Identification of an alpha 2-adrenoceptor in human coronary arteries by radioligand binding assay.

A single high affinity binding site for an alpha 2-adrenoceptor in human coronary arteries was identified by radioligand binding assay. Human coronary arteries were obtained at autopsy within 6 hours of death. A crude membrane solution was incubated with (3H)-rauwolscine at 25 degrees C for 30 min. The binding of (3H)-rauwolscine was rapidly saturable and reversible. Kd was 1.2 +/- 0.2 (SE) nM and Bmax 22 +/- 3 fmol/mg protein. This is the first study which has shown the presence of an alpha 2-adrenoceptor in human coronary arteries using a radioligand binding assay method.     

VIP receptors in pig liver cells: binding characteristics and role in degradation

The physiological role of VIP in the liver is controversial. VIP receptors are present, but their function in the metabolic regulation is uncertain. The interaction of porcine VIP with isolated cells from pig liver was studied with respect to receptor-binding, degradation and glycogenolytic action. In this model, VIP and liver showed homology of animal species. 1. Receptor-binding was heterogenous with Kd values of 10(-9) mol/l and 4 X 10(-8) mol/l, and a total amount of binding sites of 7 X 10(-11) mol per 10(9) cells. The peptide specificity showed that porcine and chicken VIP were equally potent in inhibiting receptor-bound 125I-VIP; secretin was about 30 times less potent; glucagon and somatostatin were ineffective. 2. Receptor-bound 125I-VIP was degraded since about 70% was released as radioactivity not reacting with VIP-antiserum. 3. Glucose-release was not stimulated by VIP (10(-6) mol/l) whereas the rate was increased two-fold by glucagon (10(-6) mol/l). In conclusion, VIP receptors in pig liver cells are different from other tissues regarding peptide specificity. It is suggested that receptor-binding mediates degradation of VIP by pig liver rather than metabolic effects.     

Fully automated radioligand binding filtration assay for membrane-bound receptors

Here we describe a fully automated, hands-free radioligand filtration binding assay for dopamine D3 receptors. Three separate instruments were linked in tandem to perform the following operations: The Genmate and Genesis were linked to perform liquid handling, incubation, and the scheduling operations, while an automated harvester was used to perform rapid filtration. To minimize carryover of compounds, disposable tips were used for diluting and dispensing the compounds. A custom-designed tip holder was used to handle loading and pipetting by the Genmate 96-well pipettor. The assay for 84 compounds with six concentrations that spans six logs can be completed within 4 h. The reproducibility of the individual data point (cv < 10% between duplicates) and Ki (cv < 20%) is superior to that determined by manual procedures. Ki values of various dopamine ligands determined by the hands-free procedure are similar to published values. This technology reduces hands-on time (at least 70%), minimizes exposure to radioligands (up to 95%), and improves the reproducibility of results. The technology is applicablefor high-throughput screening and rapid determination of structure-activity relationship of compounds for many other membrane-bound receptors.     

N-Methyl-D-aspartate receptor subtype-selectivity of homoquinolinate: an electrophysiological and radioligand binding study using both native and recombinant receptors

Homoquinolinate, a derivative of the endogenous NMDA agonist, quinolinate, has been shown to display higher affinity for Xenopus oocytes expressing NR2A- and NR2B-containing receptors, compared to NR2C- and NR2D-containing receptors, whilst autoradiographical experiments subsequently showed that [3H]homoquinolinate labelled a subpopulation of NMDA receptors in rat brain sections, with a similar distribution to NR2B-containing receptors. In this study, we have shown that NMDA-specific [3H]homoquinolinate binding to rat brain membranes comprised 44% of total binding with a Bmax value of 5.73 pmol/mg protein, which was inhibited by NMDA with Ki=0.867 micro m. However, NMDA-specific [3H]homoquinolinate binding was not observed for a number of human recombinant NMDA receptors investigated, suggesting that there are subtle differences between the binding sites of recombinant and native receptors. Electrophysiological experiments revealed that homoquinolinate activated human recombinant NR1a/NR2A, NR1a/NR2B and NR1a/NR2A/NR2B receptors with EC50 values of 25.2, 13.8 and 9.04 micro m, respectively, with intrinsic activities of 148, 93.3 and 125%, respectively, compared to glutamate (=100%). In contrast to an autoradiographical study, these radioligand binding and electrophysiological experiments suggest that homoquinolinate is not highly selective for NR2B-containing receptors.     

