Comparison of the pharmacological profiles of adrenergic drugs (including BRL-agonists) at [3H]prazosin and [3H]CGP-12177 binding sites in brown adipose tissue

1. In order to determine the selectivity of classical and novel adrenergic agents for alpha 1- and beta-adrenergic receptors in brown adipose tissue, the ability of these agents to compete for binding sites labelled with [3H]prazosin and [3H]CGP-12177, respectively, was investigated. 2. The beta-antagonist propranolol, known to inhibit norepinephrine-induced respiration in micromolar concentrations, bound to the [3H]CGP-12177 site with nanomolar affinity. 3. Among agonists, only isoprenaline showed high selectivity for beta-receptors, and only oxymetazoline for alpha 1-receptors. 4. Unexpectedly, the novel thermogenic agonists (BRL-agonists), shown to be potent and selective stimulators of brown fat thermogenesis, were unselective and bound only with low affinity to the [3H]CGP-12177 binding sites. 5. These results suggest that the beta-adrenergic binding site in brown adipose tissue identified here with [3H]CGP-12177 may not be the one (or not the only one) coupled to thermogenesis.     

Stimulation of nonshivering thermogenesis in the Syrian hamster by norepinephrine and β-selective adrenergic agents: A phenomenon of refractoriness

The ability of different adrenergic agents to stimulate nonshivering thermogenesis in Syrian hamsters was investigated. The hamsters were cold-acclimated to 6 °C and their thermogenic response was investigated in an open-circuit system at 24 °C. Both norepinephrine and the β₃-specific adrenergic agonist CGP-12177 induced a high rate of nonshivering thermogenesis. However, neither CGP-12177 nor other β₃-selective agonists (BRL-37344, ICI-D7114) could induce nonshivering thermogenesis fully to the extent induced by norepinephrine. It was further observed that an apparent “thermogenic refractoriness” was induced by certain adrenergic agents (isoprenaline, CGP-12177) but not by others (norepinephrine, BRL-37344, ICI-D7114). It is discussed whether the refractoriness could be secondary to effects of these agents on the vascular system. It is pointed out that the thermogenic response to adrenergic stimulation observed in the intact animal does not always fully correspond to what would be predicted from corresponding studies with isolated brown-fat cells.     

A reliable assay for beta-adrenoceptors in intact isolated human fat cells with a hydrophilic radioligand, [3H]CGP-12177

The beta-adrenergic receptors of isolated human fat cells were identified using a new hydrophilic beta-adrenergic radioligand (+/-)[3H]CGP-12177. The results were compared with those from [3H]dihydroalprenolol binding to fat cells and membranes. [3H]CGP-12177 binding to isolated fat cells showed lower nonspecific binding (less than 15% of total binding) than the lipophilic [3H]dihydroalprenolol (40-60%) at 3 times the KD. At 37 degrees C, [3H]CGP-12177 binding was rapid, reversible, of high affinity (1.2 +/- 0.3 nM) and saturable. The total number of binding sites per cell in subcutaneous adipocytes was 25,000 +/- 6,000 and was equivalent to that found using membrane fractions. Displacement of [3H]CGP-12177 bound to adipocytes by propranolol was stereoselective, consistent with competition at a single site, and had the same characteristics as in membranes. The displacement curves of the beta 1-selective antagonists (atenolol and betaxolol) were biphasic, the high affinity displacement accounting for 70% of the total binding sites. Beta-adrenergic agonists also competed with [3H]CGP-12177 binding in the order of potency: (-) isoproterenol greater than (-) norepinephrine greater than (-) epinephrine, similar to that found in membranes and in in vitro studies on the lipolytic activity of isolated fat cells. This study demonstrates that the sites specifically labeled by [3H]CGP-12177 are the physiological beta-adrenoceptors and also shows that the ligand is better than [3H]dihydroalprenolol for the accurate identification of these receptors in intact human adipocytes. The methodology, which requires biopsies of less than 1 gram of adipose tissue, can be of potential interest for clinical studies investigating the status of fat cell beta-adrenoceptors in various pathophysiological situations.     

