Detection of dihydropyridine- and voltage-sensitive intracellular Ca(2+) signals in normal human parathyroid cells.

We recently showed dihydropyridine- and voltage-sensitive Ca(2+) entry in cultured parathyroid cells from patients with secondary hyperparathyroidism. To determine whether normal parathyroid cells have a similar extracellular Ca(2+) entry system, cells were isolated from normal (non-hyperplastic) human parathyroid glands. Fluorescence signals related to the cytoplasmic Ca(2+) concentration ([Ca(2+)]I) were examined in these cells. Cells loaded with fluo-3/AM showed a transient increase in fluorescence (Ca(2+) transient) following a 10-s exposure to a 150 mM K(+) solution in the presence of millimolar concentrations of external Ca(2+). The Ca(2+) transient was reduced by dihydropyridine antagonists or 0.5 mM Cd(2+), but enhanced by FPL-64176, an L-type Ca(2+)-channel agonist. Ca(2+) transients induced by the 10-s exposure to 3.0 mM extracellular Ca(2+) ([Ca(2+)]o) were also inhibited by dihydropyridine antagonists or 0.5 mM Cd(2+). These results provide the first evidence that normal human parathyroid cells express a dihydropyridine-sensitive Ca(2+) entry system that may be involved in the [Ca(2+)]o-induced change in [Ca(2+)]I. This system might provide a compensatory pathway for negative feedback regulation of parathyroid hormone secretion under physiological conditions.

Protein kinase A-dependent modulation of Ca2+ sensitivity in cardiac and fast skeletal muscles after reconstitution with cardiac troponin.

Protein kinase A (PKA)-dependent phosphorylation of troponin (Tn)I represents a major physiological mechanism during beta-adrenergic stimulation in myocardium for the reduction of myofibrillar Ca2+ sensitivity via weakening of the interaction with TnC. By taking advantage of thin filament reconstitution, we directly investigated whether or not PKA-dependent phosphorylation of cardiac TnI (cTnI) decreases Ca2+ sensitivity in different types of muscle: cardiac (porcine ventricular) and fast skeletal (rabbit psoas) muscles. PKA enhanced phosphorylation of cTnI at Ser23/24 in skinned cardiac muscle and decreased Ca2+ sensitivity, of which the effects were confirmed after reconstitution with the cardiac Tn complex (cTn) or the hybrid Tn complex (designated as PCRF; fast skeletal TnT with cTnI and cTnC). Reconstitution of cardiac muscle with the fast skeletal Tn complex (sTn) not only increased Ca2+ sensitivity, but also abolished the Ca2+-desensitizing effect of PKA, supporting the view that the phosphorylation of cTnI, but not that of other myofibrillar proteins, such as myosin-binding protein C, primarily underlies the PKA-induced Ca2+ desensitization in cardiac muscle. Reconstitution of fast skeletal muscle with cTn decreased Ca2+ sensitivity, and PKA further decreased Ca2+ sensitivity, which was almost completely restored to the original level upon subsequent reconstitution with sTn. The essentially same result was obtained when fast skeletal muscle was reconstituted with PCRF. It is therefore suggested that the PKA-dependent phosphorylation or dephosphorylation of cTnI universally modulates Ca2+ sensitivity associated with cTnC in the striated muscle sarcomere, independent of the TnT isoform.

Dihydropyridine- and voltage-sensitive Ca2+ entry in human parathyroid cells.

Patch-clamp and fluorescence measurements of cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) were performed to directly detect extracellular Ca(2+) entry into cultured parathyroid cells from patients with secondary hyperparathyroidism. Cells loaded with fluo-3 AM or fluo-4 AM showed a transient increase in fluorescence (Ca(2+) transient) following 10 s exposure to 150 mm K(+) solution in the presence of millimolar concentrations of external Ca(2+). The Ca(2+) transient was completely inactivated after 30-40 s exposure to the high-K(+) solution, was reduced by dihydropyridine antagonists and was enhanced by FPL-64176, an L-type Ca(2+) channel agonist. The electrophysiological and pharmacological properties of the whole-cell Ca(2+) and Ba(2+) currents were similar to those of L-type Ca(2+) channels. The Ca(2+) transients induced by 10 s exposure to 3.0 mm extracellular Ca(2+) concentration ([Ca(2+)](o)) were inhibited by dihydropyridine antagonists and were partly inactivated following 30-40 s exposure to the high-K(+) solution. These results demonstrate, for the first time, that human parathyroid cells express L-type-like Ca(2+) channels that are possibly involved in the [Ca(2+)](o)-induced change in [Ca(2+)](i). This Ca(2+) entry system might provide a compensatory pathway for the negative feedback regulation of parathyroid hormone secretion, especially in hyperplastic conditions in which the Ca(2+)-sensing receptor is poorly expressed.

Troponin and titin coordinately regulate length-dependent activation in skinned porcine ventricular muscle.

We investigated the molecular mechanism by which troponin (Tn) regulates the Frank-Starling mechanism of the heart. Quasi-complete reconstitution of thin filaments with rabbit fast skeletal Tn (sTn) attenuated length-dependent activation in skinned porcine left ventricular muscle, to a magnitude similar to that observed in rabbit fast skeletal muscle. The rate of force redevelopment increased upon sTn reconstitution at submaximal levels, coupled with an increase in Ca2+ sensitivity of force, suggesting the acceleration of cross-bridge formation and, accordingly, a reduction in the fraction of resting cross-bridges that can potentially produce additional active force. An increase in titin-based passive force, induced by manipulating the prehistory of stretch, enhanced length-dependent activation, in both control and sTn-reconstituted muscles. Furthermore, reconstitution of rabbit fast skeletal muscle with porcine left ventricular Tn enhanced length-dependent activation, accompanied by a decrease in Ca2+ sensitivity of force. These findings demonstrate that Tn plays an important role in the Frank-Starling mechanism of the heart via on-off switching of the thin filament state, in concert with titin-based regulation.

Susceptibility of ammonia-oxidizing bacteria to nitrification inhibitors.

Activity of nitrification inhibitors to several typical ammonia-oxidizing bacteria isolated recently, i. e. Nitrosococcus, Nitrosolobus, Nitrosomonas, Nitrosospira and Nitrosovibrio species was assayed using 2-amino-4-methyl-trichloromethyl-1,3,5-triazine (MAST), 2-amino-4-tribromomethyl-6-trichloromethyl-1,3,5-triazine (Br-MAST), 2-chloro-6-trichloromethylpyridine (nitrapyrin) and others, and compared to confirm the adequate control of ammonia-oxidizing bacteria by the inhibitors. The order of activity of the inhibitors to 13 species of ammonia-oxidizing bacteria examined was approximately summarized as Br-MAST > or = nitrapyrin > or = MAST > other inhibitors. Two Nitrosomonas strains, N. europaea ATCC25978 and N. sp. B2, were extremely susceptible to Br-MAST, exhibiting a pI50 > or = 6.40. These values are the position logarithms of the molar half-inhibition concentration. The 16S rRNA gene sequence similarity for the highly susceptible 4 strains of genus Nitrosomonas was 94% to 100% of Nitrosomonas europaea, although those of the less susceptible 3 strains of ammonia-oxidizing bacteria, Nitrosococcus oceanus C-107 ATCC19707, Nitrosolobus sp. PJA1 and Nitrosolobus multiformis ATCC25196, were 77.85, 91.53 and 90.29, respectively. However, no clear correlation has been found yet between pI50-values and percent similarity of 16S rRNA gene sequence among ammonia-oxidizing bacteria.