Human cardiac beta-adrenergic receptors: subtype heterogeneity delineated by direct radioligand binding

Human myocardial beta-adrenergic receptors were directly identified and characterized using the high affinity antagonist radioligand [125I]iodocyanopindolol. Beta 1 and beta 2 adrenergic receptors were found to coexist in both the left ventricle and right atrium. The relative proportions of the two receptor subtypes were determined by the use of competition radioligand binding and computer modelling techniques employing the subtype selective agents atenolol (beta 1 selective) and zinterol (beta 2 selective). The left ventricle contains 86 +/- 1% beta 1 and 14 +/- 1% beta 2 adrenergic receptors while the right atrium contains 74 +/- 6% beta 1 and 26 +/- 6% beta 2 adrenergic receptors. The direct demonstration of beta 2 adrenergic receptors in the human heart, with a higher proportion in the right atrium agrees with pharmacologic data and supports the notion that chronotropic effects of adrenergic agonists in man may be mediated by both beta 1 and beta 2 adrenergic receptors.     

High throughput fluorescence polarization: a homogeneous alternative to radioligand binding for cell surface receptors

High throughput fluorescence polarization (FP) assays are described that offer a nonradioactive, homogeneous, and low-cost alternative to radioligand binding assays for cell surface receptors (G protein-coupled receptors and ligand-gated ion channels). FP assays were shown to work across a range of both peptide (vasopressin V1a and delta-opioid) and nonpeptide (beta1-adrenoceptor, 5-hydroxytryptamine3) receptors. Structure-activity relationships were investigated at beta1-receptors and were found to be consistent with radioligand binding assays. FP was shown to tolerate up to 5% DMSO with no loss in sensitivity or signal window. From a random set of 1,280 compounds, 1.9% were found to significantly interfere with FP measurement. If fluorescent or quenching compounds were eliminated (3% of all compounds), less than 0.4% of compounds were found to interfere with FP measurement. Assays could be run in 384-well plates with little loss of signal window or sensitivity compared to 96-well plate assays. New advances in FP measurement have therefore enabled FP to offer a high throughput alternative to radioligand binding for cell surface receptors.     

VIP and PACAP display different vasodilatory effects in rabbit coronary and cerebral arteries

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) are closely related peptides with wide distribution in the nervous system. The aim of the present study was to investigate functional characteristics and the influence of sex steroids on the vasodilatory effects of these two peptides in cerebral and coronary vessels from female New Zealand White (NZW) rabbits.

The localization and concentration of VIP and PACAP in cardiovascular tissue was evaluated using immunohistochemistry and radioimmunoassays. The vasodilatory effects of VIP and PACAP were investigated using myographs, allowing isometric tension recordings. In order to evaluate the influence of steroid hormones, the rabbits were ovariectomized and randomized to treatment for 4 weeks with 17β-estradiol (E₂), Norethindrone Acetate (NETA), E₂+NETA or placebo. Ring segments of the posterior cerebral artery, the right proximal coronary artery and the distal left coronary artery were examined.

The highest concentrations of VIP/PACAP were observed in cerebral and coronary arteries: 5.0/5.7 and 2.8/3.5 pmol/g, respectively. The peptides were localized in nerve fibres innervating the arteries. Both peptides produced dose-dependent vasodilatory responses in all vessels investigated. While the effects of PACAP were identical in cerebral and coronary arterial segments, the effects of VIP displayed significant differences (E_max, pI₂, Hill-slope). Treatment with sex steroids induced no changes in the vascular effects of the two peptides.

These results indicate different mechanisms of action for the vasodilating effects of the two closely related peptides VIP and PACAP in different areas of the coronary and cerebrovascular tree. Treatment with female sex steroids does not seem to change these mechanisms.     

