Transition probability cell cycle model. Part I--Balanced growth

In this study, we have identified and characterized functional alpha2-adrenergic receptor (alpha2-AR) subtypes in human corpus cavernosum and in cultured human corpus cavernosum smooth muscle cells. Analysis of total RNA, isolated from whole corpus cavernosum tissue and smooth muscle cells, by RNase protection assays, demonstrated expression of mRNA for alpha2A, alpha2B, and alpha2C adrenergic receptor subtypes in whole tissue and alpha2A and alpha2C subtypes in cultured smooth muscle cells. Binding studies with [3H]RX821002 (a highly selective and specific ligand for alpha2-adrenergic receptor) in isolated membrane fractions of human corpus cavernosum smooth muscle cells, demonstrated specific alpha2-AR binding sites with high affinity (Kd = 0.63 nM) and limited capacity (25-30 fmol/mg protein). Binding of [3H]RX821002 was displaced with the nonselective alpha-AR antagonist, phentolamine, and with the alpha-AR agonist, norepinephrine, in a dose-dependent manner, but not by the selective alpha1-AR agonist, phenylephrine. Binding of [3H]rauwolscine was also displaced by phentolamine. UK 14,304, a selective alpha2-AR agonist, inhibited forskolin-induced cyclic adenosine monophosphate (cAMP) synthesis in cultured human corpus cavernosum smooth muscle cells and induced dose-dependent contractions of tissue strips in organ bath chambers. UK 14,304-induced contractions were inhibited with alpha2-AR selective antagonists, rauwolscine and delquamine (RS 15385-197). These observations suggest that in human corpus cavernosum, norepinephrine (NE) and epinephrine may activate postsynaptic alpha2-AR subtypes, in addition to activating alpha1-AR subtypes, on smooth muscle cells, contributing to local control of human corpus cavernosum smooth muscle tone, in vivo.     

Study of isolated apparent amniogenic limb deficiency in Hungary, 1975-1984

Rat brain cortex slices and synaptosomes preincubated with [3H]noradrenaline were used to investigate whether the NMDA-evoked noradrenaline release is modulated by agonists or antagonists at presynaptic alpha 2-adrenoceptors. In experiments on slices, noradrenaline and the preferential alpha-adrenoceptor agonists talipexole (former B-HT 920) and clonidine inhibited the NMDA-evoked tritium overflow whereas the selective alpha 1-adrenoceptor agonists cirazoline and methoxamine were ineffective. The alpha 2-adrenoceptor antagonists rauwolscine and idazoxan facilitated the NMDA-evoked tritium overflow whereas the preferential alpha 1-adrenoceptor antagonist prazosin was ineffective. The concentration-response curve of talipexole for its inhibitory effect on NMDA-evoked overflow was shifted to the right by idazoxan (apparent pA2 = 7.5). The EC50 of NMDA (97 mumol/l) for its stimulating effect on tritium overflow was not substantially changed by blockade of alpha 2-autoreceptors with 1 mumol/l rauwolscine (EC50 of NMDA in the presence of the alpha 2-adrenoceptor antagonist, 155 mumol/l), but the maximal overflow of tritium was increased 2.5 fold by this rauwolscine concentration. In experiments on synaptosomes, talipexole and noradrenaline inhibited the NMDA-evoked tritium overflow. The inhibitory effect of talipexole was abolished by idazoxan which, given alone, was ineffective, as was prazosin. Talipexole did also not produce an inhibition when tritium overflow was evoked by NMDA in the presence of omega-conotoxin GVIA 0.1 mumol/l; the latter, by itself, decreased the response to NMDA by about 55%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysophosphatidylcholine potentiates vascular contractile responses by enhancing vasoconstrictor-induced increase in cytosolic free Ca2+ in rat aorta

We investigated the effects of palmitoyl-L-alpha-lysophosphatidylcholine on the contractile responses of the endothelium-denuded rat aorta to high K+, noradrenaline, UK14,304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) (a selective alpha2 adrenoceptor agonist) and phorbol 12-myristate 13-acetate (PMA). Lysophosphatidylcholine at concentrations from 10(-6) M to 10(-4) M did not contract aortic strips. However, lysophosphatidylcholine strongly potentiated the UK14,304-induced contraction. High K+ - and PMA-induced contractions were also potentiated. In contrast, the noradrenaline-induced contraction was only slightly potentiated by 10(-5) M lysophosphatidylcholine. In fura PE-3-loaded aortic strips, lysophosphatidylcholine (10(-5) M) markedly augmented the increase in both cytosolic free Ca2+ ([Ca2+]i) and contractile tension induced by UK14,304, high K+ and PMA. Nicardipine (10(-7) M) and 10(-6) M Ro-31-8220 (?1-[3-(amidinothio)propyl-1H-indoyl-3-yl]-3-(1-methyl-1H-++ +indoyl-3-yl)-maleimide-methane sulfate) strongly inhibited the increase in [Ca2+]i and contractile tension induced by UK14,304 and in the presence of these inhibitors, the enhancing effects of lysophosphatidylcholine were attenuated. However, the enhancing effect on high K+ -induced contraction was not affected by Ro-31-8220. These results suggest that lysophosphatidylcholine may cause an augmentation of the increase in [Ca2+]i induced by UK14,304 which response is depend on protein kinase C activation and in this way potentiate contractile responses in the rat aorta. Protein kinase C independent mechanisms may also be involved in the enhancing effect of lysophosphatidylcholine on smooth muscle contraction.     

