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Aims: In patients with lower urinary tract symptoms suggestive of benign prostatic obstruction the alpha1-adrenoceptor antagonist terazosin lowers blood pressure whereas only very small if any alterations were reported with the alpha1-adrenoceptor antagonist tamsulosin. Therefore, we have compared the vascular alpha1-adrenoceptor antagonism of tamsulosin and terazosin directly., Methods: Ten healthy subjects were investigated in a randomized, single-blind, three-way cross-over design and received a single dose of 0.4 mg tamsulosin, 5 mg terazosin or placebo on 3 study days at least 1 week apart. Before and 1, 3, 5, 7, 10 and 23.5 h after drug intake, alterations of diastolic blood pressure and other haemodynamic parameters in response to a graded infusion of the alpha1-adrenoceptor agonist phenylephrine were determined non-invasively., Results: At most time points tamsulosin inhibited phenylephrine-induced diastolic blood pressure elevations significantly less than terazosin (5 h time point: median difference in inhibition 35%, 95% CI: 18.7-50.3%). On the other hand, phenylephrine-induced changes of cardiac output, heart rate and stroke volume were similar during both active treatments., Conclusions: In doses equi-effective for treatment of lower urinary tract symptoms tamsulosin causes less inhibition of vasoconstriction than terazosin.

In Rat-1 fibroblasts, endothelin-1 and a protein kinase C-stimulating phorbol ester stimulated extracellular signal-regulated kinase (ERK), whereas phenylephrine, acting at stably transfected human alpha1A-adrenoceptors, inhibited basal and endothelin-1- and phorbol ester-stimulated ERK. On the other hand, phenylephrine stimulated p38 mitogen-activated protein kinase (MAPK). Anisomycin caused p38 activation and ERK inhibition quantitatively similar to those produced by phenylephrine. SB 203,580, an inhibitor of p38, significantly attenuated phenylephrine- and anisomycin-induced ERK inhibition. The ERK inhibition by phenylephrine was not affected by the cytosolic phospholipase A2 inhibitor arachidonyltrifluoromethyl ketone or the cyclooxygenase inhibitor indomethacin but was significantly attenuated by a combination of the phosphatase inhibitors Na3VO4 and okadaic acid. Neither SB 203,580 nor the phosphatase inhibitors significantly affected ERK inhibition by the adenylyl cyclase activator forskolin. We conclude that there is a previously unrecognized interaction between ERK and p38 MAPK, in which activation of p38 causes inhibition of ERK; this may at least partly involve MAPK phosphatases that inactivate ERK.

Neuropeptide Y (NPY) can cause diuresis, natriuresis, and calciuresis in rats independently of the pressure-natriuresis mechanism (A. Bischoff and M. C. Michel. Pflügers Arch. 435: 443-453, 1998). Because this is seen in systemic but not intrarenal NPY infusion, we have investigated the possible mediator of tubular NPY effects in anesthetized rats. In the present study, infusion of NPY (2 micrograms . kg-1 . min-1) enhanced renovascular resistance by approximately 8 mmHg . ml-1 . min and enhanced urine and sodium excretion by approximately 450 microliter/15 min and approximately 60-85 micromol/15 min, respectively. Acute renal denervation did not alter renovascular or tubular NPY effects, indicating that a neuronally released mediator is not involved. Treatment with the angiotensin II-receptor antagonist losartan prevented the decline of the renovascular response with time but did not modify tubular NPY effects. The bradykinin B2-receptor antagonist icatibant accelerated the decline of the renovascular NPY effects with time; concomitantly, it attenuated NPY-induced diuresis and natriuresis and abolished NPY-induced calciuresis. The converting-enzyme inhibitor ramiprilat prevented the decline of the renovascular response with time; concomitantly, it magnified the NPY-induced diuresis, natriuresis, and calciuresis. We conclude that bradykinin may be involved in NPY-induced diuresis, natriuresis, and, in particular, calciuresis.

Purpose: We compare the tolerability and blood pressure effects of 0.4 mg. tamsulosin once daily in patients with lower urinary symptoms suggestive of benign prostatic obstruction with or without concomitant disease and/or antihypertensive medication., Materials and Methods: Data from 2 open label, observational studies (study 1, 9,507 patients treated for 4 weeks and study 2, 9,858 patients treated for 12 weeks) were analyzed for global tolerability and effects on blood pressure stratifying for co-morbidity (none, diabetes, hypertension, other cardiovascular disease) and co-medication (diuretics, beta-blockers, calcium channel blockers, angiotensin converting enzyme inhibitors)., Results: Overall 90 and 95% of patients in studies 1 and 2, respectively, reported good or very good tolerability. While global tolerability was slightly reduced in patients with concomitant disease or some forms of medication (p < 0.05), it was rated as good or very good by more than 90 and 95% of patients even in those groups. In control patients, that is those with neither co-morbidity nor co-medication, the tamsulosin induced blood pressure reductions were similar to those previously reported for placebo treatment but were statistically significant (p < 0.05). Mean additional blood pressure reductions in patients with concomitant disease or medication were not more than 2 mm. Hg., Conclusions: Tamsulosin is well tolerated and has marginal effects on blood pressure in the majority of patients. It largely maintains its good global tolerability and minimal blood pressure effects in patients with cardiovascular co-morbidity or diabetes, or those on co-medication with antihypertensive agents.

Alpha1-adrenoceptors were identified in murine tissues by [3H]prazosin saturation binding studies, with a rank order of cerebral cortex > cerebellum > liver > lung > kidney > heart > spleen, with the spleen not exhibiting detectable expression. Competition binding studies were performed with 5-methylurapidil, BMY 7378, methoxamine, (+)-niguldipine, noradrenaline, SB 216469 and tamsulosin. On the basis of monophasic low-affinity competition by BMY 7378, alpha1D-adrenoceptors were not detected at the protein level in any tissue. On the basis of competition studies with the alpha1A/alpha1B-discriminating drugs, alpha1B-adrenoceptors appeared to be the predominant or even the sole subtype in murine liver, lung and cerebellum, whereas murine cerebral cortex and kidney contained approximately 30% and 50% of alpha1A-adrenoceptors, respectively. The affinities of the various competitors in the murine tissues were quite similar to those reported from other species. The ratio of high- and low-affinity sites for tamsulosin did not in all cases match the percentages of alpha1A- and alpha1B-adrenoceptors detected by the other competitors; however, the low-affinity component of the tamsulosin competition curves was abolished in the cerebral cortex of alpha1B-adrenoceptor knockout mice. Treatment with chloroethylclonidine (10 microM, 30 min, 37 degrees C) inactivated the alpha1-adrenoceptors in all tissues by >75%. When the concentration-dependent inactivation of tissue alpha1B-adrenoceptors (liver) and tissue alpha1A-adrenoceptors (cerebral cortex from alpha1B-adrenoceptor knockout mice) was compared, alpha1A-adrenoceptors were only slightly less sensitive toward chloroethylclonidine than alpha1B-adrenoceptors. We conclude that murine tissues express alpha1A- and alpha1B-adrenoceptors, which are largely similar to those in other species. However, the tissue-specific distribution of subtypes may differ from that of other species.

Neuropeptide Y (NPY) is a unique modulator of renal function that enhances urine flow and sodium excretion despite marked reductions in renal blood flow. We investigated whether the cyclooxygenase inhibitor indomethacin alters the renal NPY effects in anesthetized rats. Treatment with 5 mg/kg indomethacin i.p. lowered urinary prostaglandin excretion by approximately 85%. Systemic infusion of NPY elevated mean arterial pressure by approximately 15 mm Hg and renovascular resistance by approximately 8.0 mm Hg/ml/min, whereas the related peptide YY3-36 (PYY3-36) did not. Nevertheless, both peptides enhanced urine flow rate by approximately 250 and approximately 100 microl/15 min, respectively, and sodium excretion by approximately 15 micromol/15 min. Treatment with indomethacin did not affect NPY- and PYY3-36-induced alterations of systemic and renovascular hemodynamics but completely abolished NPY- and PYY3-36-induced diuresis and natriuresis. Endogenous creatinine clearance was not affected by any treatment. We conclude that cyclooxygenase-derived arachidonic acid metabolites are not involved in the systemic or renal hemodynamic effects of NPY and PYY3-36 but mediate NPY- and PYY3-36-induced diuresis and natriuresis.

