Donald V. Daniels

Pharmacological pleiotropism of the human recombinant α1A-adrenoceptor : implications for α1-adrenoceptor classification

Three fully‐defined α1‐adrenoceptors (α1A, α1B and α1D) have been established in pharmacological and molecular studies. A fourth α1‐adrenoceptor, the putative α1L‐adrenoceptor, has been defined in functional but not molecular studies, and has been proposed to mediate contraction of human lower urinary tract tissues; its relationship to the three fully characterized α1‐adrenoceptors is not known. In the present study, binding affinities were estimated by displacement of [3H]‐prazosin in membrane homogenates of Chinese hamster ovary (CHO‐K1) cells stably expressing the human α1A‐, α1B‐ and α1D‐adrenoceptors and were compared with affinity estimates obtained functionally in identical cells by measuring inhibition of noradrenaline (NA)‐stimulated accumulation of [3H]‐inositol phosphates. For the α1A‐adrenoceptor, binding studies revealed a pharmacological profile typical for the classically defined α1A‐adrenoceptor, such that prazosin, RS‐17053, WB4101, 5‐methylurapidil, Rec15/2739 and S‐niguldipine all displayed subnanomolar affinity. A different profile of affinity estimates was obtained in inositol phosphates accumulation studies: prazosin, WB4101, 5‐methylurapidil, RS‐17053 and S‐niguldipine showed 10 to 40 fold lower affinity than in membrane binding. However, affinity estimates were not ‘frameshifted’, as tamsulosin, indoramin and Rec15/2739 yielded similar, high affinity estimates in binding and functional assays. In contrast, results from human α1B‐ and α1D‐adrenoceptors expressed in CHO‐K1 cells gave antagonist affinity profiles in binding and functional assays that were essentially identical. A concordance of affinity estimates from the functional (inositol phosphates accumulation) studies of the α1A‐adrenoceptor in CHO‐K1 cells was found with estimates published recently from contractile studies in human lower urinary tract tissues (putative α1L‐adrenoceptor). These data show that upon functional pharmacological analysis, the cloned α1A‐adrenoceptor displays pharmacological recognition properties consistent with those of the putative α1L‐adrenoceptor. Why this profile differs from that obtained in membrane binding, and whether it explains the α1L‐adrenoceptor pharmacology observed in many native tissues, requires further investigation.

In vitroα1‐adrenoceptor pharmacology of Ro 70–0004 and RS‐100329, novel α1A‐adrenoceptor selective antagonists

It has been hypothesized that in patients with benign prostatic hyperplasia, selective antagonism of the α1A‐adrenoceptor‐mediated contraction of lower urinary tract tissues may, via a selective relief of outlet obstruction, lead to an improvement in symptoms. The present study describes the α1‐adrenoceptor (α1‐AR) subtype selectivities of two novel α1‐AR antagonists, Ro 70‐0004 (aka RS‐100975) and a structurally‐related compound RS‐100329, and compares them with those of prazosin and tamsulosin. Radioligand binding and second‐messenger studies in intact CHO‐K1 cells expressing human cloned α1A‐, α1B‐ and α1D‐AR showed nanomolar affinity and significant α1A‐AR subtype selectivity for both Ro 70‐0004 (pKi 8.9: 60 and 50 fold selectivity) and RS‐100329 (pKi 9.6: 126 and 50 fold selectivity) over the α1B‐ and α1D‐AR subtypes respectively. In contrast, prazosin and tamsulosin showed little subtype selectivity. Noradrenaline‐induced contractions of human lower urinary tract (LUT) tissues or rabbit bladder neck were competitively antagonized by Ro 70‐0004 (pA2 8.8 and 8.9), RS‐100329 (pA2 9.2 and 9.2), tamsulosin (pA2 10.4 and 9.8) and prazosin (pA2 8.7 and 8.3 respectively). Affinity estimates for tamsulosin and prazosin in antagonizing α1‐AR‐mediated contractions of human renal artery (HRA) and rat aorta (RA) were similar to those observed in LUT tissues, whereas Ro 70‐0004 and RS‐100329 were approximately 100 fold less potent (pA2 values of 6.8/6.8 and 7.3/7.9 in HRA/RA respectively). The α1A‐AR subtype selectivity of Ro 70‐0004 and RS‐100329, demonstrated in both cloned and native systems, should allow for an evaluation of the clinical utility of a ‘uroselective’ agent for the treatment of symptoms associated with benign prostatic hyperplasia.

