Michael F. Jarvis

The angiotensin hexapeptide 3–8 fragment potently inhibits [125I]angiotensin II binding to non-AT1 or -AT2 recognition sites in bovine adrenal cortex

In the present studies, ligand competition experiments were conducted to examine the ability of angiotensin II peptide agonists and nonpeptide AT1- and AT2-selective receptor antagonists to inhibit the binding of [125I]angiotensin II to bovine adrenal cortical membranes. Angiotensin II, angiotensin III, the All-(3-8) hexapeptide fragment of angiotensin II, and the AT1-selective receptor antagonist L-158,809, inhibited [125I]angiotensin II binding in a biphasic fashion indicative of a ligand interaction at more than one recognition site. Approximately 20% of low affinity [125I]angiotensin II binding was inhibited only by high micromolar concentrations of L-158,809. RG 13647 (1(-1,4-benzodioxan-2-methyl)-5-diphenylacetyl-4,5,6,7-tetra hydro-1H-imidazo- [4,5,c]-pyridine-6-carboxylic acid) represents a potent and AT2-selective analog of PD 123177 and showed weak activity in competing for [125I]angiotensin II binding with an IC50 value of 100 microM. When subsequent competition studies were conducted in the presence of 1 microM L-158,809 to block [125I]angiotensin II to the AT1 receptor subtype, the angiotensin II agonists produced monophasic inhibition curves with AII-(3-8) showing the greatest activity (IC50 = 6 nM) followed by angiotensin III (IC50 = 15 nM) much greater than angiotensin II (IC50 = 110 nM). RG 13647 was not found to significantly inhibit this portion of [125I]angiotensin II binding. These data demonstrate that bovine adrenal cortex contains both the AT1 receptor subtype, as well as, a novel class of [125I]angiotensin II recognition sites which may be analogous to the recently described angiotensin IV (AT4) receptor.

Chronic ethanol exposure selectively increases diazepam-insensitive [3H]RO15-4513 binding in mouse cerebellum

The effect of chronic ethanol exposure and withdrawal on [3H]RO15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4] benzodiazepine-3-carboxylate) binding to diazepam-sensitive and diazepam-insensitive binding sites was determined in mouse brain. Neither chronic ethanol treatment nor withdrawal significantly altered total [3H]RO15-4513 binding in mouse cortex or cerebellum. However, diazepam-insensitive [3H]RO15-4513 binding density (Bmax) in cerebellum was significantly increased immediately following chronic ethanol treatment (60%) and at 8 h following withdrawal (75%). [3H]RO15-4513 binding affinity was not significantly influenced by chronic ethanol exposure or withdrawal. These results indicate that chronic ethanol treatment and withdrawal can selectively up-regulate diazepam-insensitive [3H]RO15-4513 binding sites and suggest that this unique GABAA receptor subtype may play some role in ethanol dependence and withdrawal.

Differential expression of adenosine A2A and A2B receptor subtypes on myeloid U937 and THP-1 cells: Adenosine A2B receptor activation selectively stimulates cAMP formation and inhibition of TNFα release in THP-1 cells

The ability of adenosine to function as an inhibitory modulator of inflammatory processes has been well documented. While the activation of adenosine A2 receptors has been implicated in the anti‐inflammatory actions of adenosine, the specific mechanisms by which adenosine modulates the function of pro‐inflammatory cells is less well understood. The present studies were undertaken to further investigate the specific roles of adenosine A2 receptor subtypes (A2A and A2B) in monocyte function using two human myeloid cell lines of different developmental lineage, U937 cells (promonocytes) and THP‐1 cells (monocytes). Adenosine receptor agonists stimulated the production of cAMP in both cell lines; with an apparent A2A receptor‐mediated agonist profile (CGS 21680 = NECA ≫ CPA) for U937 cells and an apparent A2B receptor‐mediated agonist profile (NECA ≫ CPA > CGS 21680) for THP‐1 cells. NECA‐stimulated cAMP production in both cell lines could be inhibited with the adenosine receptor antagonists CGS 15943 and DPCPX in a concentration‐dependent fashion. Using RNase protection analysis, it was demonstrated that the mRNA for the adenosine A2A receptor was expressed in U937 cells, but not in THP‐ 1 cells, while the adenosine A2B receptor mRNA was detected in THP‐1 cells, but not in U937 cells. Since THP‐1 cells are widely used as a model of monocyte function, additional experiments were conducted to characterize the expression of adenosine receptor subtypes on these cells. Radioligand binding studies demonstrated the apparent lack of high affinity binding to A1, A2A, or A3 receptors on THP‐1 cell membranes. Further, the rank order potency of adenosine receptor agonists to inhibit the LPS‐stimulated release of TNFα from THP‐1 cells was consistent with the activation of adenosine A2B receptors. These results suggest the possibility that the effects of adenosine on myeloid cell function can be mediated by a differential expression of A2 adenosine receptor subtypes during developmental maturation. Further, activation of adenosine A2B receptors may represent a significant mechanism by which adenosine inhibits TNFα release from inflammatory cells. Drug Dev. Res. 44:41–47, 1998.