We examined the down-regulation of alpha-1B adrenoceptors in Madin-Darby canine kidney D1 (MDCK) cells with an emphasis on a possible role of protein kinase C. The alpha-1 adrenoceptor agonist phenylephrine (1-100 microM) concentration-dependently down-regulated alpha-1B adrenoceptors in MDCK cells. Down-regulation by 100 microM phenylephrine was detectable after 2 hr and maximal after 8 to 24 hr. The receptor down-regulation was accompanied by a decrease in phenylephrine-stimulated inositol phosphate formation but not by an altered expression of immunodetectable Gq/11 alpha subunits. Even though alpha-1B adrenoceptor and P2 purinergic receptor stimulation promote prostaglandin E2 formation, receptor down-regulation was not prevented by indomethacin (10 microM) treatment but was partly mimicked by treatment with the purinergic receptor agonists adenosine-5'-O-(3-thio)triphosphate and 2-methylthio-ATP (300 microM each). Phorbol-12-myristate-13-acetate (1-100 nM) concentration-dependently down-regulated MDCK alpha-1B adrenoceptors to a greater extent than did phenylephrine. Three protein kinase C inhibitors, H7 (100 microM), staurosporine (100 nM) and KT5926 (1 microM), markedly attenuated receptor down-regulation promoted by phorbol ester but did not affect that by phenylephrine. Two inhibitors of Ca++/calmodulin protein kinase pathways, KT5926 (1 microM) and W-7 (30 microM), also failed to prevent phenylephrine-induced down-regulation of alpha-1B adrenoceptors. We conclude that agonist-induced down-regulation of MDCK cell alpha-1B adrenoceptors is mimicked by a protein kinase C-activating phorbol ester but that the second messenger kinases protein kinase C and Ca++/calmodulin protein kinase do not mediate agonist-induced down-regulation of the alpha-1B adrenoceptor.

We have investigated the receptor subtype mediating effects of neuropeptide Y (NPY) on blood glucose concentrations. In anaesthetized rats a 120 min i.v. infusion of 2 microg/kg/min NPY lowered blood glucose by 26 mg/dl. This was mimicked by the same dose of peptide YY (PYY), NPY2-36, PYY3-36 and [Leu31,Pro34]NPY but not by NPY13-36, PYY13-36 or rat or human pancreatic polypeptide. The effects of NPY and [Leu31,Pro34]NPY were not inhibited by 10 microg/kg/min BIBP 3226. Neither treatment significantly altered plasma insulin concentrations. We conclude that NPY lowers blood glucose concentrations via a Y5 receptor subtype, i.e. a subtype which has also been implicated in stimulation of food intake.

Proliferation and immunoglobulin secretion of B lymphocytes are regulated by specific antigens and numerous accessory immunomodulatory factors. Lysophosphatidic acid (LPA) is a glycerophospholipid mediator that is released from activated blood platelets, attains high levels in serum, and exerts potent stimulatory effects on, e.g., neutrophils, monocytes, and T lymphocytes. LPA is also generated by a secretory, cytokine-inducible phospholipase A2 present in high concentrations in inflammatory exudates and septic states. We investigated effects of LPA on human Epstein-Barr virus-immortalized B lymphoblasts, a model for immunoglobulin-secreting B cells. Intracellular Ca2+ was determined with fura 2 and the formation of inositol 1,4, 5-trisphosphate by anion-exchange chromatography. LPA stimulated an increase in inositol 1,4,5-trisphosphate levels and induced a transient rise in intracellular free Ca2+ concentration from 105 +/- 17 to 226 +/- 21 nM. This Ca2+ signal resulted from Ca2+ mobilization and Ca2+ influx and was subject to homologous desensitization. Pertussis toxin inhibited these responses by approximately 70%. Furthermore, LPA stimulated a 27.5% increase in guanosine 5'-O-(3-thiotriphosphate) binding to permeabilized B lymphoblasts, which suggests the direct activation of pertussis toxin-sensitive G proteins by LPA. LPA stimulated a strong increase in the specific phosphorylation of the mitogen-activated protein kinase (immunoblot analysis) that was prevented by the MEK inhibitor PD-98059. Finally, LPA triggered a 2-fold increase in DNA synthesis ([3H]thymidine incorporation) and a 2-fold increase in B lymphoblast number and evoked a 20- to 50-fold increase in immunoglobulin formation. By RT-PCR we detected specific mRNA transcripts for the recently cloned human LPA receptor. Thus our data suggest that LPA behaves as a B cell growth factor.

Background: Acute displacement of opioids from their receptors by administration of large doses of opioid antagonists during general anesthesia is a new approach for detoxification of patients addicted to opioids. The authors tested the hypothesis that mu-opioid receptor blockade by naloxone induces cardiovascular stimulation mediated by the sympathoadrenal system., Methods: Heart rate, cardiac index, and intravascular pressures were measured in 10 patients addicted to opioids (drug history; mean +/- SD, 71 +/- 51 months) during a program of methadone substitution (96 +/- 57 mg/day). Cardiovascular variables and concentrations of catecholamine in plasma were measured in the awake state, during methohexital-induced anesthesia (dose, 74 +/- 44 microg x kg(-1) x min(-1)) before administration of naloxone, and repeatedly during the first 3 h of mu-opioid receptor blockade. Naloxone was administered initially in an intravenous dose of 0.4 mg, followed by incremental bolus doses (0.8, 1.6, 3.2, and 6.4 mg) at 15-min intervals until a total dose of 12.4 mg had been administered within 60 min; administration was then continued by infusion (0.8 mg/h)., Results: Concentration of epinephrine in plasma increased 30-fold (15 +/- 9 to 458 +/- 304 pg/ml), whereas concentration of norepinephrine in plasma only increased to a minor extent (76 +/- 44 to 226 +/- 58 pg/ml, P < 0.05). Cardiac index increased by 74% (2.7 +/- 0.41 to 4.7 +/- 1.7 min(-1) x m(-2)), because of increases in heart rate (89 +/- 16 to 108 +/- 17 beats/min) and stroke volume (+44%), reaching maximum 45 min after the initial injection of naloxone. In parallel, systemic vascular resistance index decreased (-40%). Systolic arterial pressure significantly increased (113 +/- 16 to 138 +/- 16 mmHg), whereas diastolic arterial pressure did not change., Conclusions: Despite barbiturate-induced anesthesia, acute mu-opioid receptor blockade in patients addicted to opioids induces profound epinephrine release and cardiovascular stimulation. These data suggest that long-term opioid receptor stimulation changes sympathoadrenal and cardiovascular function, which is acutely unmasked by mu-opioid receptor blockade. Because of the attendant cardiovascular stimulation, acute detoxification using naloxone should be performed by trained anesthesiologists or intensivists.

1. This study was designed to investigate the mechanism(s) of the negative inotropic effects of alpha1-adrenoceptor agonists observed in rat isolated left atria after exposure to free radicals. 2. Ouabain and calphostin C were used in contraction experiments to block the sodium pump and protein kinase C. Methoxamine-induced phospholipase C and Na+/K+ ATPase activities were measured. 3. Methoxamine (300 microM) increased contractile force by 1.6 +/- 0.2 mN in control atria but decreased contractile force in electrolysis-treated atria by 2.0 +/- 0.1 mN (P < 0.05), as determined 10 min after methoxamine addition. In contrast, the positive inotropic effects of endothelin-1 (30 nM) and isoprenaline (10 microM) were reduced from 2.6 +/- 0.3 to 1.3 +/- 0.1 mN and from 2.6 +/- 0.3 to 1.7 +/- 0.2 mN, respectively, by electrolysis treatment (P < 0.05), but not converted into a negative inotropic action. 4. In an inositol phosphate assay we observed that the stimulation of phospholipase C by methoxamine was attenuated by electrolysis when the (electrolyzed) medium from the organ bath was used, but the phospholipase C responses were restored by the use of fresh medium. However, fresh medium did not counteract the negative inotropic effect of methoxamine. Accordingly, the negative inotropic effect of methoxamine is not directly related to the impaired phospholipase C responses seen in atria subjected to electrolysis. 5. Ouabain (10 microM) and the protein kinase C inhibitor calphostin C (50 nM), completely prevented the negative inotropic effect of 300 microM methoxamine in electrolysis-treated atria. 6. Measurement of the Na+/K+ ATPase activity, revealed that in control atria, alpha1-adrenoceptor stimulation with 300 microM methoxamine, decreased the Na+/K+ ATPase activity by 14.4 +/- 7.7%. In contrast, methoxamine increased the Na+/K+ ATPase activity by 48.8 +/- 8.9% (P < 0.05) in electrolysis-treated atria. Interestingly, this increase in Na+/K+ ATPase activity was completely counteracted by calphostin C (1.4 +/- 0.1% over basal). 7. These results indicate that the negative inotropic effects of alpha1-adrenoceptor agonists, observed in rat isolated left atria exposed to free radicals, are likely to be caused by protein kinase C-mediated phosphorylation and subsequent activation of the Na+/K+ ATPase.

Neuropeptide Y (NPY) is a co-transmitter of the sympathetic nervous system including the renal nerves. The kidney expresses NPY receptors, which can also be activated by peptide YY (PYY), a circulating hormone released from gastrointestinal cells. Five subtypes of NPY receptors have been cloned, among which Y1, Y2 and Y5 appear to be involved in the regulation of renal function. NPY produces potent renal vasoconstriction in vitro in isolated interlobar arteries and in the isolated perfused kidney and in vivo upon intrarenal or systemic administration via a Y1 receptor. Nevertheless glomerular filtration rate is altered only little if at all by NPY, indicating a greater effect on the vas efferens than the vas afferens. NPY can inhibit renin release via Y1-like receptors. NPY can stimulate Na+/K+ adenosine triphosphatase (Na+/K+-ATPase) in proximal tubules via Y2 receptors and can antagonize the effects of vasopressin on isolated collecting ducts. It can also act prejunctionally to inhibit noradrenaline release via Y2 receptors. Despite the profound reductions of renal blood flow, systemic NPY infusion can cause diuresis and natriuresis; this is largely independent of pressure natriuresis mechanisms and is possibly mediated by an extrarenal Y5 receptor. Studies with the converting enzyme inhibitor ramiprilat and the bradykinin receptor antagonist icatibant indicate that bradykinin mediates, at least partly, diuretic NPY effects. NPY antagonists enhance basal renal blood flow but do not alter basal diuresis or natriuresis indicating that renovascular, but not tubular, NPY receptors may be tonically activated by endogenous NPY.

