extfieldsofstudy: Biology Chemistry Medicine

text:
Isolation and Characterization of a Novel ConusPeptide with Apparent Antinociceptive Activity*

Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds. We report the discovery and biochemical characterization of a structurally unique peptide isolated from the venom of Conus marmoreus. The new peptide, mr10a, potently increased withdrawal latency in a hot plate assay (a test of analgesia) at intrathecal doses that do not produce motor impairment as measured by rotarod test. The sequence of mr10a is NGVCCGYKLCHOC, where O is 4-trans-hydroxyproline. This sequence is highly divergent from all other known conotoxins. Analysis of a cDNA clone encoding the toxin, however, indicates that it is a member of the recently described T-superfamily. Total chemical synthesis of the three possible disulfide arrangements of mr10a was achieved, and elution studies indicate that the native form has a disulfide connectivity of Cys1-Cys4 and Cys2-Cys3. This disulfide linkage is unprecedented among conotoxins and defines a new family of Conus peptides.

Conus is a genus of predatory marine gastropods that envenomate their prey. Prey capture is accomplished through a sophisticated arsenal of peptides that target specific ion channel and receptor subtypes. Each Conus venom appears to contain a unique set of 50 -200 peptides. The structure and function of only a small minority of these peptides have been determined to date. For peptides where function has been determined, three classes of targets have been elucidated: voltage-gated ion channels, ligand-gated ion channels, and G-protein-linked receptors.
Conus peptides that target voltage-gated ion channels include those that delay the inactivation of sodium channels as well as blockers specific for sodium channels, calcium channels, and potassium channels. Peptides that target ligand-gated ion channels include antagonists of N-methyl-D-aspartate and serotonin receptors as well as competitive and non-competitive nicotinic receptor antagonists. Peptides that act on G protein receptors include neurotensin and vasopressin receptor agonists. The unprecedented targeting selectivity of conotoxins derives from specific disulfide bond frameworks combined with hypervariable amino acids within disulfide loops (see Ref. 1 for review). Due to the high potency and exquisite selectivity of the conopeptides, several are in various stages of clinical development for treatment of human disorders (2).
In this report we describe the isolation of a new peptide from the venom of the marble cone, Conus marmoreus. C. marmoreus is found in the Indo-Pacific, from India to the Marshall Islands and Fiji. It preys upon various gastropods including other cone snails (3). We previously reported the isolation and characterization of a peptide from this venom that potently inhibits voltage-gated sodium channels (4). In this report, we describe the isolation of a novel peptide that appears antinociceptive and likely represents a defining member of a new family of Conus peptides.

Materials and HPLC 1 Conditions
The venom of C. marmoreus was obtained from snails collected in the Philippines. The venom was lyophilized and stored at Ϫ70°C until use. Crude venom was extracted using previously described methods (5). Reverse phase HPLC purification was accomplished with an analytical (4.6 mm internal diameter ϫ 25 cm) Vydac C 18 column. Column pore size was 300 Å. Additional conditions are described in Fig. 1.

Reduction, Alkylation, and Peptide Sequencing
The peptide was reduced, and cysteines were carboxymethylated as described previously (6). The alkylated peptide was purified with a Vydac C 18 analytical column using a linear gradient of 0.1% trifluoroacetic acid and 0.092% trifluoroacetic acid in 60% acetonitrile. Alkylated peptide was sequenced by Edman degradation at the Peptide Core Facility at the University of Utah.

Mass Spectrometry
Electrospray ionization mass spectra were measured using a Micromass Quattro II Triple Quadrupole Mass Spectrometer with Micromass MassLynx operating system. The samples (ϳ100 pmol) were resuspended in 0.1 ml of 50% acetonitrile with 0.05% trifluoroacetic acid and automatically infused with a flow rate of 0.05 ml/min in the same solvent system. The instrument was scanned over the m/z range 50 -2,000 with a capillary voltage of 2.95 kV and a cone voltage of 64 V. The resulting data were analyzed using MassLynx software. fluorenyl)methoxycarboxyl) chemistry and standard side chain protection except on cysteine residues. Cys residues were protected in pairs with either S-trityl or S-acetamidomethyl groups. Amino acid derivatives were from Advanced Chemtech (Louisville, KY). All three possible disulfide forms of the peptide were synthesized. The peptides were removed from the resin and precipitated, and a two-step oxidation protocol was used to selectively fold the peptides as described previously (7).

mr10a Precursor cDNA Cloning
The sequence of the mr10a peptide was used to design degenerate oligonucleotide primers for use in 5Ј and 3Ј RACE amplification of the mr10a precursor cDNA. A 3Ј RACE forward primer (mr10a-F, CAG-GATCC AA(T/C) GGI GT(C/T/G) TG(T/C) TG(T/C) GG) was based on the peptide sequence NGVCCG. A 5Ј RACE reverse primer (mr10a-R, CTGGATCC GG (A/G)TG (A/G)CA (C/A/G)A(G/A) (T/C)TT (G/A)TA ICC) was based on the peptide sequence GYKLCHP. C. marmoreus venom duct RNA was prepared, and cDNA appended with 3Ј and 5Ј adapter sequences was synthesized by standard methods (8). To facilitate cloning of the RACE amplification products, the mr10a primers incorporated BamHI sites, and the 3Ј and 5Ј adapter primers contained XhoI sites. RACE amplifications were performed using a "touchdown" cycling protocol consisting of an initial denaturation of 95°C for 30 s followed by 30 cycles of 95°C for 10 s , 65°C for 15 s, decreasing 0.5°C each cycle, 72°C for 10 s, then 15 cycles of 95°C for 10 s, 50°C for 10 s, and 72°C for 10 s. Polymerase chain reaction amplifications were performed using Taq polymerase (PE Applied Biosystems, Foster City, CA), and amplification reactions were analyzed by electrophoresis on 2% agarose gels. RACE amplification products were isolated from the gel (Qiaex II DNA purification resin; Qiagen, Inc., Santa Clarita, CA), digested with BamHI and XhoI, and cloned into the plasmid vector pBluescript II SK Ϫ (Stratagene, La Jolla, CA). Plasmids containing inserts of the appropriate size were selected and sequenced on an ABI Prism 373 Fluorescent DNA Sequencer.

Experimental Animals
Adult male CF-1 mice (25-35 g) were used for all experiments. Mice were housed five per cage, maintained on a 12-h light/dark cycle, and allowed free access to food and water.

Hot Plate Test
Analgesic activity was assessed by placing mice in a plexiglass cylinder (10.2-cm diameter ϫ 30.5 cm high) on a hot plate (Mirak model HP72935, Barnstead/Thermolyne, Dubuque, IA) maintained at 55°C. Thirty minutes before the hot plate test, animals were treated with a dose of mr10a or vehicle (0.9% saline) by free-hand intrathecal injection (9) in a volume of 5 l. The time from being placed on the plate until each mouse either licked its hind paws or jumped was recorded with a stopwatch by a trained observer unaware of the treatments. An arbitrary cut-off time of 60 s was adopted to minimize tissue injury. Hot plate test data were analyzed by analysis of variance followed by Dunnett's test for multiple comparison, with p Ͻ 0.05 considered significant. Statistical analysis was performed with GraphPad Prism software (San Diego, CA). Shortly after the hot plate test, mice were placed on a 3-cm-diameter rotarod turning at 6 rpm (model 7650, Ugo Basile, Comerio, Italy). Mice were considered impaired if they fell three times in 1 min.

Electrophysiological Assays
Rana pipiens, 2.5 to 3 inches long, were used. The dissected preparation consisted of the 9th and 10th spinal nerves and their continuation down the sciatic nerve to the posterior crural nerve, freed from the skin that it innervates (see e.g. Ref. 10). Each spinal nerve was severed from the spine several millimeters proximal to its sympathetic ramus such that it could be electrically stimulated without activating sympathetic axons (see e.g. Ref. 11).
For extracellular stimulation and recording, the preparation was placed in a chamber fabricated from the silicone elastomer Sylgard (Dow Corning). The chamber was essentially a series of wells, each separated from its closest neighbor by about 1 mm. The proximal ends of the 9th and 10th nerves were placed in separate wells; the sciatic portion lay in a third well, whereas the attached lateral crural nerve spanned two additional wells, each 4 mm in diameter. All wells were filled with frog Ringer's solution (111 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 10 mM Na-HEPES (pH 7.2)). All portions of the nerve that draped over the partitions between wells and otherwise exposed to air were covered with Vaseline. One of a pair of stainless steel wire stim-ulating electrodes was placed in the well on either side of the Vaseline gap covering a spinal nerve. One of a pair of stainless steel wire recording electrodes was placed in the well on either side of the Vaseline gap covering the lateral crural nerve. Thus for both stimulation and recording, one electrode was in the same well that containing a cut end of the nerve, whereas its counterpart was in the well containing the portion of the nerve just distal (stimulation) or proximal (recording) to the cut end. The stimulating electrodes led to a Grass SIU5 stimulus isolation unit connected to a Grass S-88 stimulator. The recording electrode in the most distal well, containing the cut end of the posterior crural nerve, led to the positive input of a Grass P-55 differential preamplifier, whereas its counterpart, led to the negative input. Recordings were made with a preamplifier gain of 1000, RC-filtered (1 Hz alternating current and 1 kHz low pass) and digitized at a sampling rate of 4 kHz with a National Instruments Lab-NB board in a Macintosh Quadra 840 computer. Homemade virtual instruments (National Instruments LabVIEW) were used for data acquisition and analysis.
Supramaximal stimuli (1 ms, ϳ30 V) were applied to a spinal nerve once a minute while recording propagated compound action potentials (CAPs) reaching the other end of the preparation. Conopeptide was introduced into the well just proximal to that containing the distal cut end of the posterior crural nerve.

Oocyte Assays
Xenopus oocytes were injected with cRNA encoding human nAChR subunits as described previously (12,13). Oocytes injected with ␣ 4 ␤ 2 subunits were placed in a recording chamber (ϳ30 l), voltage-clamped, and assessed for expression of receptor using a 1-s pulse of 300 M acetylcholine (see Lu et al. (14) for details). Peptide was then added to the bath to achieve a final concentration of 10 M.

