Biomedical subjects

K N Klotz

Publications and source records attributed to K N Klotz.

At least 19 recordsLinked to original sources

PubMed1997

Adenosine A1 receptor-mediated activation of phospholipase C in cultured astrocytes depends on the level of receptor expression.

Adenosine A1 receptors induce an inhibition of adenylyl cyclase via G-proteins of the Gi/o family. In addition, simultaneous stimulation of A1 receptors and of receptor-mediated activation of phospholipase C (PLC) results in a synergistic potentiation of PLC activity. Evidence has accumulated that Gbetagamma subunits mediate this potentiating effect. However, an A1 receptor-mediated increase in extracellular glutamate was suggested to be responsible for the potentiating effect in mouse astrocyte cultures. We have investigated the synergistic activation of PLC by adenosine A1 and alpha1 adrenergic receptors in primary cultures of astrocytes derived from different regions of the newborn rat brain. It is reported here that (1) adenosine A1 receptor mRNA as well as receptor protein is present in astrocytes from all brain regions, (2) A1 receptor-mediated inhibition of adenylyl cyclase is of similar extent in all astrocyte cultures, (3) the A1 receptor-mediated potentiation of PLC activity requires higher concentrations of agonist than adenylyl cyclase inhibition and is dependent on the expression level of A1 receptor, and (4) the potentiating effect on PLC activity is unrelated to extracellular glutamate. Taken together, our data support the notion that betagamma subunits are the relevant signal transducers for A1 receptor-mediated PLC activation in rat astrocytes. Because of the lower affinity of betagamma, as compared with alpha subunits, more betagamma subunits are required for PLC activation. Therefore, only in cultures with higher levels of adenosine A1 receptors is the release of betagamma subunits via Gi/o activation sufficient to stimulate PLC. It is concluded that variation of the expression level of adenosine A1 receptors may be an important regulatory mechanism to control PLC activation via this receptor.

Adenosine↗

PubMed1995

Defense.

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Ecology↗

PubMed1994

Physical coupling of N-formyl peptide chemoattractant receptors to G protein is unaffected by desensitization.

Desensitization of N-formyl peptide chemoattractant receptors (FPR) in human neutrophils results in association of these receptors to the membrane skeleton. This is thought to be the critical event in the lateral segregation of receptors and guanyl nucleotide-binding proteins (G proteins) within the plane of the plasma membrane resulting in an interruption of the signaling cascade. In this study we probed the interaction of FPR with G protein in human neutrophils that were desensitized to various degrees. Human neutrophils were desensitized using the photoreactive agonist N-formyl-met-leu-phe-lys-N epsilon-[125I]2(p-azidosalicylamino)ethyl-1,3'- dithiopropionate (fMLFK-[125I]ASD). The interaction of FPR with protein was studied via a reconstitution assay and subsequent analysis of FPR-G protein complexes in sucrose density gradients. FPR-G protein complexes were reconstituted with solubilized FPR from partially and fully desensitized neutrophils with increasing concentrations of Gi purified from bovine brain. The respective EC50 values for reconstitution were similar to that determined for FPR from unstimulated neutrophils (Bommakanti RK et al., J Biol Chem 267: 7576-7581, 1992). We conclude, therefore, that the affinity of the interaction of FPR with G protein is not affected by desensitization, consistent with the model of lateral segregation of FPR and G protein as a mechanism of desensitization.

Affinity Labels↗

PubMed1994

Regulatory interaction of N-formyl peptide chemoattractant receptors with the membrane skeleton in human neutrophils.

