Pia A. Elustondo

The TRPM8 Protein Is a Testosterone Receptor II. FUNCTIONAL EVIDENCE FOR AN IONOTROPIC EFFECT OF TESTOSTERONE ON TRPM8

Background: TRPM8 channels are highly expressed in prostate tissues, where the role of this cold receptor is not well understood. Results: Testosterone activates TRPM8 in various cellular systems and in the planar lipid bilayers. Conclusion: TRPM8 is an ionotropic testosterone receptor. Significance: TRPM8 channels may be implicated in various physiological processes regulated by androgens. Testosterone is a key steroid hormone in the development of male reproductive tissues and the regulation of the central nervous system. The rapid signaling mechanism induced by testosterone affects numerous behavioral traits, including sexual drive, aggressiveness, and fear conditioning. However, the currently identified testosterone receptor(s) is not believed to underlie the fast signaling, suggesting an orphan pathway. Here we report that an ion channel from the transient receptor potential family, TRPM8, commonly known as the cold and menthol receptor is the major component of testosterone-induced rapid actions. Using cultured and primary cell lines along with the purified TRPM8 protein, we demonstrate that testosterone directly activates TRPM8 channel at low picomolar range. Specifically, testosterone induced TRPM8 responses in primary human prostate cells, PC3 prostate cancer cells, dorsal root ganglion neurons, and hippocampal neurons. Picomolar concentrations of testosterone resulted in full openings of the purified TRPM8 channel in planar lipid bilayers. Furthermore, acute applications of testosterone on human skin elicited a cooling sensation. Our data conclusively demonstrate that testosterone is an endogenous and highly potent agonist of TRPM8, suggesting a role of TRPM8 channels well beyond their well established function in somatosensory neurons. This discovery may further imply TRPM8 channel function in testosterone-dependent behavioral traits.

The TRPM8 Protein Is a Testosterone Receptor I. BIOCHEMICAL EVIDENCE FOR DIRECT TRPM8-TESTOSTERONE INTERACTIONS

Background: TRPM8 channels are highly expressed in prostate tissues, where the role of this cold receptor is not well understood. Results: Testosterone directly interacts with the TRPM8 protein. Conclusion: TRPM8 is a testosterone receptor. Significance: TRPM8 channels may be implicated in various physiological processes regulated by androgens. The transient receptor potential ion channel of the melastatin subfamily, TRPM8, is a major cold receptor in the peripheral nervous system. Along with the sensory neurons, the TRPM8 protein is highly expressed in the prostate epithelial cells, and this expression is regulated by androgens. Here we investigated the expression and intracellular localization of the TRPM8 channel in relationship to androgens. We performed experiments using human prostate tissues obtained from healthy individuals and patients with prostate cancer at various stages of the disease as well as in cultured cells. Using an immunohistochemistry approach, we detected an intensive colocalization pattern of the TRPM8 protein with endogenous androgens in all tissues tested, suggesting possible interactions. Co-immunoprecipitation experiments performed using cultured prostate epithelial cells, prostate cancer cells, and HEK-293 cells stably expressing TRPM8 further confirmed direct binding of the steroid hormone, testosterone, to the TRPM8 protein. Applications of picomolar concentrations of testosterone to the primary human prostate cells, endogenously expressing TRPM8, elicited Ca2+ responses and channel currents, and those were inhibited in the presence of TRPM8 antagonist, N-(2-aminoethyl)-N-(4-(benzyloxy)-3-methoxybenzyl)thiophene-2-carboxamide hydrochloride. These results indicate that the TRPM8 channel is physically associated with testosterone and suggest that, in addition to a genomic role, testosterone plays a role in direct regulation of the TRPM8 channel function.

