Richard M. Edwards

Human AT1 receptor is a single copy gene: Characterization in a stable cell line

To address conflicting reports concerning the number of angiotensin II (AII) receptor type 1 (AT1) coding loci in vertebrates, Southern blot analysis was used to determine the genomic representation of AT1 receptor genes in animals comprising a divergent evolutionary spectrum. The data demonstrate that the AT1 receptor gene is present as a single genomic copy in a broad spectrum of animals including human, monkey, dog, cow, rabbit, and chicken. In contrast, members of the rodent taxonomic order contain two genes in their genomes. These two genes may have arisen in rodents as a consequence of a gene duplication event that occurred during evolution following the branching of rodents from the mammalian phylogenetic tree. In order to investigate the properties of the human AT1 receptor in a pure cell system, the recombinant human AT1 receptor was stably expressed in mouse L cells. An isolated cell line, designated LhAT1-D6, was found to express abundant levels of recombinant receptor [430±15 fmol/mg] exhibiting high affinity [KD=0.15±0.02 nM] for [125I][SAR1, IIe8] angiotensin II (SIA). The pharmacological profile of ligands competing for [125I] SIA binding to the expressed recèptor was in accordance with that of the natural receptor. Radioligand binding of the expressed receptor was decreased in the presence of the non-hydrolyzable analog of GTP, guanosine 5′-(γ-thio) triphosphate [GTPγS]. Angiotensin II evoked a rapid efflux of45Ca2+ from LhAT1-D6 cells that was blocked by AT1 receptor specific antagonists. In addition, AII inhibited forskolin-stimulated cAMP accumulation in these cells which was blocked by the AT-1 antagonist. Thus, the LhAT1-D6 cell line provides a powerful tool to explore the human AT1 receptor regulation.

Enhanced bladder capacity and reduced prostaglandin E2-mediated bladder hyperactivity in EP3 receptor knockout mice

Nonsteroidal anti-inflammatory cyclooxygenase inhibitors that function to reduce prostaglandin E2 (PGE2) production have been widely reported as effective agents in models of urinary bladder overactivity. We therefore investigated a potential role for the PGE2 receptor, EP3, in urinary bladder function by performing conscious, freely moving cystometry on EP3 receptor knockout (KO) mice. EP3 KO mice demonstrated an enhanced bladder capacity compared with wild-type (WT) mice ( approximately 185% of WT) under control conditions, based on larger voided and infused bladder volumes. Infusion of the EP3 receptor agonist GR63799X into the bladder of WT mice reduced the bladder capacity. This was ineffective in EP3 KO mice that demonstrated a time-dependent increase in bladder capacity with GR63799X, an effect similar to that observed with vehicle in both genotypes. In addition, infusion of PGE2 into WT mice induced bladder overactivity, an effect that was significantly blunted in the EP3 KO mice. The data reported here provide the first evidence supporting a functional role for EP3 receptors in normal urinary bladder function and implicate EP3 as a contributor to bladder overactivity during pathological conditions of enhanced PGE2 production, as reported previously in overactive bladder patients.

Antihypertensive activity of the non-peptide angiotensin II receptor antagonist, SK&F 108566, in rats and dogs

SummaryThe antihypertensive activity of the nonpeptide angiotensin II receptor antagonist, SK&F 108566 (E) - α - [[2 - butyl -1 - [(4 - carboxyphenyl)methyl] -1H -imidazol-5-yl]methylene]-2-thiophene propanoic acid, was examined in rats and dogs. SK&F 108566 produced dose-dependent decreases in blood pressure in renin-dependent hypertensive rats. At 10 mg/kg intraduodenally, mean arterial blood pressure fell from between 150–160 mm Hg to approximately 124 mm Hg. A sustained infusion of SK&F 108566 at 25 μg/min intraduodenally normalized blood pressure during 3 days of infusion and for 18 h following cessation of the infusion. Evaluation of the systemic hemodynamic effects of SK&F 108566 in chronically instrumented renin-dependent hypertensive rats demonstrated that the antihypertensive effects of SK&F 108566 were accompanied by a significant increase in cardiac output with little change in stroke volume. In dogs made acutely hypertensive by an intravenous infusion of angiotensin 1, SK&F 108566 resulted in dose-dependent decreases in blood pressure. The antihypertensive activity of SK&F 108566 at 10 mg/kg p.o. was maintained for between 13–15 h, a similar duration of action as observed with enalapril (1 mg/kg, p.o.). Administration of DuP 753 (losartan) intravenously caused a small and short-lived fall in blood pressure in the angiotensin I-infused hypertensive dog. However, the active metabolite of losartan, EXP 3174, resulted in a response of longer duration. In dogs made hypertensive by placement of an ameroid constrictor on the left renal artery, SK&F 108566 (10 mg/kg, p.o.) or enalapril (1 mg/ kg, p.o.) resulted in antihypertensive responses of at least 12 h duration. The data indicate that SK&F 108566 is a long-acting antihypertensive agent in the rat and dog.