Norepinephrine inhibits neurons of the intermediate subnucleus of the lateral septum via alpha2-adrenoceptors

The physiological and pharmacological actions of norepinephrine (NE) on neurons of the intermediate subnucleus of the lateral septum (LSI) were examined using intracellular recordings in rat brain-slices. Bath-applied NE inhibited 72.5%, excited 5.5% and had no effect on 22% of LSI neurons tested; this study focused on the inhibitory effects of NE. In current clamp recordings, 100 microM NE produced a hyperpolarization of 10.82+/-0.72 mV (n=84) with a decrease in input resistance. In voltage-clamp, NE produced a direct, post-synaptic outward current of 206.8+/-22 pA (n=37) with a 64. 3+/-4.9% increase in input conductance (IC50-17.7+/-4 microM). The NE-induced inhibition was mimicked by the alpha2-agonist, UK14,304, but not by the alpha1- or beta-adrenoceptor agonists. The alpha2-agonist, clonidine, had a weak effect in LSI neurons. Interestingly, the magnitude of the UK14,304-induced response varied between cells (ranging from 29.5 to 320% of the maximal NE inhibition), possibly suggesting the involvement of alpha2A-(high affinity for UK14,304) and non-alpha2A (low affinity for UK14,304) adrenoceptor subtypes. While the alpha2-antagonists, yohimbine, rauwolscine and idazoxan blocked NE-induced inhibition in all neurons tested, the prototypical alpha1-antagonist, prazosin produced a variable degree of block (9-58%), further indicating the possible involvement of alpha2A (prazosin-insensitive) and non-alpha2A (prazosin-sensitive) receptors. However a lack of more selective pharmacological tools precludes definitive classification of the alpha2-receptor-mediated responses into different subtypes. The alpha2-receptor-mediated current in LSI neurons displayed Ba2+-sensitive inward rectification, reversed polarity near EK and was sensitive to external K+. In conclusion, NE inhibits LSI neurons via alpha2-adrenoceptor subtypes.     

American Pediatric Society John Howland Award 1998: presentation and acceptance

In the dog saphenous vein (DSV), phenylephrine (PE) responses through alpha-1 adrenoceptors receptors are antagonized by both alpha-1 and alpha-2 receptor antagonists. Furthermore, pretreatment with chloroethylclonidine (CEC) eliminates prazosin binding but reduces rauwolscine binding by half (). In new functional experiments, the effects of preincubation with phenoxybenzamine (PBZ), an irreversible alpha adrenoceptor antagonist, on responses to PE and two selective alpha-2 adrenoceptor agonists were evaluated. Also, the ability of prazosin or rauwolscine to prevent irreversible losses of responses to these agonists when coincubated with PBZ was determined. Preincubation in PBZ (10-300 nM) concentration dependently reduced PE Emax and the calculated fraction of residual receptors (q). Preincubation in PBZ (10-300 nM) increased KB values for prazosin (30 and 100 nM) but did not alter the KB value for rauwolscine (50 nM) acting at the residual receptors from control values. Coincubation of PBZ with prazosin partially prevented these PBZ actions (Emax partly restored) on responses to PE, but coincubation of rauwolscine (/=300 nM caused >50% reduction in Emax values of responses but did not alter the EC50 values for either agonist. Coincubation of rauwolscine with PBZ protected responses to alpha-2 agonists against PBZ (1 microM) effects. This study shows that PE initiates contractions at atypical alpha-1 adrenoceptors represented by all sites of PE action. Rauwolscine antagonizes PE actions but does not protect against PBZ inactivation. Typical alpha-2 adrenoceptors are distinguished from the unusual alpha-1 adrenoceptors by their lesser sensitivity to PBZ and their protection by rauwolscine from PBZ.     

Prognostic values of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 expression in bladder cancer

1. The alpha1-adrenoceptor population mediating contractile responses to noradrenaline (NA) in smooth muscles of the bladder neck from rabbit (RBN) has been characterized by use of quantitative receptor pharmacology. 2. Experiments with several 'key' alpha1-adrenoceptor antagonists of varying subtype selectivities (RS-17053, BMY 7378, indoramin, 5-methylurapidil, prazosin, REC 15/2739, SNAP 5089, terazosin, WB 4101, tamsulosin, (+)-cyclazosin and RS-100329) were conducted. Schild regression analyses yielded affinity (mean pKb) estimates of 7.1, 6.2, 8.6, 8.6, 8.4, 9.3, 7.0, 7.4, 8.9, 10.0, 7.1 and 9.3, respectively, although deviations from unit Schild regression slope question the robustness of data for RS-17053 and SNAP 5089. 3. The nature of antagonism by these agents and the profile of affinity determinations generated together suggest that a single alpha1-adrenoceptor subtype mediates contractile responses of RBN to NA. Additional studies with phenylephrine indicated also an agonist-independence of this profile. Pharmacologically, this profile was reminiscent of that described as 'alpha1L'-adrenoceptor, which has been shown to mediate contractions of several tissues including lower urinary tract (LUT) tissues of man. Furthermore, a similarity was noticed between the 'alpha1L'-adrenoceptor described here in RBN and the rabbit and human cloned alpha1a-adrenoceptor (based on data from both whole cell radioligand binding at 37 degrees C and [3H]-inositol phosphates accumulation assays), characterizations of which have been published elsewhere. 4. In conclusion, the RBN appears to provide a predictive pharmacological assay for the study of NA-induced smooth muscle contraction in LUT tissues of man.     

