The repertoire of killer cell Ig-like receptor and CD94:NKG2A receptors in T cells: clones sharing identical alpha beta TCR rearrangement express highly diverse killer cell Ig-like receptor patterns

Killer cell Ig-like receptor (KIR) and CD94:NKG2A molecules were first defined as human NK cell receptors (NKR), but now are known to be expressed and to function on subpopulations of T cells. Here the repertoires of KIR and CD94:NKG2A expression by T cells from two donors were examined and compared with their previously defined NK cell repertoires. T cell clones generated from peripheral blood of both donors expressed multiple NKR in different combinations and used the range of receptors expressed by NK cells. In both donors alpha beta T cells less frequently expressed the inhibitory receptors CD94:NKG2A and KIR2DL1 than either gamma delta T cells or NK cells. In contrast to NK cells, not all NKR(+) T cells expressed an inhibitory receptor for autologous HLA class I. This lack of specific inhibitory NKR was especially apparent on alpha beta T cells of one donor. Overall, alpha beta T cells exhibited a distinct pattern of NKR expression different from that of gamma delta T and NK cells, which expressed highly similar NKR repertoires. In one donor, analysis of TCR rearrangement revealed a dominant subset of NKR(+) T cells sharing identical TCR alpha- and beta-chains. Remarkably, among 55 T cell clones sharing the same TCR alpha beta rearrangement 18 different KIR phenotypes were seen, suggesting that KIR expression was initiated subsequently to TCR rearrangement.     

Regulation of diamine oxidase expression by beta 2-adrenoceptors in normal and hypertrophic rat kidney

The administration of preferential adrenergic receptor antagonists to uninephrectomized rats revealed the beta 2-adrenergic mediation in diamine oxidase activity increase that occurs in the remaining kidney undergoing compensatory hypertrophy. In fact, beta 1, beta 2- or beta 2, but not alpha 1-, alpha 2-, or beta 1-receptor-blocking agents prevented this enzyme enhancement. Further studies with adrenoceptor agonists, such as epinephrine (alpha 1, alpha 2, beta 1, beta 2), isoproterenol (beta 1, beta 2) or terbutaline (beta 2) showed that also in normal rat kidney diamine oxidase activity is under the control of catecholamine-beta 2-receptors through a mechanism that involves new synthesis of mRNA and protein. Theophylline, an inhibitor of phosphodiesterase, or forskolin, an activator of adenyl cyclase, increased diamine oxidase activity as does epinephrine or nephrectomy. Thus, catecholamine-triggered beta 2-receptors coupled to adenyl cyclase are involved in the regulation of diamine oxidase activity in normal and hypertrophic rat kidney.     

Reduced alpha adrenergic mediated contraction of renal preglomerular blood vessels as a function of gender and aging

As human males age, a decline in baroreflex-mediated elevation of blood pressure occurs due, at least in part, to a reduction in alpha-1 adrenergic vasoconstrictor function. Alpha adrenergic constriction is mediated by guanosine triphosphate binding Protein (G Protein) coupled signaling pathways. Alpha-1 A/C, B, and D adrenergic receptor expressions, measured by GeneChip array, are not reduced during aging in renal blood vessels of male or female rats. Alpha-1 A GeneChip expression is greater, at all ages studied, in females than in males. Prazosin binding by alpha-1 adrenergic receptors is greater in young adult female rats than in young adult male rats; however, it is reduced with aging in both male and female rats. G alpha q GeneChip expression declines while expression of adrenergic receptor kinase (GRK2) and tyrosine phosphatases (TyrP) increase with aging in male rats. The declines in alpha-1 adrenergic receptor binding and G alpha q expression and also the increases in GRK2 and TyrP expression likely relate to the age-related decline of vasoconstriction in male rats. The information that the expression of alpha-1 A adrenergic receptors is greater in female rats and (GRK2) expression does not increase during aging could relate to the gender differences in vasoconstrictor function with aging. Gene therapy to ameliorate the age-related decline in renal function could possibly reduce the need for renal dialysis. Signaling pathways such as those reviewed herein may provide an outline of the molecular pathways needed to move toward successful renal gene therapy for aging individuals.     