Synergistic neuroprotection by epicatechin and quercetin: Activation of convergent mitochondrial signaling pathways

In view of evidence that increased consumption of epicatechin (E) and quercetin (Q) may reduce the risk of stroke, we have measured the effects of combining E and Q on mitochondrial function and neuronal survival following oxygen-glucose deprivation (OGD). Relative to mouse cortical neuron cultures pretreated (24h) with either E or Q (0.1-10μM), E+Q synergistically attenuated OGD-induced neuronal cell death. E, Q and E+Q (0.3μM) increased spare respiratory capacity but only E+Q (0.3μM) preserved this crucial parameter of neuronal mitochondrial function after OGD. These improvements were accompanied by corresponding increases in cyclic AMP response element binding protein (CREB) phosphorylation and the expression of CREB-target genes that promote neuronal survival (Bcl-2) and mitochondrial biogenesis (PGC-1α). Consistent with these findings, E+Q (0.1 and 1.0μM) elevated mitochondrial gene expression (MT-ND2 and MT-ATP6) to a greater extent than E or Q after OGD. Q (0.3-3.0μM), but not E (3.0μM), elevated cytosolic calcium (Ca(2+)) spikes and the mitochondrial membrane potential. Conversely, E and E+Q (0.1 and 0.3μM), but not Q (0.1 and 0.3μM), activated protein kinase B (Akt). Nitric oxide synthase (NOS) inhibition with L-N(G)-nitroarginine methyl ester (1.0μM) blocked neuroprotection by E (0.3μM) or Q (1.0μM). Oral administration of E+Q (75mg/kg; once daily for 5days) reduced hypoxic-ischemic brain injury. These findings suggest E and Q activate Akt- and Ca(2+)-mediated signaling pathways that converge on NOS and CREB resulting in synergistic improvements in neuronal mitochondrial performance which confer profound protection against ischemic injury.

Physical and functional association of lactate dehydrogenase (LDH) with skeletal muscle mitochondria

Background: The notion of mitochondrial lactate oxidation in skeletal muscle is controversial. Results: Mitochondrial respiration increased in the presence of substrates and cofactors for the lactate dehydrogenase (LDH) reaction. Respiration was inhibited when mitochondrial pyruvate transport was blocked. Conclusion: Extra-matrix LDH is associated with muscle mitochondria. Significance: LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria. The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO2) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO2. However, subsequent addition of NAD+ significantly increased JO2, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD+-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.

Polyhydroxybutyrate targets mammalian mitochondria and increases permeability of plasmalemmal and mitochondrial membranes.

Poly(3-hydroxybutyrate) (PHB) is a polyester of 3-hydroxybutyric acid (HB) that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB). We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle.

Role of polyhydroxybutyrate in mitochondrial calcium uptake

Polyhydroxybutyrate (PHB) is a biological polymer which belongs to the class of polyesters and is ubiquitously present in all living organisms. Mammalian mitochondrial membranes contain PHB consisting of up to 120 hydroxybutyrate residues. Roles played by PHB in mammalian mitochondria remain obscure. It was previously demonstrated that PHB of the size similar to one found in mitochondria mediates calcium transport in lipid bilayer membranes. We hypothesized that the presence of PHB in mitochondrial membrane might play a significant role in mitochondrial calcium transport. To test this, we investigated how the induction of PHB hydrolysis affects mitochondrial calcium transport. Mitochondrial PHB was altered enzymatically by targeted expression of bacterial PHB hydrolyzing enzyme (PhaZ7) in mitochondria of mammalian cultured cells. The expression of PhaZ7 induced changes in mitochondrial metabolism resulting in decreased mitochondrial membrane potential in HepG2 but not in U87 and HeLa cells. Furthermore, it significantly inhibited mitochondrial calcium uptake in intact HepG2, U87 and HeLa cells stimulated by the ATP or by the application of increased concentrations of calcium to the digitonin permeabilized cells. Calcium uptake in PhaZ7 expressing cells was restored by mimicking calcium uniporter properties with natural electrogenic calcium ionophore - ferutinin. We propose that PHB is a previously unrecognized important component of the mitochondrial calcium uptake system.