Reduction in β-Adrenoceptor Density in Cultured Rat Glioma C6 Cells After Incubation with Antidepressants Is Dependent upon the Culturing Conditions Used

The hydrophilic beta-adrenoceptor ligand (-)-[3H]CGP-12177 binds to intact C6 cells with a high affinity (KD approximately 0.1 nM) and with a high degree of specificity. The binding was inhibited by DL-propranolol (Ki approximately 1 nM). Treatment of cells cultured in Dulbecco's modified Eagle medium (DMEM) without fetal calf serum for 4 days with desipramine reduced the (-)-[3H]CGP-12177 specific binding in a concentration-dependent manner, a reduction from 127 to 102 fmol/mg of protein being found at a ligand concentration of 1 nM after treatment with 10 microM desipramine. Lesser effects were seen after treatment for 1 day. A similar result was found with maprotiline, and reductions in specific binding were seen after 4 days of treatment with amitriptyline, iprindole, and citalopram. The reduction in binding-site density (measured per milligram of protein to compensate for variability in cell density per well), however, was paralleled in all cases by a reduction in the rate of cell proliferation. When C6 glioma cells were cultured in Ham's medium without fetal calf serum during the antidepressant treatment period, a higher specific binding was observed than for the DMEM-cultured cells, and 10 microM desipramine was without effect on either the (-)-[3H]CGP-12177 specific binding or cell proliferation. It is concluded that the effects of the antidepressants tested upon the density of (-)-[3H]CGP-12177 specific binding sites in intact C6 cells may be secondary to the toxicity of the compounds under the conditions used.     

Some Unique Properties of CGP-12177

Abstract

CGP-12177, like isoproterenol, has a lower affinity for desensitized receptors. Experiments were performed to study whether this property of CGP-12177 is due to its partial agonist activity or its impermeability for membranes. Reduced binding of [³H]DHA to desensitized receptors at 0°C as well as a reversal of the reduced binding in the presence of digitonin indicate a permeability barrier. A partial agonist effect of CGP-12177 was only found in intact C6 cells and neither in C6 membranes nor in S49 cells. This peculiar effect in intact C6 cells is stereospecific and not due to an inhibition of phosphodiesterases.     

Temperature-dependent binding of hydrophilic beta-adrenergic receptor ligands to intact human lymphocytes

Important differences in binding characteristics between agonists and antagonists of the beta-adrenergic receptor have been described. However, these observations have been complicated since most available antagonists are much more lipophilic than agonists. In order to separate out those binding characteristics of agonist vs. antagonist from those characteristics of lipophilic vs. hydrophilic ligands, we have studied competition of the hydrophilic ligands isoproterenol (agonist) and CGP-12177 (antagonist) with [125I]iodopindolol binding in intact human lymphocytes. Analyzing competition curves from assays performed at 13 degrees C, 25 degrees C and 37 degrees C we demonstrated that at lower temperatures there was a decrease in IC50 for isoproterenol but not for CGP-12177. Using cells preincubated with isoproterenol then extensively washed, competition curves with both isoproterenol and CGP-12177 were biphasic, and characterized by the appearance of a population of receptors with a low affinity for both hydrophilic ligands. Furthermore, at lower temperatures the biphasic nature of these curves was accentuated and was characterized by a 6-fold and 40-fold increase in the apparent KD of a population of low affinity sites for isoproterenol and CGP-12177, respectively.     

Beta-adrenergic, cAMP-mediated stimulation of proliferation of brown fat cells in primary culture. Mediation via beta 1 but not via beta 3 adrenoceptors.

The ability of adrenergic stimulation to affect the rate of DNA synthesis in mouse brown adipocyte precursor cells proliferating in primary culture was investigated. Addition of 1 microM norepinephrine to the cells at day 4 in culture (proliferating cells) significantly increased the rate of DNA synthesis, whereas no significant effect was seen at day 9 (confluent cells). The effect of norepinephrine could be mimicked by forskolin, cholera toxin, and by cAMP analogues. Specific [3H]thymidine incorporation (per unit of DNA) was reduced by norepinephrine stimulation, indicating saturation of the salvage pathway for dTTP synthesis already in unstimulated cells and implying a beta-adrenergic stimulation of dTTP synthesis. Pharmacological characterization of this effect indicated it was mediated by beta 1 receptors, with alpha 2 receptors exerting an opposing effect. Notably, the stimulation of DNA synthesis was not observed with the beta 3-specific agonist CGP-12177. In contrast, both norepinephrine and CGP-12177 were able to induce the expression of the uncoupling protein thermogenin in the confluent cultured cells. This coincided with a shift in the cAMP-elevating potential of CGP-12177, from being antagonistic to norepinephrine stimulation in proliferating cells to being itself a full agonist in confluent cells, implying occurrence of coupled beta 3 receptors as part of the differentiation process. It was concluded that brown fat precursor cells respond directly to norepinephrine stimulation with an increased DNA synthesis, and that this response is mediated via the classical beta 1 receptors. This probably represents the cellular basis for the hyperplasia observed in the tissue in physiologically recruited states.     