Characterization of postsynaptic alpha-1 and alpha-2 adrenoceptors in the feline saphenous and femoral veins [corrected

Experiments were designed to characterize the effects mediated by alpha-1 and alpha-2 adrenoceptors in saphenous and femoral veins of the cat. Ring segments of saphenous and femoral veins were mounted for isometric tension recording in modified Krebs-bicarbonate solution, gassed with 95% O2-5% CO2 and maintained at 37 degrees C. Norepinephrine (a mixed alpha 1 and alpha 2 agonist), phenylephrine (a preferential alpha 1 agonist) and clonidine (a preferential alpha 2 agonist) caused dose (concentration)-dependent contractions in saphenous and femoral veins. The maximal contractions produced by clonidine were significantly less than those produced by norepinephrine or phenylephrine in both veins. However, threshold dose and EC50 values indicated that clonidine was more potent than norepinephrine and phenylephrine. Contractile responses to these agonists were attenuated when the veins were pretreated with alpha 1-or alpha 2-adrenoceptor antagonists, prazosin and yohimbine, respectively. The contractile responses to norepinephrine and tyramine were inhibited to a greater extent by yohimbine than by prazosin in both saphenous and femoral veins, suggesting that norepinephrine released from perivascular nerve terminals activates preferentially postsynaptic alpha 2-adrenoceptors. Further examination of alpha-adrenoceptor subtypes was achieved by comparing pA2 values of prazosin and yohimbine from Arunlakshana and Schild plots. Chronic sympathetic denervation by removing lumbar sympathetic chain significantly reduced the contractile responses evoked by tyramine. Denervation did not significantly affect the concentration-response curve to phenylephrine but significantly augmented the contractile responses evoked by clonidine in both veins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of alpha 2-adrenoceptors mediating contraction of dog saphenous vein: identity with the human alpha 2A subtype

1. In the dog saphenous vein alpha 1- and alpha 2-adrenoceptors mediate noradrenaline-induced contractions in vitro. In order to study the alpha 2-adrenoceptor in isolation, alpha 1-adrenoceptors were inactivated by treatment of tissues with the alkylating agent phenoxybenzamine (3.0 microM for 30 min) in the presence of rauwolscine (1 microM) to protect alpha 2-adrenoceptors. 2. Noradrenaline-induced contractions of tissues treated with phenoxybenzamine were antagonized competitively by the selective alpha 2-adrenoceptor antagonist rauwolscine, pKB = 8.63 +/- 0.07 (means +/- s.e. mean; n = 3), consistent with an interaction at alpha 2-adrenoceptors. 3. Noradrenaline was a full agonist at alpha 2-adrenoceptors in dog saphenous vein. By use of the method of partial receptor alkylation and analysis of concentration-effect curve data by direct, operational model fitting methods, the affinity (pKA) and efficacy (tau) were 5.74 +/- 0.07 and 7.50 +/- 1.05, respectively (n = 6). Nine other agonists which were examined each had affinities higher than noradrenaline, but with the exception of the imidazoline, A-54741 (5,6-dihydroxy-1,2,3,4-tetrahydro-1-naphthyl-imidazoline) had relatively lower efficacies. 4. To compare the alpha 2-adrenoceptor in dog saphenous vein to the human recombinant subtypes, the affinities of twenty-one compounds were estimated in functional studies in the dog saphenous vein and in radioligand binding studies for the human alpha 2A, alpha 2B and alpha 2C receptor subtypes expressed in Chinese hamster lung (CHL) cells. 5. Of twenty-one compounds examined in ligand binding studies, only nine had greater than ten fold selectivity for one human receptor subtype over either of the other two. These compounds were A-54741, oxymetazoline, guanfacine, guanabenz, prazosin, spiroxatrine, tolazoline, WB 4101 and idazoxan. In dog saphenous vein, their affinities (pKA and pKB for agonists and antagonists respectively) were: A-54741 (pKA = 8.03 +/- 0.05), oxymetazoline (pKA = 7.67 +/- 0.09), guanfacine (pKA = 6.79 +/- 0.03); guanabenz (pKA = 7.02 +/- 0.13); prazosin (pKB = 5.19 +/- 0.08), spiroxatrine (pKB = 6.59 +/- 0.04), tolazoline (pKB = 6.21 +/- 0.07), WB 4101 (pKB = 7.42 +/- 0.09) and idazoxan (pKB = 7.11 +/- 0.08). 6. Comparisons of affinity estimates for these nine compounds at the receptor in dog saphenous vein and at the human recombinant subtypes suggest that the vascular receptor is most similar to the h alpha 2A subtype; correlation coefficients (r) were 0.82 (h alpha 2A), 0.24 (h alpha 2B) and 0.04 (h alpha 2C).     

