Olga Ruzsnavszky

Inhibition of TASK-3 (KCNK9) channel biosynthesis changes cell morphology and decreases both DNA content and mitochondrial function of melanoma cells maintained in cell culture.

TASK-3 channel overexpression was shown to facilitate the survival of malignantly transformed cells, possibly by providing greater hypoxia tolerance through a still unknown mechanism. Although it has been suggested previously that TASK-3 channels are expressed in the mitochondrial membranes, their role here remains elusive. In this study, a transient transfection of TASK-3 knockdown melanoma cell cultures was produced to show the significance of TASK-3 expression. Reduction of the TASK-3 protein biosynthesis induced characteristic changes in cell morphology, reduced the amount of DNA and decreased metabolic activity and mitochondrial function of melanoma cells when compared with control. These findings indicate that TASK-3 channel expression and function is indispensable for the proliferation and/or survival of the melanoma cells, as they seem to contribute to their mitochondrial functions. The significance is that, in this study, we have shown that TASK-3 channels are expressed in the mitochondria of melanoma malignum cells, and they are essential for maintaining cellular integrity and viability. The TASK-3 knockdown melanoma cell line had altered morphology, reduced DNA content, decreased metabolic activity and impaired mitochondrial function. These data indicate that TASK-3 channels are functionally present in the mitochondria of the melanoma cells, and their function is essential for the survival of these cells, thus TASK-3 channels may be the possible targets of future anticancer therapy.

Overexpression of transient receptor potential canonical type 1 (TRPC1) alters both store operated calcium entry and depolarization-evoked calcium signals in C2C12 cells

When the intracellular calcium stores are depleted, a Ca(2+) influx is activated to refill these stores. This store-operated Ca(2+) entry (SOCE) depends on the cooperation of several proteins as STIM1, Orai1, and, possibly, TRPC1. To elucidate this role of TRPC1 in skeletal muscle, TRPC1 was overexpressed in C2C12 cells and SOCE was studied by measuring the changes in intracellular Ca(2+) concentration ([Ca(2+)](i)). TRPC1 overexpression significantly increased both the amplitude and the maximal rate-of-rise of SOCE. When YM-58483, an inhibitor of TRPC1 was used, these differences were eliminated, moreover, SOCE was slightly suppressed. A decrease in the expression of STIM1 together with the downregulation of SERCA was confirmed by Western-blot. As a consequence, a reduction in maximal Ca(2+) uptake rate and a higher resting [Ca(2+)](i) following the Ca(2+) transients evoked by 120mM KCl were detected. Morphological changes also accompanied the overexpression of TRPC1. Differentiation of the myoblasts started later, and the myotubes were thinner in TRPC1-overexpressing cultures. For these changes the observed decrease in the nuclear expression of NFAT1 could be responsible. Our results suggest that enhanced expression of TRPC1 increases SOCE and has a negative effect on the STIM1-Orai1 system, indicating an interaction between these proteins.

Hypermuscular mice with mutation in the myostatin gene display altered calcium signalling

Hypermuscularity associated with naturally occurring mutations in the myostatin gene as found in Compact mice results in increased muscle mass but reduced specific force. The calcium sensitivity of the contractile apparatus as assessed on chemically skinned skeletal muscle fibres under isometric conditions is not altered in these animals. While the resting calcium concentration remains unaffected, depolarization‐evoked increases in intracellular calcium concentration are suppressed. Spontaneous calcium release events from sarcoplasmic reticulum are also decreased in frequency, amplitude and spatial spread. Our results suggest that mutations in the myostatin gene are accompanied by alterations in excitation contraction coupling, which manifest as a reduction in sarcoplasmic calcium release.

UV-B induced alteration in purinergic receptors and signaling on HaCaT keratinocytes.

Although there are a number of recognized risk factors resulting in cutaneous malignancies, very little is known about the exact mechanism. In keratinocytes different purinergic receptors have been implicated to play essential roles in deciding the fate of the cells through regulating proliferation and differentiation. While P2Y receptors seem to control the former, P2X receptors, among which the P2X(7) receptor is associated with the induction of apoptosis, are likely to be responsible for the latter. Forty mJ/cm(2) UV-B irradiation decreased the number of viable cells as assessed using MTT assay. This irradiation decreased the amount of both P2X(1) and P2Y(2) receptors and essentially destroyed the P2X(7) receptors in surviving cells. Morphology of ATP-induced Ca(2+) transients were altered in irradiated cells compared to control. The amplitude and the rate of rise of the transients were decreased and the return to resting [Ca(2+)](i) prolonged. This observation is consistent with the finding that in control cells mostly ionotropic, while in irradiated cells mostly metabotropic receptors were underlying the response to ATP. These alterations in the expression pattern of purinergic receptors and in the Ca(2+) transients could explain the observed decreased tendency for ATP-induced apoptosis and possibly contribute to the malignant transformation of keratinocytes.