Alpha adrenergic stimulation reduces cyclic adenosine 3',5'-monophosphate generation in rabbit myometrium by two mechanisms

Rabbit myometrium contains postsynaptic alpha-1, alpha-2, and beta-2 adrenoreceptors. The response to endogenous catecholamines depends on the summation of interactions at these receptors and is influenced by the hormonal environment. Estrogen treatment of ovariectomized rabbits increases the alpha adrenergic contractile response whereas progesterone treatment of estrogen primed animals results in a predominance of the beta adrenergic response, which is inhibition of contractions. Of the receptor subtypes, only the alpha-2 receptor concentration is increased at physiological estrogen concentrations. However, alpha-2 receptors have not been shown to be directly involved in myometrial contraction, which appears to be mediated solely by alpha-1 adrenergic interactions. To test whether alpha-2 receptors might indirectly affect contraction by opposing interactions at the beta receptor, we examined the ability of alpha adrenergic stimulation to reduce myometrial cyclic adenosine 3',5'-monophosphate (cAMP) generation. We find that alpha-2 receptors inhibit myometrial ade adenylate cyclase through the guanine nucleotide regulatory protein, Gi. In addition, we find that activation of alpha-1 receptors also reduces cAMP generation. This interaction, which can be demonstrated in the absence but not the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, does not appear to be mediated through Gi. These findings illustrate the complexity of adrenergic interactions in tissues containing several adrenergic subtypes.     

Activation of alpha-1 adrenergic receptors modulates the control of left/right sidedness in rat embryos.

Presomite stage rat embryos were cultured for 45-49 hr with medium containing various adrenergic agonists and antagonists. L-Norepinephrine but not D-norepinephrine (several orders of magnitude less potent than the L-isomer at alpha-1 adrenergic receptors) resulted in a dose-dependent increase of situs inversus similar to that found for phenylephrine, an alpha-1 adrenergic agonist. Prazosin, an alpha-1 adrenergic antagonist, inhibited phenylephrine-induced situs inversus in a dose-dependent manner. Neither dexmedetomidine, an alpha-2 adrenergic agonist, nor isoproterenol, a beta adrenergic agonist, caused situs inversus. These results provide pharmacological evidence that stimulation of alpha-1 but not of alpha-2 and beta adrenergic receptors modulates the control of left/right sidedness in rat embryos.     

Calcium dependent aggregation of marine sponge cells is provoked by leukotriene B4 and inhibited by inhibitors of arachidonic acid oxidation

The mechanism of agonist-induced desensitization of the beta adrenergic receptor coupled adenylate cyclase has been studied in a smooth muscle cell line, BC3H-1, which expresses both alpha and beta adrenergic receptors and nicotinic receptors. beta receptors have been investigated in intact cells using as radioligand 3HCGP-12177, an hydrophilic compound which labels only surface receptors. The treatment of BC3H-1 cells with the agonist Isoproterenol, at 37 degrees but not at 4 degrees, induced a dose dependent internalization of the beta adrenergic receptor. Agonist-induced internalization was very rapid, in the order of few minutes. beta adrenergic receptor internalization was very specific: the alpha adrenergic agonist Phenylefrine had almost no effect on beta receptor levels, while Isoproterenol treatment had no effect on the number of alpha adrenergic or nicotinic receptors expressed at the cell surface of these cells. beta adrenergic receptor internalization is probably the major mechanism responsible for catecholamine desensitization in smooth muscle cells.     

