genetic constructs were used to show that deletion of MgtC does not alter the expression or transport properties of MgtB, making the role of the companion MgtC protein unclear. (The S. typhimurium homolog of treR, which encodes a putative repressor for trehalose uptake, is encoded by a gene adjacent to mgtA, and its sequence is also reported. Finally, exteremely strong Mg(2+) regulation of the mgtA and mgtB promoters but not of the corA or treR promoters was demonstrated by cloning the appropriate DNA sequences with luxAB and measuring enhancement of light production as a function of extracellular Mg(2+) concentration. Lowering the extracellular Mg(2+) concentration from 10 mM to 1 or 10 microM elicited a transcriptional response of several thousandfold from both the mgtA and mgtB promoters.

Sequence and topology of the CorA magnesium transport systems of Salmonella typhimurium and Escherichia coli. Identification of a new class of transport protein.

The CorA Mg2+ transport systems of Salmonella typhimurium and Escherichia coli mediate both influx and efflux of Mg2+. The product of the CorA locus is sufficient for mediation of Mg2+ influx while product(s) of the unlinked CorBCD loci allow CorA to mediate efflux in addition to influx. The nucleotide sequences of the S. typhimurium and E. coli CorA loci have been determined. The locus in each species consists of a single gene expressing a protein with gel molecular masses of 42 kDa (S. typhimurium) and 39 kDa (E. coli). The predicted amino acid sequences of these proteins are each 316 amino acids in length, are 98% identical, and lack homology to any known protein. Although CorA is an integral membrane protein by biochemical criteria, its predicted amino acid sequence contains 28% charged amino acid. Membrane localization of CorA was shown to be dependent on the Sec pathway in E. coli. Hydropathy analysis predicts two C-terminal hydrophobic sequences of sufficient length to span the membrane bilayer. The membrane topology of CorA was determined by constructing deletion derivatives of CorA and genetically fusing them to BlaM or LacZ cassettes. The enzymatic activities of these hybrid proteins indicate that the N-terminal 235 amino acid residues of the CorA protein are located within the periplasmic space, comprising a single periplasmic domain. The C-terminal region of CorA is composed of three membrane-spanning segments rather than the two suggested by hydropathy plots, thus depositing the C terminus within the cytoplasm. This topology suggests that CorA functions as an oligomer since three membrane loops are most likely insufficient for any sort of membrane pore or channel. Its lack of homology to known proteins and its topology indicate that the CorA Mg2+ transporter represents a new class of membrane transport system.

Magnesium transport in Salmonella typhimurium. Regulation of mgtA and mgtB expression.

Salmonella typhimurium contains three distinct transport systems (CorA, MgtA, and MgtB) that move Mg2+ across the cytoplasmic membrane. Mutant strains containing only one of these three systems have been constructed and used to study each system in isolation. Characterization of these systems has been hampered, however, by the need to use 28Mg2+, a relatively unavailable, extremely expensive, and short lived radioisotope. This paper reports that 63Ni2+ is transported into the cell by all three of the S typhimurium Mg2+ transport systems. In a strain deficient in all three systems, uptake of 63Ni2+ was undetectable under the conditions used. Comparison of 63Ni2+ uptake kinetics and inhibition of 63Ni2+ transport by other divalent cations suggest that Ni2+ can be used as an analog of Mg2+ in the study of these three transport systems. Using 63Ni2+ to measure uptake, the effect of Mg2+ levels in the growth medium on transport by each system was tested. Transport by the CorA system was unaffected by changes in the amount of Mg2+ in the growth medium. In contrast, uptake via MgtA and MgtB was significantly increased in cells grown in 10 microM extracellular Mg2+ compared to cells grown in 10 mM Mg2+. The increases in uptake were the result of increases in Vmax without change in Km. This result suggests that, in low Mg2+ medium, cells contained higher levels of the transporters. Production of beta-galactosidase from mgtA::lacZ and mgtB::lacZ but not corA::lacZ fusions was also increased when cells were grown in low extracellular concentrations of Mg2+ indicating that the regulation occurs at the level of transcription. Expression of beta-galactosidase was also inhibited by the addition of other divalent cations including Ca2+ and Mn2+. Regulation of transcription from the mgtA and mgtB promoters was similar over the range of extracellular Mg2+ concentrations from 10 microM to 10 mM. At 1 microM, however, transcription from the mgtB promoter, as measured by beta-galactosidase levels in a mgtB::lacZ transcriptional fusion strain, was increased over 800-fold, and Ca2+ could no longer inhibit transcription effectively. In contrast, growth at 1 microM extracellular Mg2+ increased transcription from the mgtA promoter only about 30-fold and Ca2+ could still inhibit this increase. These results suggest that at least two distinct mechanisms are responsible for regulation of the mgtA and mgtB transcription in response to extracellular cation concentration.