We have studied activation of mitogen-activated protein kinase (MAP kinase) and [3H]-thymidine uptake as a marker for cellular proliferation in a proximal tubular cell line of the American Opossum kidney (OK cells). Adrenaline, serotonin, epidermal growth factor and insulin all stimulated MAP kinase and [3H]-thymidine uptake. This stimulation of cellular proliferation was markedly reduced by an inhibitor of the MAP kinase pathway, PD 98059. Epidermal growth factor stimulated MAP kinase activity more effectively than the other agonists, but all four had similar effects on OK cellular proliferation. These data indicate that amine neurotransmitters may be of similar importance for tubular regeneration as classical growth factors.

1. The aim of this study was to investigate the time-dependency of the influence of dysthyroid states on the beta-adrenoceptor system in rat heart left ventricle. Therefore, the influence of acute and chronic hyper- and hypothyroidism on beta-adrenoceptor-induced left ventricular responses, beta-adrenoceptor density, cardiac noradrenaline tissue concentrations, Gs alpha-proteins, and basal and stimulated adenylate cyclase activities was determined. 2. Hyperthyroid rats were obtained by feeding with thyroxine (T4)-containing rat-chow for 1, 4 and 8 weeks. Hypothyroidism was induced by adding 0.05% propylthiouracil (PTU) to the drinking water. Rats of varying ages were used in order to compensate for the differences in the duration of the treatments. Rats were aged 3 and 5 months at the end of the experiments. 3. Thyroxine treatment for 4 and 8 weeks increased the cardiac sensitivity to isoprenaline, but maximal induced inotropic responses were decreased. Cardiac ventricular beta-adrenoceptor density was increased only in rats treated with T4 for 1 week. This transient effect of hyperthyroidism on cardiac beta-adrenoceptor density was not observed in older rats. The PTU treatment resulted in a stable decrease of cardiac beta-adrenoceptor density. 4. Left ventricular tissue noradrenaline concentrations were unaffected by hyperthyroidism, where a decrease was observed in hypothyroid rats. Density of Gs alpha proteins was increased in hearts from chronic hyperthyroid rats. 5. These results indicate that the increased sensitivity to beta-adrenoceptor-mediated stimulation in chronic hyperthyroidism cannot be attributed to changes in cardiac beta-adrenoceptor density, but is probably caused by an enhanced content of Gs alpha. Accordingly, in hyperthyroidism, the beta-adrenoceptor system is influenced time-dependently, whereas hypothyroidism affects the beta-adrenoceptor system independent of time.

The coupling of human alpha1-adrenoceptor subtypes to protein kinase C (PKC) and PKC-related signalling events were investigated in rat-1 fibroblasts stably expressing alpha1A-, alpha1B- or alpha1D-adrenoceptors at densities of 1328+/-200, 5030+/-703 and 150+/-14 fmol/mg protein respectively. In functional assays the alpha1-adrenoceptor agonist phenylephrine significantly stimulated PKC (assessed as increased activity in the membrane fraction) in cells expressing alpha1A- or alpha1B- but not alpha1D-adrenoceptors. In immunoblot assays phorbol ester treatment enhanced membrane-associated immunoreactivity of PKCalpha, PKCdelta and PKCepsilon to a similar extent in all three cell lines. Stimulation of alpha1A- and alpha1B-adrenoceptors also increased immunoreactivity of PKCalpha, PKCdelta and PKCepsilon in the membrane fraction, while alpha1D-adrenoceptor stimulation yielded only very small and inconsistent alterations. Immunoreactivity of PKCzeta was not consistently affected by phorbol ester or phenylephrine in any of the cell lines. Stimulation of all three alpha1-adrenoceptors time- and concentration-dependently increased inositol phosphate formation. Maximum activation occurred with the order alpha1A approximately = alpha1B > alpha1D. Phenylephrine also concentration dependently elevated free intracellular [Ca2+] in all three cell lines with the order of efficacy alpha1A > alpha1B > alpha1D. In the presence of ethanol, phenylephrine stimulated phosphatidylethanol formation in alpha1A- and alpha1B-adrenoceptor-expressing cells time and concentration dependently but only weakly and inconsistently in alpha1D-adrenoceptor-expressing cells. The efficacy of phenylephrine (100 microM) relative to that of noradrenaline (100 microM) for stimulation of phosphatidylethanol formation was similar (> or = 75%) for all three subtypes. The alkylating agent phenoxybenzamine concentration dependently reduced alpha1A-adrenoceptor density and phenylephrine-stimulated Ca2+ elevations to levels seen with alpha1D-adrenoceptors but reductions of phenylephrine-stimulated phosphatidylethanol formation were weaker. We conclude that human alpha1A- and alpha1B-adrenoceptors expressed in rat-1 cells couple to activation of PKCalpha, PKCdelta and PKCepsilon but not PKCzeta; this may involve stimulation of phospholipases C and D and intracellular Ca2+ elevations. Activation of these pathways by alpha1D-adrenoceptors appears to be much weaker and was not detected consistently; this was not fully explained by weak partial agonism of phenylephrine at this subtype or by lower receptor densities. Overall the alpha1A-adrenoceptor may have the highest efficiency of stimulus-response coupling among human alpha1-adrenoceptor subtypes.

The radioligand binding properties of [3H]prazosin and [3H]tamsulosin at alpha1-adrenoceptors of several rat tissues, human prostate and cloned rat and human alpha1-adrenoceptor subtypes were compared in Tris/EDTA buffer unless otherwise indicated. The affinity of [3H]tamsulosin at tissue and cloned alpha1A- and alpha1B-adrenoceptors was somewhat greater and smaller, respectively, than that of [3H]prazosin. In most rat tissues and at cloned rat alpha1A- and alpha1B-adrenoceptors, [3H]tamsulosin had a smaller Bmax than [3H]prazosin. Studies with rat liver showed that this was due to considerably poorer labeling of agonist low affinity sites, while both radioligands detected similar numbers of agonist high affinity sites. Statistically significant differences in the number of binding sites for both ligands were not detected in HEPES or glycylglycine buffer, as the detectable receptor number for [3H]prazosin and [3H]tamsulosin tended to be smaller and greater, respectively, in these than in Tris/EDTA buffer. Among human alpha1-adrenoceptor subtypes [3H]tamsulosin labeled fewer sites than [3H]prazosin for alpha1B- but more sites for alpha1A- and alpha1D-adrenoceptors. We conclude that [3H]prazosin and [3H]tamsulosin do not detect the same number of alpha1-adrenoceptors under a variety of conditions. This should be taken into account in the interpretation of data obtained with either radioligand.

Objective: To compare the efficacy, global tolerability and blood pressure effects of tamsulosin (0.4 mg once daily) in subgroups of patients with lower urinary tract symptoms (LUTS) suggestive of benign prostatic obstruction (BPO)., Methods: Data from two open-label, observational studies (Study I: 9,507 patients treated for 4 weeks, Study II: 9,858 patients treated for 12 weeks) were analyzed to compare efficacy, global tolerability and effects on blood pressure in subgroups of patients., Results: The efficacy of tamsulosin was largely unaffected by age or previous phytotherapy; in patients with severe symptoms, the efficacy was at least as large as in those with mild or moderate symptoms. More than 90% of patients reported a good or very good tolerability; in a multivariate analysis, this was slightly reduced in patients with concomitant disease but not affected by antihypertensive co-medication or baseline blood pressure. In patients without co-morbidity or co-medication, the tamsulosin-induced blood pressure reductions were similar to those previously reported for placebo treatment; mean additional blood pressure reductions in patients with concomitant disease or medication was not more than 2 mm Hg. Patients who had previously been treated with beta-sitosterol, other plant extracts or finasteride reported tamsulosin to be more effective than their previous treatment. Patients who had previously received beta-sitosterol or other plant extracts rated the global tolerability of tamsulosin to be similar to that of their previous treatment, while those who had previously received finasteride or other alpha 1-adrenoceptor antagonists rated the global tolerability of tamsulosin to be significantly better than that of their previous treatment., Conclusions: We conclude that tamsulosin is efficacious in all types of patients with LUTS suggestive of BPO. It is globally well tolerated and has marginal effects on blood pressure, including the vast majority of patients with cardiovascular comorbidity, diabetes or on antihypertensive comedication.