Evidence for cooperative binding of (-)Isoproterenol to rat brain beta1-adrenergic receptors

In the present study, the effects of the thiol oxidising agent diamide upon the properties of rat brain beta1-adrenergic and human platelet serotonin2A receptor recognition sites have been investigated using [3H](-)CGP-12177 (in the presence of ICI-118551) and [3H]LSD as ligands. (-)Isoprenaline inhibited [3H](-)CGP-12177 binding with nH values of 0.87, 0.67, and 0.56 for added Mg2+ concentrations of 0, 2.5, and 25 mM, respectively. Pretreatment with diamide increased the nH to above unity for the inhibition of the binding by (-)isoprenaline, without a concomitant effect on the inhibition of the binding by (-)propranolol. In contrast, diamide reduced the affinity of human platelet serotonin2A-receptors for antagonists, but did not consistently induce nH values above unity for the inhibition of antagonist binding by serotonin. These results suggest that cooperative interactions reported for cardiac muscarinic receptors may also occur for beta1-adrenergic receptors in the rat brain.     

Determination of the desensitization of beta-adrenergic receptors by [3H]CGP-12177.

Isoprenaline treatment of C6-glioma cells induced a fast decrease in the number of beta-adrenergic receptors as determined by binding of [3H]CGP-12177, which paralleled the decrease in the hormonally stimulated adenylate cyclase activity. The total number of receptors, as determined by binding of (-)-[3H]dihydroalprenolol, did not decrease. Separation of the beta-adrenergic receptors on a sucrose density gradient showed that the decrease in the number of receptors detectable with CGP-12177 was due to a movement of the receptors from the plasma membrane to a vesicular cell compartment. By using both (-)-[3H]dihydroalprenolol and [3H]CGP-12177 it is thus possible to differentiate between the total number of receptors and those present at the plasma membrane in an unfractionated cell lysate.     

Evidence for the appearance of an uncoupled form of the beta-adrenergic receptor distinct from the internalized receptor

Agonist treatment of C6-glioma cells induces two altered states in beta-adrenergic receptors, a low affinity for the hydrophilic antagonist CGP-12177 and a low affinity for agonists like isoproterenol. We present evidence that, in cells not treated to inhibit receptor internalization, the two properties occur with a different time course, the low affinity for isoproterenol preceding that for CGP-12177. In that the low affinity for CGP-12177 is due to the internalization of the receptor, the results indicate that uncoupling of the receptor, indicated by the low affinity for isoproterenol, occurs while the receptor is still located on the cell surface. Removal of the agonist leads to reappearance of the receptor to the plasma membrane followed by loss of the uncoupled state.     

Labelling of Rat Brain ß-Adrenoceptors: (3H)CGP-12177 or (125I)Iodocyanopindolol?

Abstract

Binding of (¹²⁵I) iodocyanopindolol (ICYP) and (³H) CGP-12177 to rat brain homogenates was characterized and compared. ICYP was shown to bind to both ß-adrenergic and serotonin1B (5HT_1B) receptors whereas (³H)CGP-12177 only labelled the first ones. The addition of 10 μM serotonin (5HT) prevented ICYP binding to 5HT receptors and under these experimental conditions both ligands labelled a similar total number of ß-adrenoceptors in the different rat brain regions. ICYP displayed a higher affinity for cerebellar (mainly ß₂-subtype) than for cerebral cortex ß-adrenoceptors (mainly ß-subtype) suggesting a subtype selectivity. A multiple displacement binding approach using CGP-20712A, a ß₁-subtype ligand, as competitor revealed a 2.6 fold selectivity of ICYP for the ß₂-adrenoceptor subtype. On the other hand, (³H)CGP-12177 binds only to ß-adrenoceptors and is not subtype selective in the rat brain homogenate. Considering both its high specificity and its lack of subtype selectivity (³H)CGP-12177 seems to be a more suitable ligand than ICYP to non-selectively label ß-adrenoceptors in rat brain.     

