Mechanism of uptake of technetium-tetrofosmin. I: Uptake into isolated adult rat ventricular myocytes and subcellular localization.

BACKGROUND: 99mTc-labeled tetrofosmin is a new myocardial imaging agent that gives stable heart uptake. However, little is known about the mechanism of uptake in heart tissue. METHODS AND RESULTS: Uptake of 99mTc-labeled tetrofosmin has been examined in isolated adult rat ventricular myocytes. The time course of uptake, efflux rate, and the effect of metabolic and cation channel inhibitors have been assessed. The subcellular localization of radioactivity in ex vivo rat heart tissue was examined by differential centrifugation of ventricular homogenate. Uptake into rat myocytes was found to be rapid and plateaued at approximately 1.5 pmol/10(6) cells/nmol extracellular Tc-labeled tetrofosmin after 60 minutes of incubation. Uptake was temperature dependent but independent of extracellular Tc-labeled tetrofosmin concentration. Uptake at 30 minutes was inhibited by the metabolic inhibitors iodoacetic acid acid and 2,4-dinitrophenol protein but was not affected by cation channel inhibitors. Cells previously incubated with 99mTc-labeled tetrofosmin and then placed into fresh medium were found to have a slow efflux of activity; after 1 hour, 65% of activity was still cell associated. The localization of radioactivity in subcellular fractions indicated that the majority of activity was recovered with the cytosol. However, examination of the distribution of two mitochondrial enzymes indicated that this may have been artifactual. Use of carbonyl cyanide m-chlorophenyl-hydrazone or oligomycin to perturb mitochondrial membrane potential decreased or increased recovery in the mitochondrial fraction, respectively. CONCLUSIONS: 99mTc-labeled tetrofosmin uptake by myocytes is by a metabolism-dependent process that does not involve cation channel transport. The most likely mechanism for this is by potential driven diffusion of the lipophilic cation across the sarcolemmal and mitochondrial membranes.

Mechanisms of hormone-induced desensitization of adenylate cyclase.

Luteinizing hormone (LH) interacts with its plasma membrane receptor to activate the formation of cyclic AMP via the regulatory GTP binding protein (Gs). This is followed by a desensitization of that same hormonal response which is caused by an uncoupling of the LH receptor from Gs. The coupling between Gs and the adenylate cyclase catalytic unit remains intact. Treatment of Leydig and other cell types with phorbol esters mimics hormone-induced desensitization. However, differences between hormone- and phorbol ester-induced desensitization have been found. In testis Leydig cells phorbol esters, as well as uncoupling the LH receptor from Gs, also inactivates the subunit of the inhibitory GTP binding protein (Gi). These studies suggested that activation of protein kinase may be involved in the hormone-induced desensitization of adenylate cyclase. Paradoxically, it has also been found that two inhibitors of protein kinase C, sphingosine and psychosine also inhibited LH-induced cyclic AMP production. These effects were mainly found during the initial stimulatory period with LH. It is suggested that activation of adenylate cyclase may require a protein kinase C-mediated phosphorylation step which is followed by further phosphorylation resulting in uncoupling of the receptor from Gs. No direct stimulation of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3), diacylglycerol and/or activation of protein kinase C by LH in Leydig cells has been demonstrated. An alternative mechanism of protein kinase C activation has been proposed for brain cells, i.e. that involving arachidonic acid activation of protein kinase C instead of diacylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)

The inhibitory GTP-binding protein (Gi) occurs in rat Leydig cells and is differentially modified by lutropin and 12-O-tetradecanoylphorbol 13-acetate.

Luteinizing-hormone (LH)-stimulated cyclic AMP production in rat testis Leydig cells was desensitized by both LH and 12-O-tetradecanoylphorbol 13-acetate (TPA). However, TPA, but not LH, enhanced the subsequent response to cholera toxin. Treatment of the cells with pertussis toxin potentiated cyclic AMP production in both control and LH-desensitized cells, but did not potentiate further the responses obtained by TPA pretreatment. The results implicate the presence of an inhibitory GTP-binding protein (Gi), which may be inhibited by TPA. The presence of a Gi-like protein within the plasma membrane of Leydig cells was demonstrated by pertussis-toxin-catalysed [32P]ADP-ribosylation of a Mr-40000-41000 protein.