The properties of muscarinic acetylcholine receptors of porcine and human bladder detrusor were compared in radioligand binding studies using [3H]quinuclidinylbenzylate as the radioligand. The receptor affinity for the radioligand and the density of muscarinic receptors was similar in male and female pigs and in humans (Kd = 35 +/- 8 pM, Bmax = 153 +/- 30 fmol/mg protein). Atropine and subtype-selective antagonists had steep and monophasic competition curves in porcine and human detrusor with a rank order of potency of atropine >> hexahydro-sila-difenidol > or = AF-DX 116 > or = pirenzepine, indicating the presence of a homogeneous population of M2 muscarinic receptors. In female pigs bladder outflow obstruction generated by partial urethral ligation or its surgical treatment by ileum augmentation or autoaugmentation did not significantly alter expression of muscarinic receptors or of alpha2A-adrenoceptors, but the power was insufficient to exclude alterations of less than 60%. We conclude that porcine and human detrusor express muscarinic receptors of the M2 subtype; despite these qualitative similarities the use of the porcine model may be limited by large biological variance with regard to quantitative receptor expression.

Aims: A radioreceptor assay has been developed for alpha1-adrenoceptor subtypes and applied to a pharmacokinetic analysis of tamsulosin and terazosin., Methods: Young, male, healthy volunteers received 0.4 mg tamsulosin (as Omnic modified release capsules) or 5 mg terazosin (as Flotrin tablets) in a randomized, cross-over design. Before and after 1, 3, 5, 7, 10 and 23.5 h plasma was analyzed by radioreceptor assay using cloned human alpha1A-, alpha1B- and alpha1D-adrenoceptors and specific h.p.l.c. analysis., Results: Following ingestion of tamsulosin median peak plasma levels of 16 ng ml(-1) were reached after 5 h and declined to 2 ng ml(-1) at 23.5 h. The time course in the radioreceptor assay was similar, and at most time points binding to alpha1A-adrenoceptors was significantly greater than to alpha1B- and alpha1D-adrenoceptors. Following ingestion of terazosin median peak plasma levels of 91 ng ml(-1) were reached after 1 h and declined to 11 ng ml(-1) at 23.5 h. In the radioreceptor assay binding also peaked at 1 h and declined thereafter, but even after 23.5 h considerable binding activity remained detectable at all three subtypes. At most time points binding to the alpha1A- and alpha1D-adrenoceptor was significantly greater than to the alpha1B-adrenoceptor., Conclusions: We conclude that alpha1-adrenoceptor antagonist pharmacokinetics can be monitored by radioreceptor assays in a subtype-selective manner. Tamsulosin and terazosin exhibit subtype selective receptor binding ex vivo. The discordance between terazosin blood levels as determined by h.p.l.c. and radioreceptor assay at late time points indicates the possible involvement of metabolites in in vivo terazosin effects.

Objectives: This single-centre, double-blind, randomized parallel-group study compared ambulatory blood pressure (AMBP) and heart rate (HR) profiles and responses to orthostatic testing (OT) for recommended regimens of tamsulosin (TAM, modified release formulation) and terazosin (TER), two alpha1-adrenoceptor antagonists that differ in their selectivity for alpha1-adrenoceptor subtypes, pharmacokinetic properties and recommended posology for the treatment of lower urinary tract symptoms (LUTS) suggestive of benign prostatic obstruction (BPO)., Methods: 50 elderly normotensive male volunteers (mean age 68 years, range 61-78; 27 had LUTS) entered a single-blind 24-hour placebo run-in followed by a 15-day double-blind treatment in which the TER group received a 1-week treatment with 1 mg, then 1 week with 2 mg and finally a last dose of 5 mg, each dose administered once daily in the evening, while TAM subjects received 0.4 mg once daily after breakfast throughout. Subjects had AMBPs and OT while hospitalized on four occasions: during the placebo run-in and subsequent to the first dose of 1, 2 and 5 mg TER or at corresponding times under TAM. Regular OT (blood pressure after 5 min supine and after 2 min standing) was carried out between 6:00 and 6:30 a.m.; 'nocturial' OT took place whenever the subject had to leave the bed at night to void., Results: The baseline blood pressure values were slightly higher in the TAM group, but under treatment there was little difference between the treatments with regard to circadian changes in AMBP and HR. Under TER, there were 10 incidents of symptomatic hypotensive OT in 9 subjects (2 with syncope); furthermore, there were 24 events of asymptomatic exaggerated (> or = 20 mm Hg) decrease in systolic blood pressure in 12 subjects. With TAM in contrast, there was only 1 subject who experienced symptomatic hypotensive OT on 3 occasions (this subject had a previous history of vertigo and ought not to have been included); 7 subjects on TAM showed 16 incidents of asymptomatic hypotensive OT. The difference between TER and TAM was statistically significant for the number of subjects with positive symptomatic OT (p = 0.011)., Conclusion: Once-daily dosing of TAM after breakfast at a fixed dose level (0.4 mg) offers a more efficient protection against undesired cardiovascular extension effects in the normotensive elderly treated for LUTS suggestive of BPO than the usually recommended treatment schedule of TER with step-up doses (1-5 mg) administered at night.

We investigated the interaction of neuropeptide Y (NPY) with the alpha 1-adrenoceptor agonist, methoxamine, in control of mean arterial pressure, renovascular resistance and mesenteric vascular resistance in anaesthetized rats. Infusion of 3.0 but not 0.3 microgram/kg/min NPY enhanced the elevations of all three haemodynamic parameters caused by bolus injections of methoxamine (10-100 micrograms/kg). These enhancements largely involved a prolongation of the methoxamine effects. While infusion of the Y1 NPY receptor-selective antagonist, BIBP 3226 (10 micrograms/kg/min), alone did not alter methoxamine-induced vasoconstriction, it inhibited the potentiation by NPY. We conclude that NPY can potentiate methoxamine-induced vasoconstriction in vivo. This is mediated predominantly, if not exclusively, via the Y1 receptor. Endogenously released NPY does not appear to reach sufficient concentrations to cause tonic systemic vasoconstriction or potentiation thereof in the anaesthetized rat.

To investigate the functional role of different alpha1-adrenergic receptor (alpha1-AR) subtypes in vivo, we have applied a gene targeting approach to create a mouse model lacking the alpha1b-AR (alpha1b-/-). Reverse transcription-PCR and ligand binding studies were combined to elucidate the expression of the alpha1-AR subtypes in various tissues of alpha1b +/+ and -/- mice. Total alpha1-AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the alpha1b -/- as compared with +/+ mice. Because of the large decrease of alpha1-AR in the heart and the loss of the alpha1b-AR mRNA in the aorta of the alpha1b-/- mice, the in vivo blood pressure and in vitro aorta contractile responses to alpha1-agonists were investigated in alpha1b +/+ and -/- mice. Our findings provide strong evidence that the alpha1b-AR is a mediator of the blood pressure and the aorta contractile responses induced by alpha1 agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in alpha1b -/- as compared with +/+ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in alpha1b-/- mice. The alpha1b-AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different alpha1-AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.

We have compared the effects of adrenaline on activation of mitogen-activated protein kinase (MAP kinase), cyclic AMP accumulation and [3H]thymidine uptake in OK cells, a cell line derived from proximal tubules of the opossum kidney. Effects of serotonin and the direct protein kinase C activator, phorbol-12-myristate-13-acetate (PMA), were also studied. Adrenaline transiently (peak at 5 min, return to baseline by 30 min) and concentration-dependently (EC50 between 10 and 100 nM) stimulated MAP kinase activity. Maximal stimulation was approximately 100% above basal and was similar to the effects of 1 microM serotonin or 1 microM PMA. MAP kinase activation by adrenaline was inhibited by 10 microM phentolamine or 1 microM yohimbine but not significantly affected by 100 nM prazosin or 200 nM pindolol. The selective alpha2-adrenoceptor agonist UK 14,304 (10 microM) also stimulated MAP kinase activity. Activation of the 42 and 44 kDa ERK forms of MAP kinase was demonstrated by immunoblot analysis. The effect of adrenaline and UK 14,304 on MAP kinase was inhibited by pertussis toxin pretreatment and by the MAP kinase kinase inhibitor, PD 98059 (100 microM). Stimulation of MAP kinase activity was independent of cellular cAMP levels and was not affected by protein kinase C downregulation. Adrenaline, UK 14,304, serotonin, and PMA stimulated [3H]thymidine uptake, an effect inhibited by PD 98059. We conclude that adrenaline stimulates MAP kinase activity in OK-cells via alpha2-adrenoceptors and pertussis sensitive G proteins. While this occurs independently of cellular cAMP levels and protein kinase C, it involves the MEKI form of MAP kinase kinase and the ERK forms of MAP kinase. This activation results in enhanced cellular proliferation as assessed by [3H]thymidine uptake.

