Age-related skeletal muscle decline is characterized by the modification of sarcolemma ion channels important to sustain fiber excitability and to prevent metabolic dysfunction. Also, calcium homeostasis and contractile function are impaired. In the aim to understand whether these modifications are related to oxidative damage and can be reverted by antioxidant treatment, we examined the effects of in vivo treatment with an waste water polyphenolic mixture (LACHI MIX HT) supplied by LACHIFARMA S.r.l. Italy containing hydroxytirosol (HT), gallic acid, and homovanillic acid on the skeletal muscles of 27-month-old rats. After 6-week treatment, we found an improvement of chloride ClC-1 channel conductance, pivotal for membrane electrical stability, and of ATP-dependent potassium channel activity, important in coupling excitability with fiber metabolism. Both of them were analyzed using electrophysiological techniques. The treatment also restored the resting cytosolic calcium concentration, the sarcoplasmic reticulum calcium release, and the mechanical threshold for contraction, an index of excitation-contraction coupling mechanism. Muscle weight and blood creatine kinase levels were preserved in LACHI MIX HT-treated aged rats. The antioxidant activity of LACHI MIX HT was confirmed by the reduction of malondialdehyde levels in the brain of the LACHI MIX HT-treated aged rats. In comparison, the administration of purified HT was less effective on all the parameters studied. Although muscle function was not completely recovered, the present study provides evidence of the beneficial effects of LACHI MIX HT, a natural compound, to ameliorate skeletal muscle functional decline due to aging-associated oxidative stress.

Solid inclusion complex between hydroxypropyl-β-cyclodextrin (HP-β-CD) and minoxidil (MXD) was prepared by freeze-drying and characterized by yield, drug loading and dissolution rate. Moreover, the complex was formulated as alginate gel (GEL HP-β-CD)/MXD 3.5% w/w). The efficacy of the novel GEL HP-β-CD)/MXD 3.5% w/w and of MXD 3.5% w/w ethanolic/propylene-glycol solution (MXD solution) were evaluated by monitoring the hair growth of dorsal skin 1-4 weeks after depilation followed by histological analysis and gene expression in skin biopsies in male rat. Patch-clamp experiments and cell-dehydrogenase activity (CDA) were performed to evaluate the capability of the formulations to activate "in vitro" the ATP-sensitive K(+)-channels (KATP) and their effects on cell viability in skin fibroblasts. After 3 weeks, the MXD solution and MXD/HP-β-CD GEL enhanced the hair growth, respectively, of 80.1±2% and 84.3±4% vs controls. After 4 weeks, the MXD/HP-β-CD GEL significantly enhanced the hair length and bulb diameter vs others groups. The MXD/HP-β-CD GEL significantly enhanced the mRNA levels of the SUR2 and Kir6.1 subunits of the KATP channels and AKT2 vs other groups. The AR gene was down-regulated vs controls following the treatment with either MXD formulations. Either MXD (10(-4)M) formulations were effective in potentiating the KATP currents. The MXD solution and its vehicle after 9 h of incubation time, but not MXD/HP-β-CD, reduced CDA in fibroblasts. In sum, the MXD/HP-β-CD GEL shows a favorable profile following topical long-term use.