Distribution of Neutral Endopeptidase Activity along the Rat and Rabbit Nephron

Neutral endopeptidase (NEP) activity was measured in various nephron segments dissected from rat and rabbit kidney. In the rat, only the proximal straight tubule and glomerulus had measurable NEP activity of 86 ± 11.3 pmol/min/mm tubule length and 5.8 ± 1.5 pmol/min/glomerulus, respectively. In the rabbit, significant activity was observed in both the proximal convoluted tubule (70.8 ± 7.2 pmol/min/mm) and proximal straight tubule (29.6 ± 2.3 pmol/min/mm) as well as in the glomerulus (12.8 ± 2.2 pmol/min/glomerulus). In the rat proximal tubule, phosphoramidon and thiorphan inhibited NEP activity, with IC50 values of 26.6 ± 6.0 and 6.9 ± 1.6 nmol/l, respectively. Incubation of rat proximal tubules with phorbol 12-myristate 13-acetate resulted in a 50% reduction in membrane-associated NEP activity. The results demonstrate that in both the rat and rabbit NEP is restricted to the glomerulus and proximal tubule. This localized distribution of NEP and its potential regulation by the protein kinase C pathway may play a key role in determining local concentrations of important regulatory peptides in the kidney.

Modulation of bladder function by prostaglandin EP3 receptors in the central nervous system

Prostaglandin EP3 receptors in the central nervous system (CNS) may exert an excitatory effect on urinary bladder function via modulation of bladder afferent pathways. We have studied this action, using two EP3 antagonists, (2E)-3-{1-[(2,4-dichlorophenyl)methyl]-5-fluoro-3-methyl-1H-indol-7-yl}-N-[(4,5-dichloro-2-thienyl)sulfonyl]-2-propenamide (DG041) and (2E)-N-{[5-bromo-2-(methyloxy)phenyl] sulfonyl}-3-[2-(2-naphthalenylmethyl)phenyl]-2-propenamide (CM9). DG041 and CM9 were proven to be selective EP3 antagonists with radioligand binding and functional fluorescent imaging plate reader (FLIPR) assays. Their effects on volume-induced rhythmic bladder contraction and the visceromotor reflex (VMR) response to urinary bladder distension (UBD) were evaluated in female rats after intrathecal or intracerebroventricular administration. Both DG041 and CM9 showed a high affinity for EP3 receptors at subnanomolar concentrations without significant selectivity for any splice variants. At the human EP3C receptor, both inhibited calcium influx produced by the nonselective agonist PGE2. After intrathecal or intracerebroventricular administration both CM9 and DG041 dose-dependently reduced the frequency, but not the amplitude, of the bladder rhythmic contraction. With intrathecal administration DG041 and CM9 produced a long-lasting and robust inhibition on the VMR response to UBD, whereas with intracerebroventricular injection both compounds elicited only a transient reduction of the VMR response to bladder distension. These data support the concept that EP3 receptors are involved in bladder micturition at supraspinal and spinal centers and in bladder nociception at the spinal cord. A centrally acting EP3 receptor antagonist may be useful in the control of detrusor overactivity and/or pain associated with bladder disorders.

An excitatory role for peripheral EP3 receptors in bladder afferent function

The excitatory roles of EP3 receptors at the peripheral afferent nerve innervating the rat urinary bladder have been evaluated by using the selective EP3 antagonist (2E)-3-[1-[(2,4-dichlorophenyl)methyl]-5-fluoro-3-methyl-1H-indol-7-yl]-N-[(4,5-dichloro-2-thienyl)sulfonyl]-2-propenamide (DG-041). The bladder rhythmic contraction model and a bladder pain model measuring the visceromotor reflex (VMR) to urinary bladder distension (UBD) have been used to evaluate DG-041 in female rats. In addition, male rats [spontaneously hypertensive rat (SHR), Wistar-Kyoto (WKY), and Sprague-Dawley (SD)] were anesthetized with pentobarbital sodium, and primary afferent fibers in the L6 dorsal root were isolated for recording the inhibitory response to UBD following intravenous injection of DG-041. Intravenous injection of DG-041 (10 mg/kg), a peripherally restricted EP3 receptor antagonist, significantly reduced the frequency of bladder rhythmic contraction and inhibited the VMR response to bladder distension. The magnitude of reduction of the VMR response was not different in the different strains of rats (SD, SHR, and WKY). Furthermore, quantitative characterization of the mechanosensitive properties of bladder afferent nerves in SHR, WKY, and SD rats did not show the SHR to be supersensitive to bladder distension. DG-041 selectively attenuated responses of mechanosensitive afferent nerves to UBD, with strong suppression on the slow-conducting, high-threshold afferent fibers, with equivalent activity in the three strains. We conclude that sensitization of afferent nerve activity was not one of the mechanisms of bladder hypersensitivity in SHR. EP3 receptors are involved in the regulation of bladder micturition and bladder nociception at the peripheral level.

