Richard J. Hughes

Interaction of verapamil and other calcium channel blockers with alpha 1- and alpha 2-adrenergic receptors.

To determine the specificity of the previously demonstrated competition of verapamil with radioligand binding to alpha-adrenergic receptors, we examined the interaction of calcium channel blockers with alpha 1- and alpha 2-adrenergic receptors on several tissues. Verapamil competed for [3H] prazosin binding to alpha 1-adrenergic receptors and for [3H]yohimbine binding to alpha 2-adrenergic receptors in several tissues (human platelets, rat kidney and heart, and cultured muscle cells) with dissociation constants of 0.6-6 microM. The calcium channel blockers D600, D591, fendiline, and prenylamine--which are structural analogues of verapamil--also competed for [3H]yohimbine binding to human platelets. Two other calcium channel blockers, diltiazem and nifedipine, did not compete for [3H] yohimbine binding to human platelets or [3H]prazosin binding to membranes prepared from rat ventricles. We used [3H]nitrendipine binding to identify putative calcium channels on rat myocardial membranes. Nifedipine and verapamil blocked these [3H]nitrendipine-binding sites on ventricular membranes, but epinephrine and prazosin did not, indicating that the ventricular alpha 1 receptors and calcium channels are distinct. We found no specific [3H]nitrendipine binding to human platelets. We conclude that the interaction of verapamil with alpha-adrenergic receptors is not receptor subtype or tissue specific, that interaction with alpha-adrenergic receptors is not a property of all calcium channel blockers, and that the interaction of verapamil with alpha-adrenergic receptors and its interaction with calcium channels occur at at least two distinct sites.

ATP activates cAMP production via multiple purinergic receptors in MDCK-D1 epithelial cells. Blockade of an autocrine/paracrine pathway to define receptor preference of an agonist.

Extracellular nucleotides regulate function in many cell types via activation of multiple P2-purinergic receptor subtypes. However, it has been difficult to define which individual subtypes mediate responses to the physiological agonist ATP. We report a novel means to determine this by exploiting the differential activation of an autocrine/paracrine signaling pathway. We used Madin-Darby canine kidney epithelial cells (MDCK-D1) and assessed the regulation of cAMP formation by nucleotides. We found that ATP, 2-methylthio-ATP (MT-ATP) and UTP increase cAMP production. The cyclooxygenase inhibitor indomethacin completely inhibited UTP-stimulated, did not inhibit MT-ATP-stimulated, and only partially blocked ATP-stimulated cAMP formation. In parallel studies, ATP and UTP but not MT-ATP stimulated prostaglandin production. By pretreating cells with indomethacin to eliminate the P2Y2/prostaglandin component of cAMP formation, we could assess the indomethacin-insensitive P2 receptor component. Under these conditions, ATP displayed a ten-fold lower potency for stimulation of cAMP formation compared with untreated cells. These data indicate that ATP preferentially activates P2Y2 relative to other P2 receptors in MDCK-D1 cells (P2Y1 and P2Y11, as shown by reverse transcriptase polymerase chain reaction) and that P2Y2 receptor activation is the principal means by which ATP increases cAMP formation in these cells. Blockade of autocrine/paracrine signaling can aid in dissecting the contribution of multiple receptor subtypes activated by an agonist.

Extracellular ATP and cAMP as Paracrine and Interorgan Regulators of Renal Function P2Y Receptors of MDCK Cells: Epithelial Cell Regulation by Extracellular Nucleotides

1. Madin–Darby canine kidney (MDCK) cells, a well‐ differentiated renal epithelial cell line derived from distal tubule/collecting duct, respond to extracellular nucleotides by altering ion flux and the production of arachidonic acid‐derived products, in particular prostaglandin E2 (PGE2). Our work has defined the receptors and signalling events involved in such responses.

Certain beta-blockers can decrease beta-adrenergic receptor number: II. Down-regulation of receptor number by alprenolol and propranolol in cultured lymphoma and muscle cells.

We have used two different cultured cell lines—S49 lymphoma cells and BC3H-1 muscle cells—to examine the regulation of β-adrenergic receptors by receptor antagonists. Rather than an increase (“up-regulation”) of receptor number that such antagonists often produce, we found that certain β-blockers elidt a decrease (“down-regulation”) of beta-adrenergic receptors. Alprenolol and propranolol, but not sotalol or ICI 118,551, at concentrations of 10–100 nM down-regulated β-adrenergic receptors 20–70% following 16–20 hours of treatment of S49 or BC3H-1 cells. Several observations suggest that this phenomenon depends upon beta-receptor interaction, including stereoselectivity [(-)-enantiomers more potent than (+)-enantiomers], blockade of the effect by ICI 118,551, absence of down-regulation of α-adrenergic receptors hi BC3H-1 ceUs, and lack of a decrease in beta-adrenergic receptor-independent (forskolin-stimulated) cyclic AMP accumulation in S49 ceUs. The possibility of retained antagonist interfering with receptor measurement was precluded by the fact that the antagonist-induced decrease in receptor number required several hours incubation and occurred without a prominent change in receptor affinity. The ability of the β-blockers to elicit down-regulation did not correlate with hydrophobicity of the drugs. Antagonist-induced down regulation of beta-adrenergic receptors did not occur in S49 lymphoma cells that lack the α-subunit of Gg, the guanine nucleotide-binding regulatory protein, thus implying a requirement for receptor-α, interaction hi eliciting β-receptor down-regulation. The ability of certain antagonists to promote a down-regulation of β-adrenergic receptors provides a mechanism that may contribute to the pharmacological activity of these agents.

A bioluminescent assay for glycogen phosphorylase in cultured cells.

A new method for the determination of glycogen phosphorylase (1,4 alpha-D-glucose:orthophosphate alpha-glucosyltransferase, EC 2.4.1.1) in cultured cells is described. The assay utilizes bacterial luciferase (EC 2.7) in a liquid scintillation spectrometer to measure NAD(P)H formed in a coupled enzyme reaction comprising glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and phosphoglucomutase (EC 2.7.5.1). This assay is highly sensitive, easily detecting as little as 10 microU phosphorylase, fast and simple to perform. With modifications this procedure can be extended to measure other glycogenolytic enzymes and intermediates.