Biochemical Phormacolop. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Vol. 39, No. 6. pp. llll%lfl2X. 1990. Printed m Great Retain. oornZYSZ/Yo $3.00 + O.(X) @ 1990. Pergamon Press plc In the kidney many different receptor types are zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM THE AUTORADIOGRAPHIC LOCALIZATION OF ADENYLATE CYCLASE IN RAT KIDNEY USING [‘HIFORSKOLIN LYNNE R. MCMARTIN and ROGER J. SUMMERS* Department of Pharmacology, University of Melbourne, Parkville 3052, Australia zyxwvutsrqponmlkjihg (Received 7 August 1989; accepted 10 October 1989) Abstract-The localization of [ ‘Hlforskolin binding to microscope slide mounted sections of rat kidney has been examined using autoradiography. Saturation studies showed [‘Hlforskolin binding to two sites, a high affinity site (K, = 8.7 nM, B,,, = 0.14 pmol/mg protein) and a low affinity site (K, = 6.7 PM, B m&V = 11.0 pmol/mg protein). Autoradiographs showed high affinity binding (thought to identify stimulatory guanine nucleotide binding protein (Gs)-linked adenylate cyclase) to all renal structures known to possess hormone sensitive adenylate cyclase, including all tubular segments, glomeruli and blood vessels. High concentrations of binding were associated with a portion of the proximal tubule and with papillary collecting tubules and ducts. - linked to the stimulation ofadenylate cyclase, mclud- ing those which respond to parathyroid hormone @‘THt), calcitonin. glucagon, vasopressin, dopamine, histamine, serotonin, prostaglandins and noradrenaline. Only some of the physiological con- sequences of adenylate cyclase activation by these receptors are known, such as PTH stimulation of adenylate cyclase in the epithelial cells of the proxi- mal tubule which inhibits the reabsorption of phos- phate, bicarbonate, and associated fluid [l], and vasopressin stimulation of adenylate cyclase, via VZ receptors, which increases the osmotic permeability of collecting tubules to water resulting in an anti- diuretic effect (21. There are still, however, many examples of hormone-activated adenylate cyclase in the kidney for which the associated physiological responses are not known, including the vasopressin stimulated adenylate cyclase activity in the thin ascending limb of the loop of Henle [2] and glucagon stimulation of adenylate cyclase in collecting tubules [I]. The picture is further complicated by the finding that in some tubule segments different receptors activate the same adenylate cyclase pool. In the cortical segment of the thick ascending limb of the loop of Henle PTH, glucagon. calcitonin and vaso- pressin stimulate the same adenylate cyclase and presumably produce the same physlological response in these cells [2]. Much of the knowledge of hormone-responsive adenylate cyclase in the kidney is the result of adenyl- ate cyclase microassays of microdissected nephron segments. Another approach now available uses for- skolin, a diterpine compound, which stimulates hormone-sensitive adenylate cyclase via an inter- action with sites on the catalytic subunit or an associ- ated protein [3) and has been employed to investigate * To whom correspondence should be addressed. t Abbreviations used: Gs. stimulatory guanine nucleo- tide binding protein; PTH, parathyroid hormone; PGEZ, prostaglandin E?. the adenylate cyclase system in a variety of cells and tissues. The development of radiolabelled [3H]forskolin has allowed the identification and auto- radiographic localization of its binding sites in rat brain [4,5]. This study uses autoradiography to localize [3H]forskolin binding sites in a peripheral tissue, the rat kidney. The distribution of binding is discussed in relation to the known distribution of neurotransmitter- and hormone-receptors linked to adenylate cyclase in the kidney. MATERIALS AND METHODS M aterials [ 3H]Forskolin (49.2 Ci/mmol) was purchased from New England Nuclear (Boston, MA); forskolin, Napthol AS-MX phosphate solution, Fast Blue RR salt and Pyronin Y were purchased from the Sigma Chemical Co. (Poole, U.K.). Preparation of tissue sections. Female Wistar rats (240-260 g) were anaesthetized with methohexitone sodium (100 mg/kg i.p.) containing heparin sodium (50 IU/mg). Kidneys were perfused in situ with a mixture of equal parts of 0.32 M sucrose and Krebs phosphate buffer (composition mM: NaCl 118.4, KC1 4.7, MgSO., 1.2, CaClz 1.27, Na2P04 10.0, pH 7.4) via the abdominal aorta until clear of blood, followed by the same solution containing 0.1% for- maldehyde. Kidneys were removed and frozen in isopentane cooled in liquid nitrogen. Sections (10 pm) were cut using a Reichert-Jung cryostat at -18” and thaw-mounted onto cold gelatin-coated microscope slides. Labelling of slide-mounted kidney sections with (3H]forskolin. Slide-mounted sections were incu- bated at room temperature for 10 min in 50 mM Tris buffer (pH 7.7) containing 5 mM MgC12, 100mM NaCl [6] and [3H]forskolin (10 nM). To minimize the quantity of radioligand used, small aliquots of incubation medium (130 vL) were added to the slide- mounted sections. For the determination of non- specific binding, unlabelled forskolin (10 yM) was 1019