Deglycosylated mammalian beta 2-adrenergic receptors: effect on radioligand binding and peptide mapping

Mammalian beta-adrenergic receptors are glycoproteins containing both high-mannose and complex-type carbohydrate chains [G. L. Stiles, J. L. Benovic, M. G. Caron, and R. J. Lefkowitz (1984) J. Biol. Chem. 259, 8655-8663]. Endoglycosidase F treatment of beta 2-adrenergic receptors results in the removal of at least two N-linked oligosaccharides, resulting in an increased mobility of the receptor peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr 62,000 to 49,000). In the present study the properties of the deglycosylated beta 2-adrenergic receptor were assessed. Following deglycosylation, the beta 2-adrenergic receptor recognized both agonists and antagonists with the same potency order and affinities as the glycosylated form. In addition, two-dimensional gel electrophoresis peptide mapping techniques applied to control and deglycosylated beta 2-adrenergic receptors (both within and between species) demonstrated that there was a marked homology of the beta 2-adrenergic receptor between species which are closely related phylogenetically. In addition, the glycan component of the receptor did not appear to interfere with the ability of proteinases to generate accurate peptide maps.     

Adaptive changes of mesenteric arteries in pregnancy: a meta-analysis

The vascular response to pregnancy has been frequently studied in mesenteric artery models by investigating endothelial cell (EC)- and smooth muscle cell (SMC)-dependent responses to mechanical (flow-mediated vasodilation, myogenic reactivity, and vascular compliance) and pharmacological stimuli (G protein-coupled receptor responses: Gq(EC), Gs(SMC), Gq(SMC)). It is unclear to what extent these pathways contribute to normal pregnancy-induced vasodilation across species, strains, and/or gestational age and at which receptor level pregnancy affects the pathways. We performed a meta-analysis on responses to mechanical and pharmacological stimuli associated with pregnancy-induced vasodilation of mesenteric arteries and included 55 (188 responses) out of 398 studies. Most included studies (84%) were performed in Wistar and Sprague-Dawley rats (SDRs) and compared late gestation versus nonpregnant controls (80%). Pregnancy promotes flow-mediated vasodilation in all investigated species. Only in SDRs, pregnancy additionally stimulates both vasodilator Gq(EC) sensitivity (EC(50) reduced by -0.76 [-0.92, -0.60] log[M]) and Gs(SMC) sensitivity (EC(50) reduced by -0.51 [-0.82, -0.20] log[M]), depresses vasopressor Gq(SMC) sensitivity (EC(50) increase in SDRs by 0.23 [0.16, 0.31] log[M]), and enhances arterial compliance. We conclude that 1) pregnancy facilitates flow-mediated vasodilation at term among all investigated species, and the contribution of additional vascular responses is species and strain specific, and 2) late pregnancy mediates vasodilation through changes at the receptor level for the substances tested. The initial steps of vasodilation in early pregnancy remain to be elucidated.     

Evidence for the presence of GPRC6A receptors in rat mesenteric arteries

In this study, the presence of GPRC6A receptors in rat mesenteric artery was investigated. In artery homogenates, GPRC6A mRNA was detected and Western blotting showed the presence of GPRC6A protein. Immunohistochemical studies revealed GPRC6A in both endothelial cells and myocytes. In whole vessel segments, the GPRC6A activators, 300 microM l-ornithine and 100 microM Al(3+), induced endothelium-dependent myocyte hyperpolarizations sensitive to 10 microM TRAM-34, a blocker of intermediate conductance, Ca(2+)-sensitive K(+) channels (IK(Ca)). Activation of IK(Ca) with calindol (300 nM; a positive allosteric Ca(2+)-sensing receptor - CaR - modulator) was inhibited by 500 nM ouabain (inhibition of rat type 2 and type 3 Na(+)/K(+)-ATPases) but unaffected by 30 microM Ba(2+) (blockade of inwardly rectifying K(+) channels). Neither l-ornithine nor Al(3+) activated CaRs heterologously expressed in CHO or HEK293 cells. In the presence of 300 microM l-ornithine or 100 microM Al(3+), myocyte hyperpolarizations to calindol were potentiated whereas this potentiation and hyperpolarizations to l-ornithine were lost following incubation with an anti-GPRC6A antibody. It is concluded that GPRC6A receptors are present on mesenteric artery endothelial cells and myocytes and that their activation selectively opens IK(Ca) channels. This triggers a ouabain-sensitive myocyte hyperpolarization suggesting a close functional relationship between GPRC6A, the IK(Ca) channel and type 2 and/or type 3 Na(+)/K(+)-ATPases.     