Active immunization using dendritic cells mixed with tumor cells inhibits the growth of primary breast cancer

The potential role of alpha2-adrenoceptors in modulating the activity of adenylyl cyclase in the rat striatum was examined. The selective alpha2-adrenoceptor agonist, UK14,304, produced a concentration-dependent inhibition of forskolin-stimulated accumulation of cAMP in striatal slices. The effect of UK14,304 was reversed by pre-incubation of striatal slices with the selective alpha2-adrenoceptor antagonist, RX821002. To determine whether alpha2C-adrenoceptors contribute to the alpha2-adrenoceptor-induced inhibition of forskolin-stimulated cAMP accumulation, an antisense oligodeoxynucleotide directed against alpha2C-adrenoceptor mRNA (alpha(2C)AS) or a random sequence (RS) was infused directly into the striatum. The ability of alpha(2C)AS to reduce the expression of alpha2C-adrenoceptors has been previously demonstrated. Alpha2C(AS) infusions did not reduce the ability of UK14,304 to inhibit forskolin-stimulated cAMP accumulation. Instead, alpha(2C)AS significantly enhanced forskolin-stimulated cAMP accumulation on the infusion side compared to the contralateral striatum. In contrast to the effects of alpha(2C)AS, infusions of RS had no effects on forskolin-stimulated cAMP accumulation or on the ability of UK14,304 to inhibit this effect. Incubation of striatal slices from untreated rats with RX821002 could mimic the ability of alpha(2C)AS infusion to enhance forskolin-stimulated cAMP accumulation, and did so in a concentration-dependent manner. Alpha2-adrenoceptors are negatively coupled to adenylyl cyclase in the rat striatum and alpha2C-adrenoceptors appear to be under tonic activation by an endogenous ligand in striatal slices.     

Unusual alpha-adrenoceptor subtype in canine saphenous vein: comparison to mesenteric vein

1. We investigated the nature of the adrenoceptors in the dog saphenous vein (DSV) and dog mesenteric vein (DMV) to determine the nature of the unexpected interactions of phenylephrine and methoxamine with rauwolscine in the DSV, i.e. the ability of the putative alpha 2-adrenoceptor antagonist to inhibit competitively contractions to these alpha 1-agonists. Radioligand binding studies were performed in parallel with contractility studies. 2. Functionally, in the DSV, phenylephrine and methoxamine-induced, contractions were antagonized by rauwolscine with Schild slopes of -0.52 and -0.46, respectively and apparent pA2 values of 8.5 and 9.2, respectively. Such antagonism was not observed in the DMV. In the DSV, prazosin competes for [3H]-rauwolscine binding sites with a high and a low affinity binding site (Ki of 1.49 +/- 0.65 and 94.7 +/- 51 microM, n = 6, respectively). 3. Pretreatment with 100 microM chloroethylclonidine (CEC) for 15 min abolished [3H]-prazosin binding in microsomes from both veins and reduced binding (Bmax) of [3H]-rauwolscine in microsomes by 55.1 +/- 0.8% (n = 3) in the DSV but did not affect the Bmax in the DMV. CEC pretreatment in the venular rings denuded of endothelium caused persistent contraction in the DSV but not in the DMV. In the DSV, CEC appeared to interact with a single [3H]-rauwolscine binding site. In both the DSV and the DMV, CEC (100 microM) caused a significant shift in the EC50 values for phenylephrine and methoxamine. Maximum responses in the DMV were significantly attenuated while those in the DSV were unaffected when total tension was considered. 4. Studies of the functional interactions of the DSV and the DMV with WB 4101 or 5-methylurapidil (5-MU) suggested the presence of alpha 1D-adrenoceptors in the DSV and alpha 1A-adrenoceptors in the DMV. The receptors inactivated by CEC in the DMV and DSV may represent some or all of the receptors with properties of alpha 1D and alpha 1A-receptors present in the two veins. Studies of radioligand binding interactions of these two antagonists with [3H]-prazosin, were consistent with the presence of some alpha 1D-receptors in DSV and alpha 1A-receptors in DMV. These findings raise questions about the selectivity of CEC in differentiating alpha 1-adrenoceptor subtypes. 5. B-HT 920 caused contractions in the DSV smaller than those to the alpha 1-agonists but the maximum was not affected by CEC pretreatment. The EC50 values were shifted to the left after CEC. In radioligand binding studies, B-HT 920 competition for [3H]-rauwolscine binding was not significantly affected by CEC pretreatment. 6. These results suggest the presence of unusual alpha-adrenoceptors in the DSV. In addition to alpha 2-adrenoceptors, receptors recognizing rauwolscine as well as prazosin, WB 4101, phenylephrine and methoxamine and susceptible to inactivation by CEC are present. They appear to be, in part, unusual alpha 1D-adrenoceptors.     