Pharmacological characterization of AMP 579, a novel adenosine A1/A2 receptor agonist and cardioprotective

AMP 579 1S‐[1α,2β,3β,4α(S*)]‐4‐[7‐[[1‐[(3‐chloro‐2‐thienyl)methyl]propylamino]‐3H‐imidazo[4,5‐b]pyridin‐3‐yl]‐N‐ethyl‐2,3‐dihydroxy cyclopentanecarboxamide) is a novel cardioprotective adenosine agonist with the following order of affinity at adenosine receptors: A1 > A2A > A3. Agonism at A1 receptors was demonstrated in vitro in three different systems: 1) inhibition of lipolysis in rat and human isolated adipocytes, 2) restoration of insulin‐dependent glucose transport in rat adipocytes, and 3) reduction of heart rate in spontaneously beating rat right atria. Agonism at A2A receptors was reflected in vasorelaxation of porcine coronary arterial rings (IC50 = 0.3 μM); in comparison, agonism at A2B receptors was ∼100‐fold weaker, as reflected in relaxation of guinea pig aorta (IC50 = 28 μM). When given iv to conscious Sprague‐Dawley (SD) rats, AMP 579 dose‐dependently lowered free fatty acids (FFA), heart rate (HR), and mean arterial pressure (MAP), but was 25‐fold more potent at reducing FFA than at decreasing HR and MAP. In anesthetized rats undergoing myocardial ischemia‐reperfusion injury, AMP 579 (3 μg/kg + 0.3 μg/kg/min iv and 10 μg/kg + 1 μg/kg/min iv) was able to reduce infarct size by 55% and 63%, respectively, compared to control animals, when given 10 min prior to and throughout the first hour of reperfusion. These cardioprotective doses of AMP 579 caused no significant change in blood pressure or coronary blood flow. In summary, AMP 579 is a novel adenosine A1/A2A receptor agonist which causes long‐lasting reductions in FFA in vivo and has cardioprotective effects in a rat model of myocardial ischemia‐reperfusion injury at doses which have minimal hemodynamic effects. Thus, AMP 579 has significant potential for the therapy of acute myocardial infarction. Drug Dev. Res. 45:30–43, 1998.© 1998 Wiley‐Liss, Inc.

Dithiothreitol, sodium chloride, and ethylenediaminetetraacetic acid increase the binding affinity of [125I]angiotensin IV to AT4 receptors in bovine adrenal cortex

The present studies demonstrate that the sulfhydryl reducing agent, dithiothreitol (DTT), increases the specific binding of [125I]angiotensin IV ([125I]AIV) to AT4 receptors in bovine adrenal cortical membranes. Both the degree of stimulation and the pharmacological selectivity of [125I]AIV binding in the presence of DTT were quantitatively different depending on the contents of the assay buffer. Similar effects were also observed using a different sulfhydryl reducing agent, 2-mercaptoethanol (2-MCE). These sulfhydryl reducing agents (100 mM) produced a 200% increase in specific [125I]AIV binding in an assay buffer that has been used to characterize the novel AT4 receptor subtype. A much larger stimulation (700%) of specific [125I]AIV binding was found when the assay was conducted in a buffer that has been used to characterize ligand binding to the AT1 receptor. Ligand association studies indicated that 0.3 nM [125I]AIV displayed similar equilibrium kinetics and stability in both the AT4 and AT1 buffers. Ligand saturation studies indicated that [125I]AIV bound with high affinity (Kd = 6 nM) in the AT4 buffer system, but bound with lower affinity (Kd = 32 nM) in the AT1 buffer system. Removal of NaCl and EDTA from the AT4 buffer also resulted in low-affinity [125I]AIV binding (Kd = 33 nM). The subsequent inclusion of NaCl, EDTA, or DTT resulted in higher-affinity [125I]AIV binding (KdS = 3-14 nM). No significant effects on the apparent density (Bmax) of AT4 receptors were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of the adenosine A1 receptor allosteric enhancer PD 81,723 on agonist binding to brain and adipocyte membranes