Regulation of alpha- and beta-adrenergic receptor subclasses by gonadal steroids in human myometrium

Both alpha- and beta-adrenergic receptors have been identified in the human myometrium by radioligand binding. Both adrenergic receptor subclasses have been shown to mediate the contractile response of the uterus upon catecholamine stimulation: alpha-adrenergic receptors cause uterine contraction while beta-adrenergic receptors induce relaxation. We have identified alpha 1- and alpha 2-adrenergic receptors in myometrial membranes using the newly developed radiolabelled specific antagonists [3H]-prazosin and [3H]-rauwolscine. This enabled us to characterize both receptor subclasses individually. Beta adrenergic receptors were identified using the radiolabelled antagonist (-)-[3H]-dihydroalprenolol. Binding of radioligands to the myometrial membrane receptors was rapid, readily reversible, of high affinity and stereoselective. The total number of alpha 1-, alpha 2- and beta-receptors was determined by Scatchard analysis of radioligand saturation binding and the beta/beta 2-receptor ratio was determined by computer analysis of the beta 2-selective antagonist ICI 118 551) (-)-[3H]-dihydroalprenolol competition binding curves. This enabled us to study the regulation of both alpha- and beta-receptor subclasses under various physiological and pharmacological conditions in the human, i.e., during different phases of the menstrual cycle, in postmenopausal women and during depo-progestin (Medroxyprogesterone acetate) therapy. Only the alpha 2- and beta 1-adrenergic receptor concentrations were found to be subjected to gonadal steroid regulation. The number of alpha 2- and beta 1-adrenergic receptors increased concomitantly with circulating plasma oestradiol levels. This effect was counteracted by progesterone. The number of alpha 1- and beta 2-adrenergic receptors was unaffected by the gonadal steroid environment. These results are an example of the heteroregulation of membrane receptors by oestrogens and progesterone and cast new light on the regulatory mechanisms involved in uterine contractility in the human.     

Activity of cytisine and its brominated isosteres on recombinant human alpha7, alpha4beta2 and alpha4beta4 nicotinic acetylcholine receptors

Effects of cytisine (cy), 3-bromocytisine (3-Br-cy), 5-bromocytisine (5-Br-cy) and 3,5-dibromocytisine (3,5-diBr-cy) on human (h) alpha7-, alpha4beta2- and alpha4beta4 nicotinic acetylcholine (nACh) receptors, expressed in Xenopus oocytes and cell lines, have been investigated. Cy and its bromo-isosteres fully inhibited binding of both [alpha-(125)I]bungarotoxin ([alpha-(125)I]BgTx) to halpha7- and [(3)H]cy to halpha4beta2- or halpha4beta4-nACh receptors. 3-Br-cy was the most potent inhibitor of both [alpha-(125)I]BgTx and [(3)H]cy binding. Cy was less potent than 3-Br-cy, but 5-Br-cy and 3,5-diBr-cy were the least potent inhibitors. Cy and 3-Br-cy were potent full agonists at halpha7-nACh receptors but behaved as partial agonists at halpha4beta2- and halpha4beta4-nACh receptors. 5-Br-cy and 3,5-diBr-cy had low potency and were partial agonists at halpha7- and halpha4beta4-nACh receptors, but they elicited no responses on halpha4beta2-nACh receptors. Cy and 3-Br-cy produced dual dose-response curves (DRC) at both halpha4beta2- and halpha4beta4-nACh receptors, but ACh produced dual DRC only at halpha4beta2-nACh receptors. Low concentrations of cy, 3-Br-cy and 5-Br-cy enhanced ACh responses of oocytes expressing halpha4beta2-nACh receptors, but at high concentrations they inhibited the responses. In contrast, 3,5-diBr-cy only inhibited, in a competitive manner, ACh responses of halpha4beta2-nACh receptors. It is concluded that bromination of the pyridone ring of cy produces marked changes in effects of cy that are manifest as nACh receptor subtype-specific differences in binding affinities and in functional potencies and efficacies.     