The aim of our study was to characterize functionally prejunctional neuropeptide Y (NPY) receptors in human and rabbit renal cortex, as well as in human right atrium. Segments of human atrial appendages and of human and rabbit renal cortex were preincubated with [3H]noradrenaline, superfused with Krebs-Henseleit solution and stimulated electrically in superfusion chambers. The stimulation-induced outflow of radioactivity was taken as an index of endogenous noradrenaline release. The effects of subtype-selective NPY analogs on the stimulation-induced noradrenaline release were studied. NPY, its endogenous analog, peptide YY, and its C-terminal fragment, NPY13-36, but not its analog, [Leu31,Pro34]NPY, concentration dependently (1-100 nM) inhibited [3H]noradrenaline release in all tissues studied. NPY-induced inhibition of [3H]noradrenaline release in human and rabbit kidney was abolished by pretreatment with pertussis toxin. We conclude that prejunctional inhibition of noradrenaline release in human heart and human and rabbit kidney occurs through NPY receptors of the Y2 subtype, which appear to couple to a pertussis toxin-sensitive G protein.

Systemic infusion of neuropeptide Y (NPY; 1 microg kg-1 min-1) for 120 min rapidly reduced renal blood flow and increased mean arterial pressure and renovascular resistance and, at later time points (> 30 min), enhanced diuresis, natriuresis and calciuresis in anaesthetized rats. Infusion of the reported NPY antagonist PP56 (D-myo-inositol 1,2,6-triphosphate, 333 mg kg-1 min-1) slightly but significantly enhanced renal blood flow and reduced renovascular resistance over the course of the infusion period. Infusion of PP56 together with NPY (starting 30 min prior to the NPY infusion) significantly inhibited NPY-induced alterations of mean arterial pressure, renal blood flow and renovascular resistance. Coinfusion of PP56 also attenuated the renovascular effects of bolus injections of NPY (0.1-10 microg/kg) but at the highest NPY dose the antagonistic effect of PP56 could partially be overcome. In contrast to the antagonism of the vascular NPY effects, infusion of PP56 did not significantly affect NPY-induced enhancements of diuresis, natriuresis and calciuresis. Thus, PP56 is a surmountable antagonist of vascular but not tubular NPY effects. We conclude that tubular NPY effects occur largely independently of alterations of renal haemodynamics.

1. Systemic infusion of neuropeptide Y (NPY) reduces renal blood flow and can concomitantly increase diuresis, natriuresis and calciuresis in anaesthetized rats. The present study was designed to investigate whether the apparently contradictory NPY effects on renal blood flow and urine formation and composition are mediated by distinct NPY receptor subtypes. 2. NPY and its analogues, peptide YY (PYY), [Leu31, Pro34]NPY and NPY13-36, were infused in incremental doses of 0.3, 1 and 3 micrograms kg-1 min-1 for 45 min each and the results compared to those obtained in vehicle-infused rats. Renal blood flow was monitored in 1-5 min intervals, while urine excretion and composition were determined in 15 min collection periods. Plasma renin activity and aldosterone concentrations were measured at the end of the final infusion period. 3. Relative to vehicle NPY, PYY and [Leu31, Pro34]NPY dose-dependently reduced renal blood flow and increased diuresis, natriuresis and calciuresis with roughly similar potency; NPY13-36 slightly but significantly increased renal blood flow but had no effect on diuresis, natriuresis and calciuresis. None of the peptides significantly affected endogenous creatinine clearance or kaliuresis. 4. Plasma renin activity was significantly reduced by PYY. Quantitatively similar reductions were observed with NPY and [Leu31, Pro34]NPY but failed to reach statistical significance with the given number of experiments. NPY13-36 did not reduce plasma renin activity. None of the peptides significantly affected plasma aldosterone concentrations. 5. In another series of experiments infusion of PYY3-36 (2 micrograms kg-1 min-1 for 120 min) did not reduce renal blood flow but significantly enhanced diuresis and natriuresis to a similar extent as the NPY 2 micrograms kg-1 min-1. 6. In a final series of experiments the Y1-selective antagonist, BIBP 3226 (1 or 10 micrograms kg-1 min-1) dose-dependently antagonized reductions of renal blood flow elicited by bolus injections of NPY (0.1-30 micrograms kg-1). BIBP 3226 (10 micrograms kg-1 min-1) also inhibited the effects of a 120 min infusion of NPY (2 micrograms kg-1 min-1) on renal blood flow but had only minor inhibitory effects on enhancements of diuresis and did not significantly affect enhancements of natriuresis. 7. We conclude that NPY reduces renal blood via a classical Y1 subtype of NPY receptor. In contrast enhancements of diuresis, natriuresis and calciuresis occur via a distinct subtype which resembles the receptor that mediates NPY-induced enhancement of food intake.

We have used the alpha1D-adrenoceptor selective antagonist, BMY 7378, the alpha1D-selective agonists, adrenaline and phenylephrine, the alpha1A-selective antagonists, (+)-niguldipine, SB 216469 and WB4101, and the non-subtype-selective alpha1-adrenoceptor antagonist, nemonapride, to investigate the presence of alpha1D-adrenoceptors in rat tissues at the protein level. Radioligand binding studies using [3H]prazosin as the radioligand were performed in three tissues containing alpha1D-adrenoceptor mRNA, spleen, cerebral cortex and kidney, and in comparison in one tissue not containing alpha1D-adrenoceptor mRNA, liver. Cerebral cortex and kidney were also studied upon alpha1B-adrenoceptor inactivation by chloroethylclonidine treatment (10 microM, 30 min, 37 degrees C). Experiments with cloned rat alpha1-adrenoceptor subtypes transiently expressed in COS cells confirmed the known selectivity of the investigated drugs for alpha1-adrenoceptor subtypes or the lack thereof of nemonapride. Accordingly nemonapride had steep and monophasic competition curves in all native and chloroethylclonidine-treated tissues. BMY 7378 also had steep and monophasic competition curves and low affinity in all native tissues. In contrast, adrenaline and phenylephrine (in the presence of 100 microM GTP) had monophasic competition curves of low affinity in liver and spleen but biphasic competition curves in cerebral cortex and kidney. Following chloroethylclonidine treatment competition curves for adrenaline, phenylephrine, (+)-niguldipine, SB 216469 and WB 4101 remained biphasic in cerebral cortex and kidney while those for nemonapride remained monophasic. We conclude that alpha1D-adrenoceptors are not readily detectable at the protein level in a variety of rat tissues where their mRNA is expressed. The biphasic competition curves of some agonists and antagonists in chloroethylclonidine-treated rat tissues do not represent alpha1D-adrenoceptors and are not readily explained by the present alpha1A/alpha1B/alpha1D-adrenoceptor classification.

Tocolytic therapy with beta-adrenergic receptor agonists is a standard regimen to prevent preterm birth. Agonists exposure of beta-adrenergic receptors causes receptor desensitization in other organs, and this may limit the therapeutic value of beta-adrenergic receptor agonists. To study the effects of prolonged beta-adrenergic agonist treatment in human myometrium, we obtained biopsies during Caesarean section of 14 pregnant patients who had received fenoterol for at least 5 days and 14 untreated pregnant controls. The densities of total beta-adrenergic receptors, which are mainly of the beta 2-subtype as assessed by [125I]iodo-cyanopindolol binding in crude membrane fractions, were more than 50% smaller in women receiving fenoterol, whereas alpha 2-adrenergic receptor densities were similar. Gs and Gi G-protein alpha-subunit densities were unaltered as assessed by Western blotting and pertussis toxin-catalyzed [32P]ADP-ribosylation. beta-Adrenergic receptor kinase (beta ARK) activity, as determined using bovine rhodopsin as the substrate, was the same in the two groups. Adenylyl cyclase activities in the presence of guanine nucleotides, NaF, forskolin, or Mn+2 were also not altered by fenoterol treatment. The messenger RNA (mRNA) concentrations of beta 2-adrenergic receptors, beta ARK-I and glyceraldehyde-3-phosphate dehydrogenase (as a reference), as determined by quantitative PCR, were unaffected by fenoterol treatment. We conclude that tocolysis with fenoterol results in a selective down-regulation of myometrial beta-adrenergic receptors, which is not associated with a reduction in the respective mRNA concentrations or alterations of alpha 2-adrenergic receptors, Gs and Gi alpha-subunits, or beta ARK activity or mRNA.

This study aimed firstly to compare the in vivo cardiovascular effects of exogenously administered and of endogenously released noradrenaline; secondly to characterize the adrenoceptors mediating these responses; thirdly to assess the influence of parasympathetic tone on the cardiovascular effects of noradrenaline. In two randomised placebo-controlled studies, healthy, young, male volunteers received intravenous (i.v.) infusions of noradrenaline at six incremental doses of 10-160 ng/kg/min and-in order to release endogenous noradrenaline-tyramine at four incremental doses of 5-20 micrograms/kg/min. Noradrenaline and tyramine were administered in the absence and presence of alpha 1-adrenoceptor blockade with doxazosin (2 mg p.o.), alpha 2-adrenoceptor blockade with yohimbine (15 mg p.o.), selective beta 1-adrenoceptor blockade with bisoprolol (15 mg p.o.) and muscarinic receptor blockade with atropine (1.5 micrograms/kg i.v. loading dose followed by 0.15 microgram/kg/min by i.v. infusion). Vasoconstrictor effects were assessed by measurement of diastolic blood pressure (Pdiast) and myocardial effects by measurement of systolic time intervals, namely the duration of electromechanical systole corrected for heart rate (QS2c). I.v. noradrenaline increased Pdiast (delta max 17 mmHg) and this was nearly completely suppressed by doxazosin but only slightly blunted by yohimbine. Noradrenaline also slightly shortened QS2c (delta max -22 ms), and this was potentiated by both doxazosin and yohimbine and completely blocked by biosprolol. I.v. tyramine reduced Pdiast (delta max -7 mmHg), which was not affected by alpha 1-adrenoceptor blockade, and profoundly shortened QS2c (delta max -104 ms) which was significantly correlated with a marked increase in systolic blood pressure (Psyst) (delta max 57 mmHg). The shortening of QS2c and the rise in Psyst were not influenced by alpha-adrenoceptor blockade but were antagonized by bisoprolol. Atropine potentiated the blood pressure rise and the shortening of QS2c induced by i.v. noradrenaline and converted the fall in Pdiast induced by i.v. tyramine into an increase. Thus the cardiovascular effects of exogenous noradrenaline are mainly characterized by alpha 1-adrenoceptor-mediated vasoconstriction and the actions of endogenous noradrenaline (released by i.v. tyramine) by beta 1-adrenoceptor-mediated positive inotropic effects. The rise in Psyst with i.v. tyramine most likely reflects positive inotropism and not a vascular "pressor' response.

