Alpha 1D Subtypes Research Articles

The Impact of α1-Adrenoceptors Up-Regulation Accompanied by the Impairment of β-Adrenergic Vasodilatation in Hypertension

In human and animal hypertension models, increased activity of G-protein-coupled receptor kinase (GRK) 2 determines a generalized decrease of beta-adrenergic vasodilatation. We analyzed the possibility of differential changes in the expression and functionality of alpha(1A), alpha(1B), alpha(1D), beta(1), beta(2), and beta(3)-ARs also being involved in the process. We combined the quantification of mRNA levels with immunoblotting and functional studies in aortas of young and adult spontaneously hypertensive rats (SHRs) and their controls (Wistar Kyoto). We found the expression and function of beta(1)-adrenoceptors in young prehypertensive SHRs to be higher, whereas a generalized increase in the expression of the six adrenoceptors and GRK2 was observed in aortas of adult hypertensive SHRs. alpha(1D)- and beta(3)-adrenoceptors, the subtypes that are more resistant to GRK2-mediated internalization and mostly expressed in rat aorta, exhibited an increased functional role in hypertensive animals, showing two hemodynamic consequences: 1) an increased sensitivity to the vasoconstrictor stimulus accompanied by a decreased sensitivity to the vasodilator stimulus (alpha(1D)-ARs are the most sensitive to agonists, and beta(3)-ARs are the least sensitive to agonists); and 2) a slower recovery of the basal tone after adrenergic stimulus removal because of the kinetic characteristic of the alpha(1D) subtype. These functional changes might be involved in the greater sympathetic vasoconstrictor tone observed in hypertension.

Examination of alpha 1 adrenoceptor subtypes in the human ureter

Urinary stone disease is a common condition affecting up to 12% of the population. Speedy elimination of ureterolithiasis in the lower part of ureters is reported with the alpha 1-blocker. This study was carried out to characterize the alpha 1 adrenoceptors (AR) subtype gene and protein expression in the proximal, medial, and distal regions of the human ureter with the aim of facilitating stone expulsion. The study was carried out on 20 volunteers. Proximal, medial and distal ureter specimens were obtained as discarded tissues after nephroureterectomy for renal pelvic or ureteral carcinoma. Proximal ureter specimens were obtained as discarded tissues after nephrectomy for renal carcinoma. Using ureter samples, real-time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining were performed. The alpha 1d subtype mRNA was highly expressed in each ureteral region, accounting for about 54% of the total AR Messenger Ribonucleic Acid. In the proximal and medial ureter, the distribution of ARs was alpha 1d > or = alpha 1a > alpha 1b as well as that of the total ureter. The distal ureter expressed the highest amount of alpha 1d ARs. The alpha 1d subtype expression tended to be high compared with the alpha 1a subtype expression. In the distal ureter, the distribution of ARs was alpha 1d > alpha 1a > alpha 1b. Immunohistochemical staining revealed that expressions of alpha 1a-AR and alpha 1d-AR were higher than that of alpha 1b-AR. According to our results, we should try the alpha 1d AR antagonist for the conservative expulsive therapy of distal ureteral stones in the highest region of alpha 1d subtype distribution.

Increased Expression and Function of Vascular 1D-Adrenoceptors May Mediate The Prohypertensive Effects Of Angiotensin II

The peptide Angiotensin II (Ang II), part of the renin-angiotensin system (RAS), participates in the control of systemic arterial pressure. Ang II participates in increasing smooth muscle tone, and its positive effects on smooth muscle cell DNA synthesis are inhibited by treatment with prazosin, an alpha(1)-adrenoceptor agonist. Ang II also induces the expression of alpha(1)-adrenoceptor, especially the alpha(1D) subtype. Other findings suggest that the molecular signals activated by Ang II and by alpha(1D)-adrenoceptor might interweave, thus leading to the augmentation of smooth muscle tone and hypertension.