Receptor Binding Assays
-Conotoxin GVIA Binding--Conotoxin GVIA was iodinated by previously described methods (15). The binding protocol was a modification of that described in Hillyard et al. (16). Crude brain membranes from Harlan Sprague-Dawley rats were prepared as described by Catterall (17), with modifications in buffer components as described in Cruz and Olivera (15). The binding of 125 I-labeled -conotoxin GVIA to rat brain membrane was measured in a 200-l assay mix that contained approximately 10 g of membrane protein, 100,000 cpm of carrier-free -[ 125 I]conotoxin GVIA (approximately 150 pm), 0.2 mg/ml lysozyme, 0.32 M sucrose, 100 mM NaCl, and 5 mM HEPES/Tris (pH 7.4). Nonspecific binding was measured by preincubating the membrane preparation with 1 M unlabeled -conotoxin GVIA for 30 min on ice before the addition of -[ 125 I]conotoxin GVIA. mr10a was assessed for activity by preincubating the toxin for 30 min on ice. The final assay mix was then incubated at room temperature for 30 min and diluted with 1.5 ml of wash buffer containing 160 mM NaCl, 1.5 mM CaCl 2 , 2 mg/ml bovine serum albumin, 5 mM HEPES/Tris (pH 7.4). Membranes were collected on glass fiber filters (Whatman GF/C soaked in 0.1% polyethyleneamine) using a Brandell apparatus model M-24 and washed with 1.5 ml of wash buffer four times. The amount of radioactivity in the filters was then measured. The remaining assays were carried out under contract with Novascreen (Hanover, Maryland). The essentials of the procedures used are summarized below.

RESULTS
Purification and Sequencing of mr10a-Extracted crude venom from C. marmoreus was initially size-fractionated using a Sephadex G-25 column (see Fig. 1). Column fractions eluting in a range corresponding to small peptides were further purified using reverse phase HPLC utilizing conditions described under "Methods and Methods" and in Fig. 1. Throughout subsequent purification, HPLC fractions were assayed by means of intracerebral ventricular injection into mice (54). Intracerebral ventricular injection of fractions containing mr10a produced hypokinetic "sluggish" symptoms. The venom fraction was initially purified using a trifluoroacetic/acetonitrile gradient system. To obtain further purification, the new fractions were lyophilized and resuspended in 0.05% heptafluorobutyric acid (HFBA) and run on a reverse phase C 18 column using a 0.05% HFBA, acetonitrile gradient. Further purification was obtained using strong cation exchange chromatography. The fraction was then desalted using reverse phase HPLC. Final purified product is shown in Fig. 2, panel B.
The disulfide bonds of the purified peptide were reduced, and Cys residues were carboxymethylated. The alkylated peptide was then chemically sequenced and yielded NGVC-CGYKLCHOC, where O is 4-trans-hydroxyproline. Mass spectrometry of the peptide verified the sequence and indicated that Cys residues are present as disulfides and the C terminus is the free carboxyl (monoisotopic MH ϩ (Da): calculated, 1408.5; observed, 1408.5).
Peptide Synthesis-The sequence of the peptide was independently confirmed by preparation of synthetic peptide as described under "Experimental Procedures." The mr10a peptide has four Cys residues and, therefore, three possible disulfide linkages. All three disulfide bond linkages were synthesized to unequivocally identify the native configuration. Peptides were initially synthesized in linear form using pairwise protection of Cys residues (see "Experimental Procedures"). Acid cleavage from resin removed trityl-protecting FIG. 1. Purification of mr10a. A, crude venom extract was sizefractionated using a column (2.54-cm diameter ϫ 188-cm length) packed with Sephadex G-25 (dry bead diameter 20 -80 m). Elution buffer was 1.1% acetic acid at 4°C. Flow rate was ϳ18.3 ml/h. B, fractions from the indicated area in panel A (bracket) were combined, lyophilized, and resuspended in 0.1% trifluoroacetic acid and purified on a Vydak C-18 column (see "Experimental Procedures") using a linear 1% buffer B/min gradient where buffer A is 0.1% trifluoroacetic acid and buffer B is 0.092% trifluoroacetic acid, 60% acetonitrile. The gradient began at 10% buffer B. C, the material indicated in panel B (arrow) was lyophilized and resuspended in 0.05% heptafluorobutyric acid. It was then purified on a Vydac C-18 column using a linear 1% buffer B/min gradient where buffer A is 0.05% HFBA and buffer B is 0.05% HFBA, 60% acetonitrile. The gradient began at 30% buffer B. D, the material indicated in panel C (arrow) was lyophilized and dissolved in 10 mM NaH 2 PO 4 , 50% CH 3 CN (pH 2.5) (buffer A). The material was then purified using a Vydac protein-SCX (strong cation exchange) column (0.75 ϫ 5 cm) using a linear 1% B/min gradient where buffer B is the same as buffer A but with the addition of 250 mM NaCl. The flow rate was 1 ml/min in panels B, C, and D. Absorbance was monitored at 233, 214, 214, and 220 nm in panels A, B, C, and D, respectively. groups, and ferricyanide oxidation was used to close the first disulfide bridge. Iodine oxidation was subsequently used to remove S-acetamidomethyl protection groups and close the second bridge. Using this method, each possible disulfide arrangement was synthesized, i.e. Cys1-Cys2, Cys3-Cys4; Cys1-Cys3, Cys2-Cys4, and Cys1-Cys4, Cys2-Cys3. Final purified yields of each peptide were 12.5, 5.2, and 12.4%, respectively. Synthesis of each isomer was confirmed with mass spectrometry (calculated monoisotopic MH ϩ , 1408.5; observed, 1408.6, 1408.7, and 1408.6, respectively). The three forms of the peptide were distinguishable using reverse phase HPLC based on elution time. In addition, they were distinguishable by peak width, with the (Cys1-Cys4, Cys2-Cys3) form having the narrowest peak width (Fig. 2). Both the elution time and peak shape of the (Cys1-Cys4, Cys2-Cys3) disulfide form match that of the native peptide. Additionally, co-injection of each synthetic form indicates that native mr10a co-elutes only with synthetic peptide of the (Cys1-Cys4, Cys2-Cys3) configuration, providing unambiguous evidence for this disulfide linkage being native (Fig. 2).
mr10a Precursor Structure-The mr10a peptide sequence was used to design degenerate polymerase chain reaction primers for 3Ј and 5Ј RACE (rapid amplification of cDNA ends) amplification of the complete mr10a precursor cDNA. The polymerase chain reaction primers were designed to yield overlapping 3Ј and 5Ј RACE products, allowing the complete cDNA sequence to be assembled from the two sequences. Amplification of C. marmoreus cDNA gave specific products of 610 base pairs in the 3Ј RACE and 300 base pairs in the 5Ј RACE reactions. These polymerase chain reaction products were cloned, and multiple clones of both the 5Ј and 3Ј RACE products were isolated and sequenced. For both the 5Ј and 3Ј RACE products, the multiple clones all represented the same sequence, and the appropriate segments of the mr10a peptide sequence were represented by the cloned sequence. The 5Ј and 3Ј RACE product sequences were assembled to give the complete mr10a prepropeptide precursor cDNA sequence (Fig. 3). The first ATG start codon encountered from the 5Ј end of the cDNA initiates an open reading frame of 61 amino acids, encoding a protein with a structure typical of a conotoxin prepropeptide. The first 24 N-terminal amino acids compose a hydrophobic signal sequence region. The mature mr10a peptide sequence is located at the C-terminal end of the precursor sequence, immediately preceded by a basic amino acid (Arg) signaling proteolytic peptide processing. The stop codon is immediately downstream of the last cysteine residue of the mr10a peptide. A 3Ј-untranslated region of ϳ500 base pairs is terminated by a typical poly(A) ϩ addition signal (AATAAA) and a poly(A) tail.
The mr10a precursor exhibits significant sequence homology to a previously identified family of conotoxin genes, the Tsuperfamily, although the mature mr10a peptide is distinct from any of the previously isolated T-superfamily conotoxins (Fig. 4). Previously isolated T-superfamily conotoxins all share the cysteine framework CC-CC (7). The cysteine framework of the mr10a conotoxin is similar to that of the ␣-conotoxins, a large family of nicotinic receptor antagonists, yet the sequence alignment of the prepropeptides clearly indicates that mr10a FIG. 2. Elution studies of mr10a. A, ϳ200 pmol each of the three possible disulfide forms of synthetic mr10a were chromatographed using reverse phase HPLC. The disulfide connectivities are: 1, Cys1-Cys3, Cys2-Cys4; 2, Cys1-Cys4, Cys2-Cys3; 3, Cys1-Cys2, Cys3-Cys4. B, 100 pmol of native mr10a. C, ϳ200 pmol each of synthetic Cys1-Cys3, Cys2-Cys4 and native mr10a. D, ϳ200 pmol each of synthetic Cys1-Cys4, Cys2-Cys3 and native mr10a. Note that the two materials coelute. E, ϳ200 pmol each of synthetic Cys1-Cys2, Cys3-Cys4 and native mr10a. In all HPLC runs, buffer A ϭ 0.1% trifluoroacetic acid, and buffer B ϭ 0.092% trifluoroacetic acid, 60% acetonitrile. The gradient began at 15% B and increased 1% B/min. The column was an analytical C-18 (see "Experimental Procedures"). The flow rate was 1 ml/min. Absorbance was monitored at 220 nm. and ␣-conotoxins are derived from completely unrelated precursors (Fig. 4). The occurrence of the mr10a conotoxin within the T-superfamily provides a demonstration of the ability of Conus species to evolve novel toxin peptide frameworks within the same conotoxin superfamily.
Biological Activity-The biological activity of many Conus peptides can be grouped into three major categories: those that produce excitotoxic shock, those that produce paralysis, and those that inhibit sensory circuits. Intraperitoneal injection of 30 nmol of mr10a into three mice produced neither neuromuscular excitation nor muscle paralysis. We next tested the peptide for analgesic activity using mice in a hot plate assay. Native peptide (2 nmol) injected intrathecally into three mice produced a latency to first hind paw lick (a nociceptive response) of 39.5 Ϯ 13.5 s, suggestive of potent analgesic activity. Due to a limited quantity of native material, a complete doseresponse study could not be performed, and all further tests were performed with synthetic peptide.
Intrathecal administration of synthetic mr10a produced a dose-dependent (0.1-10 nmol) increase in the latency to first hind paw lick (F(3,15) ϭ 7.5, p Ͻ 0.01) in the hot plate test. At doses of 1 and 10 nmol, mr10a significantly increased the latency to lick the hind paw in this test (Fig. 5).
Motor impairment was assessed in all injected mice by means of a rotarod test (see "Experimental Procedures"). Motor impairment was not seen in any mouse injected either intrathecally or intraperitoneally. Injection of high doses of mr10a (25 nmol) by intracerebral ventricular administration resulted in akinesia and seizures in two mice tested. Further testing by intracerebral ventricular administration was not performed.
Molecular Target-mr10a was tested in a number of electrophysiological and binding assays (see "Experimental Procedures") in an effort to define its molecular target. To test effects on Na ϩ channels, we used a frog peripheral nerve preparation containing sensory fibers. mr10a (100 M) produced no appreciable effects on CAPs recorded from the posterior crural nerve, which innervates the skin (Fig. 6). Clearly evident in the traces are fast-conducting A␣-and A␤-CAPs as well as much slower conducting C-CAPs. The A-CAPs and C-CAPs in the posterior crural nerve are tetrodotoxin-sensitive and tetrodotoxin-resistant, respectively, 2 just like those seen in the frog sciatic nerve (cf. Refs. 55 and 56). It is notable that many, but not all, of the CAPs in the posterior crural nerve are sensitive to other conotoxins known to affect action potentials, 3 for example ␦-conotoxin PVIA, which targets Na ϩ channels (57), and A-conotoxin SIVA, which targets K ϩ channels (58). mr10a also was without effect on nicotinic acetylcholine receptors, the site of action of ␣-conotoxins. Ten M mr10a does not activate ␣4␤2 nicotinic acetylcholine receptors expressed in Xenopus oocytes, nor does it compete for [ 3 H]cytisine binding to putative ␣4␤2 receptors in rat brain membranes. mr10 also failed to compete for binding to many other receptor targets associated with analgesia including N-type calcium channels (the binding site of -conotoxins GVIA and MVIIA), neurotensin receptors (the molecular target of contulakin-G), ␣1, ␣2, or ␤1 adrenergic receptors, GABA A , glycine or histamine 1, central muscarine, ␦1-, 1-, or -opiate receptors, or 5HT 3 receptors and did not compete for binding to norepinephrine, serotonin, or dopamine transporters.  Gm5.1, Gm5.2, Im5.1, Tx5.1), along with two examples of ␣-conotoxin precursors (␣-SI, ␣-GI). The mr10a precursor has substantial sequence homology with T-superfamily precursor sequences, although the mature peptide has a distinct cysteine pattern, and there is little amino acid similarity. Although the mr10a conotoxin has a cysteine pattern similar to that of ␣-conotoxins, the mr10a precursor is unrelated to the ␣-conotoxin prepropeptide, and the mature toxins have no amino acid similarities other than the cysteine residues.