In Situ Investigation of Mammalian Inorganic Polyphosphate Localization Using Novel Selective Fluorescent Probes JC-D7 and JC-D8

Inorganic polyphosphate (polyP) is a polymer composed of many orthophosphates linked together by phosphoanhydride bonds. Recent studies demonstrate that in addition to its important role in the function of microorganisms, polyP plays multiple important roles in the pathological and physiological function of higher eukaryotes, including mammalians. However, due to the dramatically lower abundance of polyP in mammalian cells when comparing to microorganisms, its investigation poses an experimental challenge. Here, we present the identification of novel fluorescent probes that allow for specific labeling of synthetic polyP in vitro as well as endogenous polyP in living cells. These probes demonstrate high selectivity for the labeling of polyP that was not sensitive to a number of ubiquitous organic polyphosphates, notably RNA. Use of these probes allowed us to demonstrate the real time detection of polyP release from lysosomes in live cells. Furthermore, we have been able to detect the increased levels of polyP in cells with Parkinsons disease related mutations.

Identification of the Polyhydroxybutyrate Granules in Mammalian Cultured Cells

Poly‐3‐hydroxybutyrate (PHB) is a biological polyester present in bacteria and eukaryotic cells. Long‐chain (or storage) sPHB (up to 100,000 residues) is typically present in PHB‐accumulating bacteria and localized in specialized granules known as carbonosomes. In these organisms, sPHB plays a major role as carbon and energy storage. On the other hand, short‐chain (or complexed) cPHB (10–100 residues) is present in eukaryotic organisms, including mammals as well as in many bacteria. Previous studies indicated that cPHB is localized in various subcellular compartments of the eukaryotic organisms. Here, we used fluorescent microscopy to directly investigate the localization of PHB in mammalian cells. PHB was visualized in cultured U87 cells using fluorescent probe BODIPY 493/503. Specificity of PHB staining was confirmed by markedly decreased fluorescence of samples treated with PHB‐specific depolymerase (PhaZ7). We found that PHB is associated with granules, and that these PHB‐enriched granules do not co‐localized with mitochondria, lysosomes, or endoplasmic reticulum. These results suggest that, in mammalian cells, PHB can accumulate in the cytoplasm in granules similar to ‘energy storage’ carbonosomes found in PHB‐accumulating bacteria.

Inorganic polyphosphate (polyP) as an activator and structural component of the mitochondrial permeability transition pore.

Mitochondrial permeability transition pore (mPTP) is a large channel located in the mitochondrial inner membrane. The opening of mPTP during pathological calcium overload leads to the membrane depolarization and disruption of ATP production. mPTP activation has been implicated as a central event during the process of stress-induced cell death. mPTP is a supramolecular complex composed of many proteins. Recent studies suggest that mitochondrial ATPase plays the central role in the formation of mPTP. However, the structure of the central conducting pore part of mPTP (mPTPore) remains elusive. Here we review current models proposed for the mPTPore and involvement of polyP in its formation and regulation. We discuss the underestimated role of polyP as an effector and a putative structural component of the mPTPore. We propose the hypothesis that inclusion of polyP can explain such properties of mPTP activity as calcium activation, selectivity and voltage-dependence.

Spermine selectively inhibits high-conductance, but not low-conductance calcium-induced permeability transition pore

The permeability transition pore (PTP) is a large channel of the mitochondrial inner membrane, the opening of which is the central event in many types of stress-induced cell death. PTP opening is induced by elevated concentrations of mitochondrial calcium. It has been demonstrated that spermine and other polyamines can delay calcium-induced swelling of isolated mitochondria, suggesting their role as inhibitors of the mitochondrial PTP. Here we further investigated the mechanism by which spermine inhibits the calcium-induced, cyclosporine A (CSA) -sensitive PTP by using three indicators: 1) calcium release from the mitochondria detected with calcium green, 2) mitochondrial membrane depolarization using TMRM, and 3) mitochondrial swelling by measuring light absorbance. We found that despite calcium release and membrane depolarization, indicative of PTP activation, mitochondria underwent only partial swelling in the presence of spermine. This was in striking contrast to the high-amplitude swelling detected in control mitochondria and in mitochondria treated with the PTP inhibitor CSA. We conclude that spermine selectively prevents opening of the high-conductance state, while allowing activation of the lower conductance state of the PTP. We propose that the existence of lower conductance, stress-induced PTP might play an important physiological role, as it is expected to allow the release of toxic levels of calcium, while keeping important molecules (e.g., NAD) within the mitochondrial matrix.