Changes in the receptor profile of the aging bladder

Future demographic developments will challenge urology with a steadily increasing incidence of lower urinary tract symptoms (LUTS) derived from the aging bladder. Obstruction, instability and hypocontractility, which may be caused by changes in the receptor profile of the detrusor, are typical pathophysiologic findings in geriatric bladder dysfunction. Benign prostatic hyperplasia and diabetes mellitus are age-associated comorbidities with an additional influence on bladder receptors. Muscarinic (M(2), M(3)), purinergic (P2X, P2Y) and adrenergic receptors (alpha(1), beta(3)) are targets of efferent sympathetic and parasympathetic bladder innervation. Although the results from animal experiments are somewhat inconsistent, aging and bladder outlet obstruction (BOO) probably cause partial cholinergic denervation of the detrusor with a subsequent upregulation of muscarinic receptor sensitivity leading to bladder instability. The non-cholinergic (atropine-resistant) component of the detrusor contraction rises with aging and BOO to 50%, emphasizing the increasing impact of purinergic receptors in geriatric LUTS. alpha(1)-adrenergic receptors are modulated in the aging bladder by a shift from the predominant alpha(1a) subtype to the alpha(1d) subtype, which shows 100-fold higher affinity towards norepinephrine and increases alpha-adrenergic bladder susceptibility. No data are available on the changes in beta(3) receptor density or sensitivity with aging. Moreover, the role of sensory C-fiber receptors in geriatric LUTS remains completely unclear, although specific C-fiber blockers are already under clinical evaluation (capsaicin, resiniferatoxin).

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 alpha1A/1L-adrenoceptor (AR) partial agonist, compared with the alpha1A/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. In radioligand-binding studies Ro 115-1240 had greater affinity for alpha1A than for alpha1B and alpha1D subtypes. The potency and intrinsic activity of amidephrine and Ro 115-1240 relative to noradrenaline were determined in native and cell-based assays using human recombinant alpha1-ARs; they acted as selective alpha1A/1L-AR full and partial agonists, respectively. In anaesthetized micropigs and rabbits, amidephrine and Ro 115-1240 produced non-selective, dose-dependent increases in intraurethral and arterial blood pressures but the magnitude of the pressure increases evoked by Ro 115-1240 were about a third of those with amidephrine. In conscious micropigs both agents produced dose-dependent increases in urethral tension. Again, the magnitude of the urethral response to Ro 115-1240 was about a third of that with amidephrine. More importantly, only amidephrine produced dose-dependent increases in blood pressure and decreases in heart rate. Ro 115-1240 produced a maximum increase in urethral tension with no effect on blood pressure or heart rate. These results show that by combining selectivity for the alpha1A/1L-AR subtype with a reduction in intrinsic agonist efficacy, Ro 115-1240 has reduced haemodynamic effects while retaining to some degree the contractile effects on urethral smooth muscle. These studies indicate that Ro 115-1240 may be useful as a novel treatment for SUI.

Allosteric α1-Adrenoreceptor Antagonism by the Conopeptide ρ-TIA

A peptide contained in the venom of the predatory marine snail Conus tulipa, rho-TIA, has previously been shown to possess alpha1-adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, rho-TIA inhibited alpha1-adrenoreceptor-mediated increases in cytosolic Ca2+ concentration that were triggered by norepinephrine, but did not affect presynaptic alpha2-adrenoreceptor-mediated responses. In radioligand binding assays using [125I]HEAT, rho-TIA displayed slightly greater potency at the alpha 1B than at the alpha 1A or alpha 1D subtypes. Moreover, although it did not affect the rate of association for [3H]prazosin binding to the alpha 1B-adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by rho-TIA. N-terminally truncated analogs of rho-TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of rho-TIA (Arg4). An alanine walk of rho-TIA confirmed the importance of Arg4 for activity and revealed a number of other residues clustered around Arg4 that contribute to the potency of rho-TIA. The unique allosteric antagonism of rho-TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive alpha1-adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.

1,3-dioxolane-based ligands as a novel class of alpha1-adrenoceptor antagonists.