The mgtB Mg2+ transport locus of Salmonella typhimurium encodes a P-type ATPase.

The mgtB locus codes for one of three distinct Mg2+ transport systems of Salmonella typhimurium. The system encoded by the mgtB locus mediates Mg2+ influx only. The nucleotide sequence of a 4.6-kilobase fragment of DNA carrying mgtB has been determined. Two open reading frames were apparent. The most 5' (mgtC) could encode a hydrophobic protein of up to 25 kDa depending on which translation starts are used. A plasmid carrying this region downstream from a phage T7 promoter expresses a 22.5-kDa protein. The second open reading frame encoded a 101-kDa polypeptide (MgtB) consistent with our previous observation that a plasmid carrying the mgtB locus expresses a 102-kDa protein in maxicells. Insertions into either open reading frame abolished the ability of the plasmid to relieve the requirement for added Mg2+ and to restore Mg2+ uptake to a Mg2+ transport-deficient strain of S. typhimurium. The predicted amino acid sequence of MgtC showed no similarity to any other known protein. In contrast, the predicted sequence of MgtB indicated that it is a member of the family of cation transport P-type ATPases. Strikingly, however, MgtB was significantly more similar to eukaryotic Ca2(+)-ATPases than to prokaryotic P-type ATPases or other classes of eukaryotic P-type ATPases such as the Na+,K(+)-ATPase. MgtB is most closely related to Ca2(+)-ATPases of mammalian sarcoplasmic reticulum and yeast. A number of features of the Ca2(+)-ATPases thought to be important for cation transduction across the membrane are present in MgtB but not in other prokaryotic members of this enzyme family. Unlike the Ca2(+)-ATPases, however, which mediate efflux of cation from the cytosol, MgtB mediates influx of cation into the cytosol.

Magnesium transport in Salmonella typhimurium: genetic characterization and cloning of three magnesium transport loci.

Salmonella typhimurium strains lacking the CorA Mg2+ transport system retain Mg2+ transport and the ability to grow in medium containing a low concentration of Mg2+. Mutagenesis of a corA strain followed by ampicillin selection allowed isolation of a strain that required Mg2+-supplemented media for growth. This strain contained mutations in at least two loci in addition to corA, designated mgtA and mgtB (for magnesium transport). Strains with mutations at all three loci (corA, mgtA, and mgtB) exhibited no detectable Mg2+ uptake and required 10 mM Mg2+ in the medium for growth at the wild-type rate. A wild-type allele at any one of the three loci was sufficient to restore both Mg2+ transport and growth on 50 microM Mg2+. P22 transduction was used to map the mgt loci. The mgtA mutation was located to approximately 98 map units (cotransducible with pyrB), and mgtB mapped at about 80.5 map units (near gltC). A chromosomal library from S. typhimurium was screened for clones that complemented the Mg2+ requirement of a corA mgtA mgtB mutant. The three classes of plasmids obtained could each independently restore Mg2+ transport to this strain and corresponded to the corA, mgtA, and mgtB loci. Whereas the corA locus of S. typhimurium is analogous to the corA locus previously described for Escherichia coli, neither of the mgt loci described in this report appears analogous to the single mgt locus described in E. coli. Our data in this and the accompanying papers (M. D. Snavely, J. B. Florer, C. G. Miller, and M. E. Maguire, J. Bacteriol. 171:4752-4760, 4761-4766, 1989) indicate that the corA, mgtA, and mgtB loci of S. typhimurium represent three distinct systems that transport Mg2+.