Alpha2-adrenoceptor-mediated contractions of the porcine isolated ear artery: evidence for a cyclic AMP-dependent and a cyclic AMP-independent mechanism

1. The aim of this study was to determine the conditions under which the alpha2-adrenoceptor agonist UK14304 produces vasoconstriction in the porcine isolated ear artery. 2. UK14304 (0.3 microM) produced a small contraction of porcine isolated ear arteries which was 7.8+/-3.3% of the response to 60 mM KCl. Similar sized contractions were obtained after precontraction with either 30 nM angiotensin II, or 0.1 microM U46619 (8.2+/-1.8% and 10.2+/-2.6% of 60 mM KCl response, respectively). However, an enhanced alpha2-adrenoceptor response was uncovered if the tissue was precontracted with U46619, and relaxed back to baseline with 1-2 microM forskolin before the addition of UK14304 (46.9+/-9.6% of 60 mM KCl response). 3. The enhanced responses to UK14304 in the presence of U46619 and forskolin were not inhibited by the alpha1-adrenoceptor antagonist prazosin (0.1 microM), but were inhibited by the alpha2-adrenoceptor antagonist rauwolscine (1 microM), indicating that the enhanced responses were mediated via postjunctional alpha2-adrenoceptors. 4. In the presence of 0.1 microM U46619 and 1 mM isobutylmethylxanthine (IBMX), 1 microM forskolin produced an increase in [3H]-cyclic AMP levels in porcine isolated ear arteries. Addition of 0.3 microM UK14304 prevented this increase. 5. The enhanced UK14304 response was dependent upon the agent used to relax the tissue. After relaxation of ear arteries precontracted with 10 nM U46619 and relaxed with forskolin the UK14304 response was 46.9+/-9.6% of the 60 mM KCl response, and after relaxation with sodium nitroprusside (SNP) the response was 24.8+3.3%. However, after relaxation of the tissue with levcromakalim the UK14304 response was only 8.2+/-1.7%, which was not different from the control response in the same tissues (12.2+/-5.6%). An enhanced contraction was also obtained after relaxation of the tissue with the cyclic AMP analogue dibutyryl cyclic AMP (23.2+/-1.3%) indicating that at least part of the enhanced response to UK14304 is independent of the ability of the agonist to inhibit cyclic AMP production. 6. Relaxation of U46619 contracted ear arteries with SNP could be inhibited by the NO-sensitive guanylyl-cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) indicating that production of cyclic GMP is necessary for the relaxant effect of SNP. However, ODQ had no effect on the relaxation of tissue by forskolin, suggesting that this compound does not act via production of cyclic GMP. Biochemical studies showed that while forskolin increases the levels of cyclic AMP in the tissues, SNP had no effect on the levels of this cyclic nucleotide. 7. In conclusion, enhanced contractions to the alpha2-adrenoceptor agonist UK14304 can be uncovered in porcine isolated ear arteries by precontracting the tissue with U46619, followed by relaxation back to baseline with forskolin, SNP or dibutyryl cyclic AMP before addition of UK14304. There was a greater contractile response to UK14304 after relaxation with forskolin than with SNP or dibutyryl cyclic AMP, suggesting that cyclic AMP-dependent and- independent mechanisms are involved in the enhancement of the UK14304 response.     

Expression of two alpha 2-adrenergic receptor subtypes in human placenta: evidence from direct binding studies

Adrenergic receptors may play an important role for mediating a variety of metabolic and haemodynamic effects of catecholamines including placental blood flow. The alpha-adrenergic receptors of the human placenta were characterized in vitro by the use of [3H]rauwolscine and [3H]prazosin as radioligands. Saturation experiments would suggest that the alpha-adrenoceptors in the human placenta are alpha 2. Comparative binding studies were performed, using recently synthesized compounds (Beecham Pharmaceuticals, UK) selective for alpha 2A (BRL-44408) and alpha 2B (BRL-41992) subtypes. The results indicate that human placenta contains at least two pharmacologically distinct alpha 2-adrenoceptor subtypes with approximately 60 per cent alpha 2A and 40 per cent alpha 2B receptors. In contrast with the pattern of increasing beta-adrenoceptor density, the concentration of alpha 2-adrenoceptors in term placentae is significantly lower than in placentae from the first trimester.     