The cytoskeleton and/or membrane skeleton has been implicated in the regulation of N-formyl peptide receptors. The coupling of these chemotactic receptors to the membrane skeleton was investigated in plasma membranes from unstimulated and desensitized human neutrophils using the photoreactive agonist N-formyl-met-leu-phe-lys-N epsilon-[125I]2(p-azidosalicylamido)ethyl-1,3'- dithiopropionate (fMLFK-[125I]ASD). When membranes of unstimulated cells were solubilized in Triton-X 100, a detergent that does not disrupt actin filaments, only 50% of the photoaffinity-labeled receptors were solubilized sedimenting in sucrose density gradients at a rate consistent with previous reports. The remainder were found in the pellet fraction along with the membrane skeletal actin. Solubilization of the membranes in the presence of p-chloromercuriphenylsulfonic acid, elevated concentrations of KCl, or deoxyribonuclease I released receptors in parallel with actin. When membranes from neutrophils, desensitized by incubation with fMLFK-[125I]ASD at 15 degrees C, were solubilized, nearly all receptors were recovered in the pellet fraction. Incubation of cells with the ligand at 4 degrees C inhibited desensitization partially and prevented the conversion of a significant fraction of receptors to the form associated with the membrane skeletal pellet. In these separations the photoaffinity-labeled receptors not sedimenting to the pellet cosedimented with actin. Approximately 25% of these receptors could be immunosedimented with antiactin antibodies suggesting that N-formyl peptide receptors may interact directly with actin. These results are consistent with a regulatory role for the interaction of chemotactic N-formyl peptide receptors with actin of the membrane skeleton.

Actin Cytoskeleton↗

PubMed1994

Neutrophil chemoattractant receptors and the membrane skeleton.

Signal transduction via receptors for N-formylmethionyl peptide chemoattractants (FPR) on human neutrophils is a highly regulated process which involves participation of cytoskeletal-elements. Evidence exists suggesting that the cytoskeleton and/or the membrane skeleton controls the distribution of FPR in the plane of the plasma membrane, thus controlling the accessibility of FPR to different proteins in functionally distinct domains. In desensitized cells, FPR are restricted to domains which are depleted of G proteins but enriched in cytoskeletal proteins such as actin and fodrin. Thus, the G protein signal transduction partners of FPR become inaccessible to the agonist-occupied receptor, preventing cell activation. The mechanism of interaction of FPR with the membrane skeleton is poorly understood but evidence is accumulating that suggests a direct binding of FPR (and other receptors) to cytoskeletal proteins such as actin.

Animals↗

PubMed1994

The interaction of N-formyl peptide chemoattractant receptors with the membrane skeleton is energy-dependent.

Desensitization of N-formyl peptide chemoattractant receptors (FPR) in human neutrophils is thought to be achieved by lateral segregation of receptors and G proteins within the plane of the plasma membrane resulting in an interruption of the signalling cascade. Direct coupling of FPR to membrane skeletal actin appears to be the basis of this process; however, the molecular mechanism is unknown. In this study we investigated the effect of energy depletion on formation of FPR-membrane skeleton complexes. In addition the effect of the protein kinase C inhibitor stauroporine and the phosphatase inhibitor okadaic acid on coupling of FPR to the membrane skeleton was studied. Human neutrophils were desensitized using the photoreactive agonist N-formyl-met-leu-phe-lys-N'-[125I]2(p-azidosalicylamido)ethyl-1,3' - dithiopropionate (fMLFK-[125I]ASD) after ATP depletion with NaF or after incubation with the respective inhibitors. The interaction of FPR with the membrane skeleton was studied by sedimentation of the membrane skeleton-associated receptors in sucrose density gradients. Energy depletion of the cells markedly inhibited the formation of FPR-membrane skeleton complexes. This does not appear to be related to inhibition of protein phosphorylation due to ATP depletion because inhibition of protein kinases and phosphatases had no significant effect on coupling of FPR to the membrane skeleton. We conclude, therefore, that coupling of FPR to the membrane skeleton is an energy-dependent process which does not appear to require modification of the receptor protein by phosphorylation.

Alkaloids↗

PubMed1993

Functional molecular complexes of human N-formyl chemoattractant receptors and actin.