Atypical beta-adrenergic effects on insulin signaling and action in beta(3)-adrenoceptor-deficient brown adipocytes

Cross talk between adrenergic and insulin signaling systems may represent a fundamental molecular basis of insulin resistance. We have characterized a newly established beta(3)-adrenoceptor-deficient (beta(3)-KO) brown adipocyte cell line and have used it to selectively investigate the potential role of novel-state and typical beta-adrenoceptors (beta-AR) on insulin signaling and action. The novel-state beta(1)-AR agonist CGP-12177 strongly induced uncoupling protein-1 in beta(3)-KO brown adipocytes as opposed to the beta(3)-selective agonist CL-316,243. Furthermore, CGP-12177 potently reduced insulin-induced glucose uptake and glycogen synthesis. Neither the selective beta(1)- and beta(2)-antagonists metoprolol and ICI-118,551 nor the nonselective antagonist propranolol blocked these effects. The classical beta(1)-AR agonist dobutamine and the beta(2)-AR agonist clenbuterol also considerably diminished insulin-induced glucose uptake. In contrast to CGP-12177 treatment, these negative effects were completely abrogated by metoprolol and ICI-118,551. Stimulation with CGP-12177 did not impair insulin receptor kinase activity but decreased insulin receptor substrate-1 binding to phosphatidylinositol (PI) 3-kinase and activation of protein kinase B. Thus the present study characterizes a novel cell system to selectively analyze molecular and functional interactions between novel and classical beta-adrenoceptor types with insulin action. Furthermore, it indicates insulin receptor-independent, but PI 3-kinase-dependent, potent negative effects of the novel beta(1)-adrenoceptor state on diverse biological end points of insulin action.     

Increased number of cardiac adrenergic receptors following local heart irradiation

The effect of local X irradiation on cardiac alpha and beta receptors was studied in Wistar rats. Animals were given local heart irradiation with single doses of 15 or 20 Gy and were examined after a range of latency times of 7 to 400 days. Using the radioactive ligands [3H]CGP-12177 and [3H]prazosin, the maximal binding capacity was determined from saturation experiments. At 7 days after 20 Gy the maximal binding capacity of both alpha and beta receptors was reduced to below the level of untreated control animals. Subsequently it rose continually to a maximum of 160% of the control level for beta receptors and 130% for alpha receptors at 400 days postirradiation. The antagonist affinity as judged from the dissociation constant for [3H]CGP 12177 and [3H]prazosin did not change significantly. A similar effect was observed after 15 Gy. An increase in adrenergic receptors may represent an important pathogenetic link between early morphological and late functional changes in the pathogenesis of radiation-induced heart disease.     

Differences between the effects of noradrenaline and the beta-adrenoceptor agonist BRL 28410 in brown adipose tissue and hind limb of the anaesthetized rat

In mature (450-600 g) 21 degrees C-acclimated male rats, anaesthetized with urethane, blood flow (measured by the radioactive microsphere technique) to brown adipose tissue (BAT) was determined during the infusion of the beta-adrenoceptor agonist BRL 28410 or noradrenaline bitartrate at doses chosen to give similar increases in whole body oxygen uptake. Blood flow to BAT during BRL 28410 infusion was only about one third of that found during noradrenaline infusion although increases in whole body thermogenesis were similar (55 and 77% for BRL 28410 and noradrenaline, respectively). This suggests that BAT may be less involved in the thermogenic response to BRL 28410 than to noradrenaline. In a separate experiment using slightly smaller rats (350-500 g) hind limb oxygen uptake was measured in situ using a venous bypass preparation. BRL 28410, at a dose having a maximum effect on whole body thermogenesis (53% increase), had no effect on oxygen delivery to the hind limb but significantly increased oxygen extraction by 33% (p less than 0.001). In contrast, noradrenaline, also at a dose that maximally increased whole body thermogenesis, led to a 35% decrease in oxygen delivery to the hind limb and no change in oxygen extraction. For the thermogenic beta-agonist BRL 28410 the hind limb, and presumably muscular tissue in general, may be contributing to thermogenesis.     

