ATP shows more potential as a urinary biomarker than acetylcholine and PGE2 , but its concentration in urine is not a simple function of dilution.

AIMS: To determine whether the amount of ATP, prostaglandin E2 (PGE2 ), and acetylcholine (ACh) in voided urine are influenced enough by that released within the lower urinary tract (LUT) for them to be useful biomarkers of bladder function. METHODS: Participants without LUT symptoms collected total urine voids at 15, 30, 60, and 120 min (20 males/23 females) and 240 min (18 males/26 females) following the previous void. Aliquots of urine were immediately frozen at -20°C and later used to measure ATP (luciferin-luciferase), PGE2 (enzyme-linked immunosorbent assay), ACh (mass spectrometry), creatinine (colorimetric), and lactose dehydrogenase (colorimetric). RESULTS: The amount of ATP in voided urine correlated strongly with the rate of urine production, suggesting that the majority, if not all, the ATP in voided urine has an LUT, and likely bladder, origin. In contrast, there appeared to be no significant net LUTs release of creatinine or ACh into the urine. PGE2 was intermediate with an LUT component that increased with urine production rate and contributed about 25% of the total at 1 ml/min in women but a smaller fraction in men. CONCLUSION: Whereas the majority of the ATP measured within the voided urine originates in the LUT, ACh reflects that extracted from the plasma in the kidneys and PGE2 is a mixture of both sources. ATP has the most potential as a biomarker of benign bladder disorders. Expressing urinary ATP concentration relative to creatinine concentration is questioned in light of these results.

ATP release from freshly isolated guinea-pig bladder urothelial cells: a quantification and study of the mechanisms involved.

OBJECTIVES: To quantify the amount of ATP released from freshly isolated bladder urothelial cells, study its control by intracellular and extracellular calcium and identify the pathways responsible for its release. MATERIALS AND METHODS: Urothelial cells were isolated from male guinea-pig urinary bladders and stimulated to release ATP by imposition of drag forces by repeated pipetting. ATP was measured using a luciferin-luciferase assay and the effects of modifying internal and external calcium concentration and blockers of potential release pathways studied. RESULTS: Freshly isolated guinea-pig urothelial cells released ATP at a mean (sem) rate of 1.9 (0.1) pmoles/mm(2) cell membrane, corresponding to about 700 pmoles/g of tissue, and about half [49 (6)%, n = 9) of the available cell ATP. This release was reduced to a mean (sem) of 0.46 (0.08) pmoles/mm(2) (160 pmoles/g) with 1.8 mm external calcium, and was increased about two-fold by increasing intracellular calcium. The release from umbrella cells was not significantly different from a mixed intermediate and basal cell population, suggesting that all three groups of cells release a similar amount of ATP per unit area. ATP release was reduced by ≈ 50% by agents that block pannexin and connexin hemichannels. It is suggested that the remainder may involve vesicular release. CONCLUSIONS: A significant fraction of cellular ATP is released from isolated urothelial cells by imposing drag forces that cause minimal loss of cell viability. This release involves multiple release pathways, including hemichannels and vesicular release.

Anti-proliferative actions of T-type calcium channel inhibition in Thy1 nephritis.

Aberrant proliferation of mesangial cells (MCs) is a key finding in progressive glomerular disease. TH1177 is a small molecule that has been shown to inhibit low-voltage activated T-type Ca(2+) channels (TCCs). The current study investigates the effect of TH1177 on MC proliferation in vitro and in vivo. The effect of Ca(2+) channel inhibition on primary rat MC proliferation in vitro was studied using the microculture tetrazolium assay and by measuring bromodeoxyuridine incorporation. In vivo, rats with Thy1 nephritis were treated with TH1177 or vehicle. Glomerular injury and average glomerular cell number were determined in a blinded fashion. Immunostaining for Ki-67 and phosphorylated ERK were also performed. The expression of TCC isoforms in healthy and diseased tissue was investigated using quantitative real-time PCR. TCC blockade caused a significant reduction in rat MC proliferation in vitro, whereas L-type inhibition had no effect. Treatment of Thy1 nephritis with TH1177 significantly reduced glomerular injury (P < 0.005) and caused a 49% reduction in glomerular cell number (P < 0.005) compared to the placebo. TH1177 also reduced Ki-67-positive and pERK-positive cells per glomerulus by 52% (P < 0.01 and P < 0.005, respectively). These results demonstrate that TH1177 inhibits MC proliferation in vitro and in vivo, supporting the hypothesis that TCC inhibition may be a useful strategy for studying and modifying MC proliferative responses to injury.

A separate pool of cardiac phospholemman that does not regulate or associate with the sodium pump: multimers of phospholemman in ventricular muscle.