Magnesium transport in Salmonella typhimurium: expression of cloned genes for three distinct Mg2+ transport systems.

In Salmonella typhimurium, the corA, mgtA, and mgtB loci are involved in active transport of Mg2+ (S. P. Hmiel, M. D. Snavely, C. G. Miller, and M. E. Maguire, J. Bacteriol. 168:1444-1450, 1988; S. P. Hmiel, M. D. Snavely, J. B. Florer, M. E. Maguire, and C. G. Miller, J. Bacteriol. 171:4742-4751, 1989). In this study, the gene products coded for by the corA, mgtA, and mgtB genes were identified by using plasmid expression in Escherichia coli maxicells. Complementation was assessed by introducing plasmids into a Mg2+-dependent corA mgtA mgtB strain and determining the ability of the plasmid to restore growth on medium without a Mg2+ supplement. Complementing plasmids containing corA expressed a 42-kilodalton (kDa) protein. This protein was not expressed by plasmids containing insertions or deletions that eliminated complementation. A plasmid containing mgtA expressed 37- and 91-kDa gene products. Data obtained with subclones and insertions in this plasmid indicated that plasmids expressing only the 91-kDa polypeptide complemented; plasmids that did not express this protein did not complement regardless of whether they expressed the 37-kDa protein. Plasmids carrying mgtB expressed a single protein of 102 kDa whose presence or absence correlated with the ability of the plasmid to complement the Mg2+-dependent triple mutant. Fractionation of labeled maxicells demonstrated that the 42-kDa corA, the 91-kDa mgtA, and the 102-kDa mgtB gene products are all tightly associated with the membrane, a location consistent with involvement in a transport process. These data provide further support the for existence of three distinct systems for Mg2+ transport in S. typhimurium.

Magnesium transport in Salmonella typhimurium: 28Mg2+ transport by the CorA, MgtA, and MgtB systems.

Three loci in Salmonella typhimurium (corA, mgtA, and mgtB) code for components of distinct Mg2+ transport systems (S. P. Hmiel, M. D. Snavely, J. B. Florer, M. E. Maguire, and C. G. Miller, J. Bacteriol. 171:4742-4751, 1989). Strains carrying one wild-type and two mutant alleles of the three loci were constructed to study the kinetics and specificity of ion transport of each system in isolation. The transport systems had different Km and Vmax values for Mg2+ uptake, and each was inhibited by other divalent cations in a distinct rank order of potency: for CorA, Mg2+ greater than Mn2+ greater than Co2+ greater than Ni2+ greater than Ca2+; for MgtA, Zn2+ greater than or equal to Mg2+ greater than Ni2+ approximately Co2+ greater than Ca2+; and for MgtB, Mg2+ approximately Ni2+ approximately Ni2+ greater than Mn2+ much greater than Ca2+. Other differences among the three systems were apparent. The CorA transport system functioned as a Mg2+-Mg2+ exchange system, mediating both efflux and influx of Mg2+. Neither the MgtA nor the MgtB system could mediate Mg2+ efflux. Transport via the MgtB system was very temperature sensitive; Mg2+ was transported at 37 degrees C but not at 20 degrees C. The MgtA and the MgtB transport systems were found to be regulated by the extracellular concentration of Mg2+.

Magnesium transport in Salmonella typhimurium: characterization of magnesium influx and cloning of a transport gene.