Expression of anti-Mullerian hormone receptor on the appendix testis in connection with urological disorders

The female internal sex organs develop from the paramesonephric (Mullerian) duct. In male embryos, the regression of the Mullerian duct is caused by the anti-Mullerian hormone (AMH), which plays an important role in the process of testicular descent. The physiological remnant of the Mullerian duct in males is the appendix testis (AT). In our previous study, we presented evidence for the decreased incidence of AT in cryptorchidism with intraoperative surgery. In this report, the expression of the anti-Mullerian hormone receptor type 2 (AMHR2), the specific receptor of AMH, on the AT was investigated in connection with different urological disorders, such as hernia inguinalis, torsion of AT, cysta epididymis, varicocele, hydrocele testis and various forms of undescended testis. The correlation between the age of the patients and the expression of the AMHR2 was also examined. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect the receptors mRNA and protein levels, respectively. We demonstrate that AMHR2 is expressed in the ATs. Additionally, the presence of this receptor was proven at the mRNA and protein levels. The expression pattern of the receptor correlated with neither the examined urological disorders nor the age of the patients; therefore, the function of the AT remains obscure.

Differential Effects of Phosphatase Inhibitors on the Calcium Homeostasis and Migration of HaCaT Keratinocytes

Changes in intracellular calcium concentration ([Ca2+]i) as well as in the phosphorylation state of proteins have been implicated in keratinocyte wound healing revealed in scratch assays. Scratching confluent HaCaT monolayers decreased the number of cells displaying repetitive Ca2+ oscillations as well as the frequency of their Ca2+-transients in cells close to the wounded area and initiated migration of the cells into the wound bed. In contrast, calyculin-A (CLA) and okadaic acid (OA), known cell permeable inhibitors of protein phosphatase-1 and 2A, increased the level of resting [Ca2+]i and suppressed cell migration and wound healing of HaCaT cells. Furthermore, neither CLA nor OA influenced how scratching affected Ca2+ oscillations. It is assumed that changes in and alterations of the phosphorylation level of Ca2+-transport and contractile proteins upon phosphatase inhibition mediates cell migration and wound healing.

Cannabinoid signalling inhibits sarcoplasmic Ca2+ release and regulates excitation–contraction coupling in mammalian skeletal muscle

Marijuana was found to cause muscle weakness, although the exact regulatory role of its receptors (CB1 cannabinoid receptor; CB1R) in the excitation–contraction coupling (ECC) of mammalian skeletal muscle remains unknown. We found that CB1R activation or its knockout did not affect muscle force directly, whereas its activation decreased the Ca2+‐sensitivity of the contractile apparatus and made the muscle fibres more prone to fatigue. We demonstrate that CB1Rs are not connected to the inositol 1,4,5‐trisphosphate pathway either in myotubes or in adult muscle fibres. By contrast, CB1Rs constitutively inhibit sarcoplasmic Ca2+ release and sarcoplasmic reticulum Ca2+ ATPase during ECC in a Gi/o protein‐mediated way in adult skeletal muscle fibres but not in myotubes. These results help with our understanding of the physiological effects and pathological consequences of CB1R activation in skeletal muscle and may be useful in the development of new cannabinoid drugs.

Myostatin Deficient Mice Display Altered Calcium Signaling

Myostatin, a member of the transforming growth factor β family was shown to be a potent negative regulator of skeletal muscle growth. It is strongly expressed in skeletal muscle and myostatin deficient mice have a great increase in muscle mass. Yet, the physical performance of these animals is not improved or even suppressed as compared to control mice. To understand the possible underlying mechanisms we investigated the calcium homeostasis of skeletal muscle fibers from mice with the compact myostatin mutation (MstnCmpt-dlAbc).Resting membrane potentials were essentially identical on control and MstnCmpt-dlAbc mice (−79.2±0.3 vs. −78.0±0.3 mV). Action potentials (AP), evoked by field stimulation through a pair of platinum electrodes on single intact flexor digitorum brevis (FDB) muscle fibers, on the other hand, were significantly shorter on MstnCmpt-dlAbc animals. The average time between the peak and 90% repolarization of the AP corresponded to 0.92±0.03 vs. 0.78±0.02 ms on control and on MstnCmpt-dlAbc fibers, respectively. The other parameters of the APs were essentially identical.The resting and the depolarization-evoked changes in intracellular Ca2+ concentration ([Ca2+]i) were measured on single FDB fibers with the fluorescent dye Fura-2. While resting [Ca2+]i was higher (57.6±1.4 vs. 65.4±2.8 nM), the amplitude of the KCl-evoked calcium transients was smaller (360±49 vs. 215±44 nM) in MstnCmpt-dlAbc mice. Similar experiments were conducted on Fluo-3 loaded fibers using a confocal microscope and repeated (at 100 Hz for 200 ms) field stimulation. AP-evoked calcium transients were again smaller (2.75±0.15 vs. 2.20±0.14) on MstnCmpt-dlAbc fibers. Elementary calcium release events were detected using Fluo-3 on permeabilized FDB fibers. No significant difference in the parameters of sparks was observed between control and MstnCmpt-dlAbc mice.These results indicate that an alteration in calcium signaling might underlie the reduced muscle performance in myostatin deficient mice.