DISCUSSION
We describe the characterization of a novel peptide with apparent potent antinociceptive activity isolated from the venom of the mollusk-hunting species, C. marmoreus. Like many Conus peptides, mr10a is rich in disulfides, with 4 of 13 residues being Cys residues. Two other groups of Conus peptides were previously shown to have four Cys residues, the ␣-conotoxins (59) and T-superfamily conotoxins (7). All ␣-conotoxins and T-superfamily conotoxins characterized to date (1,7,60) have Cys1-Cys3, Cys2-Cys4 connectivity. In contrast, mr10a has Cys1-Cys4, Cys2-Cys3 connectivity, a pattern unprecedented among Conus peptides. In addition to the novel disulfide bond connectivity, mr10a bears little if any sequence similarity to the ␣-conotoxins or other T-superfamily peptides, and clearly represents a new class of Conus peptide (Table I).
Conus peptides are initially translated from mRNA as prepropeptide precursors that are subsequently processed into the small mature neuroactive toxins. Conopeptides can be grouped into superfamilies based on the signal sequences of the precursors and on the disulfide framework of the mature toxin. Thus, in the O-superfamily for example there are -conotoxins (Ca 2ϩ channel antagonists), O-conotoxins (Na ϩ channel blockers), ␦-conotoxins (peptides that delay inactivation of Na ϩ channels), and -conotoxins (K ϩ channel blockers). Peptides in these four families share a highly conserved signal sequence as well as the same disulfide framework. Thus, the polypeptides belonging to the same superfamily can be processed to mature conotoxins that are biochemically and pharmacologically diverse.
The mr10a peptide described in this report provides a new twist to this paradigm. Analysis of a cDNA clone of mr10a clearly indicated that this peptide is a member of the T-superfamily. In members of the O-superfamily, although there is hypermutation of toxin sequences, the disulfide connectivity is conserved. In contrast, the previously identified T-superfamily conotoxins versus mr10a have both a divergent arrangement of Cys residues and, most surprisingly, a different disulfide bond linkage. This is the first known example of such a divergent disulfide connectivity within members of a Conus peptide superfamily. Thus, the mr10A peptide defines a distinct branch of the T-conopeptide superfamily clearly different from T-superfamily peptides previously characterized. It seems likely that more than one pharmacological family of Conus peptides will compose each branch.
Although the purpose for which C. marmoreus employs mr10a is unknown, it is possible that this is an example of a peptide that inhibits specific neuronal circuits in prey. We demonstrate in this report that mr10a produces inhibition of withdrawal response when tested in a mouse hot plate assay at intrathecal doses that do not produce gross motor impairment or impair performance on the rotarod test. We have postulated elsewhere that a "nirvana cabal" of peptides is used to inhibit sensory circuitry of the fish prey of piscivorous Conus species that use a net strategy (61); the discovery of the mr10a peptide raises the possibility that the mollusk-hunting C. marmoreus uses a similar nirvana cabal strategy.
Due to the potency and selectivity of Conus peptides, several are now in various stages of clinical trials. Two Conus peptides are being developed for the treatment of pain. The most advanced is -conotoxin MVIIA (ziconotide), an N-type calcium channel blocker (62). -Conotoxin MVIIA, isolated from Conus magus, is approximately 1000 times more potent than morphine, yet does not produce the tolerance or addictive properties of opiates. -Conotoxin MVIIA has completed Phase III clinical trials in humans and now awaits FDA approval as a new therapeutic agent. -Conotoxin MVIIA is introduced into human patients by means of an implantable, programmable pump with a catheter threaded into the intrathecal space. Preclinical testing is being carried out on another Conus peptide, contulakin-G, isolated from Conus geographus (63); contulakin is an agonist of neurotensin receptors but, interestingly, appears significantly more potent than neurotensin in inhibiting pain in in vivo assays. Contulakin-G is being investigated for use in post-surgical pain. For a review of conotoxins and therapeutic applications, see Jones and Bulaj (2) and Adams et al. (65). The mechanism of action of mr10a is unknown, but results of the present study indicate that its mechanism is distinct from ␣-, -, ␦-, or A-conotoxins as well as contulakin-G. In addition, it fails to act at numerous other receptor sites associated with analgesia.


extfieldsofstudy: Biology Medicine

text:
GATA-6 Activates Transcription of Surfactant Protein A*

Surfactant protein A (SP-A) is a member of the collectin family of innate host defense molecules expressed primarily in respiratory epithelial cells of the lung. SP-A concentrations are influenced by both cell-specific and ubiquitous nuclear proteins that regulate SP-A gene transcription in a cell-selective and temporally regulated manner. In this work, a consensus GATA-binding site (GBS) was identified at positions −69 to −64 of the mouse SP-A gene. The transcriptional activity of wild-type SP-A reporter constructs in HeLa cells was increased 5–10-fold when cotransfected with a GATA-6 expression plasmid. Deletion of the GBS completely blocked transactivation by GATA-6. Transfection of a construct expressing GATA-6-engrailed fusion protein inhibited basal expression of the SP-A/chloramphenicol acetyltransferase construct in MLE-15 cells. Nuclear extract proteins from MLE-15 cells bound to the GBS in the mouse SP-A gene, and a supershifted band was detected with a GATA-6-specific antibody. Transactivation of the wild-type SP-A constructs by GATA-6 increased transcriptional activity 7–10-fold, whereas thyroid transcription factor-1 (TTF-1) increased the activity of these constructs 12–18-fold. The effects of cotransactivating with both GATA-6 and TTF-1 expression constructs were additive. However, mutation of the TTF-1-binding sites alone or in combination decreased GATA-6 transactivation. Likewise, mutation of the GBS blocked TTF-1 activation of the SP-A promoter. In situ hybridization demonstrated GATA-6 mRNA in the peripheral epithelial cells of fetal mouse lung, consistent with the sites of SP-A expression. GATA-6 is expressed in respiratory epithelial cells and binds to acis-acting element in the SP-A gene promoter, activating the transcriptional activity of the gene.

Surfactant protein A (SP-A) is a member of the collectin family of innate host defense molecules expressed primarily in respiratory epithelial cells of the lung. SP-A concentrations are influenced by both cell-specific and ubiquitous nuclear proteins that regulate SP-A gene transcription in a cell-selective and temporally regulated manner. In this work, a consensus GATA-binding site (GBS) was identified at positions ؊69 to ؊64 of the mouse SP-A gene. The transcriptional activity of wildtype SP-A reporter constructs in HeLa cells was increased 5-10-fold when cotransfected with a GATA-6 expression plasmid. Deletion of the GBS completely blocked transactivation by GATA-6. Transfection of a construct expressing GATA-6-engrailed fusion protein inhibited basal expression of the SP-A/chloramphenicol acetyltransferase construct in MLE-15 cells. Nuclear extract proteins from MLE-15 cells bound to the GBS in the mouse SP-A gene, and a supershifted band was detected
with a GATA-6-specific antibody. Transactivation of the wild-type SP-A constructs by GATA-6 increased transcriptional activity 7-10-fold, whereas thyroid transcription factor-1 (TTF-1) increased the activity of these constructs 12-18-fold. The effects of cotransactivating with both GATA-6 and TTF-1 expression constructs were additive. However, mutation of the TTF-1-binding sites alone or in combination decreased GATA-6 transactivation. Likewise, mutation of the GBS blocked TTF-1 activation of the SP-A promoter. In situ hybridization demonstrated GATA-6 mRNA in the peripheral epithelial cells of fetal mouse lung, consistent with the sites of SP-A expression. GATA-6 is expressed in respiratory epithelial cells and binds to a cis-acting element in the SP-A gene promoter, activating the transcriptional activity of the gene. 1 is a 28 -36-kDa member of the collectin family of innate defense molecules (for review, see Refs. [1][2][3]. In humans, SP-A is expressed at high levels by respiratory epithelial cells of the lung, being secreted by sub-sets of tracheobronchial, bronchiolar, and alveolar cells in vivo. SP-A binds to phospholipids, contributing to tubular myelin formation, and influences surfactant phospholipid secretion and re-uptake in vitro (1)(2)(3). Targeted gene inactivation of mouse SP-A suggests that its primary role is in host defense. SP-A binds to the surface of various respiratory pathogens or directly activates macrophages, enhancing opsonization and clearance of bacteria and viruses from the lung (1)(2)(3)(4)(5)(6)(7). Although SP-A does not play a critical role in surfactant function or homeostasis in vivo, SP-A is required for the formation of tubular myelin in the alveolus (8 -10).