The benzoylthiophene analog, PD 81,723, has been shown to allosterically enhance agonist binding and functional activation of the mammalian adenosine (ADO) A(1) receptor subtype by putatively maintaining the receptor in a high affinity state. The present studies were conducted to evaluate the ability of PD 81,723 to enhance the binding of [3H]cyclohexyladenosine ([3H]CHA) to A(1) receptors of neural (cerebral cortex) and non-neural (adipocyte) origin in three different species; rat, guinea pig and dog. PD 81, 723 (0.3-100 microM) produced a concentration-dependent enhancement of [3H]CHA binding to rat brain A(1) receptors. These effects were also species-dependent with larger enhancements (150-200% of control) observed in guinea pig and dog brain membranes as compared to the rat (120% of control). In contrast, PD 81,723 did not produce any enhancement of [3H]CHA binding to A(1) receptors in adipocyte membranes from any of the species examined. Additional binding studies were conducted using pharmacological manipulations that have previously been shown to enhance the allosteric effects of PD 81,723. In the presence of 1 mM GTP, the allosteric effects of PD 81,723 (15 microM) were increased in rat, guinea pig and dog brain membranes, however, in adipocyte membranes from each species, no significant alteration in agonist binding was observed. Similarly, the A(1) receptor selective antagonist 8-cyclopentyl-1, 3-dipropylxanthine (added to effectively reduce the intrinsic antagonist properties of PD 81,723) was found to enhance the allosteric effects of PD 81,723 (15 microM) in brain, but produce no alteration of agonist binding in adipocyte membranes from each species. Examination of the dissociation kinetics of [3H]CHA binding from rat brain and adipocyte membranes revealed that PD 81,723 (15 microM) differentially slowed agonist dissociation from brain, but not adipocyte, membranes. Taken together, the present data support the hypothesis that in tissues that are sensitive to PD 81,723, this benzyolthiophene functions to maintain the A(1) receptor in a high-affinity state and that the relative proportions of high-affinity A(1) receptors present in specific tissues may contribute, at least in part, to the apparent differential effects of PD 81,723 on agonist binding. The tissue specific modulation of A(1) receptor function by PD 81,723 also illustrates the possibility that the locus of allosteric modulation by PD 81,723 may be manifest via a specific, but indirect and tissue-dependent, interaction with the A(1) receptor.

Pharmacological characterization of the rat cerebellar endothelinB (ETB) receptor using the novel agonist radioligand [125I]BQ3020

A novel linear peptide fragment of endothelin-1 (ET-1), N-acetyl-[Ala11,15]ET-1[6-21] (BQ3020) has been identified as a potent and ETB-selective agonist. The present studies were conducted in order to characterize the binding of [125I]BQ3020 to the ETB receptor in rat cerebellum. [125I]BQ3020 (0.1 nM) bound with high specificity (90% of total binding) and selectivity for the ETB receptor. ET-1, ET-2, and ET-3 inhibited 0.1 nM [125I]BQ3020 binding with equivalent affinity (Ki values = 55-110 pM). The sarafotoxins S6a, S6b, and S6c also potently inhibited [125I]BQ3020 binding (Ki values = 55-2000 pM). The ETA-selective antagonist, BQ123 (100 microM) did not significantly inhibit [125I]BQ3020 binding. Ligand saturation studies indicated that [125I]BQ3020 labeled a single class of recognition sites with very high affinity (Kd = 31 pM) and limited capacity (Bmax = 570 fmol/mg protein). High affinity 0.1 nM [125I]BQ3020 binding was reduced by 40-50% in the presence of 1 mM guanine nucleotides. Additional competition studies indicated that ET-1 and ET-2 produced biphasic inhibition curves in competing for 0.5 nM [125I]BQ3020. The high affinity component of the ET-1 inhibition curve was subsequently eliminated in the presence of 1 mM GTP-gamma-S The guanine nucleotide sensitivity of [125I]BQ3020 binding offers the possibility that different functional consequences of ETB receptor activation may be mediated by multiple affinity states of the receptor. The present data demonstrate that [125I]BQ3020 is a potent and selective agonist for the ETB receptor that can discriminate high and low affinity states of the ETB receptor.

[125I]BQ3020, a novel endothelin agonist selective for the endothelinB receptor subtype

The endothelins represent a family of three (ET-1, ET-2, and ET-3) isopeptides that are derived from vascular endothelial cells and function as potent vasoconstrictors. Two endothelin receptor subtypes, ETA and ETB, have been cloned and are structurally similar to the large family of guanine nucleotide-binding protein (G-protein) linked receptors. The ET receptor subtypes can also be distinguished by their different pharmacological profiles.. A novel linear peptide fragment of endothelin-1 (ET-1), N-acetyl-[Ala11,15]ET-1[6-21] (BQ3020) has been identified as a potent and ETB-selective agonist. [125I]BQ3020 binds with high affinity (Kd = 31 pM) and limited capacity (Bmax = 570 fmol/mg protein) to a single class of recognition sites in rat cerebellum. High affinity [125I]BQ3020 binding is reduced in the presence of guanine nucleotides indicating an interaction with high and low affinity states of the ETB receptor. This protocol describes the use of [125I]BQ3020 as a specific radioligand for the ETB receptor.