We have investigated the affinity and selectivity of tamsulosin and its metabolites, M1, M2, M3, M4 and AM1, at the tissue and the cloned alpha-1 adrenoceptor subtypes in the radioligand binding and the functional studies. In the radioligand binding studies, the compounds competed for [3H]prazosin binding to the rat liver and kidney alpha-1 adrenoceptors, with the rank order of potency tamsulosin approximately M4 > M1 > M2 approximately M3 > > AM1 with the latter having a negligible affinity. All compounds differentiated cloned alpha-1 adrenoceptor subtypes with the rank order of potency of alpha-1A > or = alpha-1D > alpha-1B, except for M4 which had the highest affinity for the alpha-1D adrenoceptor. The compounds also concentration-dependently antagonized phenylephrine-induced contractions in the rabbit aorta and prostate. The resulting apparent pA2 values were very similar to those at the cloned rat alpha-1A adrenoceptor. We conclude that most tamsulosin metabolites are high potency antagonists at the alpha-1 adrenoceptors and retain the alpha-1A over the alpha-1B adrenoceptor selectivity of tamsulosin.

We have quantified and characterized alpha 1-, alpha 2- and beta-adrenoceptor subtypes in porcine bladder detrusor and bladder neck, human bladder detrusor, and porcine and human prostate. alpha 1-, alpha 2- and beta-adrenoceptor were identified in radioligand binding studies using [3H]prazosin, [3H]RX 821002 and [125I]iodocyanopindolol, respectively, as the radioligands. In porcine male and female detrusor and bladder neck and male prostate, adrenoceptors were detected in the order of abundance beta > alpha 2 >> alpha 1 (not detectable), with no major difference between the sexes or between detrusor and bladder neck. In human detrusor and prostate the order of abundance was beta > alpha 2 >> alpha 1 (not detectable) and beta >> alpha 1 > alpha 2, respectively. The alpha 2-adrenoceptors in all tissues were homogeneously of the alpha 2A-subtype as evidenced by competition binding studies with yohimbine, prazosin, ARC 239 and oxymetazoline. The beta-adrenoceptors represented a mixed population with a dominance of the beta 2-subtype in all tissues as demonstrated by competition binding with ICI 118,551 and CGP 20,712A. We conclude that pigs may be a suitable model for studies of detrusor function with respect to adrenoceptor expression. They may be less suitable for studies of bladder neck or prostate function.

Objective: Apparent cyclosporin A (CSA) blood levels, as determined by fluorescence polarization immunoassay (FPIA) and enzyme-multiplied immunoassay technique (EMIT), were compared in CSA-treated patients with various degrees of liver dysfunction., Methods: FPIA and EMIT were performed in parallel according to test manufacturer instructions in blood from kidney (n = 82), liver (n = 96) and heart transplant (n = 20) patients., Results: The precision of both techniques was greatest in patients with the highest blood levels, and at each blood level greater for the FPIA than for the EMIT. Apparent CSA blood levels, as determined by EMIT, were typically approximately 70% of those determined by FPIA, indicating greater cross-reaction of the antibody in the FPIA with CSA metabolites. However, the ratio of values determined with EMIT and FPIA was very similar in kidney, liver and heart transplant patients. Among liver transplant patients it was also very similar in those without major alterations of hepatic function and in those with impaired excretory (increased bilirubin and gamma GT) or synthetic (i.e., reduced thromboplastin time) function. Extended storage of blood samples for up to 10 days did not affect apparent CSA blood level estimates by EMIT in a clinically relevant manner., Conclusions: We conclude that the greater specificity of the antibody in the EMIT for the CSA parent compound does not translate into a clinically relevant advantage for CSA monitoring.

1. We have compared the expression of protein kinase C (PKC) activity and immuno-detectable isoenzymes in cytosolic and membrane extracts of rat and human cardiovascular tissues (heart, kidney, aorta, saphenous vein). Experiments were performed in raw extracts and upon combined diethylaminoethylcellulose (DEAE) and phenylsepharose column chromatography. 2. PKC activity that bound to DEAE mostly eluted with 200 mM NaCl. DEAE-purified PKC from all tissues except rat kidney bound almost quantitatively to phenylsepharose and eluted with 0.5-0 M NaCl. 3. Immunoblots with an antibody against classical PKCs and the activator profile for phosphatidylserine, diolein and Ca2+ revealed that the PKC from rat kidney, which did not bind to phenylsepharose, was most probably due to a proteolytically-generated, constitutively active PKC which is not under the control of a regulatory subunit. 4. Studies in the reference tissue, rat brain, demonstrated that all PKC isoenzymes investigated (classical PKCs alpha, beta, gamma, new PKCs delta, epsilon, eta, theta, and atypical PKCs zeta, lambda, iota) have similar DEAE and phenylsepharose chromatography elution profiles. In the functional assay an inhibitor of all known PKC isoenzymes, bisindolylmaleimide, and a specific inhibitor of classical PKCs, Gö 6976, both inhibited PKC from rat brain completely and with high potency indicating that the functional assay preferentially detects classical PKC isoenzymes. 5. Each PKC isoenzyme had a tissue-specific expression profile which was similar in rat and man. The classical PKC alpha, the new PKCs delta and epsilon and all atypical PKCs were detectable in most tissues, whereas the PKC beta and PKC gamma were not detected in any pheripheral tissue; PKC eta and PKC theta were found in some tissues. 6. We conclude that combined DEAE and phenylsepharose chromatography is useful to enrich and detect PKC isoenzymes; no major species differences in tissues-specific expression patterns appear to exist between rat and man.

Neuropeptide Y (NPY 1-36) binds to Y1 and Y2 receptors with similar affinity. No endogenous molecular form of NPY with selectivity for Y1 or Y2 receptors has been described so far. We report the presence of an endogenous fragment of NPY in porcine brain, NPY 3-36, which lacks the amino-terminal dipeptide Tyr-Pro of NPY 1-36. NPY 3-36 accounts for 35% of NPY-like immunoreactivity in porcine brain. We have compared binding of NPY 3-36 and NPY 1-36 in model systems of Y1-like (SK-N-MC cells) and Y2-like receptors (CHP234 cells). NPY 3-36 and NPY 1-36 had similarly high affinity for Y2-like receptors on CHP234 cells, but NPY 3-36 had a 1000-fold lower affinity than NPY 1-36 for Y1-like receptors on SK-N-MC cells. Thus amino-terminal cleavage of NPY 1-36 generating NPY 3-36 converts an unselective Y1/Y2 receptor ligand into a highly Y2 selective ligand. This may be a means of fine tuning NPY biological actions.

1. The affinity of the alpha 1-adrenoceptor antagonist SB 216469 (also known as REC 15/2739) has been determined at native and cloned alpha 1-adrenoceptor subtypes by radioligand binding and at functional alpha 1-adrenoceptor subtypes in isolated tissues. 2. In radioligand binding studies with [3H]-prazosin, SB 216469 had a high affinity at the alpha 1A-adrenoceptors of the rat cerebral cortex and kidney (9.5-9.8) but a lower affinity at the alpha 1B-adrenoceptors of the rat spleen and liver (7.7-8.2). 3. At cloned rat alpha 1-adrenoceptor subtypes transiently expressed in COS-1 cells and also at cloned human alpha 1-adrenoceptor subtypes stably transfected in Rat-1 cells, SB 216469 exhibited a high affinity at the alpha 1a-adrenoceptors (9.6-10.4) with a significantly lower affinity at the alpha 1b-adrenoceptor (8.0-8.4) and an intermediate affinity at the alpha 1d-adrenoceptor (8.7-9.2). 4. At functional alpha 1-adrenoceptors, SB 216469 had a similar pharmacological profile, with a high affinity at the alpha 1A-adrenoceptors of the rat vas deferens and anococcygeus muscle (pA2 = 9.5-10.0), a low affinity at the alpha 1B-adrenoceptors of the rat spleen (6.7) and guinea-pig aorta (8.0), and an intermediate affinity at the alpha 1D-adrenoceptors of the rat aorta (8.8). 5. Several recent studies have concluded that the alpha 1-adrenoceptor present in the human prostate has the pharmacological characteristics of the alpha 1A-adrenoceptor subtype. However, the affinity of SB 216469 at human prostatic alpha 1-adrenoceptors (pA2 = 8.1) determined in isolated tissue strips, was significantly lower than the values obtained at either the cloned alpha 1a-adrenoceptors (human, rat, bovine) or the native alpha 1A-adrenoceptors in radioligand binding and functional studies in the rat. 6. Our results with SB 216469, therefore, suggest that the alpha 1-adrenoceptor mediating contractile responses of the human prostate has properties which distinguish it from the cloned alpha 1a-adrenoceptor or native alpha 1A-adrenoceptor. Since it has previously been shown that the receptor is not the alpha 1B- or alpha 1D-adrenoceptor, the functional alpha 1-adrenoceptor of the human prostate may represent a novel receptor with properties which differ from any of the alpha 1-adrenoceptors currently defined by pharmacological means.