Differentiation of Alpha-Adrenergic Receptors Using Pharmacological Evaluation and Molecular Modeling of Selective Adrenergic Agents

Abstract

Subtypes of alpha adrenergic receptors were studied using selective adrenergic agonists. A-53693, A-54741, and related compounds were evaluated for their affinity for alpha receptor subtypes using radioligand binding techniques. Efficacy and potency were also evaluated using in vitro bioassays of alpha-1 receptors in rabbit aorta smooth muscle and alpha-2 receptors in the phenoxybenzamine-pretreated canine saphenous vein. Active and inactive compounds were then submitted for computer-assisted molecular modeling evaluation to ascertain the structural requirements for optimal potency and selectivity. Rigid catecholamines such as A-53693 display a high degree of selectivity for alpha-2 compared to alpha-1 receptors, probably because of the unique regions of space at the ligand binding site occupied by active compounds. Imidazolines such as A-54741 also interact with extremely high affinity and potency for alpha-2 receptors, and to a lesser extent at alpha-1 receptors. The spatial domains occupied by phenethylamines and imidazolines differ, each having unique regions of permissable space at alpha receptors. Compounds such as A-53693 and A-54741 are extremely useful probes of the molecular interactions of alpha agonistic compounds which will help in the design of even more selective drugs for alpha adrenergic receptors.     

Cadmium effects on ros production and DNA damage via adrenergic receptors stimulation: role of Na+/H+ exchanger and PKC

The objective of the present study was to elucidate the events that are involved in reactive oxygen species (ROS) production and DNA damage after adrenergic receptors stimulation by cadmium, in relation to cAMP, protein kinase C (PKC) and Na+/H+ exchanger (NHE). Cadmium (50 microM) caused increased levels of ROS with a concomitant increase in DNA damage in digestive gland of Mytilus galloprovincialis. Either the use of EIPA, a NHE blocker, or calphostin C, the inhibitor of PKC, reduced cadmium effects. Cells treated with alpha1-, alpha2-, beta- and beta1- adrenergic antagonists together with cadmium reversed cadmium alone effects, while the respective adrenergic agonists, phenylephrine and isoprenaline, mimic cadmium effects. Moreover, cadmium caused an increase in the levels of cAMP in digestive gland cells that were reversed after NHE and PKC inhibition as well as in the presence of each type of adrenergic antagonist. The different sensitivity of alpha1-, alpha2-, beta-, beta1- adrenergic receptors on ROS, cAMP production and DNA damage possibly leads to the induction of two signaling pathways that may be interacting or to the presence of a compensatory pathway that acts in concert with the alpha- and beta- adrenergic receptors. In these signaling pathways PKC and NHE play significant role.     

Marked differences in the efficiency of expression of distinct alpha beta T cell receptor heterodimers

Ag recognition by most T lymphocytes is mediated by clonally distributed alpha beta heterodimeric receptors. A major fraction of TCR diversity is believed to be due to the random coexpression in individual T cells of the products of independently rearranging alpha- and beta-genes (combinatorial diversity). However, analysis of cell surface receptors on transfected T hybridoma cells synthesizing various sets of alpha- and beta-chains revealed marked differences in the efficiency of expression of certain alpha beta-pairs. Specifically, using the functionally rearranged gene products of the 2B4 cytochrome c specific T hybridoma (V beta 3, V alpha 11.2) and BW5147 parent lymphoma (V beta 1, V alpha BW), a hierarchy of expression efficiency relative to indirectly measured precursor chain levels in the cell was shown to be 2B4 alpha-BW beta greater than 2B4 alpha - 2B4 beta greater than BW alpha - BW beta greater than BW alpha - 2B4 beta. The estimated difference between the best expressed and worst expressed pairs is on the order of 50-fold. For the beta-chain, the primary determinant of expression efficiency with a given alpha-chain appears to be the V segment, as a second V beta 1-chain with distinct D and J regions from BW beta was expressed with the same pattern. These data imply that alpha- and beta-chains do not form well-expressed TCR in a random manner and that limitations on the useful combinatorial association of these chains may significantly affect the functional T cell repertoire.     