Tumor necrosis factor alpha inhibits contractions to sympathetic nerve stimulation by a nitric oxide-dependent mechanism

Gram-negative sepsis and administration of tumor necrosis factor alpha (TNF alpha) are associated with hypotension and peripheral neuropathies suggestive of impaired sympathetic neurotransmission. We examined the effect of TNF alpha on the responses of the bovine pulmonary artery (BPA) to transmural sympathetic nerve stimulation (SNS). BPA contracted to SNS (0.5-32 Hz, 5-10 V, 2-msec duration, 2-msec delay) in a frequency-dependent manner. The contractions of the BPA to SNS were mediated by norepinephrine and activation of postsynaptic alpha 1-adrenoceptors, since they were attenuated by prazosin. Maximum contraction of the BPA to SNS was significantly enhanced (148 +/- 37% increase, n = 6) after inhibition of nitric oxide synthase with L-NG-monomethylarginine (LNMMA, 500 microM), an effect abrogated by L-arginine (1 mM). TNF alpha (0.0042, 0.042, and 0.42 micrograms/ml) selectively inhibited contractions of the BPA to SNS without affecting the contraction of the BPA to exogenous norepinephrine. In BPA incubated with LNMMA (5-500 microM), TNF alpha facilitated rather than inhibited SNS. TNF alpha increased the formation of amperiometrically measured free nitric oxide in bovine adrenal chromaffin cells in primary culture. The data show that in the absence of LNMMA, TNF alpha releases free nitric oxide from a sympathetic neuron and selectively inhibits the contractions of the BPA to SNS. In BPA in which nitric oxide synthase I is inhibited by LNMMA, TNF alpha amplifies the contractions to SNS, even in the absence of endothelium. Thus, TNF alpha can modify vascular smooth muscle tone by affecting SNS. TNF alpha inhibits SNS at the level of the neuron by a mechanism involving the L-arginine-nitric oxide pathway. TNF alpha-induced suppression of SNS and neurotransmission may contribute to the hypotension and peripheral neuropathy of sepsis.     

Characteristics of adrenoceptors in the human radial artery: clinical implications

OBJECTIVES: The radial artery has been suggested to be spastic. Endogenous and exogenous catecholamines and the use of beta-blockers may be related to radial artery spasm, but the characteristics of adrenoceptors in this artery are unknown. This study was designed to characterize the alpha- and beta-adrenoceptor in the human radial artery. METHODS: Ring segments of the radial artery (n = 59) taken from patients undergoing coronary artery bypass grafting were studied in organ chambers. Alpha-adrenoceptor agonists (norepinephrine, methoxamine, and UK14304) and antagonists (phentolamine hydrochloride [INN: phentolamine], prazosin, and yohimbine) were used to characterize the alpha-adrenoceptor. Beta-adrenoceptor function was studied in U46619-precontracted rings in response to isoproterenol (INN: isoprenaline). RESULTS: Norepinephrine induced 6.9 +/- 0.6 gm (80.6% +/- 6.8% of the contraction by 100 mmol/L KCl), and this was almost fully inhibited by phentolamine hydrochloride (10 micromol/L, p < 0.0001). The contraction force induced by methoxamine (2.9 +/- 0.8 gm) was abolished by 0.5 micromol/L prazosin (p = 0.017). The contraction force induced by UK14304 (1.7 +/- 0.4 gm) was abolished by 1 micromol/L yohimbine. In contrast to the porcine coronary artery used as the control (fully relaxed to isoproterenol), radial artery rings did not have significant relaxation (1.1% +/- 0.8%). CONCLUSIONS: The human radial artery is an alpha-adrenoceptor-dominant artery with little beta-adrenoceptor function. The use of beta-blockers will not likely evoke the spasm of the radial artery. Furthermore, the radial artery has a dominant alpha1-adrenoceptor function, but the postjunctional alpha2-adrenoceptor is also functional. Circulating catecholamines will mainly contract the human radial artery by activation of the alpha1-adrenoceptors and to a lesser extent also by alpha2-adrenoceptors.     

Norepinephrine-induced contraction of isolated rabbit bronchial artery: role of alpha 1- and alpha 2-adrenoceptor activation

The contractile effect of norepinephrine (NE) on isolated rabbit bronchial artery rings (150-300 microns in diameter) and the role of alpha 1- and alpha 2-adrenoceptors (AR) on smooth muscle and endothelium were studied. In intact arteries, NE increased tension in a dose-dependent manner, and the sensitivity for NE was further increased in the absence of endothelium. In intact but not in endothelium-denuded arteries, the response to NE was increased in the presence of both indomethacin (Indo; cyclooxygenase inhibitor) and NG-nitro-L-arginine methyl ester [L-NAME; nitric oxide (NO) synthase inhibitor], indicating that two endothelium-derived factors, NO and a prostanoid, modulate the NE-induced contraction. The alpha 1-AR antagonist prazosin shifted the NE dose-response curve to the right, and phenylephrine (alpha 1-AR agonist) induced a dose-dependent contraction that was potentiated by L-NAME or removal of the endothelium. The sensitivity to NE was increased slightly by the alpha 2-AR antagonists yohimbine and idazoxan, and this effect was abolished by Indo or removal of the endothelium. Similarly, contractions induced by UK-14304 (alpha 2-AR agonist) were potentiated by Indo or removal of the endothelium. These results suggest that NE-induced contraction is mediated through activation of alpha 1- and alpha 2-ARs on both smooth muscle and endothelium. Activation of the alpha 1- and alpha 2-ARs on the smooth muscle causes contraction, whereas activation of the endothelial alpha 1- and alpha 2-ARs induces relaxation through release of NO (alpha 1-ARs) and a prostanoid (alpha 2-ARs).     