Brown adipose tissue (BAT) is controlled by the sympathetic nervous system (SNS) and has the ability to dissipate energy through uncoupling protein-1 (UCP-1), influencing energy expenditure. Besides BAT, the SNS influences bone, and recent studies have demonstrated a positive correlation between BAT activity and bone. Nerve Growth Factor (NGF) genes are expressed in brain, white adipose tissue (WAT), BAT and bone where it coordinates brain and body reactions to challenges. Similarly Osteocalcin besides its role in bone metabolism acts as an hormone regulating glucose metabolism and brain. We previously showed that NGF and its receptor p75NTR genes are highly expressed in BAT versus brain in mice, suggesting that NGF may act as a regulator of energy. To further investigate the role of NGF and osteocalcin in bone and energy regulation we analyzed NGF and its receptor p75NTR and Osteocalcin mRNA from 3 months old mice after cold exposure. UCP-1 was used as positive control. Mice were divided into three groups: room temperature (RT), cold stress for 6h and 5 days (n = 5 for each group). Mice as control group were all placed at RT (23 °C) for 5 days, while the cold groups were placed at 4 °C for the abovementioned times. The mice were subsequently sacrificed and the interscapular BAT, bone and brain were analyzed for mRNA extraction. The exposure of 6 h to cold stress enhanced mRNA levels of UCP-1 and NGF genes by 3 and 2.5 fold vs controls, respectively, reducing mRNA of p75NTR by 19 fold. The UCP-1 gene was still up-regulated after 5 days of cold stress, the NGF gene was not affected and mRNA of the p75NTR gene was reduced by 7 fold vs controls. The mRNA NGF/NGFR genes were not affected in bone or brain following cold stress. The mRNA levels of osteocalcin in bone were upregulated following 6h and 5 days cold exposure vs controls. Osteocalcin gene in brain was downregulated following cold stress. Our study shows that NGF mRNA expression significantly increases after 6 hours of cold stress however with minor extent with respect to UCP-1. The enhanced mRNA level of NGF is observed in parallel with the decrease of NGF receptor gene expression. We found no change in NGF and p75NTR expression in brain or bone, but osteocalcin genes were upregulated in bone following cold stress. These results suggest that during cold stress when BAT-dependent thermogenesis is required, NGF activity is required, and osteocalcin may exert a local protective effect on bone mass.

We investigated on the role of the genes encoding for the ATP-sensitive K+-channel(KATP) subunits(SUR1-2A/B, Kir6.2) in the atrophy induced "in vitro" by staurosporine (STS) in different skeletal muscle phenotypes of mouse. Patch-clamp and gene expression experiments showed that the expression/activity of the sarcolemma KATP channel subunits was higher in the fast-twitch than in the slow-twitch fibers. After 1 to 3h of incubation time, the STS(2.14×10-6M) treatment enhanced the expression/activity of the SUR2B, SUR1 and Kir6.2 subunit genes, but not SUR2A, in the slow-twitch muscle fibers, induced the caspase-3-9, Atrogin-1 and Murf-1 gene expression without affecting protein content. After 3 to 6h, the STS-related atrophy markedly down-regulated the SUR2B, SUR1 and Kir6.2 genes reducing the KATP currents and reduced the protein content/muscle weight ratio of the slow-twitch muscle by -36.4±6%(p<0.05). After 6 to 24h, no additional changes of the SUR1-2B and Kir6.2 gene expression and muscle protein were observed. In the fast-twitch muscles, STS mildly affected the atrophic genes and protein content, but potentiated the KATP currents down-regulating the Bnip-3 gene. Diazoxide(250-500×10-6M), a SUR1-2B/Kir6.2 channel opener, prevented the protein loss induced by STS in the slow-twitch muscle after 6h showing an EC50 of 1.35×10-7M and Emax of 75%, down-regulated the caspase-9 gene and enhanced the KATP currents. The enhanced expression/activity of the SUR2B, SUR1 and Kir6.2 genes are cytoprotective against STS-induced atrophy in the slow-twitch muscle; their reduced expression/activity is associated with proteolysis and atrophy in skeletal muscle