1,3-Dioxolane-based compounds (2-14) were synthesized, and the pharmacological profiles at alpha(1)-adrenoceptor subtypes were assessed by functional experiments in isolated rat vas deferens (alpha(1A)), spleen (alpha(1B)), and aorta (alpha(1D)). Compound 9, with a pA(2) of 7.53, 7.36, and 8.65 at alpha(1A), alpha(1B), and alpha(1D), respectively, is the most potent antagonist of the series, while compound 10 with a pA(2) of 8.37 at alpha(1D) subtype and selectivity ratios of 162 (alpha(1D)/alpha(1A)) and 324 (alpha(1D)/alpha(1B)) is the most selective. Binding assays in CHO cell membranes expressing human cloned alpha(1)-adrenoceptor subtypes confirm the pharmacological profiles derived from functional experiments, although the selectivity values are somewhat lower. Therefore, it is concluded that 1,3-dioxolane-based ligands are a new class of alpha(1)-adrenoceptor antagonists.

Ligand design for alpha(1) adrenoceptors.

An area of continuing interest in medicinal chemistry is the design, synthesis and pharmacological evaluation of ligands which bind at adrenoceptor subtypes, which include alpha(1A), alpha(1B), alpha(1D); alpha(2A), alpha(2B), alpha(2C); beta(1), beta(2), beta(3) and possibly beta(4) subtypes. The selective blockade or stimulation of these receptor subtypes is of on-going pharmacological and medicinal interest. However, the design principles for ligand differentiation at these subtypes still need further development. This review focuses on alpha(1) adrenoceptors with a concentration on literature over the past five years. Structural, physiological and therapeutic aspects of the alpha(1A), alpha(1B) and alpha(1D) subtypes are discussed together with ligands binding to these receptor subtypes. Approaches to alpha(1) adrenoceptor ligand design based on known ligands and on receptor docking are evaluated. A new combined approach using pharmacophores and receptor docking affords possibilities for deeper insights into achieving small molecule binding selectivity.

Hypotension, Autonomic Failure, and Cardiac Hypertrophy in Transgenic Mice Overexpressing the α1B-Adrenergic Receptor

alpha(1)-Adrenergic receptors (alpha(1A), alpha(1B), and alpha(1D)) are regulators of systemic arterial blood pressure and blood flow. Whereas vasoconstrictory action of the alpha(1A) and alpha(1D) subtypes is thought to be mainly responsible for this activity, the role of the alpha(1B)-adrenergic receptor (alpha(1B)AR) in this process is controversial. We have generated transgenic mice that overexpress either wild type or constitutively active alpha(1B)ARs. Transgenic expression was under the control of the isogenic promoter, thus assuring appropriate developmental and tissue-specific expression. Cardiovascular phenotypes displayed by transgenic mice included myocardial hypertrophy and hypotension. Indicative of cardiac hypertrophy, transgenic mice displayed an increased heart to body weight ratio, which was confirmed by the echocardiographic finding of an increased thickness of the interventricular septum and posterior wall. Functional deficits included an increased isovolumetric relaxation time, a decreased heart rate, and cardiac output. Transgenic mice were hypotensive and exhibited a decreased pressor response. Vasoconstrictory regulation by alpha(1B)AR was absent as shown by the lack of phenylephrine-induced contractile differences between ex vivo mesenteric artery preparations. Plasma epinephrine, norepinephrine, and cortisol levels were also reduced in transgenic mice, suggesting a loss of sympathetic nerve activity. Reduced catecholamine levels together with basal hypotension, bradycardia, reproductive problems, and weight loss suggest autonomic failure, a phenotype that is consistent with the multiple system atrophy-like neurodegeneration that has been reported previously in these mice. These results also suggest that this receptor subtype is not involved in the classic vasoconstrictory action of alpha(1)ARs that is important in systemic regulation of blood pressure.

Analogues of prazosin that bear a benextramine-related polyamine backbone exhibit different antagonism toward alpha1-adrenoreceptor subtypes.