BACKGROUND: Phospholemman regulates the plasmalemmal sodium pump in excitable tissues. RESULTS: In cardiac muscle, a subpopulation of phospholemman with a unique phosphorylation signature associates with other phospholemman molecules but not with the pump. CONCLUSION: Phospholemman oligomers exist in cardiac muscle. SIGNIFICANCE: Much like phospholamban regulation of SERCA, phospholemman exists as both a sodium pump inhibiting monomer and an unassociated oligomer. Phospholemman (PLM), the principal quantitative sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. Much like phospholamban, which regulates the related ATPase SERCA, PLM is reported to oligomerize. We investigated subpopulations of PLM in adult rat ventricular myocytes based on phosphorylation status. Co-immunoprecipitation identified two pools of PLM: one not associated with the sodium pump phosphorylated at Ser(63) and one associated with the pump, both phosphorylated at Ser(68) and unphosphorylated. Phosphorylation of PLM at Ser(63) following activation of PKC did not abrogate association of PLM with the pump, so its failure to associate with the pump was not due to phosphorylation at this site. All pools of PLM co-localized to cell surface caveolin-enriched microdomains with sodium pump α subunits, despite the lack of caveolin-binding motif in PLM. Mass spectrometry analysis of phosphospecific immunoprecipitation reactions revealed no unique protein interactions for Ser(63)-phosphorylated PLM, and cross-linking reagents also failed to identify any partner proteins for this pool. In lysates from hearts of heterozygous transgenic animals expressing wild type and unphosphorylatable PLM, Ser(63)-phosphorylated PLM co-immunoprecipitated unphosphorylatable PLM, confirming the existence of PLM multimers. Dephosphorylation of the PLM multimer does not change sodium pump activity. Hence like phospholamban, PLM exists as a pump-inhibiting monomer and an unassociated oligomer. The distribution of different PLM phosphorylation states to different pools may be explained by their differential proximity to protein phosphatases rather than a direct effect of phosphorylation on PLM association with the pump.

Esmolol cardioplegia: the cellular mechanism of diastolic arrest.

AIMS: Esmolol, an ultra-short-acting beta-blocker, acts as a cardioplegic agent at millimolar concentrations. We investigated the mechanism by which esmolol induces diastolic ventricular arrest. METHODS AND RESULTS: In unpaced Langendorff-perfused rat hearts, esmolol (0.03-3 mmol/L) had a profound negative inotropic effect resulting in diastolic arrest at 1 mmol/L and above. This inhibition of contraction was maintained during ventricular pacing. At 3 mmol/L, esmolol also abolished action potential conduction. To determine the cellular mechanism for the negative inotropism, we measured contraction (sarcomere shortening) and the calcium transient (fura-2 fluorescence ratio; Ca(tr)) in electrically-stimulated rat ventricular myocytes at 23 and 34 degrees C. The decrease in contraction (by 72% at 23 degrees C, from 0.16 +/- 0.01 to 0.04 +/- 0.01 microm, P < 0.001) was similar to that of isolated hearts and was caused by a large decrease in Ca(tr) (from 0.13 +/- 0.02 to 0.07 +/- 0.02, P < 0.001). There was no additional effect on myofilament Ca(2+) sensitivity. Esmolol's effects on contraction and Ca(tr) were not shared or altered by the beta-blocker, atenolol (1 mmol/L). Sarcoplasmic reticulum inhibition with thapsigargin did not alter the inhibitory effects of esmolol. Whole-cell voltage-clamp experiments revealed that esmolol inhibited the L-type calcium current (I(Ca,L)) and the fast sodium current (I(Na)), with IC(50) values of 0.45 +/- 0.05 and 0.17 +/- 0.025 mmol/L, respectively. CONCLUSION: Esmolol at millimolar concentrations causes diastolic ventricular arrest by two mechanisms: at 1 mmol/L (and below), the pronounced negative inotropic effect is due largely to inhibition of L-type Ca(2+) channels; additionally, higher concentrations prevent action potential conduction, probably due to the inhibition of fast Na(+) channels.

The rate of loss of T-tubules in cultured adult ventricular myocytes is species dependent.

In this study, we compared the rate of detubulation of adult mouse and rat ventricular myocytes over a 72 h culture period. The T-tubule density was measured in the following two ways: (i) as whole-cell capacitance in voltage-clamped myocytes relative to cell area; and (ii) using di-8-ANEPPS staining and confocal microscopy. In adult rat ventricular myocytes, whole-cell capacitance/area was significantly reduced from 47 +/- 3 fF microm(2) (mean +/- s.e.m.; n = 16) in freshly isolated (control) cells to 36 +/- 2 fF microm(2) (n = 20) after 72 h in culture. The T-tubular density, as assessed optically using di-8-ANEPPS staining, at 48 h was significantly reduced to 70 +/- 7% (n = 14) compared with control cells. The T-tubular density was further reduced after 72 h in culture to 43 +/- 7% (n = 10) compared with control cells. In contrast, in mouse myocytes neither whole-cell capacitance relative to cell area nor optical assessment of T-tubules showed any significant reduction in capacitance/cell area or T-tubule density after 72 h of culture. Expression of caveolin-3 (CAV-3) (a marker of T-tubule development) was also measured, and a significant reduction was observed in CAV-3 expression in rat myocytes at 48 (80 +/- 5.5%; n = 6) and 72 h (66 +/- 9.5%; n = 6) compared with control cells. The expression of CAV-3 in mouse myocytes was not significantly reduced even at 72 h. When rat ventricular myocytes were paced in culture for 72 h they exhibited no significant improvement in T-tubule density or CAV-3 expression compared with non-paced cultured cells. In rat myocytes, sarcomere length shortening was significantly reduced in myocytes cultured for 48 (4.96 +/- 0.72%; n = 26) and 72 h (4.32 +/- 0.80%; n = 26) compared with freshly isolated cells (7.12 +/- 0.56%; n = 18). Mouse myocytes, after 24 h in culture, were unable to follow external pacing. These results suggest that detubulation in quiescent culture is slower in the mouse than the rat and that this loss of T-tubules profoundly affects excitation-contraction coupling in rat myocytes.