Emerging evidences suggest that Ca(2+)activated-K(+)-(BK) channel is involved in the regulation of cell viability. The changes of the cell viability observed under hyperkalemia (15 mEq/L) or hypokalemia (0.55 mEq/L) conditions were investigated in HEK293 cells expressing the hslo subunit (hslo-HEK293) in the presence or absence of BK channel modulators. The BK channel openers(10(-11)-10(-3)M) were: acetazolamide(ACTZ), Dichlorphenamide(DCP), methazolamide(MTZ), bendroflumethiazide(BFT), ethoxzolamide(ETX), hydrochlorthiazide(HCT), quercetin(QUERC), resveratrol(RESV) and NS1619; and the BK channel blockers(2 x 10(-7)M-5 x 10(-3)M) were: tetraethylammonium(TEA), iberiotoxin(IbTx) and charybdotoxin(ChTX). Experiments on cell viability and channel currents were performed using cell counting kit-8 and patch-clamp techniques, respectively. Hslo whole-cell current was potentiated by BK channel openers with different potency and efficacy in hslo-HEK293. The efficacy ranking of the openers at -60 mV(Vm) was BFT> ACTZ >DCP ≥RESV≥ ETX> NS1619> MTZ≥ QUERC; HCT was not effective. Cell viability after 24 h of incubation under hyperkalemia was enhanced by 82+6% and 33+7% in hslo-HEK293 cells and HEK293 cells, respectively. IbTx, ChTX and TEA enhanced cell viability in hslo-HEK293. BK openers prevented the enhancement of the cell viability induced by hyperkalemia or IbTx in hslo-HEK293 showing an efficacy which was comparable with that observed as BK openers. BK channel modulators failed to affect cell currents and viability under hyperkalemia conditions in the absence of hslo subunit. In contrast, under hypokalemia cell viability was reduced by -22+4% and -23+6% in hslo-HEK293 and HEK293 cells, respectively; the BK channel modulators failed to affect this parameter in these cells. In conclusion, BK channel regulates cell viability under hyperkalemia but not hypokalemia conditions. BFT and ACTZ were the most potent drugs either in activating the BK current and in preventing the cell proliferation induced by hyperkalemia. These findings may have relevance in disorders associated with abnormal K(+) ion homeostasis including periodic paralysis and myotonia.

Bone mass, metabolism and reproduction are regulated coordinately. The bone-derived osteocalcin(Ost) favors insulin sensitivity, male fertility and neurogenesis. The neurotrophins BDNF and NGF are involved in energy and bone metabolism. NGF regulates fertility elevating LH in the female, Ost-/- mice show obesity and high LH in spite of decreased testosterone. To investigate the NGF-osteocalcin interaction we analyzed by RT-PCR the mRNA levels of NGF, BDNF, Ost. and their receptors p75NTR/NTRK1, TRKb and Gprc6a respectively in adipose WAT/BAT, reproductive organs, brain and bone(positive controls) of 3 months old female and male mice. Here, the mRNA levels of NGF and p75NTR are 50% higher in BAT than the brain. NGF and its receptors are down-regulated in WAT and bone in both genders. Osteocalcin and Gprc6a are up-regulated in bone and brain, downregulated in BAT/WAT. BDNF and TRKb expression in bone is higher than brain, but lower in BAT/WAT; TRKb is down-regulated in bone and up-regulated in adipose tissue. NGF is up-regulated in the ovaries/uterus, but down-regulated in the testes. The mRNA levels of p75NTR is respectively 300%, 100% and 50% higher in testis, ovaries and uterus than the brain. NTRK1 is down-regulated in all tissues. The Gprc6a is expressed in the testes, not in the ovaries and uterus. BDNF and TRKb are down-regulated in the sexual organs. Therefore, the up-regulation of NGF and related-receptors in fat is consistent with NGF as an energy regulator. The inverse correlation of NGF and BDNF in fat and bone, shows these exerting opposite effects on leptin with BDNF regulating bone. The up-regulation of p75NTR in the testes match the Gprc6a expression, and is responsible for higher LH in the Ost-/- mice. The animal care was performed in accordance with the DIRECTIVE 2010/63/EU. The protocol was approved by the Ethics Committee of the University of Bari, Italy.

Therapeutic monoclonal antibodies (mAbs) have high efficacy in treating TNF α-related immunological diseases. Other than neutralizing TNF α, these IgG1 antibodies exert Fc receptor-mediated effector functions such as the complement-dependent cytotoxicity (CDC) and antibody-dependent cell cytotoxicity (ADCC). The crystallizable fragment (Fc) of these IgG1 contains a single glycosylation site at Asn 297/300 that is essential for the CDC and ADCC. Glycosylated antibodies lacking core fucosylation showed an improved ADCC. However, no structural data are available concerning the ligand-binding interaction of these mAbs used in TNF α-related diseases and the role of the fucosylation. We therefore used comparative modeling for generating complete 3D mAb models that include the antigen-binding fragment (Fab) portions of infliximab, complexed with TNF α (4G3Y.pdb), the Fc region of the human IGHG1 fucosylated (3SGJ) and afucosylated (3SGK) complexed with the Fc receptor subtype Fcγ RIIIA, and the Fc region of a murine immunoglobulin (1IGT). After few thousand steps of energy minimization on the resulting 3D mAb models, minimized final models were used to quantify interactions occurring between Fcγ RIIIA and the fucosylated/afucosylated Fc fragments. While fucosylation does not affect Fab-TNF α interactions, we found that in the absence of fucosylation the Fc-mAb domain and Fcγ RIIIA are closer and new strong interactions are established between G129 of the receptor and S301 of the Chimera 2 Fc mAb; new polar interactions are also established between the Chimera 2 Fc residues Y299, N300, and S301 and the Fcγ RIIIA residues K128, G129, R130, and R155. These data help to explain the reduced ADCC observed in the fucosylated mAbs suggesting the specific AA residues involved in binding interactions.