The influx of Mg2+ in Salmonella typhimurium LT-2 was studied by both kinetic and genetic techniques. Wild-type cells grown in a high MgSO4 concentration (10 mM) exhibited a Km of 15 microM for Mg2+ influx, with a Vmax of 0.25 nmol of Mg2+ per min per 10(8) cells. The apparent Km decreased to 3 microM, and the Vmax increased 60% after growth in a low MgSO4 concentration (10 microM). Co2+ was a simple competitive inhibitor (Ki = 30 microM) of Mg2+ influx in cells grown in high Mg2+ concentrations but blocked only a portion of the Mg2+ influx in cells grown in low Mg2+ concentrations. Co2+ influx exhibited kinetics similar to those of Mg2+ influx (Km = 30 microM; Vmax = 0.5 nmol of Co2+ per min per 10(8) cells) but was not affected by growth conditions. Co2+ influx was competitively inhibited by both Mg2+ and Mn2+. Mutations affecting Mg2+ uptake were isolated by selection for spontaneous resistance to toxic levels of Co2+. One class of mutants designated corA mapped at 84 min near metE with the following gene order: corA, metE, zie-3161::Tn10, pepQ. A second class designated corB mapped at 98 min near pyrB. Mg2+ influx was decreased in a corA mutant strain (relative to that of the wild type) when grown in high Mg2+ concentrations but was restored when grown in low Mg2+ concentrations. Co2+ transport was completely abolished by the corA mutation under all growth conditions. Recombinant plasmids carrying the corA region from either Escherichia coli K-12 or S. typhimurium complemented the corA mutation in S. typhimurium, restoring uptake of both Co2+ and Mg2+ and conferring sensitivity to Co2+. The S. typhimurium corA gene was localized to a restriction fragment of approximately 1.5 kilobases.

Subtype-selective down-regulation of rat renal cortical alpha- and beta-adrenergic receptors by catecholamines.

In the current studies, we have explored agonist-mediated down-regulation of adrenergic receptors in vivo. We infused catecholamines from sc implanted osmotic minipumps and examined the effects of the resultant increases in circulating levels of catecholamines on rat renal cortical alpha- and beta-adrenergic receptor subtypes, as assessed in radioligand binding studies. Infusion of epinephrine or norepinephrine (at 150 micrograms/kg X h) elevated plasma levels of each catecholamine 10- to 20-fold and decreased renal cortical alpha 1-receptor number about 50% without changing alpha 2-receptor number. Isoproterenol infusion (150 micrograms/kg X h) raised plasma levels of this catecholamine, but had no effect on the number of either alpha 1- or alpha 2-receptors. Renal cortical beta-adrenergic receptor number was decreased by infusion of all three catecholamines. However, the beta 1- and beta 2-adrenergic receptors were altered selectively by the different agonists. Infusion of norepinephrine decreased both beta 1- and beta 2-receptor number, but was more effective for the beta 1-receptors; this result was somewhat at variance with that we previously reported for rats bearing transplanted pheochromocytomas. The decrease in beta-receptor number due to epinephrine infusion was largely due to loss of the renal cortical beta 2-receptors. Infusion of isoproterenol decreased the number of both beta 1- and beta 2-receptors (69% and 75%, respectively). Infusion of norepinephrine maximally decreased the number of alpha 1-, beta 1-, and beta 2-receptors within 2 days, and the t 1/2 for receptor loss was about 12 h. beta-Receptors lost in response to isoproterenol infusion could not be recovered in a pellet prepared by high speed centrifugation of the supernatant derived from the preparation of renal cortical membranes. These results indicate that adrenergic receptor subtypes are differentially down-regulated by elevated levels of circulating catecholamines and that this differential loss of receptors depends on the nature of the receptor subtype, the agonist, and perhaps also whether catecholamines are infused rather than increased by pheochromocytoma.

A new approach to determine rates of receptor appearance and disappearance in vivo. Application to agonist-mediated down-regulation of rat renal cortical beta 1- and beta 2-adrenergic receptors.