When human neutrophils become desensitized to formyl peptide chemoattractants, the receptors (FPR) for these peptides are converted to a high affinity, GTP-insensitive form that is associated with the Triton X-100-insoluble membrane skeleton from surface membrane domains. These domains are actin and fodrin-rich, but G protein-depleted suggesting that FPR shuttling between G protein-enriched and depleted domains may control signal transduction. To determine the molecular basis for FPR interaction with the membrane skeleton, neutrophil subcellular fractions were screened for molecules that could bind photoaffinity-radioiodinated FPR solubilized in Triton X-100. These receptors showed a propensity to bind to a 41- to 43-kDa protein band on nitrocellulose overlays of SDS-PAGE-separated cytosol and plasma membrane fractions of neutrophils. This binding, as well as FPR binding to purified neutrophil actin, was inhibited 50% by 0.6 microM free neutrophil cytosolic actin. Addition of greater than 1 microM G-actin to crude or lectin-purified Triton X-100 extracts of FPR from neutrophil membranes increased the sedimentation rate of a significant fraction of FPR two to three fold as measured by velocity sedimentation in Triton X-100-containing linear sucrose density gradients. Addition of anti-actin antibodies to FPR extracts caused a concentration-dependent immunoprecipitation of at least 65% of the FPR. More than 40% of the immunoprecipitated FPR was specifically retained on protein A affinity matrices. Membrane actin was stabilized to alkaline washing when membranes were photoaffinity labeled. Conversely, when purified neutrophil cytosolic actin was added to membranes or their digitonin extracts, after prior depletion of actin by an alkaline membrane wash, photoaffinity labeling of FPR was increased two- to fourfold with an EC50 of approximately 0.1 microM actin. We conclude that FPR from human neutrophils may interact with actin in membranes to form Triton X-100-stable physical complexes. These complexes can accept additional G-actin monomers to form higher order molecular complexes. Formation of FPR-actin complexes in the neutrophil may play a role in the regulation of chemoattractant-induced activation or actin polymerization.

Actins↗

PubMed1993

A carboxyl-terminal tail peptide of neutrophil chemotactic receptor disrupts its physical complex with G protein.

The binding of G protein to the N-formyl peptide receptor of human neutrophils was investigated with site-specific synthetic peptides. Peptide CT336(322) (322RALTEDSTQTSDTAT336) from the carboxyl-terminal tail region of the receptor competed with the receptor for binding to bovine Gi protein. The peptide competition was assayed by dissociation of a GTP-sensitive, rapidly sedimenting (7S) form of receptor-G protein complex as analyzed by velocity sedimentation on linear sucrose density gradients. An IC50 of 590 microM was determined for CT336(322) peptide. A control peptide, with the reverse sequence, rCT322(336) (336TATDSTQTSDETLAR322), did not perturb the sedimentation of the reconstituted receptor-G protein complex up to the highest tested concentration, 3 mM. Other peptides tested, corresponding to central portions of the predicted intracellular loop regions CII140(127) (127VLHPVWTQNHRTVS140) and CIII239(227) (227KIHKQGLIKSSRP239) of the receptor, failed to dissociate the reconstituted receptor-G protein complex. Control peptides from the extracellular region EII184(170) (170KTGTVACTFNFSPWT184) and an unrelated sequence matching a portion of neutrophil cytochrome b, CYT306(296) (296KVVITKVVTHPFKTIE306), were also ineffective. Our results suggest that the cytoplasmic tail of the formyl chemotactic peptide receptor is involved in its coupling to the signal-transducing G protein.

Amino Acid Sequence↗

PubMed1993

Cytoskeletal regulation of chemotactic receptors: molecular complexation of N-formyl peptide receptors with G proteins and actin.

Signal transduction via receptors for N-formylmethionyl peptide chemoattractants (FPR) on human neutrophils is a highly regulated process. It involves direct interaction of receptors with heterotrimeric G-proteins and may be under the control of cytoskeletal elements. Evidence exists suggesting that the cytoskeleton and/or the membrane skeleton determines the distribution of FPR in the plane of the plasma membrane, thus controlling FPR accessibility to different proteins in functionally distinct membrane domains. In desensitized cells, FPR are restricted to domains which are depleted of G proteins but enriched in cytoskeletal proteins such as actin and fodrin. Thus, the G protein signal transduction partners of FPR become inaccessible to the agonist-occupied receptor, preventing cell activation. We are investigating the molecular basis for the interaction of FPR with the membrane skeleton, and our results suggest that FPR, and possibly other receptors, may directly bind to cytoskeletal proteins such as actin.

Actins↗

PubMed1992

2-Alkynyl derivatives of adenosine and adenosine-5'-N-ethyluronamide as selective agonists at A2 adenosine receptors.