Bone mass, metabolism and reproduction are regulated coordinately. The bone-derived osteocalcin (Ost) favors insulin sensitivity, male fertility and neurogenesis. The neurotrophins BDNF/NGF and oxytocin (Oxt) are involved in energy and bone metabolism. NGF regulates fertility elevating LH in female, Ost-/- mice show obesity and high LH in spite of decreased testosterone. To investigate the NGF/BDNF-Oxt-Ost interactions we analyzed by RT-PCR the mRNA levels of NGF, BDNF, Oxt, Ost and their receptors p75NTR/NTRK1, TRKb, Oxtr and Gprc6a in brain, bone, WAT/BAT and reproductive organs, of 3 months old female and male mice. Brain and bone were used as positive controls respectively. NGF and p75NTR expression is 50% higher in BAT than brain. NGF and its receptors are downregulated in WAT and bone in both genders. Ost and Gprc6a are upregulated in bone and brain, down-regulated in BAT/WAT. BDNF and TRKb expression in bone is higher than brain, but lower in BAT/WAT; TRKb is downregulated in bone and up-regulated in adipose tissue. NGF is up-regulated in ovaries/uterus, but down-regulated in the testes. p75NTR is respectively 300%, 100% and 50% higher in testis, ovaries and uterus than brain. NTRK1 is downregulated in all tissues. The Gprc6a is expressed in testes, not in ovaries and uterus. BDNF and TRKb are downregulated in sexual organs. Oxt is markedly expressed in brain and with minor extend in bone in either genders, while Oxtr in ovaries although a significant expression level is observed in fat and bone. The up-regulation of NGF and related-receptors in fat is consistent with NGF as an energy regulator. The inverse correlation of NGF and BDNF in fat and bone, shows these exerting opposite effects on leptin with BDNF regulating bone. The up-regulation of p75NTR in testes matches the Gprc6a expression in the same organ. Gene correlation analysis shows the existence of an interaction between NGF and osteocalcin. Therefore, we speculate that NGF can be a physiologic mediator of osteocalcin both peripherally regulating steroid production in Leydig cell in the testosterone deficient OST-/-, and centrally thought the sprouting of new synapses in this cognitively impaired mice. The pattern of expression of these molecules and their receptors show a similar trend with Ost, NGF, Oxt and BDNF genes highly expressed in brain of both genders, while their receptors were expressed in the reproductive organs showing a gender expression profile. These data add evidences that the signalling of bone metabolism and reproduction are released from CNS to act on peripheral tissues.

Several 6-substituted 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one (THAI) derivatives were synthesized and evaluated for their activity as cholinesterase (ChE) inhibitors. The most potent inhibitors were identified among 6-(2-phenylethyl)-THAI derivatives, and in particular compounds 12b and 12d proved to be very active against human BChE (IC50 = 13 and 1.8 nM, respectively), with 1000-fold selectivity over AChE. Structure-activity relationships highlighted critical features (e.g., ring fusion [4,3-b], integrity of the lactam CONH function) and favorable physicochemical properties of the 6-(2-phenylethyl) group (i.e., optimal position, size and lipophilicity of phenyl substituents). The effects of a number of compounds against NMDA-induced SH-SY5Y neuronal cell injury were also evaluated. Treatment with 12b increased cell viability in SH-SY5Y cells pretreated with 250 μM NMDA, with significant effects (P < 0.05) at concentrations between 0.5 and 5 μM. These findings suggest that THAI can be used as a scaffold for developing new drug leads for the treatment of Alzheimer-type neurodegeneration syndrome.