Overcoming apoptosis: new methods for improving protein-expression systems

This study was designed to further discriminate alpha1-adrenoceptor subtypes in rat aorta and prostate using functional experiments. Responses induced by phenylephrine were equilibrated in both tissues. The pA2 values and slope factors of several alpha1-antagonists were assessed using concentration-response curves. The antagonists used were prazosin, WB-4101, 5-methylurapidil (5-MU), HV-723, and tamsulosin. In addition, the effects of chloroethylclonidine (CEC) and nifedipine on phenylephrine-induced contractions were investigated. A high pA2 value for prazosin was observed in both tissues (aorta 9.84, prostate 9.19) and the ranking of each drug's pA2 value is as follows: tamsulosin > prazosin > WB-4101 > HV-723 > 5-MU in the aorta, and tamsulosin > prazosin > 5-MU > WB-4101 = HV-723 in the prostate. A significant difference between the pA2 value of each drug except for tamsulosin in the aorta and in prostate was observed (p < 0.01). Inhibition of contraction by pretreatment with CEC was 83.9 +/- 2.42% in the aorta, and 6.17 +/- 0.94% in the prostate. On the other hand, inhibition of maximal response by pretreatment with nifedipine (1 micromol/l) was 35.1 +/- 2.2% in the aorta and 24.5 +/- 3.1% in the prostate. A good correlation between these pA2 values and pKi values for recombinant human alpha1b-adrenoceptor expressed in CHO cells (aorta) and alpha1a-subtypes of CEC pretreated rat hippocampus (prostate) were observed. In conclusion, these results suggest that: (1) the contraction of these two tissues is mediated by alpha1H-adrenoceptor with a high affinity for prazosin; (2) alpha1H-adrenoceptors correspond to alpha1b-(aorta) and alpha1a-subtypes (prostate), and (3) each alpha1-adrenoceptor subtype in the aorta and prostate may be alpha1b-(aorta) and alpha1a-subtypes (prostate), respectively.     

Cardiovascular effects of oxymetazoline and UK14,304 in conscious and pithed rats

Relatively selective alpha 2-adrenoceptor agonists have proven useful in a variety of therapeutic situations including hypertension, glaucoma and withdrawal from opiate addiction. In particular, oxymetazoline (OXY) and UK14,304 (UK) have been used in subclassifying alpha 2-adrenoceptors and imidazoline receptors. We evaluated the cardiovascular effects of OXY and UK in conscious and pithed rats in the presence and absence of efaroxan (EFA), idazoxan (IDA) and rauwolscine (RAU). Both OXY or UK (1, 5 and 10 micrograms/kg, i.v.) increased blood pressure (BP) and reduced heart rate (HR) in conscious rats. In pithed rats, OXY and UK each increased BP to a greater extent than that observed in conscious rats, but HR was not affected. BP increases following sympathetic nerve stimulation in the pithed rats were not affected by OXY but were reduced by UK at 0.1 Hz and 0.3 Hz. HR responses to nerve stimulation in pithed rats were reduced after OXY at all frequencies, but only at 0.1 Hz following UK. EFA, IDA and RAU inhibited the pressor responses of UK, with EFA being most potent. OXY-induced pressor responses were inhibited by all three antagonists, RAU being the least potent. HR responses to either OXY or UK were not affected by the antagonists. Taken together, the data suggest that: 1) alpha 2-adrenoceptors contribute less to the vascular response to OXY than to UK based upon the antagonistic effect of RAU; 2) prejunctional I1 receptors maybe more prevalent in the heart than in vascular tissue based upon the response to OXY in pithed rats. Thus, the heterogeneity among receptors mediating cardiac and vascular responses are complex.     

Dopamine releases endothelium-derived relaxing factor via alpha 2-adrenoceptors in canine vessels: comparisons between femoral arteries and veins

1. We investigated the role of vascular smooth muscle alpha-adrenoceptor subtypes in the vasoconstrictor response of femoral arteries and veins to dopamine and whether the vasoconstriction is modified by endothelium-dependent relaxation mediated via the activation of alpha 2-adrenoceptors in ring preparations of femoral arteries and veins from mongrel dogs. 2. Dopamine contracted both arteries and veins in a dose-dependent manner and this contraction was inhibited by pretreatment with phentolamine or prazosin. Pretreatment with yohimbine shifted the dose-response curve for dopamine to the right in femoral veins, but not in arteries. 3. Phenylephrine contracted femoral arteries and veins in a dose-dependent manner and this contraction was inhibited by pretreatment with prazosin. 4. Clonidine produced a bell-shaped dose-response curve in femoral veins and this curve was shifted upwards by pretreatment with NG-nitro-L-arginine (L-NNA). In contrast, femoral arteries were not affected by clonidine. NG-Nitro-L-arginine potentiated contractile responses to dopamine in both veins and arteries. This potentiation was inhibited by yohimbine or by the removal of the endothelium in both arteries and veins. 5. These results suggest that dopamine contracts femoral arteries via stimulation of alpha 1-adrenoceptors and contracts femoral veins via stimulation of both alpha 1- and alpha 2-adrenoceptors and that these contractions are attenuated by the vasodilator action of dopamine via alpha 2-adrenoceptor-mediated release of endothelium-derived relaxing factor.     