The aim of this study was to characterize the properties of endothelin (ET)-receptor subtypes mediating inositol phosphate (IP)-formation in rat kidney and their regulation during ontogenesis. In renal cortical slices of adult rats (12-16 weeks old) ET's concentration-dependently increased IP-formation with an order of potency ET-1 >> ET-3. While the non-selective ET-receptor antagonist bosentan (10 microM) completely suppressed ET-induced IP-formation, the ETA-receptor antagonist BQ-123 (10 microM) inhibited it only by 70%, the ETB-receptor antagonist IRL 1038 (1 microM) by 25%; combined application of BQ-123 + IRL 1038 caused complete inhibition of ET-1-induced IP-formation. Pretreatment of isolated renal cells with pertussis toxin (PTX, 500 ng/ml) overnight did not attenuate but significantly increased ET-1-induced IP-formation. Ontogenetic studies in renal sites from neonatal, 1, 2, 3, 6, 12 and 24 weeks old rats revealed that ET-1-induced IP-formation maturation-dependently declined being highest in neonatal rats (increase: 169% over basal) and lowest in 24 weeks old rats (increase: 47% over basal). This decline in ET-induced IP-formation was accompanied by a decrease in renal ET-receptor number and the amount of immunodetectable Gq/11 (assessed by Western-blotting using the QL-antiserum). Moreover, ET-receptor subtypes changed during the maturation process: from neonates to 12 weeks old rats number and functional responsiveness of ETA-receptors declined, while that of ETB-receptors increased. We conclude that in adult rat renal cortex ET-induced IP-formation is mediated by activation of both ETA- and ETB-receptors and does not involve a PTX-sensitive G-protein. ET-induced IP-formation declines during the maturation process; this is associated with a decrease in ET-receptor number and the immunodetectable amount of Gq/11.

Background: The present study investigated biochemical and functional consequences of chronic activation of the inhibitory Gi alpha-coupled adenylyl cyclase pathway in the heart., Methods and Results: Rats (220 to 260 g) were treated with 4-day infusions of the M-cholinoceptor agonist carbachol (9.6 mg/kg per day) or vehicle. An additional group that received the beta-adrenoceptor agonist isoprenaline (2.4 mg/kg per day) served as control. The main finding was that chronic infusion of carbachol led to a marked increase in isoprenaline- or forskolin-induced arrhythmia in electrically driven papillary muscles (in vitro). Compared with control, the potency of isoprenaline and forskolin to induce arrhythmia in cardiac preparations from carbachol-treated rats was increased 36- and 2.2-fold and the efficacy was increased 7.3- and 2.3-fold, respectively. The potency of carbachol to antagonize the isoprenaline- and forskolin-induced arrhythmia was decreased 30-fold. These changes were accompanied by a decrease in left ventricular M-cholinoceptor density by 15% (P < .05) and a decrease in pertussis toxin-sensitive G proteins (Gi alpha) by 26% (P < .05) without a decrease in the corresponding mRNAs. beta-Adrenoceptor density and basal and stimulated adenylyl cyclase activity remained unchanged. In contrast, isoprenaline infusion induced a decrease in arrhythmogenic potency of forskolin (P = NS), which was accompanied by a decrease in beta-adrenoceptor density, an increase in Gi alpha protein and mRNA levels, and a decrease in basal and stimulated adenylyl cyclase activity., Conclusions: Chronic parasympathetic activation sensitizes the myocardium to cAMP-induced arrhythmia. These changes may be due to quantitative alterations in functional Gi alpha.

1. To elucidate a possible role of species differences in the classification of alpha 1-adrenoceptor subtypes, we have characterized the alpha 1-adrenoceptors in guinea-pig spleen, kidney and cerebral cortex and in bovine cerebral cortex using concentration-dependent alkylation by chloroethylclonidine and competitive binding with 5-methlurapidil, methoxamine, (+)-niguldipine, noradrenaline, oxymetazoline, phentolamine, SDZ NVI-085, tamsulosin and (+)-tamsulosin. Rat liver alpha 1B-adrenoceptors were studied for comparison. Chloroethylclonidine-sensitivity and (+)-niguldipine affinity were also compared at cloned rat and bovine alpha 1a-adrenoceptors. 2. Chloroethylclonidine concentration-dependently inactivated alpha 1-adrenoceptors in all five tissues. While chloroethylclonidine inactivated almost all alpha 1-adrenoceptors in rat liver and guinea-pig kidney and brain, 20-30% of alpha 1-adrenoceptors in guinea-pig spleen and bovine brain were resistant to alkylation by 10 microM chloroethylclonidine. With regard to concentration-dependency guinea-pig kidney and brain were approximately 10 fold less sensitive than guinea-pig spleen or rat liver. 3. In rat liver, all drugs tested competed for [3H]-prazosin binding with steep and monophasic curves. Drug affinities were relatively low and resembled most closely those of cloned rat alpha 1b-adrenoceptors. 4. In guinea-pig spleen, all drugs tested competed for [3H]-prazosin binding with steep and monophasic curves. Drug affinities were relatively low and resembled most closely those of cloned rat alpha 1b-adrenoceptors. 5. In guinea-pig kidney most drugs tested competed for [3H]-prazosin binding with steep and monophasic curves and had relatively low drug affinities close to those of cloned rat alpha 1b- and alpha 1d-adrenoceptors. However, noradrenaline and tamsulosin had consistently biphasic competition curves recognizing 36-39% high and 61-64% low affinity sites. 6. In guinea-pig cerebral cortex, all drugs tested competed for [3H]-prazosin binding with shallow and biphasic curves. While most drugs recognized approximately 25% high affinity sites, tamsulosin and noradrenaline recognized approximately 50% high affinity sites. Drug affinities at the high and low affinity sites except those for tamsulosin and noradrenaline resembled those at cloned alpha 1a- and alpha 1b-adrenoceptors, respectively. 7. In bovine cerebral cortex all drugs tested except for noradrenaline competed for [3H]-prazosin binding with shallow and biphasic curves. All drugs recognized approximately 70% high affinity sites. Drug affinities at the high and low affinity sites resembled those at cloned alpha 1a- and alpha 1b-adrenoceptors, respectively. Noradrenaline competition curves in bovine cerebral cortex were steep and monophasic. 8. When cloned rat and bovine alpha 1a-adrenoceptors transiently expressed in COS cells were studied in a direct side-by-side comparison, both species homologues had similar chloroethylclonidine-sensitivity and (+)-niguldipine affinity. 9. We conclude that properties of bovine alpha 1A- and alpha 1B-adrenoceptors are very similar to those of other species such as rat. alpha 1-Adrenoceptor subtypes in guinea-pigs resemble alpha 1A- and alpha 1B-adrenoceptors in other species but chloroethylclonidine sensitivity and competition binding profiles of noradrenaline and tamsulosin are not compatible with previously established alpha 1-adrenoceptor subtype classification.

1. We have previously shown that among alpha 1-adrenoceptor antagonists used or investigated for the treatment of benign prostatic hyperplasia, tamsulosin discriminates alpha 1-adrenoceptor subtypes in rat tissues whereas alfuzosin and naftopidil do not. We now expand these studies to additional drugs (doxazosin, terazosin) being used and/or investigated for this purpose, and have evaluated all of these drugs at cloned subtypes and in human prostate. 2. Competition binding studies were performed with [3H]-prazosin in membrane samples from rat spleen, kidney and cerebral cortex and human prostate and with cloned alpha 1-adrenoceptors expressed in COS cells. Doxazosin and terazosin did not discriminate alpha 1-adrenoceptor subtypes in rat kidney and cerebral cortex. In contrast, the subtypes present in the tissues were well discriminated by the alpha 1A-adrenoceptor-selective reference drug WB 4101. 3. Alfuzosin, doxazosin, naftopidil and terazosin did not discriminate cloned alpha 1-adrenoceptor subtypes transiently expressed in COS cells whereas tamsulosin and WB 4101 did. 4. In human prostate, alfuzosin, doxazosin, naftopidil and terazosin did not discriminate the alpha 1-adrenoceptor subtypes present in this tissue whereas tamsulosin and the alpha 1A-adrenoceptor-selective reference drugs WB 4101, phentolamine and 5-methylurapidil did. Based on data with the alpha 1A-adrenoceptor-selective drugs, human prostate contains alpha 1A- and alpha 1B-adrenoceptors in an approximate 70:30% ratio. 5. We conclude that tamsulosin, in common with WB 4101, but in contrast to alfuzosin, doxazosin, naftopidil, and terazosin is selective for alpha 1A-adrenoceptors which appear to dominate in the human prostate; the therapeutic relevance of this selectivity remains to be assessed in clinical studies.