Surfactant protein A (SP-A)
SP-A gene expression is regulated in a complex manner at the level of gene transcription and mRNA stability (for review, see Ref. 11). Like surfactant proteins B and C, SP-A transcription is dependent upon the action of thyroid transcription factor-1 (TTF-1), a homeodomain protein of the Nkx family of transcription factors (12)(13)(14). TTF-1 regulates the mouse and baboon SP-A genes, enhancing reporter gene transcription in vitro (15,16).
Recently, in situ hybridization studies demonstrated that GATA-5 and GATA-6 are expressed in developing mouse lung (17). The GATA family of transcription factors includes at least six polypeptides whose temporal and spatial expression is highly distinct. GATA-6 mRNA was detected in respiratory epithelial cells of developing lung tubules, consistent with a potential role in lung cell differentiation or gene regulation. Embryonic stem cells with a targeted disruption of the Gata-6 gene failed to contribute to bronchial epithelial cells in the lung, supporting the concept that GATA-6 may play a role in the differentiation of subsets of lung cells (18). The recent finding that GATA-6 activates TTF-1 reporter gene transcription provides further support for the concept that GATA-6 plays a role in the regulation of respiratory epithelial cell differentiation or function, either regulating TTF-1 expression or directly influencing transcription of surfactant proteins (19). This study was designed to test whether GATA family members influence mouse SP-A gene transcription in vitro. Cell transfection and electrophoretic mobility shift assay of native and mutant mouse SP-A gene sequences demonstrated that GATA-6 activates SP-A gene transcription in vitro.

MATERIALS AND METHODS
Plasmid Construction and Site-directed Mutagenesis-The SP-A constructs used in this study are presented in Fig. 1. The mouse SP-A sequences used to generate the various constructs were isolated and cloned as described previously (20). The murine SP-A gene promoter construct pCPA0.9 (Ϫ907 to ϩ45) was made by isolating a 952-base pair SspI-PstI fragment from pCPA1.4 and using linker ligation to create a 5Ј-HindIII site. The product was cloned into pCPA-0 to generate pCPA0.9. The SP-A promoter construct pCPA0.45 was generated by PCR using linker primers containing 5Ј-HindIII and 3Ј-PstI sites to create fragment Ϫ399 to ϩ45. The promoter constructs pCPA0.1 (Ϫ70 to ϩ45) and pCPA0.1m (Ϫ60 to ϩ45) were generated by PCR using linker primers containing 5Ј-HindIII and 3Ј-PstI sites. The TTF-1 sitedirected mutants were generated by PCR using pCPA0.3 (Ϫ256 to ϩ45) as template, as described previously (15). Oligomers were made to each of the three of the TTF-1-binding sites, replacing each with a restriction enzyme sequence. The TTF-1 site located at positions Ϫ159 to Ϫ154 was changed to a SalI site; the site located at positions Ϫ135 to Ϫ130 was changed to a NcoI site; and the TTF-1 site at positions Ϫ125 to Ϫ120 was changed to a BamHI restriction site. These oligomers were then used in PCRs with pCPA0.3 as template. These products were digested as appropriate and cloned into pCPA-0. The mutation of the GATAbinding site was generated by PCR using pCPA0.45 as template and replacing the 6-base pair GATA-binding site (GBS), TGATAG, with an EcoRI site, GAATTC. Additional constructs from positions Ϫ907 and Ϫ399 to ϩ45 were generated from the 5Ј-end by digesting the linkerligated HindIII site and from the 3Ј-end using a BstEII site within the flanking region 5Ј to the TTF-1-binding elements (TBEs).
Cell Culture, Transfections, and Reporter Gene Assays-HeLa cells were cultured in Dulbecco's modified Eagle's medium and 10% fetal calf serum. MLE-15 cells were cultured in HITES medium (RPMI 1640 containing 10 mM HEPES, 5 g/ml insulin, 10 g/ml transferrin, 3 ϫ 10 Ϫ8 M sodium selenite, 1 ϫ 10 Ϫ8 M ␤-estradiol, and 200 mM L-glutamine) and transferred to Dulbecco's modified Eagle's medium and 10% fetal calf serum as described previously (15). MLE-15 cells express TTF-1, GATA-6, SP-A, and surfactant protein B and C mRNAs. Cells were transfected using calcium precipitates prepared with 7.5 pmol of test plasmid and 4 pmol of pCMV/␤-galactosidase. For GATA-6 transactivation experiments in HeLa cells, each 10-cm dish was treated with a precipitate prepared using 7.5 pmol of promoter/CAT fusion plasmid, 4 pmol of pCMV/␤-galactosidase, and 1 pmol of either the empty expression vector (pCMV1.1) or an expression vector containing the entire GATA-6 open reading frame (pCMV/GATA-6, a kind gift from Dr. J. Molkentin, Children's Hospital Medical Center, Cincinnati, OH). Cells were maintained at 37°C for 48 -72 h, and the lysates were assayed for ␤-galactosidase and CAT activities as described previously (15). MLE-15 cells were also transiently transfected with an expression plasmid (pcDNA3/GATA-6-engrailed) that contains the DNA-binding domain of GATA-6 and the inhibitory regulatory domain encoded by the Drosophila engrailed gene. All data are presented as means Ϯ S.D.
Preparation of Nuclear Extracts-MLE-15 and HeLa nuclear extracts were prepared using a modified mini-extract procedure. Nuclear extraction procedures were performed with ice-cold reagents. Confluent monolayers from six 10-cm-diameter dishes were washed twice with 10 FIG. 2. Transactivation of SP-A promoter constructs by GATA-6. A, the CAT activity of SP-A promoter constructs was determined in HeLa cells cotransfected with an empty pCMV expression plasmid (pCMV1.1) or with a GATA expression vector (pCMV/GATA-6). CAT activity is presented as -fold transactivation of the respective construct by GATA-6 compared with basal levels without GATA-6 (set as 1 for each construct). Data are representative of at least three separate experiments with triplicate plates for each condition. The pCPA-0 control plasmid does not have SP-A gene sequences and was described previously (20). B, a representative CAT assay is presented. Each construct was transfected onto three separate plates of HeLa cells. Forty-eight hours after transfection, cells were collected and lysed, and CAT activity was determined as described under "Materials and Methods." Following thin-layer chromatography to separate acetylated from unacetylated [ 4 C]chloramphenicol, the thin-layer chromatogram was exposed to XAR film for 16 h.

FIG. 1. Schematic representation of SP-A promoter constructs.
The 5Ј-position of each SP-A promoter construct is depicted on the left. The 3Ј terminus of plasmid constructs was at map position ϩ45. Closed ovals represent TBEs described previously (15). Open ovals depict potential GATA-binding sites. Site-specific mutations of the TBE (*) and GBS (#) are indicated. ml of phosphate-buffered saline (pH 7.2) and harvested by scraping into 1 ml of phosphate-buffered saline, and the cells were pelleted in a chilled 1.5-ml microcentrifuge tube at 3000 rpm for 5 min. The pellet was washed once in phosphate-buffered saline and re-pelleted as described above. The cell pellet was resuspended in 1 packed cell volume of fresh lysis buffer (10 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl 2 , 0.2% (v/v) Nonidet P-40, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride), and the cells were lysed during a 5-min incubation with occasional vortexing. A nuclear pellet was obtained by microcentrifugation at 3000 rpm for 5 min and was resuspended in 1 packed nuclear volume of fresh extract buffer (20 mM Hepes (pH 7.9), 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl 2 , 25% (v/v) glycerol, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride). Nuclei were extracted during a 10-min incubation with occasional gentle vortexing. Extracted nuclei were pelleted in a microcentrifuge at 14,000 rpm for 10 min. The supernatant recovered was saved as the extracted nuclear protein. These nuclear extracts were quick-frozen and stored at Ϫ80°C.
Synthetic Oligonucleotides-Single-stranded oligonucleotides were annealed at a concentration of 10 mM in annealing buffer (10 mM Tris (pH 7.5), 10 mM MgCl 2 , and 50 mM NaCl) in a 95°C dry heat block and then slowly cooled to room temperature. The A 260 was determined, and dilutions of this mixture were made in TE buffer (10 mM Tris (pH 8.0) and 1 mM EDTA). These double-stranded oligomers were gel-purified for labeling. For use as a probe in EMSA, 20 l of the annealed oligomer was gel-purified using 4% Bio-Gel and a MERmaid kit (Bio 101, Inc.) as specified by the manufacturer. The A 260 was determined, and 1.5 pmol of annealed and gel-purified oligonucleotide was end-labeled using [␥-32 P]ATP and T4 polynucleotide kinase. End-labeled probe was purified from unincorporated nucleotide using an Amersham Pharmacia Biotech Nick column and recovered in 400 l of TE buffer.
In Situ Hybridization-In situ hybridization analyses for GATA-6 mRNA were performed on tissues from fetal mice on day 17.5 of gestation using 35 S-labeled cDNA probes following methods similar to those used previously (20). A 3.1-kilobase pair mouse GATA-6 cDNA template was utilized to generate 35 S-labeled probe, which was reduced to an average size of 200 base pairs by alkaline hydrolysis.

RESULTS
Cotransfection of HeLa cells with the pCMV/GATA-6 expression vector and SP-A/CAT expression constructs containing sequences from positions Ϫ907 to ϩ45 of the mouse SP-A gene (20) resulted in activation of reporter gene expression in all constructs (Fig. 2). Comparison of the mouse (20) and rat (21) SP-A sequences demonstrated a conserved GBS at positions Ϫ69 to Ϫ64 of the mouse gene (Fig. 3). Deletion of region Ϫ69 to Ϫ64 abrogated GATA-6-dependent activity, suggesting that the stimulatory effects of GATA-6 were dependent on an intact GBS located at positions Ϫ69 to Ϫ64 (Fig. 2, see pCPA0.1m). Although both GATA-6 and GATA-5 expression vectors stimulated the activity of the pCPA0.9 construct, GATA-6 was consistently more active than GATA-5 (data not shown). HeLa cells do not express GATA-6 mRNA, likely accounting for the more marked activation of the SP-A promoter construct (5-10fold) by GATA-6 in HeLa cells. The pCPA0.9 and pCPA0.45 constructs were also cotransfected with pCMV/GATA-6 in MLE-15 pulmonary adenocarcinoma cells. Whereas the basal activity of the SP-A/CAT constructs was considerably higher in MLE-15 cells than in HeLa cells, SP-A promoter activity was stimulated only 2-3-fold by cotransfection with the GATA-6 expression vector (data not shown), a finding likely related to the presence of endogenous GATA-6 activity in this cell line (19).
GATA-6-engrailed Repressor-To test for endogenous GATA activity in MLE-15 cells, the 0.45-kilobase pair SP-A/CAT construct was cotransfected into MLE-15 cells with pCMV/GATA-6-engrailed. The latter contains the GATA-6 DNA-binding domain, but the activation domain has been replaced by the repressor domain of engrailed, thereby inhibiting GATA-6-dependent transcription. pCMV/GATA-6-engrailed inhibited SP-A/CAT activity in the MLE-15 cells in a dose-dependent manner, supporting the concept that endogenous GATA activity, at least in part, contributed to the higher activity of SP-A/CAT reporter constructs in the MLE-15 cell line (Fig. 4).
EMSA of the GBS-Inspection of the 5Ј-flanking region of the mouse and rat SP-A genes revealed the presence of an extensive region of homology that included the sequence located at positions Ϫ69 to Ϫ64 (TGATAG) (Fig. 3). This sequence is homologous to a consensus GATA-binding site. To more precisely define the function of this element and to determine whether GATA-6 bound to the region, EMSA was performed with extracts prepared from adult mouse type II epithelial cells and MLE-15 cells. Migration of the 32 P-labeled GATA-6 oligonucleotide was retarded by either mouse type II