The MDCK cell line is made up of populations of cells with diverse resistive and transport properties

The MDCK cell line has been used as a model for the renal distal tubule. When grown on permeable supports, the cells form tight junctions and transport sodium and calcium. Widely different values have, however, been obtained for resistance and transports by different investigators. Since the MDCK cell line is genetically heterogeneous, one potential reason for observed differences is the use of different populations of cells. Dilutional techniques have been used to obtain eleven MDCK cell clones. The clones exhibit a range of values for resistance (300-4000 ohms cm2), net Ca2+ transport (3.2-81 pmol/cm2/min) and net Na+ transport (0.4-6.0 nmol/cm2/min). To determine if the characteristics of clonal cell lines changed with increasing passage number, resistance was measured at relatively early and late passage number. Resistance increased with increasing passage number in two of six lines and decreased in one. These studies demonstrate that differences between previous studies may be due to differences in cell population, length of time since passage, and passage number. Clonal cell lines should provide a useful tool to investigate ion transport and mechanisms of regulation of transport in this model of renal distal tubule.     

Absence of alpha-2 adrenergic effects on cAMP production in a genital tract smooth muscle cell line

This study sought to evaluate alpha-2 and beta adrenergic modulation of cAMP production in the DDT1 MF-2 transformed smooth muscle myocyte. After stimulation with forskolin or adrenergic agonists with or without subtype specific antagonists, cAMP production was determined. These experiments confirmed an increase of cAMP in response to forskolin, isoproterenol, epinephrine, and norepinephrine; the adrenergic stimulation was inhibited by propranolol. On the other hand, the alpha-2 agonist clonidine did not inhibit cAMP production. Likewise, alpha-2 receptor blockade did not increase cAMP production in response to epinephrine. These studies, therefore, suggest that the DDT1 MF-2 myocyte does not contain a significant population of functional alpha-2 adrenergic receptors.     

A point mutation in the seventh hydrophobic domain of the alpha 2 adrenergic receptor increases its affinity for a family of beta receptor antagonists.

Previous studies have shown that differences in subtype-specific ligand binding between alpha 2 and beta 2 adrenergic receptors are largely determined by the seventh hydrophobic domain. Here, we report that a single amino acid substitution (Phe412----Asn) in the seventh hydrophobic domain of the alpha 2 adrenergic receptor reduces affinity for the alpha 2 antagonist yohimbine by 350-fold and increases affinity for beta antagonist alprenolol by 3000-fold. The affinity of this mutant receptor alpha 2F----N for several alpha and beta adrenergic receptor agonists and antagonists was determined. Beta adrenergic receptor antagonists containing an oxygen atom linking the amino side chain with the aromatic ring bound to alpha 2F----N with high affinity, while the beta receptor antagonist sotalol, which lacks this oxygen, bound with low affinity. These data suggest that the Asn residue is involved in conferring specificity for binding to a specific class of beta receptor antagonists.     

beta-Adrenergic stimulated lipolysis in pony adipocytes is exclusively via a beta2-subtype and is not affected by lactation

Catecholamines are important lipolytic agents in horses and ponies but the nature of the adrenergic receptor subtype distribution in their adipocytes is uncertain. A first objective was to identify the beta-adrenergic receptor subtype(s) present in adipocytes from horses and ponies. A second objective was to evaluate if the lipolytic responsiveness of isolated adipocytes to beta-adrenergic agonists is altered during lactation, a condition known to affect markedly maternal fat metabolism. Isoproterenol and salbutamol elicited strong lipolytic responses in adipocytes isolated from horse and pony subcutaneous adipose tissue. There were weak lipolytic responses to norepinephrine, dobutamine and BRL37344. The weak lipolytic response to NE compared to isoproterenol or salbutamol suggests an antilipolytic action from alpha2-adrenergic receptors. The relative order of potency for the beta-adrenergic agonists was isoproterenol>/=salbutamol>dobutamine=BRL37344. There was expression of beta2-adrenergic receptor mRNA in pony and horse adipose tissues, as estimated by relative RT-PCR, but no expression of mRNAs for beta1- or beta3-adrenergic receptors. Early lactation did not alter the lipolytic responses to beta-adrenergic agonists, nor the expression of beta2-adrenergic receptor mRNA. Thus, these results indicate a dominant if not exclusive presence of beta2-adrenergic receptors in pony and horse adipocytes that is not affected by lactation.     