Signal transduction by membrane receptors in viable electropermeabilized cells: isoproterenol-stimulated cyclic AMP synthesis in C6 glioma cells

The activity of beta-adrenergic receptors at the plasma membrane level was investigated in viable, electropermeabilized C6 glioma cells. Electric field pulses were applied directly to the plated cells without any previous proteinase treatment. The affinity for isoproterenol and the density of the beta-adrenergic receptors, as judged from the number of [3H]CGP-12177 binding sites, were not affected by the electropermeabilization whereas the isoproterenol-stimulated cAMP accumulation was transiently impaired. This decrease in activity is due to an electropermeabilization-induced GTP leak. Normal activity could be obtained either by treating the cells by the electric field in a GTP-containing buffer, or by spontaneous recovery of the cells after the resealing of the plasma membrane, with a delay depending on the temperature. The activity of the receptors was not affected by the structural organization of the membrane associated to its electropermeabilization.     

Desensitization of the beta-adrenergic receptor-coupled adenylate cyclase in cultured mammalian cells. Receptor sequestration versus receptor function

Human A431 and rat glioma C6 cells exposed to isoproterenol underwent a time- and dose-dependent loss of isoproterenol-stimulated adenylate cyclase activity. Desensitization was accompanied by sequestration of beta-adrenergic receptors, which became less accessible to the hydrophilic antagonist 3H-labeled 4-(3-tert-butylamino-2-hydroxypropoxy)benzimidazole-2-one hydrochloride ([3H]CGP-12177) and redistributed from the heavier density plasma membrane fraction to a lighter density membrane fraction. Prior treatment of the cells with concanavalin A or phenylarsine oxide blocked sequestration of the receptors but not desensitization of the agonist-stimulated adenylate cyclase. The membranes from such pretreated cells were exposed to alkali to inactivate adenylate cyclase, and the receptors were transferred to a foreign adenylate cyclase by membrane fusion with polyethylene glycol. beta receptors from desensitized cells exhibited a reduced ability to maximally stimulate the foreign adenylate cyclase, but remained accessible to [3H]CGP-12177 in the fused membranes. When isoproterenol-treated cells were washed free of agonist, there was a time-dependent recovery of agonist responsiveness and [3H]CGP-12177-binding sites. Using the fusion technique, the receptors recovered their functional activity in the resensitized cells. In concanavalin A-treated cells, desensitization and resensitization appeared to occur in the absence of receptor sequestration. Finally, membranes from desensitized cells pretreated with concanavalin A were fused with polyethylene glycol and assayed for agonist-stimulated adenylate cyclase. There was no reversal of the desensitized state. Thus, the primary, essential step in the desensitization process is a reduction in functional activity of the beta-adrenergic receptor. In contrast, sequestration of the receptors is not a prerequisite, but a secondary event during desensitization.     

Adenylate cyclase uncoupled beta-adrenergic receptors in salamander proximal tubules

The isolated perfused proximal tubule of the neotenic salamander Ambystoma tigrinum responds with either a hyperpolarization or depolarization of both the basolateral cell membrane and transepithelial potentials following the addition of 10(-5) M isoproterenol to the bath superfusate. Both responses were blocked by 10(-6) M propranolol but neither response was mimicked by 10(-4) M cAMP. beta-Adrenergic binding studies of individual microdissected proximal tubules using (-)-[3H]CGP-12177 as a hydrophyllic radioligand and (+/-)-timolol (0.1 mM) as the displacer drug revealed two distinct populations of proximal tubules possessing either low (KD = 153.8 nM; Bmax = 110.2 fM/mm) or high affinity (KD = 12.0 nM: Bmax = 3.9 fM/mm) binding characteristics. Competition studies indicated that the bound (-)-[3H]CGP-12177 behaved as a typical beta-adrenergic ligand, being displaced by (-)-isoproterenol but not by (+)-isoproterenol or (-)-phenylephrine. However, neither appeared to be coupled to the adenylate cyclase system. These data suggest the presence of functional beta-adrenergic receptors that do not appear to be coupled to the adenylate cyclase system.     

Rapid and reversible disappearance of beta-adrenergic cell surface receptors

The water soluble beta-adrenergic ligand [3H]CGP-12177 was used to measure the cell surface receptors in intact cells. In two cell lines, C6 glioma and WEHI 7 lymphoma cells, -50% of the cell surface receptors disappear within minutes of incubation of the cells with isoproterenol. The receptors can still be detected in homogenates and reappear on the cell surface when cells are washed and reincubated at 37 degrees C. The data agree with a disappearance of the receptors from the cell surface by an agonist-mediated endocytosis.     

Atypical beta-adrenergic receptor in 3T3-F442A adipocytes. Pharmacological and molecular relationship with the human beta 3-adrenergic receptor.