Molecular cloning, genomic characterization and expression of novel human α1A-adrenoceptor isoforms

We have isolated and characterized from human prostate novel splice variants of the human α1A‐adrenoceptor, several of which generate truncated products and one isoform, α1A‐4, which has the identical splice site as the three previously described isoforms. Long‐PCR on human genomic DNA showed that the α1A‐4 exon is located between those encoding the α1A‐1 and α1A‐3 variants. CHO‐K1 cells stably expressing α1A‐4 showed ligand binding properties similar to those of the other functional isoforms as well as agonist‐stimulated inositol phosphate accumulation. Quantitative PCR analyses revealed that α1A‐4 is the most abundant isoform expressed in the prostate with high levels also detected in liver and heart.

A single column method for the assay of adenylate cyclase

An improved, one-step method for the separation of cyclic AMP from other nucleotides on disposable columns of neutral aluminum oxide is described. The method consists of several modifications of an established assay for adenylate cyclase. These modifications were designed to increase the sensitivity of the method, to decrease the time required for column preparation, and to eliminate the variable elution patterns for cyclic AMP that are obtained when using aluminum oxide from different commercial sources. Uniform elution patterns and high recoveries (approximately 80%) of cyclic AMP were obtained when 0.1 M ammonium acetate was used to elute cyclic AMP instead of Tris-HCl buffer. Prior to column chromatography, the adenylate cyclase reactions were terminated with the addition of hydrochloric acid and the mixtures were heated to degrade acid-labile nucleotides that would otherwise elute with cyclic AMP from aluminum oxide columns. Disposable polypropylene columns, fabricated with a reservoir and fast-flow filters, were used for column chromatography. Low blank values, generally less than 15 dpm/assay tube, were obtained when the acidified reaction mixtures were applied directly to aluminum oxide columns without prior neutralization. The proposed method should be useful for the routine assay of adenylate cyclase activity.

A separation method for the assay of adenylylcyclase, intracellular cyclic AMP, and cyclic-AMP phosphodiesterase using tritium-labeled substrates

A method for the separation of cyclic AMP from adenosine and polyvalent adenine nucleotides is described. The method consists of the sequential elution of adenosine and cyclic AMP from a single column of acidic aluminum oxide (alumina) with dilute hydrochloric acid and ammonium acetate. Adenosine, adenine, xanthine, and hypoxanthine are rapidly eluted with the application of 0.005 N hydrochloric acid while cyclic AMP remains adsorbed to the alumina. A subsequent application of 0.1 M ammonium acetate elutes more than 90% of the cyclic AMP. Under these conditions, polyvalent nucleotides (AMP, ADP, and ATP) remain adsorbed to the alumina. The method permits the measurement of adenylylcyclase activity using [3H]ATP as the labeled substrate. The same technique can be used to measure the accumulation of cyclic AMP in intact cells after labeling the ATP pool with [3H]adenine. With slight modification, the technique can be used to measure the activity of cyclic-AMP phosphodiesterase using [3H]cyclic AMP as the substrate. The proposed technique provides rapid, highly reproducible assays using inexpensive, disposable columns.

Comparative pharmacology of recombinant human M3 and M5 muscarinic receptors expressed in CHO‐K1 cells

Affinity estimates were obtained for several muscarinic antagonists against carbachol‐stimulated [3H]‐inositol phosphates accumulation in Chinese hamster ovary (CHO‐K1) cells stably expressing either human muscarinic M3 or M5 receptor subtypes. The rationale for these studies was to generate a functional antagonist affinity profile for the M5 receptor subtype and compare this with that of the M3 receptor, in order to identify compounds which discriminate between these two subtypes. The rank order of antagonist apparent affinities (pKB) at the muscarinic M5 receptor was atropine (8.7)tolterodine (8.6)=4‐diphenylacetoxy‐N‐methylpiperidine (4‐DAMP, 8.6)>darifenacin (7.7)zamifenacin (7.6)>oxybutynin (6.6)=para‐fluorohexahydrosiladifenidol (p‐F‐HHSiD, 6.6)>pirenzepine (6.4)methoctramine (6.3)=himbacine (6.3)>AQ‐RA 741 (6.1). Antagonist apparent affinities for both receptor subtypes compare well with published binding affinity estimates. No antagonist displayed greater selectivity for the muscarinic M5 subtype over the M3 subtype, but himbacine, AQ‐RA 741, p‐F‐HHSiD, darifenacin and oxybutynin displayed between 9‐ and 60 fold greater selectivity for the muscarinic M3 over the M5 subtype. This study highlights the similarity in pharmacological profiles of M3 and M5 receptor subtypes and identifies five antagonists that may represent useful tools for discriminating between these two subtypes. Collectively, these data show that in the absence of a high affinity M5 selective antagonist, affinity data for a large range of antagonists is critical to define operationally the M5 receptor subtype.