Inhibition of human mesangial cell proliferation by targeting T-type calcium channels.

BACKGROUND: Aberrant glomerular mesangial cell (MC) proliferation is a common finding in renal diseases. T-type calcium channels (T-CaCN) play an important role in the proliferation of a number of cell types, including vascular smooth muscle cells. The hypothesis that T-CaCN may play a role in the proliferation of human MC was investigated. METHODS: The presence of T-CaCN in primary cultures of human MC was examined using voltage clamping and by RT-PCR. The effect of calcium channel inhibitors, and of siRNA directed against the Cav3.2 T-CaCN isoform, on MC proliferation was assessed using the microculture tetrazolium assay and nuclear BrdU incorporation. RESULTS: Human MC express only the Cav3.2 T-CaCN isoform. Co-incubation of MC with a T-CaCN inhibitor (mibefradil, TH1177 or Ni(2+)) results in a concentration-dependent attenuation of proliferation. This effect cannot be attributed to direct drug-induced cytotoxicity or apoptosis and is not seen with verapamil, an L-type channel blocker. Transfection of MC with siRNA results in knockdown of T-CaCN Cav3.2 mRNA and a clear attenuation of MC proliferation. CONCLUSIONS: These results demonstrate for the first time an important role for T-CaCN in human MC proliferation. This could potentially lead to a novel therapy in the treatment of proliferative renal diseases.

FXYD1 phosphorylation in vitro and in adult rat cardiac myocytes: threonine 69 is a novel substrate for protein kinase C.

FXYD1 (phospholemman), the primary sarcolemmal kinase substrate in the heart, is a regulator of the cardiac sodium pump. We investigated phosphorylation of FXYD1 peptides by purified kinases using HPLC, mass spectrometry, and Edman sequencing, and FXYD1 phosphorylation in cultured adult rat ventricular myocytes treated with PKA and PKC agonists by phosphospecific immunoblotting. PKA phosphorylates serines 63 and 68 (S63 and S68) and PKC phosphorylates S63, S68, and a new site, threonine 69 (T69). In unstimulated myocytes, FXYD1 is approximately 30% phosphorylated at S63 and S68, but barely phosphorylated at T69. S63 and S68 are rapidly dephosphorylated following acute inhibition of PKC in unstimulated cells. Receptor-mediated PKC activation causes sustained phosphorylation of S63 and S68, but transient phosphorylation of T69. To characterize the effect of T69 phosphorylation on sodium pump function, we measured pump currents using whole cell voltage clamping of cultured adult rat ventricular myocytes with 50 mM sodium in the patch pipette. Activation of PKA or PKC increased pump currents (from 2.1 +/- 0.2 pA/pF in unstimulated cells to 2.9 +/- 0.1 pA/pF for PKA and 3.4 +/- 0.2 pA/pF for PKC). Following kinase activation, phosphorylated FXYD1 was coimmunoprecipitated with sodium pump alpha(1)-subunit. We conclude that T69 is a previously undescribed phosphorylation site in FXYD1. Acute T69 phosphorylation elicits stimulation of the sodium pump additional to that induced by S63 and S68 phosphorylation.

The role of voltage gated T-type Ca2+ channel isoforms in mediating "capacitative" Ca2+ entry in cancer cells.

The mechanism by which Ca2+ enters electrically non-excitable cells is unclear. The sensitivity of the Ca2+ entry pathway in electrically non-excitable cells to inhibition by extracellular Ni2+ was used to direct the synthesis of a library of simple, novel compounds. These novel compounds inhibit Ca2+ entry into and, consequently, proliferation of several cancer cell lines. They showed stereoselective inhibition of proliferation and Ca2+ influx with identical stereoselective inhibition of heterologously expressed Cav3.2 isoform of T-type Ca2+ channels. Proliferation of human embryonic kidney (HEK)293 cells transfected with the Cav3.2 Ca2+ channel was also blocked. Cancer cell lines sensitive to our compounds express message for the Cav3.2 T-type Ca2+ channel isoform, its delta25B splice variant, or both, while a cell line resistant to our compounds does not. These observations raise the possibility that clinically useful drugs can be designed based upon the ability to block these Ca2+ channels.