We have developed a method for the assessment of agonist-induced down-regulation of receptors in vivo in terms of rates of receptor appearance and disappearance. This method involves computer-assisted analysis of the kinetics of receptor loss during agonist infusion and of the recovery of receptor number upon the removal of the agonist. These kinetics are analyzed in terms of a steady state model that allows estimation of the rate constants for receptor appearance, kappa ap, and receptor disappearance, kappa dp. Several tests establish that the model can fit experimentally derived data very well. In testing this model, we examined the in vivo down-regulation and recovery of rat renal cortical membrane beta 1- and beta 2-adrenergic receptors in response to infusion of the agonist isoproterenol from subcutaneously implanted osmotic minipumps. During recovery from down-regulation, the beta 1-receptors have a t 1/2 of 45 hr and a kappa ap of 1.6%/hr, and the beta 2-adrenergic receptors a t 1/2 of 18 hr and a kappa ap of 3.9%/hr. During down-regulation, the t 1/2 for both receptors is 12 hr, while kappa ap for beta 1-receptors and beta 2-receptors are 3 and 2.3%/hr, respectively. To the extent that the kinetics of recovery from down-regulation reflect "basal" receptor metabolism, the data indicate that enhanced receptor clearance of both receptor subtypes from the plasma membrane contribute to down-regulation, but changes in rates of receptor appearance may occur as well. The use of this computer modeling technique for defining kinetics of changes in receptor number from one steady state level to another should provide a generally useful means to assess hormone and neurotransmitter receptor metabolism in vivo.

Radioligand binding and functional assays demonstrate postsynaptic alpha 2-receptors on proximal tubules of rat and rabbit kidney.

We have explored the localization of renal alpha 2-adrenoceptors by radioligand binding techniques and studies of 22Na+ influx into isolated renal tubular cells. Initially, we validated the use of the alpha 2-adrenergic antagonist [3H]rauwolscine as a high-affinity (Kd = 2.5 +/- 0.65 nM) probe that binds to a single class (Hill slope = 0.94 +/- 0.06) of alpha 2-adrenoceptors in rat renal cortical membranes (Bmax = 340 +/- 50 fmol/mg). [3H]Rauwolscine and [3H]yohimbine identified an identical number of sites, but [3H]rauwolscine bound with a several-fold higher affinity. Treatment of rats with 6-hydroxydopamine depleted renal norepinephrine greater than 80% but failed to alter the number or affinity of renal cortical alpha 2-receptors, thus suggesting that these alpha 2-receptors are not located on presynaptic sites. Further evidence for the postsynaptic localization of these receptors was obtained by in vitro autoradiographic studies, which showed that [3H]rauwolscine sites were preferentially located on proximal tubular cells, and by functional assays with isolated rabbit proximal tubular cells. In the latter studies, Na+-H+ antiport activity was stimulated up to 2-fold by the selective alpha 2-adrenergic agonist guanabenz, and this stimulation was blocked by yohimbine but not by the alpha 1-blocker prazosin. Although amiloride was able to block this response to guanabenz, radioligand binding studies demonstrated that amiloride can compete for [3H]rauwolscine sites at the concentrations used to block Na+-H+ antiport activity. These data suggest that alpha 2-adrenoceptors are present on proximal tubular cells in rats and rabbits and that these receptors appear to stimulate Na+-H+ antiport activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Quinidine is a competitive antagonist at alpha 1- and alpha 2-adrenergic receptors.