Expression of ligand binding properties for an atypical beta-adrenergic receptor (beta-AR) subtype was studied during the adipose differentiation of murine 3T3-F442A cells and compared with that of the human beta 3-AR expressed in Chinese hamster ovary cells stably transfected with the human beta 3-AR gene (CHO-beta 3 cells) Emorine, L. J., Marullo, S., Briend-Sutren, M. M., Patey, G., Tate, K., Delavier-Klutchko, C., and Strosberg, A. D. (1989) Science 245, 1118-1121). 3T3-F442A adipocytes exhibited high and low affinity binding sites for (-)-4-(3-t-butylamino-2-hydroxypropoxy) [5,7-3H]benzimidazole-2-one ((-)-[3H]CGP-12177) (KD = 1.2 and 38.3 nM) and (-)-[125I]iodocyanopindolol ([125I]CYP) (KD = 47 and 1,510 pM). The high affinity sites corresponded to the classical beta 1- and beta 2-AR subtypes whereas the KD values of the low affinity sites for the radioligands were similar to those measured in CHO-beta 3 cells (KD = 28 nM and 1,890 pM for (-)-[3H]CGP12177 and [125I]CYP, respectively). These low affinity sites were undetectable in preadipocytes but represented about 90% of total beta-ARs in adipocytes. The atypical beta-AR and the human beta 3-AR add similarly low affinities (Ki = 3-5 microM) for (+/-)-(2-(3-carbamoyl-4-hydroxyphenoxy)ethylamino-3)-(4-(1-methyl- 4- trifluormethyl-2-imidazolyl)-phenoxy)-2-propanol methane sulfonate (CGP20712A) or erythro-(+/-)-1-(7-methylindan-4-yloxy)-3-isopropylaminob utan-2-ol (ICI118551), highly selective beta 1- and beta 2-AR antagonists, respectively, in agreement with the poor inhibitory effect of the compounds on (-)-isoproterenol (IPR)-stimulated adenylate cyclase activity. Atypical beta-AR and beta 3-AR had an affinity about 10-50 times higher for sodium-4-(2-[2-hydroxy-2-(3-chlorophenyl)ethylamino]propyl)phenoxyace tate sesquihydrate (BRL37344) than the beta 1-AR subtype. This correlates with the potent lipolytic effect of BRL37344 in adipocytes. The rank order of potency of agonists in functional and binding studies was BRL37344 greater than IPR less than (-)-norepinephrine greater than (-)-epinephrine both in 3T3 adipocytes and CHO-beta 3 cells. As in CHO-beta 3 cells, the classical beta 1- and beta 2-antagonists CGP12177, oxprenolol, and pindolol were partial agonists in adipocytes. Although undetectable in preadipocytes, a major mRNA species of 2.3 kilobases (kb) and a minor one of 2.8 kb were observed in adipocytes by hybridization to a human beta 3-AR specific probe.(ABSTRACT TRUNCATED AT 400 WORDS)     

Norepinephrine-induced sustained inward current in brown fat cells: alpha(1)-mediated by nonselective cation channels

The nature of the sustained norepinephrine-induced depolarization in brown fat cells was examined by patch-clamp techniques. Norepinephrine (NE) stimulation led to a whole cell current response consisting of two phases: a first inward current, lasting for only 1 min, and a sustained inward current, lasting as long as the adrenergic stimulation was maintained. The nature of the sustained current was here investigated. It could be induced by the alpha(1)-agonist cirazoline but not by the beta(3)-agonist CGP-12177A. Reduction of extracellular Cl(-) concentration had no effect, but omission of extracellular Ca(2+) or Na(+) totally eliminated it. When unstimulated cells were studied in the cell-attached mode, some activity of approximately 30 pS nonselective cation channels was observed. NE perfusion led to a 10-fold increase in their open probability (from approximately 0.002 to approximately 0.017), which persisted as long as the perfusion was maintained. The activation was much stronger with the alpha(1)-agonist phenylephrine than with the beta(3)-agonist CGP-12177A, and with the Ca(2+) ionophore A-23187 than with the adenylyl cyclase activator forskolin. We conclude that the sustained inward current was due to activation of approximately 30 pS nonselective cation channels via alpha(1)-adrenergic receptors and that the effect may be mediated via an increase in intracellular free Ca(2+) concentration.