VIP and PACAP receptors coupled to adenylyl cyclase in human lung cancer: a study in biopsy specimens

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are important neuropeptides in the control of lung physiology. Both of these commonly bind to specific G protein coupled receptors named VPAC(1)-R and VPAC(2)-R, and PAC(1)-R (with higher affinity for PACAP). VIP and PACAP have been implicated in the control of cell proliferation and tumor growth. This study examined the presence of VIP and PACAP receptors in human lung cancer samples, as well as the functionality of adenylyl cyclase (AC) stimulated by both peptides. Results from RT-PCR and immunoblot experiments showed the expression of VPAC(1)-, VPAC(2)- and PAC(1)-R in lung cancer samples. Immunohistochemical studies showed the expression of VPAC(1) and VPAC(2) receptors. These receptors were positively coupled to AC, but the enzyme activity was impaired as compared to normal lung. There were no changes in Galpha(s) or Galpha(i) levels. Present results contribute to a better knowledge of VIP/PACAP actions in lung cancer and support the interest for the development of VIP/PACAP analogues with therapeutic roles.     

Comparison of rat and human left ventricle beta-adrenergic receptors: subtype heterogeneity delineated by direct radioligand binding

Beta adrenergic receptors were identified in rat myocardial left ventricle and human papillary muscle by using the antagonist radioligand 3H-dihydroalprenolol. The number (37.3 and 44.5 fmol/mg of protein, respectively in rat and man), and the KD (1.6 and 2.8 nM, respectively in rat and man) of beta receptors were not significantly different. Adrenergic receptors of both beta 1 and beta 2 subtypes were found to coexist in the left ventricle. The relative proportions of the two beta receptor subtypes were determined by the use of competition radioligand selective binding and computer modelling techniques employing the subtype selective antagonists ICI 118,551 (beta 2 selective) and atenolol (beta 1 selective) in rat or metoprolol (beta 1 selective) in man. The rat left ventricle contained about 74% beta 1 and 26% beta 2 adrenergic receptors, human left ventricle papillary muscles contained about 69% beta 1 and 31% beta 2. Human and rat left ventricles contain both beta 1 and beta 2 adrenergic receptors with similar affinities. Rat might be a model for the study of human myocardial beta adrenergic receptors.     

Leptin receptors are expressed in coronary arteries, and hyperleptinemia causes significant coronary endothelial dysfunction

Obesity is associated with marked increases in plasma leptin concentration, and hyperleptinemia is an independent risk factor for coronary artery disease. As a result, the purpose of this investigation was to test the following hypotheses: 1) leptin receptors are expressed in coronary endothelial cells; and 2) hyperleptinemia induces coronary endothelial dysfunction. RT-PCR analysis revealed that the leptin receptor gene is expressed in canine coronary arteries and human coronary endothelium. Furthermore, immunocytochemistry demonstrated that the long-form leptin receptor protein (ObRb) is present in human coronary endothelium. The functional effects of leptin were determined using pressurized coronary arterioles (<130 microm) isolated from Wistar rats, Zucker rats, and mongrel dogs. Leptin induced pharmacological vasodilation that was abolished by denudation and the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester and was absent in obese Zucker rats. Intracoronary leptin dose-response experiments were conducted in anesthetized dogs. Normal and obese concentrations of leptin (0.1-3.0 microg/min ic) did not significantly change coronary blood flow or myocardial oxygen consumption; however, obese concentrations of leptin significantly attenuated the dilation to graded intracoronary doses of acetylcholine (0.3-30.0 microg/min). Additional experiments were performed in canine coronary rings, and relaxation to acetylcholine (6.25 nmol/l-6.25 micromol/l) was significantly attenuated by obese concentrations of leptin (625 pmol/l) but not by physiological concentrations of leptin (250 pmol/l). The major findings of this investigation were as follows: 1) the ObRb is present in coronary arteries and coupled to pharmacological, nitric oxide-dependent vasodilation; and 2) hyperleptinemia produces significant coronary endothelial dysfunction.     