Although quinidine is known to have antiadrenergic effects in the cardiovascular system, the precise mechanism by which it exerts these effects is not well defined. We asked whether quinidine binds directly to adrenergic receptors. Radioligand-binding assays were used to identify alpha 1-adrenergic receptors [( 3H]prazosin-binding sites) on membranes prepared from rat heart and kidney, alpha 2-adrenergic receptor [( 3H]yohimbine-binding sites) on human platelets and rat kidney membranes, and beta-adrenergic receptors [( 125I]iodocyanopindolol-binding sites) on rat heart and kidney membranes. Although it did not effectively compete for binding to beta-adrenergic receptors, quinidine competed for binding to alpha 1- and alpha 2-adrenergic receptors and yielded equilibrium dissociation constants of 0.3-3 microM. Two other antiarrhythmic agents, lidocaine and procainamide, did not compete for binding to alpha-adrenergic receptors. Further experiments demonstrated that the interactions of quinidine with the cardiac alpha 1- and platelet alpha 2-adrenergic receptors were competitive and reversible. We conclude that that antiadrenergic actions of quinidine can be explained by occupancy and competitive blockade of alpha 1- and alpha 2-adrenergic receptors.

alpha 1- and beta 2-adrenergic receptor expression in the Madin-Darby canine kidney epithelial cell line.

The Madin-Darby canine kidney (MDCK) cell line, derived from distal tubule/collecting duct, expresses differentiated properties of renal tubule epithelium in culture. We studied the expression of adrenergic receptors in MDCK to examine the role of catecholamines in the regulation of renal function. Radioligand-binding studies demonstrated, on the basis of receptor affinities of subtype-selective adrenergic agonists and antagonists, that MDCK cells have both alpha 1- and beta 2-adrenergic receptors. To determine whether these receptor types were expressed by the same cell, we developed a number of clonal MDCK cell lines. The clonal lines had stable but unique morphologies reflecting heterogeneity in the parent cell line. Some clones expressed only beta 2-adrenergic receptors and were nonmotile, whereas others expressed both alpha 1- and beta 2-receptors and demonstrated motility on the culture substrate at low cell densities. In one clone, alpha- and beta-receptor expression was stable for more than 50 passages. Catecholamine agonists increased phosphatidylinositol turnover by activating alpha-adrenergic receptors and cellular cyclic adenosine monophosphate accumulation by activating beta-adrenergic receptors. Guanine nucleotide decreased the affinity of isoproterenol for the beta 2-receptor but did not alter the affinity of epinephrine for the alpha 1-receptor. These results show that alpha 1- and beta 2-receptors can be expressed by a single renal tubular cell and that the two receptors behave as distinct entities in terms of cellular response and receptor regulation. Heterogeneity of adrenergic receptor expression in MDCK clones may reflect properties of different types of renal tubule cells.

Selective down-regulation of adrenergic receptor subtypes in tissues from rats with pheochromocytoma.

We have used an animal model of pheochromocytoma and radioligand-binding techniques to examine the effects of high levels of circulating norepinephrine and dopamine on adrenergic receptor subtypes in several peripheral tissues. New England Deaconess Hospital rats with transplanted pheochromocytomas were hypertensive and had levels of plasma norepinephrine and dopamine 50-fold greater than those of controls. The number of beta-adrenergic receptors in membranes prepared from the renal cortex and the left ventricle from these rats was decreased about 50%, but the animals had no significant decrease in the overall number of beta-adrenergic receptors in pulmonary membranes. beta-Receptor affinity was unaltered in animals with pheochromocytoma. Competition for [125I]iodocyanopindolol binding to beta-receptors by subtype-selective agents indicated a selective decrease of about 80% in the number of beta 1-adrenergic receptors in renal cortical and pulmonary membranes, without a decrease in beta 2-adrenergic receptor number. Rats with pheochromocytoma also had about a 70% decrease in the number of alpha 1-adrenergic receptors in membranes from renal cortex and lung, but no significant decrease in the number of alpha 1-adrenergic receptors in hepatic membranes and no decrease in the number of alpha 2-adrenergic receptors in renal cortical and hepatic membranes. These results indicate that rats in which pheochromocytomas are transplanted show adrenergic receptor subtype- and tissue-specific down-regulation. Although the selective down-regulation of alpha 1- and beta 1-adrenergic receptors may reflect a response to the preponderance of norepinephrine in these animals, the results indicate that different tissues and different adrenergic receptor subtypes may have varying susceptibility to down-regulation in response to increased circulating catecholamines in vivo.