ATP-sensitive-K+ (KATP) channels couple metabolism to the electrical activity of the cells. This channel is associated with glycolytic enzymes to form complexes regulating the channel activity in various tissues. The pyruvate-kinase (PK) enzyme is an antigen in the Paediatric Autoimmune Neuropsychiatric Disorders Associated Streptococcal infection known as PANDAS which is characterized by an abnormal production of auto-antibodies against PK. Here, the effects of the anti-pyruvate kinase antibody (anti-PK-ab) on the muscle and neuronal KATP channels were investigated in native rat skeletal muscle fibres and human neuroblastoma cell-line (SH-SY5Y), respectively. Furthermore, the interaction of PK with the inwardly rectifier potassium channel (Kir6.1/Kir6.2) subunits of the KATP channels was investigated by co-immunoprecipitation experiments in mouse brain using the anti-PK-ab. Patch-clamp experiments showed that the short-term incubation (1 h) of the fibres with the anti-PK-ab at the dilutions of 1:500 and 1:300 enhanced the KATP current of 19.6% and 33.5%, respectively. As opposite, the long-term incubation (24 h) of the fibres with the anti-PK-ab at the dilutions of 1:500 and 1:300 reduced the KATP current of 16% and 24%, respectively, reducing the diameter with atrophy. The direct application of the anti-PK-ab to the excised patches in the absence of intracellular ATP caused channel block, while in the presence of nucleotide channel opened. In neuronal cell line, in the short-term the anti-PK-ab potentiated KATP currents without affecting survival, while in the long-term the anti-PK-ab reduced KATP currents inducing neuronal death. Opening/blocking actions of the anti-PK antibodies on the KATP channels were observed, the blocking action causes fibre atrophy and neuronal death. We demonstrated that PK and Kir subunits are physically/functionally coupled in neurons. The KATP/PK complex can be proposed a novel target in the autoimmune diseases associated with anti-PK production as in PANDAS.

The periodic paralysis (PP) are rare autosomal-dominant disorders associated to mutations in the skeletal muscle sodium, calcium, and potassium channel genes characterized by muscle fiber depolarization with un-excitability, episodes of weakness with variations in serum potassium concentrations. Recent advances in thyrotoxic PP and hypokalemic PP (hypoPP) confirm the involvement of the muscle potassium channels in the pathogenesis of the diseases and their role as target of action for drugs of therapeutic interest. The novelty in the gating pore currents theory help to explain the disease symptoms, and open the possibility to more specifically target the disease. It is now known that the fiber depolarization in the hypoPP is due to an unbalance between the novel identified depolarizing gating pore currents (Igp) carried by protons or Na+ ions flowing through aberrant alternative pathways of the mutant subunits and repolarizing inwardly rectifying potassium channel (Kir) currents which also includes the ATP-sensitive subtype. Abnormal activation of the Igp or deficiency in the Kir channels predispose to fiber depolarization. One pharmacological strategy is based on blocking the Igp without affecting normal channel gating. It remains safe and effective the proposal of targeting the KATP, Kir channels, or BK channels by drugs capable to specifically open at nanomolar concentrations the skeletal muscle subtypes with less side effects.

The molecular composition and drug responses of calcium-activated K(+) (BK) channels of skeletal muscle are unknown. Patch-clamp experiments combined with transcript scanning of the Kcnma1 gene encoding the alpha subunit of the BK channel were performed in rat slow-twitch soleus (Sol) and fast-twitch flexor digitorum brevis (FDB) skeletal muscles. Five splicing products of the Kcnma1 gene were isolated from Sol and FDB: the e17, e22, +29 aa, Slo27 and Slo0 variants. RT-PCR analysis demonstrated that the expression of e22 and Slo0 were 80-90% higher in FDB than Sol, whereas the expression of Slo27 was 60% higher in Sol than FDB, and the +29 aa variant was equally expressed in both muscle types. No beta 1-4 subunits were detected. In Sol, a large BK current with low Ca(2+) sensitivity was recorded. The BK channel of Sol also showed a reduced response to BK channel openers, such as NS1619, acetazolamide and related drugs. In FDB, a reduced BK current with high Ca(2+) sensitivity and an enhanced drug response was recorded. The total BK RNA content, which was 200% higher in Sol than in FDB, correlated with the BK currents in both muscles. Drug responses primarily correlated with e22 and Slo0 expression levels in FDB and to Slo27 expression in Sol muscle. In conclusion, phenotype-dependent BK channel biophysical and pharmacological properties correlated with the expression levels of the variants in muscles. These findings may be relevant to conditions affecting postural muscles, such as prolonged bed-rest, and to diseases affecting fast-twitch muscles, such as periodic paralysis. Down-regulation or up-regulation of the variants associated with pathological conditions may affect channel composition and drug responses.