Hybrid tetraaamine disulfides 4-9 were synthesized by combining the structural features of prazosin (1), a competitive alpha1-adrenoreceptor antagonist, and benextramine (2), an irreversible alpha1/alpha2-adrenoreceptor antagonist, and their biological profiles at alpha1-adrenoreceptor subtypes were assessed by functional experiments in isolated rat vas deferens (alpha1A), spleen (alpha1B), and aorta (alpha1D). To verify the role of the disulfide moiety on the interaction with alpha1-adrenoreceptor subtypes, carbon analogues 10-15 were included in this study. All quinazolines lacking the disulfide bridge behaved, like 1, as competitive antagonists, whereas all polyamine disulfides displayed a nonhomogeneous mechanism of inhibition at the three subtypes since they were, like 2, noncompetitive antagonists at the alpha1A and alpha1B subtypes while being, unlike 2, competitive antagonists at the alpha1D. In particular, the blocking effects were characterized by a decrease of the maximal response to noradrenaline that was affected only slightly by washings. Probably the alpha1A and alpha1B subtypes bear in the binding pocket a suitable thiol function that would suffer an interchange reaction with the disulfide moiety of the antagonist and which is missing, or not accessible, in the alpha1D subtype. Polyamines 8, 9, and 14, among others, emerged as promising tools for the characterization of alpha1-adrenoreceptors, owing to their receptor subtype selectivity. Finally, the effect of nonbasic substituents on the phenyl ring of prazosin analogues 16-28 on potency and selectivity for the different subtypes can hardly be rationalized.

Cardiovascular α<sub>1</sub>-adrenoceptor subtypes: functions and signaling

alpha-Adrenoceptors (alpha1AR) are G protein-coupled receptors and include alpha1A, alpha1B, and alpha1D subtypes corresponding to cloned alpha1a, alpha1b, and alpha1d, respectively. alpha1AR mediate several cardiovascular actions of sympathomimetic amines such as vasoconstriction and cardiac inotropy, hypertrophy, metabolism, and remodeling. alpha1AR subtypes are products of separate genes and differ in structure, G protein-coupling, tissue distribution, signaling, regulation, and functions. Both alpha(1A)AR and alpha(1B)AR mediate positive inotropic responses. On the other hand, cardiac hypertrophy is primarily mediated by alpha(1A)AR. The only demonstrated major function of alpha(1D)AR is vasoconstriction. alpha1AR are coupled to phospholipase C, phospholipase D, and phospholipase A2; they increase intracellular Ca2+ and myofibrillar sensitivity to Ca2+ and cause translocation of specific phosphokinase C isoforms to the particulate fraction. Cardiac hypertrophic responses to alpha1AR agonists might involve activation of phosphokinase C and mitogen-activated protein kinase via Gq x alpha1AR subtypes might interact with each other and with other receptors and signaling mechanisms.

Heterogeneity of vascular smooth muscle alpha-adrenoceptors in canine blood vessels.

1. For the past decade, using radioligand binding, contractility and immunohistochemical techniques, we have been characterizing vascular smooth muscle (VSM) adrenoceptors in four functionally different canine blood vessels, namely the dog aorta (DAO), dog mesenteric artery (DMA), dog mesenteric vein (DMV) and dog saphenous vein (DSV). 2. This communication briefly reviews our findings (with a special emphasis on alpha-adrenoceptor subtypes), which showed that none of the four canine vessels showed the same complement of alpha-adrenoceptor subtypes. 3. All four vessels elicited alpha 1-adrenoceptor contractile responses (antagonized by prazosin), but alpha 2-adrenoceptor responses (sensitive to rauwolscine), were found only in DMV and DSV. 4. Pharmacological characterization using alpha 1-adrenoceptor subtype-selective antagonists showed that DAO contains mainly alpha 1B-adrenoceptors (some alpha 1D-adrenoceptors) and DMA has primarily alpha 1A/1L-adrenoceptors, alpha 1-Adrenoceptors in DMV are primarily of the alpha 1D subtype, but either a new or unusual alpha 1-adrenoceptor subtype was revealed in the DSV.

Alpha2D/A-adrenergic receptor gene induction in the retina by phorbol ester: involvement of an AP-2 element.