In the search for more selective A2-receptor agonists and on the basis that appropriate substitution at C2 is known to impart selectivity for A2 receptors, 2-alkynyladenosines 2a-d were resynthesized and evaluated in radioligand binding, adenylate cyclase, and platelet aggregation studies. Binding of [3H]NECA to A2 receptors of rat striatal membranes was inhibited by compounds 2a-d with Ki values ranging from 2.8 to 16.4 nM. 2-Alkynyladenosines also exhibited high-affinity binding at solubilized A2 receptors from human platelet membranes. Competition of 2-alkynyladenosines 2a-d for the antagonist radioligand [3H]DPCPX and for the agonist [3H]CCPA gave Ki values in the nanomolar range, and the compounds showed moderate A2 selectivity. In order to improve this selectivity, the corresponding 2-alkynyl derivatives of adenosine-5'-N-ethyluronamide 8a-d were synthesized and tested. As expected, the 5'-N-ethyluronamide derivatives retained the A2 affinity whereas the A1 affinity was attenuated, resulting in an up to 10-fold increase in A2 selectivity. A similar pattern was observed in adenylate cyclase assays and in platelet aggregation studies. A 30- to 45-fold selectivity for platelet A2 receptors compared to A1 receptors was found for compounds 8a-c in adenylate cyclase studies.

Adenosine↗

PubMed1992

Reconstitution of a physical complex between the N-formyl chemotactic peptide receptor and G protein. Inhibition by pertussis toxin-catalyzed ADP ribosylation.

Photoaffinity-labeled N-formyl chemotactic peptide receptors from human neutrophils solubilized in octyl glucoside exhibit two forms upon sucrose density gradient sedimentation, with apparent sedimentation coefficients of approximately 4 and 7 S. The 7 S form can be converted to the 4 S form by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with an EC50 of approximately 20 nM, suggesting that the 7 S form may represent a physical complex of the receptor with endogenous G protein (Jesaitis, A. J., Tolley, J. O., Bokoch, G. M., and Allen, R. A. (1989) J. Cell Biol. 109, 2783-2790). To probe the nature of the 7 S form, we reconstituted the 7 S form from the 4 S form by adding purified G protein. The 4 S form, obtained by solubilizing GTP gamma S-treated neutrophil plasma membranes, was incubated with purified (greater than 95%) Gi protein from bovine brain (containing both Gi alpha 1 and Gi alpha 2) or with neutrophil G protein (Gn), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC50 of 7 S complex formation induced by the two G proteins was 70 +/- 25 and 170 +/- 40 nM for Gn and Gi, respectively. No complexation was measurable when bovine transducin (Gt) was used up to 30 times the EC50 for Gn. The EC50 for Gi was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 microM GTP gamma S to the reconstituted 7 S complex caused a complete revision of the receptor to the 4 S form, and anti-Gi peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gi prevented formation of the 7 S form even at 20 times the concentration of unribosylated Gi normally used to attain 50% conversion to the 7 S form. These observations suggest that the 7 S species is a physical complex containing N-formyl chemotactic peptide receptor and G protein.

Adenosine Diphosphate Ribose↗

PubMed1992

Reduction of postischemic leukocyte-endothelium interaction by adenosine via A2 receptor.

The adhesion of leukocytes to the endothelium of postcapillary venules hallmarks a key event in ischemia-reperfusion injury. Adenosine has been shown to protect from postischemic reperfusion injury, presumably through inhibition of postischemic leukocyte-endothelial interaction. This study was performed to investigate in vivo by which receptors the effect of adenosine on postischemic leukocyte-endothelium interaction is mediated. The hamster dorsal skinfold model and fluorescence microscopy were used for intravital investigation of red cell velocity, vessel diameter, and leukocyte-endothelium interaction in postcapillary venules of a thin striated skin muscle. Leukocytes were stained in vivo with acridine orange (0.5 mg kg-1 min-1 i.v.). Parameters were assessed prior to induction of 4 h ischemia to the muscle tissue and 0.5 h, 2 h, and 24 h after reperfusion. Adenosine, the adenosine A1-selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA), the A2-selective agonist CGS 21,680, the non-selective adenosine receptor antagonist xanthine amine congener (XAC), and the adenosine uptake blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) were infused via jugular vein starting 15 min prior to release of ischemia until 0.5 h after reperfusion. Adenosine and CGS 21,680 significantly reduced postischemic leukocyte-endothelium interaction 0.5 h after reperfusion (p less than 0.01), while no inhibitory effect was observed with CCPA. Coadministration of XAC blocked the inhibitory effects of adenosine. Infusion of NBTI alone effectively decreased postischemic leukocyte-endothelium interaction. These findings indicate that adenosine reduces post-ischemic leukocyte-endothelium interaction via A2 receptor and suggest a protective role of endogenous adenosine during ischemia-reperfusion.