The 2H-1,4-benzoxazine derivatives are novel drugs structurally similar to nucleotides; however, their actions on the pancreatic beta-cell ATP-sensitive-K(+)(KATP) channel and on glucose disposal are unknown. Therefore, the effects of the linear/branched alkyl substituents and the aliphatic/aromatic rings at position 2 of the 2H-1,4-benzoxazine nucleus on the activity of these molecules against the pancreatic beta-cell KATP channel and the Kir6.2C36 subunit were investigated using a patch-clamp technique. The effects of these compounds on glucose disposal that followed glucose loading by i.p. GTT and on fasted glycemia were investigated in normal mice. The 2-n-hexyl analog blocked the KATP(IC50=10.1x10(-9)M) and Kir6.2C36(IC50=9.6x10(-9)M) channels which induced depolarization. In contrast, the 2-phenyl analog was a potent opener(DE50=0.04x10(-9)M), which induced hyperpolarization. The ranked order of the potency/efficacy of the analog openers was 2-phenyl>2-benzyl>2-cyclohexylmethyl. The 2-phenylethyl and 2-isopropyl analogs were not effective as blockers/openers. The 2-n-hexyl (2-10 mg kg(-1)) and 2-phenyl analogs (2-30 mg kg(-1)) reduced and enhanced the glucose AUC curves, respectively, following the glucose loading in mice. These compounds did not affect the fasted glycemia as is observed with glibenclamide. The linear alkyl chain and the aromatic ring at position 2 of the 1,4-benzoxazine nucleus are the determinants, which respectively confer the KATP channel blocking action with glucose lowering effects and the opening action with increased glucose levels. The opening/blocking actions of these compounds mimic those that were observed with ATP and ADP. The results support the use of these compounds as novel anti-diabetic drugs.

The involvement of ATP-sensitive K+ (K-ATP) channels in the atrophy of slow-twitch (MHC-I) soleus (SOL) and fast-twitch (MHC-IIa) flexor digitorum brevis (FDB) muscles was investigated in vivo in 14-day-hindlimb-unloaded (14-HU) rats, an animal model of disuse, and in vitro in drug-induced muscle atrophy. Patch-clamp and gene expression experiments were performed in combination with measurements of fibre diameters used as an index of atrophy, and with MHC labelling in 14-HU rats and controls. A down-regulation of K-ATP channel subunits Kir6.2, SUR1 and SUR2B with marked atrophy and incomplete phenotype transition were observed in SOL of 14-HU rats. The observed changes in K-ATP currents were well correlated with changes in fibre diameters and SUR1 expression, as well as with MHC-IIa expression. Half of the SOL fibres of 14-HU rats had reduced diameter and K-ATP currents and were labelled by MHC-I antibodies. Non-atrophic fibres were labelled by MHC-IIa (22%) antibodies and had enhanced K-ATP currents, or were labelled by MHC-I (28%) antibodies but had normal current. FDB was not affected in 14-HU rats and this is related to the high expression/activity of Kir6.2/SUR1 subunits characterizing this muscle phenotype. The long-term incubation of the control muscles in vitro with the K-ATP channel blocker glibenclamide (10-6 m) reduced the K-ATP currents with atrophy and these effects were prevented by the K-ATP channel opener diazoxide (10-4 m). The in vivo down-regulation of SUR1, and possibly of Kir6.2 and SUR2B, or their in vitro pharmacological blockade activates atrophic signalling in skeletal muscle. All these findings suggest a new role for the K-ATP channel as a molecular sensor of atrophy.