Interaction of verapamil and other calcium channel blockers with alpha 1- and alpha 2-adrenergic receptors.

To determine the specificity of the previously demonstrated competition of verapamil with radioligand binding to alpha-adrenergic receptors, we examined the interaction of calcium channel blockers with alpha 1- and alpha 2-adrenergic receptors on several tissues. Verapamil competed for [3H] prazosin binding to alpha 1-adrenergic receptors and for [3H]yohimbine binding to alpha 2-adrenergic receptors in several tissues (human platelets, rat kidney and heart, and cultured muscle cells) with dissociation constants of 0.6-6 microM. The calcium channel blockers D600, D591, fendiline, and prenylamine--which are structural analogues of verapamil--also competed for [3H]yohimbine binding to human platelets. Two other calcium channel blockers, diltiazem and nifedipine, did not compete for [3H] yohimbine binding to human platelets or [3H]prazosin binding to membranes prepared from rat ventricles. We used [3H]nitrendipine binding to identify putative calcium channels on rat myocardial membranes. Nifedipine and verapamil blocked these [3H]nitrendipine-binding sites on ventricular membranes, but epinephrine and prazosin did not, indicating that the ventricular alpha 1 receptors and calcium channels are distinct. We found no specific [3H]nitrendipine binding to human platelets. We conclude that the interaction of verapamil with alpha-adrenergic receptors is not receptor subtype or tissue specific, that interaction with alpha-adrenergic receptors is not a property of all calcium channel blockers, and that the interaction of verapamil with alpha-adrenergic receptors and its interaction with calcium channels occur at at least two distinct sites.

Characterization of alpha-adrenergic receptor subtypes in the rat renal cortex. Differential regulation of alpha 1- and alpha 2-adrenergic receptors by guanyl nucleotides and Na.

We have used the subtype-selective radioligands [3H]prazosin (an alpha 1-adrenergic antagonist) and [3H]yohimbine (an alpha 2-adrenergic antagonist) to examine alpha-adrenergic receptors in rat renal cortical membranes. Under the conditions used in this study, [3H]prazosin bound only to alpha 1-adrenergic receptors, whereas [3H]yohimbine bound only to alpha 2-adrenergic receptors; the two radioligands were completely selective and did not bind to a common site. The ratio of alpha 2- to alpha 1-adrenergic receptors was about 3:1. Guanyl nucleotides decreased the affinity of epinephrine at both receptor subtypes, but this effect was greater at the alpha 2-receptor and, according to computer analysis, occurred through different mechanisms at the two receptor subtypes. NaCl decreased the affinity of epinephrine at both alpha-receptor subtypes; this effect was more Na+-selective at alpha 2- than at alpha 1-receptors. Guanyl nucleotides and NaCl were additive in decreasing the affinity of epinephrine at the alpha 1-receptor but were synergistic at the alpha 2-receptor. In addition, NaCl increased specific binding of [3H] yohimbine but had no effect on the binding of [3H]prazosin. This enhancement of [3H] yohimbine binding was Na+-specific and fully reversible, and represented an increase in maximal binding capacity. Although binding of epinephrine to both alpha 1- and alpha 2-receptors could be modulated by guanyl nucleotides, we were unable to detect inhibition by epinephrine of basal or hormone-stimulated adenylate cyclase activity. Thus, separate alpha 1- and alpha 2-adrenergic receptors can be detected in the rat renal cortex and binding to both receptor subtypes can be regulated by guanyl nucleotides and Na+ X Na+ may directly interact with alpha 2- but not alpha 1-adrenergic receptors in the renal cortex. Our findings with renal cortical membranes indicate that regulation of agonist binding at alpha-adrenergic receptor subtypes by guanyl nucleotides and Na+ is not limited to alpha 2-adrenergic receptors, as previously reported with other tissues.