FIG. 3. Conservation of a GATA-binding site in the rat and mouse SP-A genes. Region
Ϫ70 to Ϫ1 of the mouse SP-A gene was 90% identical to region Ϫ70 to Ϫ1 of the rat SP-A gene (21). A consensus GATA-binding site is depicted in boldface, and the TATAAA sequence is boxed. Nucleotide differences are underlined. cell or MLE-15 cell nuclear extracts. Co-incubation of nuclear extracts with antibody to GATA-6 caused a clear supershift, consistent with the binding of GATA-6 to the EMSA probe containing the GBS (Ϫ64 to Ϫ69) (Fig. 5).
Site-directed Mutagenesis of the GBS-Mutation of the GBS (Ϫ64 to Ϫ69) by substitution of the sequence GAATTC for TGATAG in the pCPA0.45 construct resulted in complete inhibition of the GATA-6-dependent activation of the SP-A reporter construct (Fig. 6, compare pCPA0.45-G with pCPA0.45). Thus, the element that mediates the stimulatory effects of GATA-6 is located at positions Ϫ64 to Ϫ69 in the 5Ј-region of the SP-A gene.
Effects of GATA-6 and TTF-1 on SP-A Gene Transcription-Since Nkx2 and GATA family members are known to participate in the regulation of a number of genes in target tissues, we tested whether TTF-1 (Nkx2.1) influences GATA-6 activation of the SP-A/CAT reporter constructs in vitro. Cotransfection of TTF-1 and GATA-6 expression plasmids in HeLa cells markedly increased the activity of the cotransfected pCPA0.45/CAT constructs (25-fold) (Fig. 7). The effect of cotransfection with TTF-1 and GATA-6 expression plasmids was approximately additive (Fig. 7). To further clarify the nature of the effects of GATA-6 and TTF-1, SP-A/CAT constructs containing mutations in TTF-1-binding sites were cotransfected with pCMV/ GATA-6 in HeLa cells. Mutation of three TTF-1 sites, singly or in combination, markedly reduced GATA-6-and TTF-1dependent transactivation of the pCPA0.45 constructs, supporting the concept that optimal GATA-6-dependent activity required the binding of TTF-1 to TTF-1-binding elements in this region of the promoter (Fig. 8A). Likewise, mutation of the GBS blocked TTF-1 transactivation of the pCPA0.45 construct (Fig. 8B). Thus, TTF-1 and GATA-6 transactivation depends on both intact GATA-and TTF-1-binding sites.
GATA-6 Transactivation Requires a Single GBS at Positions Ϫ69 to Ϫ64 -Since other potential GATA-binding sites were  2 and 3; and B, lanes 1 and 2) or type II cell (B, lanes 3 and 4) nuclear extracts. In B (lanes 2 and 4), an antibody to GATA-6 (␣-GATA-6) was incubated with the nuclear extracts and labeled probe. The position of the major retarded band is depicted by an arrowhead (A  and B), and the position of a supershifted band is depicted by an arrow (B). The autoradiogram is representative of two separate experiments for each condition. The plus sign indicates the presence of the respective nuclear extract, antibody, or probe in the incubation mixture. cold GATA oligo, unlabeled GATA oligonucleotide; 32 P-WT GATA, 32 P-labeled wild-type GATA. noted upstream of positions Ϫ69 to Ϫ64, the larger SP-A construct, containing additional consensus GATA-binding sites, was tested. Cotransfection of pCMV/GATA-6 with pCPA0.9 alone or with a GBS mutation at gene positions Ϫ69 to Ϫ64 demonstrated that GATA-6 activity was mediated by the proximal GBS (Fig. 9).
Localization of GATA-6 mRNA in Fetal Mouse Lung-GATA-6 mRNA was localized in developing mouse lung by in situ hybridization (Fig. 10). On day 17.5, GATA-6 mRNA was detected in peripheral airway epithelial cells overlapping with the sites of expression of SP-A and TTF-1 mRNAs, providing further support for the potential role of GATA-6 in the regulation of expression of SP-A in prenatal lung development. Unlike TTF-1 or SP-A, GATA-6 mRNA was not confined to respiratory epithelial cells in the lung, being detected in epithelial, mesenchymal, and vascular tissues at this time in development. DISCUSSION GATA-6 mRNA was detected in respiratory epithelial cells in fetal lung at sites coincident with those of SP-A mRNA and protein. GATA-6 bound to an element (GBS) located at positions Ϫ64 to Ϫ69 in the 5Ј-flanking region of the SP-A gene, enhancing the activity of the SP-A gene reporter constructs. Mutation of the GBS or transfection of MLE-15 cells with a construct expressing a GATA-6-engrailed fusion protein inhibited the activity of the SP-A promoter constructs. Mutagenesis of the GBS inhibited TTF-1-dependent activation of the SP-A promoter, and the activity of the GBS was influenced by mutations in any of three distinct TTF-1 regulatory elements located within the 5Ј-flanking region of the SP-A gene. Taken together, these findings support a role for GATA-6 in the reg-ulation of the transcriptional activity of the mouse SP-A gene.
GATA-6 was consistently more active than GATA-5 in the activation of the SP-A promoter construct. Although both GATA-5 and GATA-6 are detected in developing lung (18), only GATA-6 is expressed in respiratory epithelial cells, prior to and coincident with SP-A mRNA and protein. Previous studies demonstrated that GATA-6 and surfactant protein mRNAs are expressed in freshly isolated adult type II epithelial cells (19). In contrast, GATA-5 is expressed primarily in the mesenchyme of developing lung, as assessed by in situ hybridization, at sites that do not overlap with SP-A (17). In the present study, GATA-6 was also detected by EMSA in MLE-15 cells and mouse type II cells. Type II epithelial cells isolated from adult mice and immortalized MLE-15 cells express TTF-1, GATA-6, and various surfactant protein mRNAs (19). Early in lung morphogenesis, the distribution of GATA-6 and TTF-1 mRNAs is similar, with both being detected in progenitor cells of the respiratory epithelium. TTF-1 plays a critical role in lung morphogenesis and in the regulation of surfactant proteins in respiratory epithelial cells. Therefore, GATA-6 may influence SP-A expression indirectly, by enhancing TTF-1 gene transcription, or directly, by binding to and activating the promoters of the surfactant protein genes (19). Recent studies from this laboratory demonstrated that GATA-6 also directly influences both surfactant protein B and C transcription. 2 GATA-6 is a member of the zinc finger family of transcription factors. The family members GATA-1 and GATA-2 were initially identified as important factors in hematopoietic precur- sor cells, where they play a role in differentiation and gene expression during erythropoiesis. More recently, other GATA family members were identified in non-hematopoietic tissues during embryonic development. GATA-4, -5, and -6 mRNAs were detected in various tissues in the embryo, including heart, gastrointestinal tract, and lung (22,23). In the present work, both GATA-6 and TTF-1 mRNAs were detected at overlapping sites in respiratory epithelial cells of developing lung (24), although GATA-6 mRNA was detected in both epithelial and mesenchymal compartments in the lung. GATA-6 bound to the SP-A promoter in a region of the SP-A gene containing a number of TTF-1-binding sites that were previously shown to be FIG. 8. GATA-6 and TTF-1 do not transactivate SP-A promoter constructs with site-specific mutations of TTF-1-or GATA-binding sites. A, constructs were cotransfected into HeLa cells with pCMV/ GATA-6. The relative CAT activity of the respective constructs is presented as -fold induction compared with constructs transfected without pCMV/GATA-6. pCPA0.45 is the wild-type construct, and pCPA0.45T designates constructs mutagenized in the respective TBEs (see Fig. 1). B, constructs were cotransfected into HeLa cells with the expression plasmid pCMV/TTF-1. The relative CAT activity of the respective constructs is presented as -fold activation after transfection with pCMV/ TTF-1 compared with constructs transfected without pCMV/TTF-1. Data are representative of at least three separate experiments.
FIG. 9. The proximal GBS is required for transactivation by GATA-6. Constructs were cotransfected into HeLa cells with pCMV/ GATA-6. The relative CAT activity of the respective constructs is presented as -fold induction compared with constructs transfected without pCMV/GATA-6. pCPA0.9 contains sequences from positions Ϫ907 to ϩ45 of the mouse SP-A promoter. pCPA0.9-G contains the mutation of the GBS at positions Ϫ69 to Ϫ64. pCPA0.9-TG contains mutations of the GBS at positions Ϫ69 to Ϫ64 and mutations in TBE-1, -3, and -4 (see Fig. 1). Data are representative of two separate experiments, each performed in triplicate.
FIG. 10. GATA-6 mRNA in embryonic day 17.5 mouse lung. In situ hybridization analyses were performed with radiolabeled antisense mouse GATA-6 (A) or sense riboprobe (B) and exposed to film for 6 weeks. Specific hybridization was observed in peripheral lung buds, acinar epithelium, and blood vessels. No hybridization was observed in the larger conducting airways. Magnification is approximately ϫ200.
involved in SP-A gene transcription (15). Mutation of any of the TTF-1-binding sites in the proximal region of the promoter inhibited GATA-6 stimulation of the SP-A/CAT constructs, suggesting that the effects of GATA-6 were dependent upon TTF-1 and its interaction with TTF-1-binding elements in the SP-A gene.
It is unclear whether TTF-1 and GATA-6 factors function independently or are involved in a higher order DNA-protein complex that may be involved in SP-A gene regulation. In analogous models, GATA-4 and Nkx2.5 appear to directly interact in regulating expression of the atrial natriuretic peptide gene promoter (25,26) and of the ␣-actin gene promoter in cardiac tissue (27). In those studies, Nkx2.5 and GATA-4 formed a complex that activated gene transcription. Whereas Durocher et al. (25) suggested that GATA-4 transactivation required DNA binding to a proximal GBS in the atrial natriuretic peptide gene promoter, Shiojima et al. (26) suggested that the synergistic activity of Nkx2.5 and GATA-4 did not require direct binding of GATA-4 to DNA. In the present study, mutation of either the GBS or TTF-1-binding sites in the mouse SP-A gene reduced transactivation by the other factor. It remains unclear whether direct or indirect interactions between TTF-1 and GATA-6 mediate their cooperative effects on SP-A promoter activity. We were unable to demonstrate direct binding or coprecipitation of TTF-1 and GATA-6 in MLE-15 cell extracts using antibodies to GATA-6 or TTF-1 (data not shown). It is possible that the respective antibodies bind epitopes required for protein-protein interactions.
In vivo, GATA-6 and SP-A mRNAs are co-localized in developing lung, and both are expressed in isolated adult type II epithelial cells (19). The early expression of GATA-6 in the development of respiratory tubules supports its potential role in lung morphogenesis and gene regulation. Similarly, GATA-6 mRNA are expressed at the highest levels in developing progenitor cells of the intestinal epithelium and developing cardiomyocytes (27,28), suggesting a critical role of GATA-6 in developing tissues. Recent findings in chimeric mice generated from GATA-6 Ϫ/Ϫ and GATA-6 ϩ/ϩ embryonic stem cells demonstrated that GATA-6 Ϫ/Ϫ cells do not contribute to the formation of the bronchiolar epithelium in chimeric mice (18). In contrast, GATA-6 Ϫ/Ϫ embryonic stem cells are fully competent to contribute to other fetal tissues. This finding suggests a unique role for GATA-6 in the morphogenesis of bronchiolar tissues. Further clarification of the role of GATA-6 in lung morphogenesis and gene regulation will require site-specific alterations in GATA-6 since mice lacking GATA-6 fail to survive to stages in which lung development is initiated (18).
SP-A is subjected to precise temporal, spatial, and tissuespecific regulation mediated, at least in part, at the transcriptional level. Although TTF-1 plays a critical role in lung cellselective expression of SP-A, mutations of the TBEs lead to enhanced SP-A reporter gene transcription in vitro. This enhanced gene transcription is also influenced by combinatorial interactions of other transcription factors. For example, B-Myb binds to the murine SP-A promoter and influences SP-A gene transcription, with the highest transactivation occurring with phosphorylated B-Myb (29). In contrast to findings with the rat and mouse SP-A genes, expression of rabbit, baboon, and human SP-A genes is enhanced by cAMP. Both basal and cAMP transcription of the rabbit and primate SP-A genes require binding of TTF-1, upstream factors 1a and b, unknown members of the steroid receptor family, and Kruppel family mem-bers (16, 30 -32). cAMP stimulates phosphorylation of TTF-1, enhancing transactivation of a baboon SP-A gene (30). It is therefore increasingly apparent that SP-A gene transcription is controlled by complex interactions among cis-acting elements and multiple transcription factors, including GATA-6.
This study demonstrates a role for GATA-6 in the regulation of the transcriptional activity of the mouse SP-A gene. Coexpression of GATA-6 and SP-A mRNAs during development and in adult type II epithelial cells supports a potential role for GATA-6 in the regulation of SP-A production, therefore influencing host defense and inflammatory responses in postnatal lung.