Unconventional binding sites and receptors for VIP and related peptides PACAP and PHI/PHM: an update

The 28-amino-acid neuropeptide VIP and related peptides PACAP and PHI/PHM modulate virtually all of the vital functions in the body. These peptides are also commonly recognized as major regulators of cell growth and differentiation. Through their trophic and cytoprotective functions, they appear to play major roles in embryonic development, neurogenesis and the progression of a number of cancer types. These peptides bind to three well-characterized subtypes of G-protein coupled receptors: VPAC1 and VPAC2 share a common high affinity in the nanomolar range for VIP and PACAP; a third receptor type, PAC1, has been characterized for its high affinity for PACAP but its low affinity for VIP. Complex effects and pharmacological behaviors of these peptides suggest that multiple subtypes of binding sites may cooperate to mediate their function in target cells and tissues. In this complex response, some of these binding sites correspond to the definition of the conventional receptors cited above, while others display unexpected pharmacological and functional properties. Here we present potential clues that may lead investigators to further characterize the molecular nature and functions of these atypical binding species.     

Involvement of protein kinases on the upregulation of endothelin receptors in rat basilar and mesenteric arteries

Endothelin(B) (ET(B)) receptors are upregulated in experimental stroke or after 24 hrs of organ culture. This upregulation is manifested both as stronger contraction and as an increase in ET(B) receptor messenger RNA (mRNA) levels. The present study was designed to evaluate the importance of protein kinases (c-Jun N-terminal kinase [JNK], protein kinase C [PKC], and extracellular signal-regulated kinase [ERK1/2]) in ET(B) receptor upregulation after organ culture. Rat basilar and mesenteric arteries were incubated for 24 hrs in Dulbecco's modified Eagle's medium (DMEM) with or without the PKC inhibitor, RO-31-7549; the ERK1/2 inhibitor, SB386023; or the JNK inhibitor, SP600125, added 3, 6, or 12 hrs after initiation of incubation. Subsequently, vessel segments were mounted in myographs and the contractile responses to ET-1 and sarafotoxin 6c were studied. The ET(B) and ET(A) receptor mRNA levels were determined with a real-time polymerase chain reaction (PCR). The cellular localization and protein level of ET(B) receptors were evaluated by immunohistochemistry. The PKC and ERK1/2 inhibitors attenuated the contraction induced by S6c in the basilar arteries more than in the mesenteric arteries. The efficiency of the inhibitors was proportional to the incubation time. Real-time PCR showed a decrease in the ET(B) receptor mRNA levels in arteries treated with PKC or ERK inhibitors. The JNK inhibitor had a significant inhibitory effect on ET(B) receptor upregulation in the basilar arteries. Immunohistochemistry revealed that the ET(B) receptor upregulation occured in the smooth-muscle cells and that it had the same pattern as in the quantitative PCR. Our results show that the PKC, ERK1/2, and JNK are more important for the upregulation of contractile ET(B) receptors in cerebral arteries compared with mesenteric arteries. ERK1/2 seems to be more important for the ET(B) receptor upregulation, as compared with PKC and JNK. The evaluation of the time dependency suggests that the phenomenon can be reversed even after its initiation.     

Action of agonists and antagonists on adrenergic receptors in isolated porcine coronary arteries

The adrenergic receptors of porcine coronary arteries were investigated in helically cut strips of small (less than or equal to 0.5 mm outer-diameter (od), medium (0.8-1.2 mm od), large (1.5-2.5 mm od), and very large (greater than 4 mm od) coronary arteries. Both the beta1 agonist dobutamine and the beta2 agonist terbutaline relaxed coronary arteries partially contracted by 25 mM of KCl. Dobutamine contracted small coronary arteries at 10(-5) M concentrations, then relaxed them at 10(-4) M. The beta1-adrenoceptor antagonist metoprolol contracted coronary arteries relaxed by either dobutamine or terbutaline, but the beta2 antagonist H35/25 did so only in high and probably nonselective concentrations. Alpha1-adrenoreceptor stimulating concentrations of phenylephrine did not contract any of the arteries. Metoprolol and high concentrations of H35/25 further contracted large coronary arteries partially contracted by 25 mM potassium. These contractions were blocked by verapamil and papaverine but not by atropine, phentolamine, yohimbine, mepyramine, or methysergide. This seems to indicate that beta-adrenergic receptors in porcine coronary arteries are beta1-receptors, or closely resemble beta1-receptors. They differ from many other beta1-receptors, however, in that they are stimulated by terbutaline. Alpha1 adrenoreceptors seem not to be present in these porcine coronary arteries to a significant extent. Metoprolol and high concentrations of H35/25 have a direct contractile effect in large porcine coronary artery that is not mediated by alpha-adrenergic, muscarinic, histaminergic, or serotonergic receptors but requires verapamil-sensitive calcium.     