Functional assessment of alpha 1-adrenoceptor subtypes in porcine coronary artery

1. alpha 1-Adrenoceptors are known to play an important role in vasoconstriction in response to adrenergic stimulation. However, the functional importance of alpha 1-adrenoceptor subtypes at the epicardial coronary artery remains unclear. We examined alpha 1-adrenoceptor subtypes by comparing functional affinities for alpha-adrenoceptor antagonists on noradrenaline (NA)-induced vasoconstriction in porcine denuded right coronary arteries. 2. Noradrenaline induced a dose-dependent vasoconstriction in incubated vessel rings. Prazosin and phentolamine were potent and competitive antagonists for NA-induced contraction (pA2 10.27 and 9.03, respectively). In contrast, the selective alpha 2-adrenoceptor antagonist yohimbine had a low affinity (pA2 6.13). Two selective alpha 1A-adrenoceptor antagonists, WB 4101 and 5-methyl urapidil, were potent and competitive antagonists of alpha 1-adrenoceptor-induced contraction (pA2 10.67 and 8.90, respectively) and the selective alpha 1D-adrenoceptor antagonist BMY 7378 had a low affinity (pA2 6.06). Noradrenaline-induced contraction was insensitive to the alkylating effects of chlorethylclonidine. These observations indicate that the vasoconstriction is predominantly mediated by the alpha 1A-adrenoceptor subtype. This was also supported by a good correlation between pA2 values from the present study and reported binding affinities (pKi) of various alpha-adrenoceptor antagonists with cloned human alpha 1A-adrenoceptors (r = 0.98), but not for alpha 1B- or alpha 1D-adrenoceptor subtypes (r = 0.77 and 0.41, respectively). 3. Our results indicate that the alpha 1A-adrenoceptor is the main functional receptor subtype in porcine denuded coronary arteries.     

Characterization of alpha1-adrenoceptor subtypes mediating contractions to phenylephrine in rat thoracic aorta, mesenteric artery and pulmonary artery

1. The subtype of alpha1-adrenoceptor mediating contractions to phenylephrine of the rat thoracic aorta, mesenteric artery and pulmonary artery were investigated by use of antagonists which show selectivity between the cloned alpha1-adrenoceptor subtypes in binding studies. 2. Cumulative concentration-contraction curves for phenylephrine were competitively antagonized in the rat thoracic aorta by prazosin (pA2 9.9), WB4101 (pA2 9.6), 5-methylurapidil (pA2 8.1), benoxathian (pA2 9.2) and indoramin (pA2 7.4). These compounds were also competitive antagonists in the mesenteric and pulmonary arteries (except for 5-methylurapidil in the pulmonary artery), (prazosin pA2 9.9 and 9.7; WB4101 pA2 9.8 and 9.6; 5-methylurapidil pA2 7.9 and pK(B) estimate 8.0; benoxathian pA2 8.8 and 9.3; indoramin pA2 7.2 and 7.5, respectively). 3. RS 17053 was not a competitive antagonist in any blood vessel as Schild plot slopes were greater than unity. The pK(B) estimates for RS 17053 were 7.1 in aorta, 7.0 in the mesenteric artery and 7.7 in the pulmonary artery. 4. The alpha1D-subtype selective antagonist BMY 7378 appeared to be non-competitive with shallow Schild plot slopes. The data were better fitted with two lines in all tissues, with Schild plot slopes that were no longer different from unity, except in the pulmonary artery. The higher affinity site for BMY 7378 in the aorta had a pA2 of 9.0, while it was 8.8 and 8.9 in the mesenteric and pulmonary arteries, respectively. 5. MDL73005EF acted in a non-competitive manner in all three blood vessels, with shallow Schild plot slopes. The pK(B) estimates for MDL73005EF were 8.4 in aorta, 7.5 in the mesenteric artery and 8.0 in the pulmonary artery. 6. In all three blood vessels the functionally determined antagonist affinity estimates correlated best with published pKi values for their displacement of [3H]-prazosin binding on membranes expressing cloned alpha1d-adrenoceptors compared with alpha1a- or alpha1b-adrenoceptors. The antagonist affinity estimates in the aorta, mesenteric and pulmonary arteries correlated highly with their previously published pA2 values in rat aorta (alpha1D) and less well with those for alpha1A- and alpha1B-adrenoceptors mediating contraction of the rat epididymal vas deferens and rat spleen, respectively. 7. The results of this study suggest that the contraction to phenylephrine of the rat thoracic aorta, mesenteric artery and pulmonary artery are mediated in part via the alpha1D-subtype of adrenoceptor. The data for both BMY 7378 and MDL73005EF in all three blood vessels are consistent with receptor heterogeneity. However, the identity of the second site is unclear.