Although the function of metallothionein (MT), a 6-to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging experiments with live endothelial cells show that agents that increase cytoplasmic Ca 2؉ act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT ؊͞؊ ) unless endogenous NO synthesis is blocked. These results are the first application of intramolecular green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO production. In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed. Metallothioneins (MT) are 6-to 7-kDa intracellular cysteine-rich (30 mol%) metal binding proteins whose function remains elusive (1). A critical role for MT in protection against toxic non-essential metals such as cadmium is apparent (2), and MT appears to act as an antioxidant under a variety of conditions (3). More recently, in vitro data support the hypothesis that MT is a critical link between cellular redox state and metal ion homeostasis (4-6). In this regard, cysteines of metal thiolate clusters confer unique redox sensitivity to an otherwise redox inert metal ligand (e.g., zinc) and facilitate the potential for MT to participate in intracellular signal transduction pathways (7). In the current study, we examine this latter novel hypothesis in intact cells and tissue.We chose to study the interaction of MT and the free radical, nitric oxide (NO), because (i) the bioregulatory targets of NO usually contain cysteines and͞or metals at their active or allosteric site (8); (ii) NO (or a secondary product) reacts with MT in vitro, leading to the release of zinc (9) or cadmium (10); (iii) NO can form stable EPR-detectable complexes with MT in vitro (11); and (iv) MT can reduce the sensitivity of cells to potential toxic levels of NO (12). Application of a chimeric construct (called FRET-MT) based on a recently described cameleon for calmodulin (13) revealed an NO-induced conformational change in MT, indicative of metal release, thereby providing the first demonstrations of (i) changes in intramolecular FRET (fluorescence resonance energy transfer) of a native protein; and (ii) metal release from MT in response to physiologic stimuli in intact cells. Furthermore, the lack of myogen...

Administration of the ovarian hormone relaxin to nonpregnant rats vasodilates the renal circulation comparable to pregnancy. This vasodilation is mediated by endothelin (ET), the ET(B) receptor, and nitric oxide. Furthermore, endogenous relaxin mediates the renal vasodilation and hyperfiltration that occur during gestation. The goal of this study was to investigate whether myogenic reactivity of small renal and mesenteric arteries is reduced in relaxin-treated rats comparable to the pregnant condition. Relaxin or vehicle was administered to virgin female Long-Evans rats for 5 days at 4 microg/h, thereby producing midgestational blood levels of the hormone. The myogenic responses of small renal arteries (200-300 microm in diameter) isolated from these animals were evaluated in an isobaric arteriograph system. Myogenic reactivity was significantly reduced in the small renal arteries from relaxin-treated compared with vehicle-treated rats. The reduced myogenic responses were mediated by the ET(B) receptor and nitric oxide since the selective ET(B) receptor antagonist RES-701-1 and the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester restored myogenic reactivity to virgin levels. The influence of relaxin was not limited to the renal circulation because myogenic reactivity was also reduced in small mesenteric arteries isolated from relaxin-treated rats. Thus relaxin administration to nonpregnant rats mimics pregnancy, insofar as myogenic reactivity of small renal and mesenteric arteries is reduced in both conditions.

We tested the hypothesis that endothelin acting through the endothelial ET(B) receptor subtype and the nitric oxide (NO) pathway accounts for reduced myogenic reactivity of the renal resistance vasculature during pregnancy. Small renal arteries (100-200 microm) were isolated from virgin and midterm pregnant rats when gestational renal hyperfiltration and vasodilation are maximal in this species. Myogenic reactivity (the adjustment of arterial diameter in response to a change in transmural pressure) was assessed with a pressurized myograph system. A rapid increase in transmural pressure from 60 to 80 mmHg resulted in a 2.4% diameter increase in vessels from virgin compared with an 8.1% increase in arteries from midgestation rats (n = 8 each, P < 0.05). Thus myogenic reactivity is markedly reduced during pregnancy. Incubation with the NO synthase inhibitors, an ET(B) receptor subtype antagonist (RES-701-1), the nonselective ET(A/B) receptor blocker (SB-209670), or endothelial removal abrogated the reduced myogenic reactivity of vessels from gravid rats without affecting myogenic reactivity in arteries from virgin animals. Thus the endothelium mediates the reduced myogenic reactivity of small renal arteries of midgestation rats most likely through the ET(B) receptor subtype and NO pathway.

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