The alpha2-adrenergic receptor (alpha2-AR) expressed in the bovine retina has been demonstrated to be of the alpha2D subtype. The bovine alpha2D-adrenergic receptor (alpha2D/A-AR) gene has been cloned and characterized. This report describes the induction of this gene by phorbol- 12, 13-myristate acetate (PMA), an activator of protein kinase C (PKC). Treatment of the bovine retina for 60 min with PMA (1 micrometer) resulted in significant and similar increases in alpha2D/A-AR mRNA level and gene transcription. This indicates that PMA causes alpha2D/A-AR gene induction and that this induction takes place directly at the transcriptional level. In C6 cells, treatment with PMA at a concentration which was as low as 0.1 micrometer induced endogenous alpha2D/A-AR mRNA after 60 min. Luciferase reporter assays in C6 cells mapped the PMA-responsive element to a region between -247 bp and -163 bp on the alpha2D/A-AR promoter. Electrophoretic mobility shift assays showed an increased binding of nuclear factor(s) from PMA-treated bovine retina to this promoter region. Competition assays indicate that an AP-2 element may be involved in the PMA-dependent induction. These findings demonstrate for the first time, the direct induction of the alpha2D/A-AR gene by PMA and support a role for an AP-2 element in the induction mechanism.

Novel adrenoceptor antagonists with a tricyclic pyrrolodipyridazine skeleton.

A still unknown tricyclic heterocyclic system (5) was synthesized from 6-hydroxy-2-methylpyridazin-3-one and its structure identified as 2,8-dichloro-6-methylpyrrolo[1,2-b:3,4-d']dipyridazin-5(6H)- one by spectroscopic investigations. Selective condensation of 5 with 2-[4-(2-substituted-phenyl)piperazin-1-yl]ethylamine gave the 2-arylpiperazinylethylamino-8-chloro derivatives 6a-c, which were investigated in binding studies toward the three alpha1-adrenergic and 5-HT1A-serotonergic receptor subtypes. They displayed high potency on all the assays and some selectivity for alpha1a and alpha1d subtypes.

PATTERNS OF MESSENGER RNA EXPRESSION FOR alpha 1-ADRENOCEPTOR SUBTYPES IN HUMAN CORPUS CAVERNOSUM

Purpose The present study evaluated the expression of alpha 1-adrenoceptor subtypes in human corpus cavernosum. Materials and Methods The mRNA encoding alpha 1a, and alpha 1b and alpha 1d subtypes were assessed by RNA-directed complementary cDNA synthesis followed by Taq DNA amplification. The level of alpha 1 mRNA was calculated in arbitrary optical density units and normalized with respect to the length of the respective cDNA fragments. Results We found that alpha 1a, alpha 1b and alpha 1d adrenoceptor subtypes are expressed in human corpus cavernosum, with a predominant expression of the alpha 1a subtype. Conclusion These results suggest that alpha 1a-adrenoceptor subtype is important and that understanding the biochemical and functional characteristics of this subtype may lead to the development of specific antagonists in the treatment of impotence.

Decrease in Apparent Alphax1-Adrenoceptor-G Protein Coupling During Maturation in Rat Aorta