Relaxation of Renal Arterioles by Parathyroid Hormone and Parathyroid Hormone-Related Protein

The effects of parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) on renal arteriolar tone and proximal tubule adenylate cyclase were examined. In both afferent and efferent arterioles, PTH produced concentration-dependent relaxation of norepinephrine-induced tone with EC50 values of 8.7 and 9.9 nmol/l, respectively. PTHrP also produced relaxations that were indistinguishable from PTH. In proximal convoluted tubules PTH and PTHrP stimulated adenylate cyclase to the same extent and with similar potencies. The PTH antagonist, bPTH(7-34), totally blocked PTH-induced arteriolar relaxation but had no effect on proximal tubule adenylate cyclase. The results demonstrate that PTH and PTHrP are potent relaxants of glomerular arterioles and that PTH receptors present on the renal microvasculature may differ from those present on proximal tubules.

Inhibition of Vasopressin-Sensitive cAMP Accumulation by α2-Adrenoceptor Agonists in Collecting Tubules Is Species Dependent

The ability of alpha 2-adrenoceptor agonists to inhibit vasopressin (VP)-stimulated cAMP accumulation in collecting tubules and to inhibit the antidiuretic effect of VP in rats is clearly established. However, in other species, such as the dog, alpha 2-adrenoceptor-induced inhibition of VP action has not been convincingly demonstrated. In the present study, we examined the effects of epinephrine and other alpha 2-adrenoceptor agonists on VP-stimulated cAMP accumulation in inner medullary collecting tubule cells and/or cortical collecting tubules from a number of species. Epinephrine, oxymetazoline, clonidine, and guanabenz inhibited VP-induced cAMP formation in rat inner medullary collecting tubule cells with IC50s ranging from 10 to 30 nM. However, epinephrine or guanabenz had no effect on VP-stimulated cAMP formation in cells from dog, pig, rhesus monkey, or human inner medulla. Similarly, epinephrine inhibited VP-induced cAMP accumulation in cortical collecting tubules dissected from rat kidneys but not from dog or rabbit kidneys. We conclude that there is a marked species difference in the ability of alpha 2-adrenoceptor agonists to inhibit VP-induced cAMP formation at the tubular level. This may explain the difficulty in demonstrating an alpha 2-adrenoceptor agonist-induced inhibition of VP action in other species such as dog and man.

Response of Isolated Intracerebral Arterioles to Endothelins

The effects of endothelin-1, -2 and -3 (ET-1, -2, -3) on lumen diameter of intracerebral arterioles isolated from rat brain were examined. All three ETs produced concentration-dependent decreases in lumen diameter with EC50 values of 0.7, 1.5 and 58 mmol/l for ET-1, ET-2 and ET-3, respectively. The calcium channel antagonist, nicardipine, had no significant effect on ET-1-induced contractions except at a concentration of 1 mumol/l which attenuated the maximum response to ET-1, but had no effect on the EC50 value. The potent vasoconstrictor action of ET on intracerebral arterioles suggest an important role for this peptide in the regulation of the cerebral microvasculature.

Basolateral, but not apical, ATP inhibits vasopressin action in rat inner medullary collecting duct.

Previous studies have shown that basolateral ATP inhibits vasopressin action in the renal collecting tubule. Although there is evidence for an apical P2Y2 receptor in this tubule segment, it is not known whether apical ATP has similar effects. In the rat inner medullary collecting duct basolateral, but not apical, ATP (0.1-100 microM) reversibly inhibited vasopressin-induced increases in water permeability with an IC50 of 1.09 microM. Basolateral UTP, but not ADP, alpha,beta-methylene-ATP or 2-methylthio-ATP also inhibited vasopressin action. It is concluded that basolateral but not apical P2Y2 receptors inhibit vasopressin action in the collecting duct.