Here we investigated on the role of the calcium activated K(+)-channels(BKCa) on the regulation of the neuronal viability. Recordings of the K(+)-channel current were performed using patch-clamp technique in human neuroblastoma cells (SH-SY5Y) in parallel with measurements of the cell viability in the absence or presence of the BKCa channel blockers iberiotoxin(IbTX) and tetraethylammonium (TEA) and the BKCa channel opener NS1619. Protein kinase C/A (PKC, PKA) activities in the cell lysate were investigated in the presence/absence of drugs. The whole-cell K(+)-current showed a slope conductance calculated at negative membrane potentials of 126.3 pS and 1.717 nS(n = 46) following depolarization. The intercept of the I/V curve was -33 mV. IbTX(10(-8) - 4 × 10(-7) M) reduced the K(+)-current at +30 mV with an IC50 of 1.85 × 10(-7) M and an Imax of -46% (slope = 2.198) (n = 21). NS1619(10-100 × 10(-6) M) enhanced the K(+)-current of +141% (n = 6), at -10 mV(Vm). TEA(10(-5)-10(-3) M) reduced the K(+)-current with an IC50 of 3.54 × 10(-5) M and an Imax of -90% (slope = 0.95) (n = 5). A concentration-dependent increase of cell proliferation was observed with TEA showing a maximal proliferative effect(MPE) of +38% (10(-4) M). IbTX showed an MPE of +42% at 10(-8) M concentration, reducing it at higher concentrations. The MPE of the NS1619(100 × 10(-6) M) was +42%. The PKC inhibitor staurosporine (0.2-2 × 10(-6) M) antagonized the proliferative actions of IbTX and TEA. IbTX (10 × 10(-9) M), TEA (100 × 10(-6) M), and the NS1619 significantly enhanced the PKC and PKA activities in the cell lysate with respect to the controls. These results suggest that BKCa channel regulates proliferation of the SH-SY5Y cells through PKC and PKA protein kinases

The molecular composition and drug response of calcium-activated-K+ (BK) channels of skeletal muscle are unknown. Patch-clamp experiments in parallel with transcript scanning of the Kcnma1 gene encoding the alpha subunit of the BK channel were performed in slow-twitch soleus (Sol) and fast-twitch flexor-digitorum-brevis (FDB) rat skeletal muscles. Five splicing products of the Kcnma1 gene were isolated in Sol and FDB such as the e17, e22, +29 aa, Slo27 and Slo0 variants. RT-PCR showed that the expression of the e22 and Slo0 were 80-90% higher in FDB with respect to Sol; while the expression of Slo27 was 60% higher in Sol with respect to FDB; the +29aa variant was equally expressed. No beta1-4 subunits were detected. In Sol, a large BK current with low Ca2+-sensitivity was recorded. The BK channel of Sol also showed a reduced response to the BK channel openers such as NS1619, acetazolamide and related drugs. In FDB, a reduced BK current with high Ca2+-sensitivity and enhanced drug response was recorded. The total BK-RNA content, which was 200% more abundant in Sol than in FDB, was correlated to the BK currents in both muscles. The drug responses were mostly correlated to the e22 and Slo0 expression levels in the FDB, while to the Slo27 expression in the Sol muscle. In conclusion, the phenotype-dependent BK channel biophysical and pharmacological properties were correlated to the variants expression levels in the muscles. This can be relevant in conditions affecting postural muscles such as the prolonged bed-rest, and in diseases affecting fast-twitch muscle such as the periodic paralysis. Down-regulation or up-regulation of these variants associated with pathological conditions may affects the channel composition and drug response.

Formulazione farmaceutica comprendente 3,4-diidrossi-6- [18F] -fluoro-L-fenilalanina e almeno un agente tampone in un veicolo acquoso, in cui la formulazione ha un valore di pH compreso tra 4,0 e 5,5, preferibilmente tra 4,5 e 5,0, più preferibilmente circa 5 e usi corrispondenti nei metodi di imaging diagnostico.

L' invenzione ha per oggetto una bevanda che utilizza il succo idrico delle olive, ovvero il succo delle olive senza l'olio. Tale succo, che consiste nell'acqua di vegetazione delle olive, contiene moltissimi polifenoli utilissimi per la nostra salute, mentre in tutti i procedimenti per l'estrazione dell'olio extravergine di oliva è considerato un prodotto di scarto e quindi eliminato.