Cloning, sequencing, and expression of the gene encoding the porcine alpha 2-adrenergic receptor. Allosteric modulation by Na+, H+, and amiloride analogs

The gene for an alpha 2-adrenergic receptor has been cloned from a porcine genomic library, using as a probe a 0.95-kilobase Pst fragment of the gene for the human platelet alpha 2-adrenergic receptor. The identity of the cloned porcine gene was confirmed initially on the basis of partial amino acid sequence information obtained following cyanogen bromide digestion of homogeneous preparations of porcine brain alpha 2-adrenergic receptors. The deduced amino acid sequence for the porcine receptor, when compared to other members of the family of guanine nucleotide-binding protein-coupled receptors, shares the same overall structural characteristics and most closely resembles the human platelet C10 alpha 2-adrenergic receptor (greater than 93% homology). The putative porcine alpha 2-receptor gene was expressed in the COS-M6 cell line. Transfected cells display saturable [3H]yohimbine binding. The KD for [3H]yohimbine, determined in digitonin-solubilized preparations, is 5.8 nM. The selectivity of agonists and antagonists in competing for [3H]yohimbine binding to membranes prepared from the transfected cells is characteristic of the alpha 2A subtype of adrenergic receptors. The porcine alpha 2-receptor also was expressed permanently in LLC-PK1 porcine kidney cells at a level of 100 pmol/mg protein. The alpha 2-agonist UK14304 is able to attenuate forskolin or vasopressin-stimulated cAMP accumulation by at least 50% in these cells. Allosteric modulation of [3H] yohimbine binding by Na+, H+, and 5-amino-substituted analogs of amiloride also was demonstrated for the alpha 2-receptor expressed in COS-M6 cells. Moreover, these modulatory effects were quantitatively similar to those observed for homogeneous preparations of the alpha 2-receptor purified from porcine brain cortex. Retention of the effects of cations and amiloride analogs in transiently expressed alpha 2-receptors supports the interpretation that the allosteric sites for these agents reside in the alpha 2-receptor molecule itself.     

Hepatocyte growth factor upregulates α2β1 integrin in Madin-Darby canine kidney cells: Implications in tubulogenesis

It has been well established that hepatocyte growth factor (HGF) induces branching tubule formation of Madin-Darby canine kidney (MDCK) cells cultured in collagen gel. Tubulogenesis per se requires the involvement of cell proliferation, migration, focalization proteolysis, cell-cell interaction and differentiation. However, signaling pathways and proteins involved in HGF-induced tubulogenesis by MDCK cells have not been thoroughly studied. Because cell-matrix interactions play important roles in tubulogenesis, we analyzed whether HGF altered the expression of extracellular matrix receptor (alpha2, alpha3, beta1 and alphavbeta3 integrin). We found that among those proteins examined, alpha2beta1 integrin levels were enhanced by HGF. HGF-induced upregulation of alpha2beta1 integrin was mediated via upregulation of alpha2 integrin mRNA abundance. Cycloheximide blocked the HGF-induced increase in alpha2 integrin mRNA expression. To understand the signaling pathways leading to an HGF-induced increase in alpha2beta1 integrin levels, PD98059 (MEK1 inhibitor), LY294002 (PI3-kinase inhibitor), and GF109203X (PKC inhibitor) were used. We found that PD98059 blocked the HGF-induced increase in alpha2beta1 integrin expression. Furthermore, 5E8 (specific anti-alpha2beta1 integrin antibody) was employed to elucidate the potential role of HGF-induced upregulation of alpha2beta1 integrin in branching morphogenesis. 5E8 did not alter HGF-induced scattering effects but disrupted HGF-induced branching tubulogenesis in collagen gel via inhibition of cell-cell interactions and growth. Taken together, HGF upregulates alpha2beta1 integrin expression via an indirect pathway, the results of which contribute to the regulation of cell-cell interactions and cell growth during branching morphogenesis in collagen gel.