text:
Calcium signal transmission between ryanodine receptors and mitochondria.

Control of energy metabolism by increases of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) may represent a fundamental mechanism to meet the ATP demand imposed by heart contractions, but the machinery underlying propagation of [Ca(2+)] signals from ryanodine receptor Ca(2+) release channels (RyR) to the mitochondria remains elusive. Using permeabilized cardiac (H9c2) cells we investigated the cytosolic [Ca(2+)] ([Ca(2+)](c)) and [Ca(2+)](m) signals elicited by activation of RyR. Caffeine, Ca(2+), and ryanodine evoked [Ca(2+)](c) spikes that often appeared as frequency-modulated [Ca(2+)](c) oscillations in these permeabilized cells. Rapid increases in [Ca(2+)](m) and activation of the Ca(2+)-sensitive mitochondrial dehydrogenases were synchronized to the rising phase of the [Ca(2+)](c) spikes. The RyR-mediated elevations of global [Ca(2+)](c) were in the submicromolar range, but the rate of [Ca(2+)](m) increases was as large as it was in the presence of 30 microm global [Ca(2+)](c). Furthermore, RyR-dependent increases of [Ca(2+)](m) were relatively insensitive to buffering of [Ca(2+)](c) by EGTA. Therefore, RyR-driven rises of [Ca(2+)](m) appear to result from large and rapid increases of perimitochondrial [Ca(2+)]. The falling phase of [Ca(2+)](c) spikes was followed by a rapid decay of [Ca(2+)](m). CGP37157 slowed down relaxation of [Ca(2+)](m) spikes, whereas cyclosporin A had no effect, suggesting that activation of the mitochondrial Ca(2+) exchangers accounts for rapid reversal of the [Ca(2+)](m) response with little contribution from the permeability transition pore. Thus, rapid activation of Ca(2+) uptake sites and Ca(2+) exchangers evoked by RyR-mediated local [Ca(2+)](c) signals allow mitochondria to respond rapidly to single [Ca(2+)](c) spikes in cardiac cells.

Excitation-contraction coupling involves local interactions between dihydropyridine receptors (DHPR) 1 located in the plasma membrane and ryanodine receptor Ca 2ϩ release chan-nels (RyR) located in the sarcoplasmic reticulum Ca 2ϩ stores. The mechanism for excitation-contraction coupling differs between skeletal and cardiac muscle. Conformational coupling between DHPR and RyR is utilized in skeletal muscle, whereas RyR respond to local [Ca 2ϩ ] signals established by Ca 2ϩ entering through DHPR in cardiac muscle. RyR-mediated [Ca 2ϩ ] signals lead to activation of the contractile machinery (for review see Ref. 1) and may also be utilized to activate mitochondrial energy metabolism to meet the ATP requirements of muscle contraction (2)(3)(4). This regulatory pathway depends on propagation of [Ca 2ϩ ] c signals to the mitochondria and, in turn, activation of the Ca 2ϩ -sensitive mitochondrial dehydrogenases. However, significant mitochondrial Ca 2ϩ uptake does not occur until [Ca 2ϩ ] c exceeds 1 M in studies carried out with permeabilized cardiac myocytes (5) and the threshold [Ca 2ϩ ] c for mitochondrial Ca 2ϩ uptake is 300 -500 nM in intact myocytes (6). Moreover, the rate of activation of mitochondrial Ca 2ϩ uptake is slow at submicromolar [Ca 2ϩ ] levels (7,8). Thus, RyR-mediated increases of global [Ca 2ϩ ] c that peak in the submicromolar range are not expected to yield rapid rises of [Ca 2ϩ ] m . Importantly, [Ca 2ϩ ] peaks at substantially higher levels in the vicinity of activated RyR than the peak of the global [Ca 2ϩ ] c rise (9,10). Therefore, if mitochondria are located in sufficient proximity to experience the local [Ca 2ϩ ] gradients, propagation of RyR-mediated [Ca 2ϩ ] signals to the mitochondria may result from local coupling between these sites. Recent data on the distance between mitochondria and adjacent Ca 2ϩ release units in cardiac myocytes are in support of the idea that RyR and mitochondria are sufficiently close to each other to establish local [Ca 2ϩ ] communication (11). Significantly, local [Ca 2ϩ ] signaling between IP 3 receptor Ca 2ϩ release channels and mitochondria has also been shown in many cell types (12)(13)(14)(15)(16).
Most of the studies carried out in recent years to investigate propagation of RyR-mediated [Ca 2ϩ ] c responses to the mitochondria used fluorescence measurements of [Ca 2ϩ ] m in intact cardiac cells. Some reports showed that changes of [Ca 2ϩ ] m appear in association with changes in the frequency of [Ca 2ϩ ] c spiking (6,(17)(18)(19), whereas other studies showed beat-to-beat control of [Ca 2ϩ ] m in myocytes (20 -23). In smooth muscle cells, [Ca 2ϩ ] m was also demonstrated to closely follow the kinetics of [Ca 2ϩ ] c rise evoked by activation of RyR (24). Though strategies have been developed for loading of the mitochondria with Ca 2ϩsensitive probes, it is difficult to eliminate the cytosolic tracer component in many cell types, particularly in cardiac myocytes. Thus, the concern was raised that the cytosolic contribution from Ca 2ϩ -sensitive dyes could contribute to the beat-to-beat fluctuations reported in [Ca 2ϩ ] m (e.g. Ref. 6). Studies that evaluated mitochondrial Ca 2ϩ uptake by investigating the effect of mitochondrial inhibitors on RyR-linked [Ca 2ϩ ] c signals also provided conflicting data on the contribution of mitochondria to single [Ca 2ϩ ] c transients (Refs. 6, 17 versus 25). Electron probe microanalysis studies carried out to determine the changes of total mitochondrial calcium showed the presence (26) as well as the absence of changes (27) during the contractile cycle. Nevertheless, membrane potential (⌬⌿ m ) and mitochondrial NAD(P)H responses, which depended on mitochondrial Ca 2ϩ uptake, have been reported during RyR-mediated [Ca 2ϩ ] c spikes (28,29). This suggests that propagation of [Ca 2ϩ ] c increases to the mitochondria can regulate Ca 2ϩ -dependent mitochondrial functions.
The present study was undertaken to determine whether a privileged Ca 2ϩ transfer is involved in the Ca 2ϩ coupling between RyR and mitochondria and to address how mitochondrial Ca 2ϩ transport is controlled during RyR-mediated [