We have previously shown that the adrenoceptor agonist norepinephrine (NE) is more potent in eliciting contraction in aortas from 1-month-old Fischer 344 rats than it is in older animals. In the present study, we examined alpha1-adrenoceptor-guanine nucleotide regulatory binding protein (G protein) coupling in aortic membranes in order to investigate the mechanism for the age-dependent reduced responsiveness of aorta to NE. We used the guanosine 5'(betagamma-imido)triphosphate (Gpp[NH]p)-induced shift in agonist binding affinity as a measure of the efficiency of alpha11-adrenoceptor-G protein coupling. The binding of NE was assessed by measuring the displacement of 2-[beta-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl] tetralone ([125]-HEAT) by NE in aortic membranes. In 1-, 6-, and 24-month-old rat aortas, two apparent binding sites were detected in the competition isotherms for NE. This heterogeneous binding pattern was independent of Gpp(NH)p at all ages, and is likely to be due to a heterogeneous receptor population (alpha(1a), alpha(1b), and alpha(1d) subtypes). In 1-month-old rats, the high affinity binding of NE to alpha,-adrenoceptors was sensitive to Gpp(NH)p, indicating a significant interaction between the receptor and G protein. This Gpp(NH)p-sensitive high affinity binding was not observed in aortas from 6- or 24-month-old animals. Despite the lack of Gpp(NH)p-sensitive high affinity binding of agonist in 6- or 24-month-old aortas, NE was still able to induce maximal contraction in these aortas, albeit, with a relatively low potency. A partial reduction in alpha1-adrenoceptor-G protein coupling between 1 and 6 months of age can explain the observed decrease in ago- nist potency and the loss of Gpp(NH)p-sensitive high affinity binding of NE. This phenomenon can be explained as a reduction of allosteric coupling between the bindings of ligand and G protein to the receptor, that has been formulated in the ternary complex model. Computer simulation using the simple ternary complex model shows that manipulating the reciprocal coupling factor alone can lead to a loss of Gpp(NH)p-sensitive high affinity agonist binding, along with a reduction in agonist potency for contraction without altering the maximal response. Thus, a change in the relative expression of different alpha,-adrenoceptor subtypes, which we have previously observed in the aorta, and which possess diverse intrinsic allosteric couplings, may be speculated to be the mechanism for the apparent reduction of alpha,-adrenoceptor-G protein coupling during maturation.

Effects of imidazoline derivatives on cholinergic motility in guinea-pig ileum: involvement of presynaptic alpha2-adrenoceptors or imidazoline receptors?

The present study investigates the possibility that imidazoline receptors mediate modulation of cholinergic motor functions of the guinea-pig ileum. For this purpose, the effects of a series of compounds with known affinity for alpha2-adrenoceptors and/or imidazoline recognition sites were examined on the cholinergic twitch contractions evoked by electrical field stimulation (0.1 Hz) of longitudinal muscle-myenteric plexus preparations. Additional experiments were carried out on ileal strips preincubated with [3H]choline, superfused with physiological salt solution containing hemicholinium-3, and subjected to electrical field stimulation (1 Hz). The stimulation-induced outflow of radioactivity was taken as an index of endogenous acetylcholine release. Alpha-methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline and xylazine caused a concentration-dependent inhibition of twitch responses (IC50 from 0.13 to 1.05 microM; Emax from 85.9 to 92.5%). Rilmenidine and agmatine were less potent in reducing the twitch activity, and the latter compound acted also with low intrinsic activity (IC50=44.9 microM; Emax=35.5%). In interaction experiments, the inhibitory action of clonidine on twitch responses was competitively antagonized by RX 821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline), idazoxan, rauwolscine, yohimbine and BRL 44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)-methyl] -4,5-dihydroimidazoline), whereas prazosin (10 microM), ARC 239 (2-(2,4-(O-methoxy-phenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl- 1,3-(2H,4H)-isoquinolindione; 10 microM) and BRL 41992 (1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo[c,f]imidazol[1,5-a]a zepine; 10 microM) were without effect. Rauwolscine antagonized the inhibitory effects of various agonists on ileal twitch activity in a competitive manner and with similar potency. Agmatine and idazoxan did not significantly modify the twitch contractions when tested in the presence of alpha2-adrenoceptor blockade by rauwolscine (3 microM) or RX 821002 (1 microM). Linear regression analysis showed that the affinity values of antagonists correlated with their affinity at the alpha2A and alpha2D binding sites as well as at previously classified alpha2A/D adrenoceptor subtypes, whereas no significant correlation was obtained when comparing the potency estimates of agonists and antagonists with the affinity at I1 or I2 binding sites. When tested on the electrically induced outflow of tritium, alpha-methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline, xylazine and rilmenidine yielded inhibitory concentration-response curves which were shifted rightward to a similar extent in the presence of rauwolscine (3 microM). In the absence of further drugs, agmatine significantly reduced the evoked tritium outflow at the highest concentrations tested (10 and 100 microM), whereas idazoxan (up to 100 microM) was without effect. When RX 821002 (1 microM) was added to the superfusion medium, neither agmatine nor idazoxan modified the evoked outflow of radioactivity. The results argue against modulation by imidazoline receptors of acetylcholine release from myenteric plexus nerve terminals. They provide evidence that compounds endowed with imidazoline-like structures affect the cholinergic motor activity of the guinea-pig ileum by interacting with presynaptic alpha2-adrenoceptors belonging to the alpha2D subtype.