Adenosine↗

PubMed1992

G-protein-coupled A1 adenosine receptors in coated vesicles of mammalian brain: characterization by radioligand binding and photoaffinity labelling.

A1 adenosine receptors in coated vesicles have been characterized by radioligand binding and photoaffinity labelling. Saturation experiments with the antagonist 8-cyclopentyl-1,3-[3H]dipropyl-xanthine ([3H]DPCPX) gave a Kd value of 0.7 nM and a Bmax value of 82 +/- 13 fmol/mg protein. For the highly A1-selective agonist 2-chloro-N6-[3H]cyclopentyladenosine ([3H]CCPA) a Kd value of 1.7 nM and a Bmax value of 72 +/- 29 fmol/mg protein was estimated. Competition of agonists for [3H]DPCPX binding gave a pharmacological profile with R-N6-phenylisopropyladenosine (R-PIA) > CCPA > S-PIA > 5'-N-ethylcarboxamidoadenosine (NECA), which is identical to brain membranes. The competition curves were best fitted according to a two-site model, suggesting the existence of two affinity states. GTP shifted the competition curve for CCPA to the right and only one affinity state similar to the low affinity state in the absence of GTP was detected. The photoreactive agonist 2-azido-N6-125I-p-hydroxyphenylisopropyladenosine ([125I]AHPIA) specifically labelled a single protein with an apparent molecular weight of 35,000 in coated vesicles, which is identical to A1 receptors labelled in brain membranes. Therefore, coated vesicles contain A1 adenosine receptors with similar binding characteristics as membrane-bound receptors, including GTP-sensitive high-affinity agonist binding. Photoaffinity labelling data suggest that A1 receptors in these vesicles are not a processed receptor form. These results confirm that A1 receptors in coated vesicles are coupled to a G-protein, and it appears that the A1 receptor systems in coated vesicles and in plasma membranes are identical.

Affinity Labels↗

PubMed1992

Characterization of adenosine A1 receptor in a cell line (28A) derived from rabbit collecting tubule.

We have previously reported that in several renal cell types, adenosine receptor agonists inhibit adenylyl cyclase and activate phospholipase C via a pertussis toxin-sensitive G protein. In the present study, in 28A cells, both of these adenosine receptor-mediated responses were inhibited by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a highly selective A1 adenosine receptor antagonist. The binding characteristics of the adenosine A1 receptor in the 28A renal cell line were studied using the radiolabeled antagonist [3H]DPCPX to determine whether two separate binding sites could account for these responses. Saturation binding of [3H]DPCPX to 28A cell membranes revealed a single class of A1 binding sites with an apparent Kd value of 1.4 nM and maximal binding capacity of 64 fmol/mg protein. Competition experiments with a variety of adenosine agonists gave biphasic displacement curves with a pharmacological profile characteristic of A1 receptors. Comparison of [3H]DPCPX competition binding data from 28A cell membranes with rabbit brain membranes, a tissue with well-characterized A1 receptors, reveals that the A1 receptor population in 28A cells has similar agonist binding affinities to the receptor population in brain but has a considerably lower density. Addition of guanosine 5'-triphosphate (100 microM) to 28A cell membranes caused the competition curves to shift from biphasic to monophasic, indicating that the A1 receptors exist in two interconvertible affinity states because of their coupling to G proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

PubMed1992

Effects of adenosine on histamine release from human lung fragments.

The actions of adenosine on histamine release of human lung fragments were investigated. Histamine release was stimulated either with the calcium ionophore A23187 or with concanavalin A. Adenosine and its analogue 5'-N-ethylcarboxamidoadenosine alone had no significant effect on basal release or on the release elicited by A 23187 or concanavalin A. However, in the presence of the adenosine receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyloxy]-phenyl]-1, 3-dipropylxanthine (XAC), which itself did not affect the release, adenosine increased the stimulated histamine release. On the other hand, in the presence of the nucleoside transport inhibitor S-(p-nitrobenzyl)-6-thioinosine (NBTI), adenosine caused a reduction in stimulated histamine release. NBTI itself caused a stimulation of release. Thus, a stimulatory effect of adenosine was seen in the presence of XAC, whereas an inhibitory effect was unmasked by NBTI. From these data it is concluded that adenosine exerts two opposing effects on histamine release in the human lung which neutralize each other: it inhibits release via a site antagonized by XAC, which presumably represents an A2 adenosine receptor, and it stimulates release via a mechanism that is blocked by NBTI, suggesting that adenosine needs to reach the interior of cells to exert this effect. The slight stimulatory effect of NBTI alone demonstrates that trapping intracellularly formed adenosine inside mast cells leads to sufficient concentrations of adenosine to stimulate histamine release. These findings suggest an important bimodal role of adenosine in regulating histamine release in the human lung.