EXPERIMENTAL PROCEDURES
Cell Culture-H9c2 cells (obtained from ATCC) were cultured in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 1 mM pyruvate in humidified air (CO 2 5%) at 37°C. For imaging experiments cells were plated onto poly-D-lysine-coated glass coverslips, and for PCR assays cells were cultured in 75-cm 2 flasks. Cells were grown for 3-5 days for studies with myoblasts (subconfluent cultures), whereas cells were grown to reach confluency (1 week on average) and subsequently for an additional 3-7 days to allow differentiation for studies with myotubes.
Fluorescence Imaging Measurements-Prior to loading with fluorescent dyes, the cells were preincubated for 30 min in an extracellular medium (2% bovine serum albumin/ extracellular medium) consisting of 121 mM NaCl, 5 mM NaHCO 3 , 10 mM Na-HEPES, 4.7 mM KCl, 1. For permeabilized cell measurements the dye-loaded cells were washed three times with a Ca 2ϩ -free extracellular buffer, containing 120 mM NaCl, 5 mM KCl, 1 mM KH 2 PO 4 , 0.2 mM MgCl 2 , 0.1 mM EGTA, and 20 mM Hepes/NaOH, pH 7.4, and then permeabilized with 20 g/ml digitonin for 4 min in an intracellular medium composed of 120 mM KCl, 10 mM NaCl, 1 mM KH 2 PO4, 20 mM Hepes/Tris, pH 7.2, supplemented with 2 mM MgATP, 20 M EGTA/Tris, and 1 g/ml each of antipain, leupeptin, pepstatin. The intracellular medium was passed through a Chelex column prior to the addition of protease inhibitors to reduce ambient Ca 2ϩ . To detect perimembrane [Ca 2ϩ ] ([Ca 2ϩ ] pm ), cells were labeled with 0.5-4 M fura-C18 during the permeabilization. After permeabilization the cells were washed with the same buffer without digitonin. Measurements were performed in intracellular medium that contained ϳ300 nM [Ca 2ϩ ] after the addition of 2 mM ATP, 2 mM succinate, and protease inhibitors. During measurements of ⌬⌿ m , 20 nM TMRE was present in the buffer to maintain the fluorescence labeling achieved during the loading with TMRE.
Images were acquired using an Olympus IX70 inverted microscope (40ϫ, UApo340, NA 1.35 oil immersion objective) fitted with a cooled CCD camera (PXL, Photometrics) under computer control. The computer also controlled a scanning monochromator (DeltaRAM, PTI) to select the excitation wavelength. Excitation at 340 and 380 nm was used for fura2 and fura-C18, 380 nm was used for mitoGFP, 360 nm was used for NAD(P)H, and 545 nm was used for rhod2 and TMRE. Using multiwavelength beamsplitter/emission filter combinations (Chroma Technology Corp., Brattleboro, VT) simultaneous detection of fura-C18 and rhod2 fluorescence or NADH and rhod2 fluorescence was achieved.
Experiments were carried out with at least three different cell preparations, and 20 -60 cells were monitored in each experiment. Traces represent single cell responses unless indicated otherwise.
Reverse Transcriptase-PCR Amplification and Sequencing-Total RNA was prepared from H9c2 cells using the RNeasy Mini Kit (Qiagen). First strand cDNA was synthesized from 0.5-1 g of RNA using MuLV reverse transcriptase (Perkin-Elmer). cDNAs were amplified using Am-pliTaq DNA polymerase (Perkin-Elmer). PCR reactions (100 l) contained first strand cDNA template, 150 nM of each primer, 0.2 mM of each dNTP, 2 mM MgCl 2 , 1ϫ PCR buffer II, and 2.5 units of AmpliTaq DNA polymerase. Reactions were carried out in a Perkin-Elmer Gene-Amp PCR System 9600 for 35 cycles, after an initial 105 s at 95°C, melting for 15 s at 95°C, and anneal-extending for 30 s at the appropriate temperature (50 -60°C). This was followed by a final extension for 7 min at 72°C. Primers to distinguish between rat cardiac and skeletal muscle ␣ 1 subunits were selected by (GCG Prime program) and corresponded to a region in the fourth domain. The cardiac forward primer (3955-3973) was as follows: 5Ј-TCCTCATCCTGCTCAACAC-3Ј. The cardiac reverse primer (4403-4420) was as follows: 5Ј-AATACCT-GCATCCCAATC-3Ј. The skeletal forward primer (1297-1314) was as follows: 5Ј-ATGAACCACATATCGGAC-3Ј. The skeletal reverse primer (1665-1682) was as follows: 5Ј-ATCAGCAAAGCCACATAC-3Ј.
Degenerate primers were used to detect the three RyR isoforms and corresponded to a region in the membrane-spanning domains M3 and M4 (30) 100 l of PCR product was fractionated on a 1-2% low melting temperature agarose gel (Life Technologies, Inc.), and individual bands were purified using a QIAquick gel extraction kit (Qiagen). The purified PCR product was sequenced by the Molecular Core Facility at UMDNJ.