AMPLIFICATION OF α1D-ADRENOCEPTOR MEDIATED CONTRACTIONS IN RAT AORTIC RINGS PARTIALLY DEPOLARISED WITH KCL

Partial depolarisation of smooth muscle in endothelium-denuded rat aortic ring preparations, by increasing physiological buffer KC1 concentrations from 4.7 to 14.7 mM, produced a leftward shift of concentration response curves (CRCs) to the alpha 1-adrenoceptor agonist noradrenaline (NA), phenylephrine and methoxamine, without changing maximal responses, whereas maximal responses to clonidine (CLON), also an alpha 1-agonist in this tissue were considerably increased. Partial depolarisation did not alter responses to 10 nM NA or 100 nM CLON in Ca2+(-free) buffer, but significantly increased the contractions obtained on adding Ca2+ back in the presence of the agonists. The potentiation of NA (2.5 and 5 nm) contractions by partial depolarisation was prevented by the voltage-operated Ca2+ channel (VOCC) antagonist nifedipine (NIF, 1 microM). NIF did not significantly affect NA CRCs in 4.7 mM KCl, whereas responses in 14.7 mM KCl were significantly decreased, indicating VOCC recruitment by NA only in the latter condition. Initial depletion of intracellular Ca2+ stores with 1 microM thapsigargin (THAP) in Ca2+(-free) buffer did not alter NA CRCs subsequently obtained in normal Ca2+. However, after THAP-pretreatment, these NA responses (in both 4.7 and 14.7 mM KC1) were attenuated by NIF, indicating that VOCCs were activated by NA in THAP-treated tissues. SKF 96365 (SKF, 30 microM), which can block VOCC and non-VOCC routes of extracellular Ca2+ influx, inhibited NA responses in 4.7 mM and 14.7 mM KCl, possibly implying a role for both types of Ca2+ entry in contractions. However, the greater inhibitory effects of SKF in THAP-pretreated tissues, probably reflected the mobilisation of VOCCs by NA following THAP exposure, because SKF was shown separately to block VOCC-mediated contractions in tissues depolarised with 100 mM KCl alone. 10 microM niflumic acid, an inhibitor of Ca2+(-activated) Cl- channels, did not affect responses to NA in 4.7 mM or 14.7 mM KC1, suggesting that VOCC opening induced by NA in 14.7 mM KCl was not due to depolarisation produced by alpha 1-adrenoceptor induced Cl- efflux. CRCs for NA were unaffected by pretreatment of rings with 100 ng ml-1 pertussis toxin (PT), suggesting a lack of involvement of PT-sensitive G proteins in the contractions obtained either in 4.7 or 14.7 mM KCl. BMY 7378 (100 microM), a selective antagonist for alpha 1D-adrenoceptors, competitively inhibited NA contractions with apparent pKB values of 8.7 +/- 0.2 and 8.4 +/- 0.1 in 4.7 mM and 14.7 mM KCl, respectively. Pretreatment of rings with chloroethylclonidine (100 microM), an irreversible antagonist of alpha 1B-and alpha 1D-adrenoceptors, produced similar rightward shifts in CRCs to NA by 3.2 +/- 0.2 and 3.7 +/- 0.3 log concentration units in 4.7 mM and 14.7 mM KCl, respectively, without changing maximal responses. Inositol phosphate (IP) turnover produced by NA in aortic rings was not significantly different in 4.7 mM compared with 14.7 mM KCl. As a whole, these results suggest that partial depolarisation of the rat aorta with KCl enhances alpha 1-adrenoceptor mediated contractions predominantly via the alpha 1D-subtype, and by a mechanism to be identified which allows greater recruitment of VOCCs by NA. In addition, the ability of THAP-pretreatment also to enhance VOCC activation by NA suggests that Ca2+ release from, or prevention of its reuptake into, intracellular stores may contribute to those processes leading to VOCC opening.