Adenosine↗

PubMed1991

Carbamazepine distinguishes between adenosine receptors that mediate different second messenger responses.

The mechanism of the therapeutic and prophylactic effects of carbamazepine (CBZ) in affective psychoses is unknown but may in part be related to the potent competitive interaction of CBZ with adenosine-binding sites in the brain. The anticonvulsant and sedative properties of CBZ are reminiscent of the effects evoked by adenosine-agonists and contrast sharply with the opposite actions of adenosine-antagonists like caffeine. However, indirect evidence suggests an antagonist- rather than an agonist-like activity of CBZ at adenosine-receptors. We have used various model systems, in which adenosine receptor subtypes mediate different second messenger-responses, to investigate this-apparent paradox. CBZ was found to antagonize the A1-receptor-mediated inhibition of cyclic AMP accumulation in cultured astroblasts and in GH3-cells. Furthermore, CBZ also inhibits the adenosine-induced increase in the level of cyclic AMP in cultured astroblasts, which is mediated by low-affinity A2b-receptors. In contrast, CBZ does not block the inhibition elicited by adenosine-agonists of the agonist-induced increased formation of inositolphosphates in human neutrophils, which is mediated by high-affinity A2a-receptors. The specific antagonism by CBZ of A1- but not of high-affinity A2a-receptors was further supported by binding experiments using rat brain membranes. These results suggest that the paradox of CBZ's antagonistic effects at adenosine-receptors might be at least partially reconciled by a selective antagonistic action of CBZ at A1 receptors but not at high-affinity A2a-receptors.

Adenosine↗

PubMed1991

Comparison of A1 adenosine receptors in brain from different species by radioligand binding and photoaffinity labelling.

Radioligand binding to A1 adenosine receptors at brain membranes from seven species was investigated. The antagonist 8-cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX) bound with affinities between 0.17 nM in sheep brain and 2.1 nM in guinea pig brain. Competition of several antagonists for [3H]DPCPX binding showed that the most potent compounds were DPCPX with Ki values of 0.05 nM in bovine brain and 1.1 nM in guinea pig brain and xanthine amine congener (XAC) with Ki values of 0.03 nM in bovine brain and 5.5 nM in guinea pig brain. The differences in affinity of the agonist radioligand 2-chloro-N6-[3H]cyclopentyl-adenosine ([3H]CCPA) were less pronounced, ranging from a KD value of 0.12 nM (hamster brain) to 0.42 nM (guinea pig brain). Agonist competition for [3H]DPCPX binding of photoaffinity labelling, however, exhibited marked species differences. N-Ethylcarboxamidoadenosine (NECA) and S-N6-phenylisopropyladenosine (S-PIA) showed 20 to 25-fold different KD values in different species. NECA had a particularly high affinity in guinea pig brain and was only two-fold less potent than R-PIA. Thus, the difference from the "classical" A1 receptor profile (R-PIA greater than -NECA greater than S-PIA) is not sufficient to speculate that A1 receptor subtypes may exist that are coupled to different effector systems. Our data show that these difference can easily be explained by species differences.

Affinity Labels↗

PubMed1991

Mechanism of A2 adenosine receptor activation. I. Blockade of A2 adenosine receptors by photoaffinity labeling.

It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxy-phenylisopropyladenosine [(R)-AHPIA] into the A1 adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of cellular cAMP levels [Mol. Pharmacol. 30:403-409 (1986)]. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenylate cyclase stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies indicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase stimulation of up to 50% of the control value. Similarly, the activation via these 10-20% of receptors occurs with a half-life that is only 2 times longer than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenylate cyclase stimulation. These observations require a modification of the models of receptor-adenylate cyclase coupling, which is described in the accompanying paper [Mol. Pharmacol. 39:524-530 (1991)].

Adenosine↗