The cytoskeletal localization of inhibitory guanine-nucleotide-binding (Gi) proteins and the coupling of these proteins to formyl peptide receptors were studied in myeloid differentiated human leukemia (HL-60) cells. Treatment of HL-60 cells with cytochalasin B or botulinum C2 toxin, which leads to the disruption of microfilaments, increased the binding of the stable GTP analogue guanosine 5'[gamma-thio]triphosphate (GTPS[S]) to permeabilized cells by about 30%. In contrast, the microtubule-disrupting agents colchicine and vinblastine, and cytochalasin B treatment of isolated HL-60 membranes did not affect GTP[S] binding. The stimulatory effect of cytochalasin B treatment was concentration and time dependent, with maximal increases observed at 5 micrograms/ml cytochalasin B and an incubation time of 10 min, and was counteracted by the F-actin-stabilizing toxin phalloidin. Cytochalasin B treatment increased the amount of G proteins activated by chemoattractant receptors by about 25%. Furthermore, the number of Gi-protein-coupled receptors for the chemoattractant, N-formyl-Met-Leu-Phe, was increased by about 25% upon cytochalasin B treatment. Based on these functional data, which suggest an association of G proteins with actin filaments, the Triton X-100 (1%)-insoluble cytoskeleton was analyzed for the presence of G proteins. Gia subunits were detected in the cytoskeleton preparations, both by specific antisera and by pertussis-toxin -catalyzed ADP-ribosylation. Cytochalasin B pretreatment depleted the cytoskeleton in Gialpha, with an approximately 20% concomitant increase in membrane Gialpha content. In conclusion, evidence is presented that part of the cellular Gia is localized at actin filaments in HL-60 cells. After filament disruption, these Gia subunits seem to be translocated to the plasma membrance, where they can productively interact with chemoattractant receptors.

We have studied the contractile effects of the sympathetic transmitter noradrenaline and its cotransmitter neuropeptide Y (NPY) given alone and in combination on isolated rat mesenteric resistance vessels (200-300 microns diameter). Noradrenaline and NPY each concentration-dependently contracted rat mesenteric microvessels (EC50 approximately equal to 800 nM and 10 nM, respectively), but noradrenaline caused considerably greater maximal effects than NPY (14.3 mN vs. 3.5 mN). A low antagonistic potency of yohimbine indicated that the response to noradrenaline did not involve alpha 2-adrenoceptors, and the subtype-selective antagonists 5-methylurapidil, tamsulosin and chloroethylclonidine indicated mediation via an alpha 1A-adrenoceptor. Shallow Schild regressions for prazosin and 5-methylurapidil indicated that an alpha 1-adrenoceptor subtype with relatively low prazosin affinity might additionally be involved. Studies with the NPY analogues PYY, [Leu31, Pro34] NPY and NPY18-36 demonstrated that NPY acted via a Y1 NPY receptor. In addition to its direct vasoconstricting effects NPY also lowered the noradrenaline EC50 but did not appreciably affect maximal noradrenaline responses indicating possible potentiation. The potentiating NPY response occurred with similar agonist potency as the direct contractile NPY effects and also via a Y1 NPY receptor. The Ca2+ entry blocker nitrendipine (300 nM) reduced direct contractile responses to noradrenaline and NPY but did not affect the potentiation response to NPY.

Pharmacological and molecular cloning techniques have identified six human subtypes of alpha-adrenoceptors which are designated alpha 1A, alpha 1D, alpha 2A, and alpha 2C. At the protein level human kidney expresses predominantly alpha 2A-adrenoceptors while other alpha 2-adrenoceptor subtypes or alpha 1-adrenoceptors have not been detected consistently in radioligand binding studies. However, the presynaptic receptors, which inhibit noradrenaline release in the human kidney, appear to belong to the alpha 2C-subtype. Intrarenal infusion of the nonselective alpha-adrenoceptor antagonist, phentolamine, and of the selective alpha 2-adrenoceptor antagonist, yohimbine, but not of the selective alpha 1-adrenoceptor antagonist, doxazosin, increase renal blood flow and renin release in hypertensive patients undergoing diagnostic renal angiography. Thus, alpha 2- but not alpha 1-adrenoceptors appear to mediate a tonic renal vasoconstriction and inhibition of renin release. Effects of systemically given alpha 1-adrenoceptor agonists and antagonists are difficult to interpret on a mechanistic level since direct effects in the kidney and indirect effects due to baroreflex activation and peripheral presynaptic and central sympatholytic actions may at least partially offset each other. Moreover, some of these drugs may additionally act independent of alpha-adrenoceptors, for example, via imidazoline recognition sits. The net result in a given subject may depend on the endogenous sympatho-adrenal tone. Thus, for each target population of interest, effects have to be described empirically for each drug.

We have compared muscarinic acetylcholine receptor (mAChR) coupling to phospholipase C (PLC) and increases in cytoplasmic Ca2+ concentration [Ca2+]i in human embryonic kidney (HEK) cells, stably expressing either the human m3 or m2 receptor subtype. In m3 mAChR-expressing cells, carbachol stimulated inositol phosphate (InsP) formation and increased [Ca2+]i with EC50 values of about 2 microM and 30 nM, respectively. Maximal inositol 1,4,5-trisphosphate (InsP3) production (about fourfold) was rapid (15 s) and stable for 2 min. Maximal increases in [Ca2+]i were 300-350 nM and mainly, almost 90%, due to influx of extracellular Ca2+. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was not significantly different from that of carbachol. All m3 mAChR-mediated responses were pertussis toxin (PTX)-insensitive. In m2 mAChR-expressing cells, carbachol stimulated InsP formation and increased [Ca2+]i with EC50 values of about 20 microM and 7 microM, respectively. Maximal InsP formation was only 10-15% of that observed in m3 mAChR-expressing cells, whereas maximal elevations of [Ca2+]i were similar in both cell types. Formation of InsP3 was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR activation, [Ca2+]i increases induced by m2 mAChR activation were exclusively due to Ca2+ mobilization from intracellular stores. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was 50% and 20% of the efficacy of carbachol, respectively. PTX treatment did not affect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediated increases in [Ca2+]i to 50%. In conclusion, m3 and m2 mAChRs stably expressed in HEK cells can induce similar cellular responses; however, they do so by activating apparently distinct signalling pathways. While coupling of m2 mAChR to PLC occurs in a PTX-insensitive manner, coupling to mobilization of Ca2+ from intracellular stores is partly PTX-sensitive and this may occur at least partly independent of PLC activation.

We have compared a new commercially available non-radioactive protein kinase C (PKC) activity assay based on the fluorescent [A9,10K11]glycogen synthase1-11 analogue C1-peptide with a classical radioactive assay based on myelin basic protein4-14 (MBP4-14) and other substrates. The C1-peptide had lower affinity for PKC from rat brain than substrates such as MBP4-14, [S25]PKC alpha 19-31, and [A9,10K11,12]glycogen synthase1-12. The sensitivity of the C1-peptide-based assay was considerably lower than that of the MBP4-14-based assay. The C1-peptide was readily degraded in an ATP-independent manner by crude and DEAE-column chromatography-purified cytosolic extracts from rat brain, rat kidney, SK-N-MC and L929 cells. In rat kidney this degradation was not prevented by many common protease inhibitors. Phenylsepharose column chromatography separated the C1-peptide degrading activity from PKC. We conclude that the C1-peptide-based fluorescent PKC assay is applicable to highly purified PKC preparations but has low sensitivity and is not applicable to crude extracts due to substrate degradation.

Two alpha 1-adrenoceptor subtypes (alpha 1A and alpha 1B) have been detected in various tissues by pharmacological techniques, and three distinct cDNAs encoding alpha 1-adrenoceptor subtypes have been cloned. The profile of an increasing number of subtype-selective compounds at cloned and endogenous receptors recently has facilitated alignment between cloned and pharmacologically defined alpha 1-adrenoceptor subtypes. Thus, alpha 1a-adrenoceptors (previously designated alpha 1c), alpha 1b-adrenoceptors and alpha 1d-adrenoceptors (previously designated alpha 1a, alpha 1d or alpha 1a/d) are now recognized. Since the alpha 1d-adrenoceptor shares characteristics with both alpha 1A- and alpha 1B-adrenoceptors, tissues previously reported to express alpha 1A- and/or alpha 1B-adrenoceptors may additionally contain alpha 1d-adrenoceptors. This article reviews the features of all three subtypes and discusses possible pitfalls in their pharmacological identification.