Characterization of alpha-adrenoceptors in canine mesenteric vein.

The alpha-adrenergic receptors (alpha-ARs) in canine mesenteric vein (DMV) were studied by using nonselective agonists and selective antagonists in functional studies and in ligand binding to classify the subtypes present. Based on functional studies of phenylephrine (PE)-induced contractions and ligand-binding interactions of [3H]-prazosin with prazosin (PR), WB 4101 (WB), 5-methylurapidil (5-MU), BMY 7378, and SK&F 105854, and pretreatment with chloroethylclonidine (CEC), DMV alpha1-ARs resembled the alpha1D subtype. However, the affinity of PR assessed in functional and ligand-binding studies was less (pK(B,D,i) < or = 9) than expected from previous characterization of cloned rat or human alpha1-AR (pKi > or = 10). Interactions with 5-MU, BMY 7378, or SK&F 105854 suggested the presence of some alpha1-ARs that were not typical of alpha1D-AR and that binding and functional interactions did not yield corresponding results. PR binding was abolished by treatment with CEC, contractile responses to PE were reduced in Emax, and the concentration-effect curve shifted to the left, as previously reported. DMVs contracted in response to alpha2-AR agonists and were studied when contractions were potentiated by increasing extracellular KCl to 20 mM. Rauwolscine (RAU) had K(B) values at these sites consistent with K(D) values in binding studies. CEC had no effect on RAU binding in DMV. Ligand-binding studies to [3H]-RAU sites did not reveal a clear identification of subtype, but these alpha2-ARs were clearly not alpha2B-ARs. We conclude that canine mesenteric vein contains alpha1D-like ARs, but with significant differences, and an unclassifiable alpha2-AR. There may also be a smaller population of other, not alpha1D-like ARs, receptors, mediating responses to PE and binding of prazosin.

Alpha-Methylnoradrenaline-induced contractions in rat aorta are mediated via alpha 1D-adrenoceptors.

The subtype(t) of alpha-adrenoceptor-mediating contractions to alpha-methynoradrenaline in the rat aorta has been investigated by using alpha-adrenoceptor-selective competitive antagonists and the alpha 1-adrenoceptor selective agonist, phenylephrine, for comparison. alpha-Methylnoradrenaline and phenylephrine elicited concentration-dependent contractions in the endothelium-denuded and endothelium-intact aortic rings with similar potencies and maximal effects. alpha-Methylnoradrenaline- and phenylephrine-induced contractions in endothelium-denuded aortic rings were competitively antagonized by prazosin (pA2 = 9.38 and 9.13; respectively) and rauwolscine (pA2 = 7.19 and 6.60, respectively). This confirms that there is an alpha 1- and a non alpha 2-adrenoceptor response to alpha-methylnoradrenaline in the rat aorta. The subtype selective alpha 1D-adrenoceptor antagonist, BMY 7378, was found to antagonize contractions to alpha-methylnoradrenaline and phenylephrine competitively in endothelium-denuded and endothelium-intact aortic rings. The pA2 values of BMY 7378 against alpha-methylnoradrenaline (8.39 and 8.41; endothelium-intact and endothelium-denuded, respectively) and phenylephrine (8.64 and 8.76; endothelium-intact and endothelium-denuded, respectively), are consistent with its published functional potency and clonal alpha 1D-adrenoceptor binding affinity. In addition, contractions to alpha-methylnoradrenaline and phenylephrine in endothelium-denuded aortic rings, were potently inhibited by WB 4101 with pA2 values of 9.75 and 9.25, respectively. The high pA2 values for WB 4101 indicate that the alpha 1B-adrenoceptor subtype does not seem to participate in alpha-methylnoradrenaline (and phenylephrine) induced contractions in this artery. These results suggest that the alpha 1D-subtype plays a determining role in rat aorta contractions induced by alpha-methylnoradrenaline.