RESULTS AND DISCUSSION
Calcium Signaling in H9c2 Myoblasts and Myotubes-In subconfluent cultures, H9c2 myoblasts displayed a spindle or polygonal shape, and multinucleated myotubes were not present ( Fig. 1, upper row of images). Depolarization by high [K ϩ ] (60 mM KCl) or activation of RyR with caffeine did not elicit a [Ca 2ϩ ] c signal, whereas an IP 3 -linked agonist, vasopressin (VP), induced large [Ca 2ϩ ] c spikes in most of the cells (Fig. 1, i-v). Myoblast cultures grown to reach confluency and subsequently for an additional 3-7 days to allow differentiation contained a number of long, multinucleated myotubes ( Fig. 1, lower row of images). Depolarization resulted in a rise in [Ca 2ϩ ] c in all of the cells in these differentiated cultures (Fig. 1, vii). This [Ca 2ϩ ] c rise was prevented by the dihydropyridine Ca 2ϩ channel inhibitor, nifedipine (10 M, not shown). The addition of caffeine also caused a [Ca 2ϩ ] c rise, which was particularly large in the multinucleated cells (Fig. 1, viii). The rise in [Ca 2ϩ ] c evoked by VP added after caffeine was relatively small in differentiated cells (Fig. 1, iv-v versus ix-x). In these experiments, depletion of intracellular Ca 2ϩ stores evoked by caffeine before the addition of VP might explain the decreased VP-dependent [Ca 2ϩ ] c response in the differentiated cells. However, the relatively small effect of VP was also apparent when the hormone was added to naive cells (data not shown).
To identify the Ca 2ϩ channels that were involved in the depolarization-and caffeine-induced [Ca 2ϩ ] c responses in myotubes, the expression of voltage-operated L-type Ca 2ϩ channels and RyR were examined by reverse transcriptase-PCR. Isotype-specific regions of the ␣ 1 subunit of L-type Ca 2ϩ channel mRNA were amplified by reverse transcriptase-PCR. Fig. 2 shows that differentiation of the myoblasts was associated with substantial increases in the mRNA encoding both cardiac and skeletal isoforms of the L-type channels. This is consistent with previous studies (31)(32)(33). RyR mRNA was not detected in myoblasts but it appeared after differentiation (Fig. 2). The reverse transcriptase-PCR results suggest the presence of one isotype that shows 85, 76, and 80% sequence identity to human RyR1, RyR2, and RyR3, respectively. Taken together, these data provide evidence for expression of the Ca 2ϩ channels that are hallmarks of the myotube phenotype in differentiated H9c2 cells and also suggest that up-regulation of the expression of these Ca 2ϩ channels occurring during differentiation accounts for the appearance of [Ca 2ϩ ] c signals in response to depolarization and to RyR activators in differentiated cells.
Propagation of RyR-mediated [Ca 2ϩ ] Signals to the Mitochondria in Permeabilized Myotubes-To study whether the Ca 2ϩ signals mediated by the activation of RyR are relayed to the mitochondria, [Ca 2ϩ ] m was studied in rhod2-loaded, permeabilized H9c2 cells. Compartmentalization of rhod2 in the mitochondria is facilitated by the net positive charge of the dye, and the cytosolic dye component is eliminated during cell permeabilization. In myoblasts, ryanodine or caffeine caused no rise in [Ca 2ϩ ] rhod2 , whereas the addition of IP 3 yielded large responses in most of the cells (Fig. 3A, upper row). By contrast, myotubes displayed large increases of [Ca 2ϩ ] rhod2 in response to ryanodine (Fig. 3A, v) or caffeine (viii). Images obtained at higher spatial resolution revealed a pattern of [Ca 2ϩ ] rhod2 similar to the distribution of GFP targeted to the mitochondria (Fig. 3, B versus C). Moreover, the decrease of [Ca 2ϩ ] rhod2 resulting from uncoupler-induced dissipation of the mitochondrial membrane potential was also associated with the structures that displayed the caffeine-induced [Ca 2ϩ ] rhod2 rise (Fig.  3B, iii versus iv). These data suggest that activation of RyR in the myotubes brings about increases of [Ca 2ϩ ] m .
Several intramitochondrial dehydrogenases are activated by elevated [Ca 2ϩ ] m , and this activation can be monitored fluorometrically through changes in the pyridine nucleotide redox state. Caffeine-induced increases of [Ca 2ϩ ] rhod2 were associated with an increase in NAD(P)H fluorescence, reflecting the activation of Ca 2ϩ -sensitive mitochondrial dehydrogenases (Fig.  4A). The increase in NAD(P)H fluorescence was transient, but the addition of Ca 2ϩ to the permeabilized cells elicited a further rise of [Ca 2ϩ ] rhod2 and a second transient in NAD(P)H. The fluorescence increase associated with maximal reduction of pyridine nucleotides was obtained by the addition of rotenone to block oxidation of NADH by the respiratory chain. These data provide evidence that the rise of [Ca 2ϩ ] m coupled to activation of RyR exerts control over the Ca 2ϩ -sensitive steps of mitochondrial metabolism in permeabilized myotubes.
To further investigate the signal transmission machinery between RyR and mitochondria, rhod2-loaded permeabilized cells were exposed to fura-C18, a dye that allows measurements of [Ca 2ϩ ] immediately adjacent to cellular membranes (16,34,35 (Fig. 4B). Relaxation of the FIG. 2. PCR products from cardiac myoblasts and myotubes. RNA was isolated from myoblasts (non-diff) and differentiated H9c2 cells (diff) as described under "Experimental Procedures." PCR products generated by primers designed to amplify sequences of the specific genetic messages for cardiac and skeletal muscle L-type Ca 2ϩ channels and RyR were fractionated on agarose gel. Negative controls (no RNA but with reverse primer) were performed and showed no bands. [Ca 2ϩ ] m spikes was slower than the fall of [Ca 2ϩ ] pm , and in some cells an effectively sustained rise of [Ca 2ϩ ] m was coupled to a baseline-spike pattern of the caffeine-induced [Ca 2ϩ ] pm oscillations ( Fig. 4D and presumably, C). Treatment with supramaximal caffeine caused spikes in [Ca 2ϩ ] pm and [Ca 2ϩ ] m displaying a prolonged decay phase (Fig. 4, B and D). The addition of mitochondrial uncoupler caused a small increase in [Ca 2ϩ ] pm but induced a rapid decay of the caffeine-induced elevation of [Ca 2ϩ ] m (Fig. 4B). Rapid reversal of the caffeineinduced rise of [Ca 2ϩ ] m was also evoked by Ru360, a drug reported to inhibit mitochondrial Ca 2ϩ uptake without inhibition of RyR (36). Consistent with this report, the Ru360-induced decay of [Ca 2ϩ ] m was observed in the absence of inhibition of RyR-mediated [Ca 2ϩ ] pm oscillations (Fig. 4D). Furthermore, Ru360 added before supramaximal caffeine did not affect the magnitude of the [Ca 2ϩ ] pm increase but abolished the rise in [Ca 2ϩ ] m (Fig. 4, E and F). Coupled oscillations of [Ca 2ϩ ] pm and [Ca 2ϩ ] m also occurred in permeabilized myotubes exposed to other activators of the RyR, such as Ca 2ϩ and ryanodine (Fig. 5, A and B). Taken together, these data show that activation of RyR in permeabilized myotubes yields oscillatory Ca 2ϩ release and reuptake that is associated with rapid activation of mitochondrial Ca 2ϩ uptake to yield [Ca 2ϩ ] m signals.
Local  were observed in the absence of any caffeine-induced [Ca 2ϩ ] pm increase. After washout of the Ca 2ϩ buffer, the caffeine-induced Ca 2ϩ release brought about a [Ca 2ϩ ] pm rise and an augmented [Ca 2ϩ ] m increase (Fig. 6) response by a slow Ca 2ϩ buffer like EGTA suggests that the spatial separation between RyR and mitochondrial Ca 2ϩ uptake sites is probably in the range of 100 nm rather than Ͻ20 nm (10,16). This conclusion is in agreement with recent measurements of the distances between Ca 2ϩ release units and mitochondria in ventricular myocardium (11).
In an effort to assess the magnitude of the local [Ca 2ϩ ] increases to which the mitochondrial Ca 2ϩ uptake sites are exposed during RyR-mediated Ca 2ϩ release, rates of mitochondrial Ca 2ϩ uptake were measured with varying concentrations of added medium Ca 2ϩ and compared with the rate of Ca 2ϩ uptake obtained during caffeine-induced Ca 2ϩ release. Fig. 7 shows that the addition of Ca 2ϩ led to dose-dependent increases in mitochondrial Ca 2ϩ uptake rates. Half-maximal stimulation was attained at Ϸ20 M [Ca 2ϩ ] c , and maximal activation appeared to require at least 50 M [Ca 2ϩ ] c . Importantly, mitochondrial Ca 2ϩ uptake was not limited by the mitochondrial membrane potential (⌬⌿ m ) under the substrate conditions used in these experiments (2 mM succinate, 2 mM MgATP), because only very small depolarizations were evoked by the addition of large pulses of Ca 2ϩ (Fig. 7, left panel). When caffeine-induced mitochondrial Ca 2ϩ uptake was studied under ] m changes were recorded simultaneously in a permeabilized myotube. Cells were exposed to submaximal and maximal concentrations of caffeine and then to uncoupler (unc; 1 M carbonyl cyanide p-trifluoromethoxyphenylhydrazone ϩ 2.5 g/ml oligomycin). C, time courses of [Ca 2ϩ ] m changes were recorded in a permeabilized myotube exposed to caffeine and Ru360 (Calbiochem, 2 M). D, time courses of [Ca 2ϩ ] pm and [Ca 2ϩ ] m changes were recorded simultaneously in a permeabilized myotube. Cells were exposed to caffeine and Ru360 (2 M). E and F, mean increases of [Ca 2ϩ ] pm and [Ca 2ϩ ] m in naive and Ru360-pretreated myotubes challenged with supramaximal caffeine. Myotubes were exposed to Ru360 (10 M for 3 min) or solvent and then to caffeine (20 mM). The mean traces for [Ca 2ϩ ] pm and [Ca 2ϩ ] m shown in E are from separate runs using cells from the same culture. All myotubes in the field were included for calculation of the mean responses. Calculated magnitude of the [Ca 2ϩ ] pm and [Ca 2ϩ ] m responses shown in F are mean Ϯ S.E. of values from 19 (control) and 16 (Ru360-pretreated) cells from two different cell cultures. Fluorescence of NAD(P)H and compartmentalized rhod2 are shown as fluorescence arbitrary units. the same conditions in permeabilized myotubes, the Ca 2ϩ uptake rate was similar to that achieved with 30 M added free [Ca 2ϩ ] in the bulk medium (Fig. 7). Thus, the local [Ca 2ϩ ] c rise sensed by the mitochondrial Ca 2ϩ uptake sites appears to be in the range of 30 M.
Although the demonstration of efficient Ca 2ϩ transfer from RyR to the mitochondrial matrix in H9c2 myotubes does not prove that the same functional organization occurs in myocytes of the beating heart, there are a number of observations that suggest that mitochondria are exposed to local [Ca 2ϩ ] gradients generated by RyR in cardiac myocytes. The distance between mitochondria and RyR estimated from our [Ca 2ϩ ] flux studies in the presence of EGTA/Ca 2ϩ buffer as well as the large magnitude of perimitochondrial [Ca 2ϩ ] rises are consistent with the data from morphological measurements in cardiac myocytes showing proximity of mitochondria and calcium release units (11). Moreover, a recent study demonstrated large [Ca 2ϩ ] m elevations in association with caffeine-induced [Ca 2ϩ ] c spikes in skinned myocytes. 3 Therefore, it is likely that a privileged pathway of calcium signaling also exists between RyR and mitochondria in cardiac myocytes.  (Fig. 8A). At higher caffeine concentrations, the interspike period decreased (Fig. 8, B  and F), and the fraction of cells showing more prolonged [Ca 2ϩ ] pm transients increased (Fig. 8, C and E). The prolonged responses at high levels of caffeine presumably reflect sustained activation of RyR, and the decay phase was because of depletion of the caffeine-sensitive Ca 2ϩ store. The gradual decrease in the amplitude of spikes during [Ca 2ϩ ] pm oscillations observed in a few cells (e.g. Figs. 8B and 9A) may also reflect depletion of the Ca 2ϩ store. Moreover, bleaching of the fluorescent probe or dissociation from the membranes that occurred during the measurements may also contribute to the decrease in spike amplitudes during [Ca 2ϩ ] pm oscillations. In most cells, spikes of [Ca 2ϩ ] pm were associated with transients of [Ca 2ϩ ] m that displayed a cell-specific pattern of the decay phase. Increases in the oscillation frequency established by successive caffeine additions did not affect the amplitude and upstroke kinetics of the individual spikes (Fig. 8D). Thus, the [Ca 2ϩ ] pm and [Ca 2ϩ ] m spiking triggered by activators of the RyR display the fundamental features of the frequency modulation. Interestingly, in a few cells the [Ca 2ϩ ] m oscillations associated with uniform [Ca 2ϩ ] pm oscillations were found to begin with a relatively small initial [Ca 2ϩ ] m spike (e.g. Fig. 9B; spike at 200 s versus spikes at 250 and 300 s). This result suggests that the Ca 2ϩ coupling between RyR and mitochondrial Ca 2ϩ uptake sites was optimized during the first spikes, and this process may include a decrease in the distance between RyR and mitochondria or sensitization of the mitochondrial Ca 2ϩ uptake sites.
Mechanisms Underlying Rapid Ca 2ϩ Efflux from the Mitochondria-The temporal pattern of the [Ca 2ϩ ] m spikes recorded in individual cells displaying similar [Ca 2ϩ ] pm spikes was heterogeneous. In particular, the decay phase of the [Ca 2ϩ ] m spikes showed cell-to-cell variability. To identify the mechanisms that are responsible for the decay phase, the effect of inhibitors of the two main mitochondrial Ca 2ϩ efflux pathways (Ca 2ϩ exchanger, permeability transition pore) (37) on [Ca 2ϩ ] m oscillations was studied (Fig. 9). Both pathways have 3 S.-S. Sheu, personal communication. been suggested to play a role in physiological Ca 2ϩ regulation in cardiac myocytes (38,39). An inhibitor of the Ca 2ϩ exchanger, CGP37157, did not affect the shape of the [Ca 2ϩ ] pm spikes or the frequency of [Ca 2ϩ ] pm and [Ca 2ϩ ] m spikes but slowed the decay phase of [Ca 2ϩ ] m spikes (Fig. 9, A and C). In contrast, an inhibitor of the permeability pore, cyclosporin-A, failed to affect the decay phase of the [Ca 2ϩ ] m spikes (Fig. 9, B  and C)  each heartbeat is associated with a [Ca 2ϩ ] m rise, complete reversal between consecutive [Ca 2ϩ ] c spikes would require that the relaxation of [Ca 2ϩ ] m be at least an order of magnitude faster than that which occurs in caffeine-stimulated permeabilized H9c2 myotubes. Interestingly, the relaxation phases shown in our study display cell-specific differences and increases in the oscillation frequency are not associated with major changes in the relaxation rate of [Ca 2ϩ ] m spikes. Although inhibition of mitochondrial Ca 2ϩ efflux did not result in any major changes in the pattern of RyR-driven [Ca 2ϩ ] c spiking, a plethora of data suggests that mitochondrial uptake and release of Ca 2ϩ is important in IP 3 -linked cytosolic Ca 2ϩ signaling (35, 40 -47). As such, rapid extrusion of Ca 2ϩ from mitochondria may contribute to the control of cytosolic effector systems and may also contribute to the refilling of the RyRsensitive Ca 2ϩ store. Thus, translation of RyR-driven [Ca 2ϩ ] c signals into brief [Ca 2ϩ ] m transients may have multiple roles in mitochondrial and cytosolic [Ca 2ϩ ] regulation.
Conclusions-This study provides direct evidence that calcium signal propagation from RyR to the mitochondria may occur without significant changes in global [Ca 2ϩ ] c . Thus, it appears that this signaling pathway, which is sufficient to yield an essentially maximal activation of mitochondrial Ca 2ϩ uptake, is established by local communication between the RyR and mitochondrial uptake sites. Remarkably, the physiological regulatory cascade initiated by plasma membrane depolarization in the heart includes privileged communication between DHPR and RyR as well as between RyR and mitochondrial Ca 2ϩ uptake sites, albeit with lesser proximity in the latter case. One function of this pathway is to enhance mitochondrial oxidative metabolism to increase ATP formation, as demonstrated by our finding that the RyR-mediated [Ca 2ϩ ] m signal yields activation of Ca 2ϩ -sensitive mitochondrial dehydrogenases. It appears to be characteristic to the mitochondrial calcium signaling displayed by heart cells that the RyR-driven large spikes of [Ca 2ϩ ] m can be reversed very rapidly owing to activation of the mitochondrial Ca 2ϩ exchanger. The rapid mitochondrial turnover of Ca 2ϩ may protect against sequestration of large amounts of Ca 2ϩ in the mitochondria under physiological conditions and may also contribute to cytosolic calcium signaling on a beat-to-beat basis. All these properties of the signal detection and processing by the mitochondria suggest that the mitochondrial calcium signaling pathway may have an important role in adjusting the activity of energy metabolism to the needs of the contractile machinery.