Pharmacological characteristics of Ro 115-1240, a selective alpha1A/1L-adrenoceptor partial agonist: a potential therapy for stress urinary incontinence.

To describe the preclinical pharmacology of Ro 115–1240, a peripherally acting selective α1A/1L‐adrenoceptor (AR) partial agonist, compared with the α1A/1L‐AR full agonist amidephrine, as AR agonists have some utility in the treatment of stress urinary incontinence (SUI) but are limited by undesirable cardiovascular and central nervous system side‐effects.

Functional characterization of rat submaxillary gland muscarinic receptors using microphysiometry

Muscarinic cholinoceptors (MChR) in freshly dispersed rat salivary gland (RSG) cells were characterized using microphysiometry to measure changes in acidification rates. Several non‐selective and selective muscarinic antagonists were used to elucidate the nature of the subtypes mediating the response to carbachol. The effects of carbachol (pEC50=5.74±0.02 s.e.mean; n=53) were highly reproducible and most antagonists acted in a surmountable, reversible fashion. The following antagonist rank order, with apparent affinity constants in parentheses, was noted: 4‐DAMP (8.9)=atropine (8.9)>tolterodine (8.5)>oxybutynin (7.9)>S‐secoverine (7.2)>pirenzepine (6.9)>himbacine (6.8)>AQ‐RA 741 (6.6)>methoctramine (5.9). These studies validate the use of primary isolated RSG cells in microphysiometry for pharmacological analysis. These data are consistent with, and extend, previous studies using alternative functional methods, which reported a lack of differential receptor pharmacology between bladder and salivary gland tissue. The antagonist affinity profile significantly correlated with the profile at human recombinant muscarinic M3 and M5 receptors. Given a lack of antagonists that discriminate between M3 and M5, definitive conclusion of which subtype(s) is present within RSG cells cannot be determined.

Pharmacological characterization of canine bradykinin receptors in prostatic culture and in isolated prostate

The objective of this study was to characterize pharmacologically bradykinin (Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe‐Arg, BK) receptors in the canine prostate. Primary cultures of canine prostate stromal (PS) and epithelial cells (PE) were established and then characterized using cell‐specific antibodies (actin, vimentin and cytokeratin). Cultured cells were assayed for BK receptors using fluorometric imaging plate reader assays. In addition, isolated strips of the canine prostate were studied for BK‐induced isometric contraction. PS cells were labeled only with anti‐actin and ‐vimentin antibodies, while the anti‐cytokeratin antibodies labeled only the PE cells. In cultured prostate cells, the BK receptor 2 (B2)‐preferring agonist BK induced mobilization of intracellular Ca2+ in a concentration‐dependent manner with potencies (log[EC50]∣PE, pEC50) of 8.72±0.12 in PS and 8.75±0.06 in PE cells. In contrast, the BK receptor 1 (B1)‐selective agonist [des‐Arg9]BK (Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe) did not elicit any significant effect (pEC50<5) on Ca2+ responses. BK agonism (10 nM) was inhibited by HOE‐140 (D‐arginyl‐L‐arginyl‐L‐prolyl‐trans‐4‐hydroxy‐L‐prolylglycyl‐3‐(2‐thienyl)‐L‐alanyl‐L‐seryl‐D‐1,2,3,4‐tetrahhydro‐3‐isoquinolinecarbonyl‐L‐(2a,3b,7ab)‐octahydro‐1H‐indole‐2‐carbonyl‐L‐arginine), a B2‐selective antagonist, with a log[IC50] (pIC50) of 8.11±0.19 and 9.23±0.20 in PS and PE cells, respectively. [des‐Arg10]HOE‐140 (D‐arginyl‐L‐arginlyl‐L‐prolyl‐trans‐4‐hydroxy‐L‐prolylglycyl‐3‐(2‐thienyl)‐L‐alanyl‐L‐seryl‐D‐1,2,3,4‐tetrahydro‐3‐isoquinolinecarbonyl‐L‐(2a, 3b,7ab)‐octahydro‐1H‐indole‐2‐carbonyl), a B1‐selective antagonist, displayed weak antagonism with pIC50 values of 4.87±0.23 and 6.38±0.16 in PS and PE cells, respectively. Isolated tissue strips of the canine prostate contracted to BK (10 μM) but not to [des‐Arg9]BK (10 μM). BK‐induced contractility was attenuated by HOE‐140 (1 μM). In conclusion, canine prostates express functional B2 receptors, with no apparent B1 receptor subtypes.