Characterization of human and rodent native and recombinant adenosine A2B receptors by radioligand binding studies

Adenosine A_2B receptors of native human and rodent cell lines were investigated using [³H]PSB-298 [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine] in radioligand binding studies. [³H]PSB-298 showed saturable and reversible binding. It exhibited a K_D value of 60 ± 1 nM and limited capacity (B_max = 3.511 fmol per milligram protein) at recombinant human adenosine A_2B receptors expressed in human embryonic kidney cells (HEK-293). The addition of sodium chloride (100 mM) led to a threefold increase in the number of binding sites recognized by the radioligand. The curve of the agonist 5′-N-ethylcarboxamidoadenosine (NECA) was shifted to the right in the presence of NaCl, while the curve of the antagonist PSB-298 was shifted to the left, indicating that PSB-298 may be an inverse agonist at A_2B receptors. Adenosine A_2B receptors were shown to be the major adenosine A₂ receptor subtype on the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 cells. Binding studies at rat INS-1 cells (insulin secreting cell line) demonstrated that [³H]PSB-298 is a selective radioligand for adenosine A_2B binding sites in this cell line.     

alpha 1-Adrenergic receptors in guinea pig myocardium: identification by binding of a new radioligand, (3H)-prazosin.

Binding of (³H)-prazosin to adrenoceptors in guinea pig myocardial membranes was rapid, readily reversible, stereospecific and saturable. By Scatchard analysis (n = 6) Bmax was 58 fmol of (³H)-prazosin bound/mg protein and the K_D was 0.58 nm. The Hill number was 1.05. Adrenergic agonists competed with (³H)-prazosin as follows: (?)adrenaline > (?)noradrenaline > (?)phenylephrine ? ($\text{+}$)isoprenaline > (+)noradrenaline; antagonists competed in the order: non-radioactive prazosin > phentolamine ? piperoxan > yohimbine > sulpiride > propranolol. The K_D for beta-adrenoceptors assessed by (?³H)-dihydroalprenolol was 0.86 nM and the Bmax (96 fmol/mg protein) was almost twice that of alpha-adrenoceptors. (³H)-prazosin appears to be a useful radioligand for the study of post-synaptic (alpha₁) adrenoceptors in myocardial tissue.     

The binding of 3H-acetylcholine to cholinergic receptors in bovine cerebral arteries

Cholinergic receptor sites in bovine cerebral arteries were analyzed using radioligand binding techniques with the cholinergic agonist, 3H-acetylcholine (ACh), as the ligand. Specific binding of 3H-ACh to membrane preparations of bovine cerebral arteries was saturable, of two binding sites, with dissociation constant (KD) values of 0.32 and 23.7 nM, and maximum binding capacity (Bmax) values of 67 and 252 fmol/mg protein, respectively. Specific binding of 3H-ACh was displaced effectively by muscarinic cholinergic agents and less effectively by nicotinic cholinergic agents. IC50 values of cholinergic drugs for 3H-ACh binding were as follows: atropine, 38.5 nM; ACh, 59.8 nM; oxotremorine, 293 nM; scopolamine 474 nM; carbamylcholine, 990 nM. IC50 values of nicotinic cholinergic agents such as nicotine, cytisine and alpha-bungarotoxin exceeded 50 microM. Choline acetyltransferase activity was 1.09 nmol/mg protein/hour in the cerebral arteries. These findings suggest that the cholinergic nerves innervate the bovine cerebral arteries and that there are at least two classes of ACh binding sites of different affinities on muscarinic receptors in these arteries.