No change in myonuclear number during muscle unloading and reloading.

Muscle fibers are the cells in the body with the largest volume, and they have multiple nuclei serving different domains of cytoplasm. A large body of previous literature has suggested that atrophy induced by hindlimb suspension leads to a loss of "excessive" myonuclei by apoptosis. We demonstrate here that atrophy induced by hindlimb suspension does not lead to loss of myonuclei despite a strong increase in apoptotic activity of other types of nuclei within the muscle tissue. Thus hindlimb suspension turns out to be similar to other atrophy models such as denervation, nerve impulse block, and antagonist ablation. We discuss how the different outcome of various studies can be attributed to difficulties in separating myonuclei from other nuclei, and to systematic differences in passive properties between normal and unloaded muscles. During reload, after hindlimb suspension, a radial regrowth is observed, which has been believed to be accompanied by recruitment of new myonuclei from satellite cells. The lack of nuclear loss during unloading, however, puts these findings into question. We observed that reload led to an increase in cross sectional area of 59%, and fiber size was completely restored to the presuspension levels. Despite this notable growth there was no increase in the number of myonuclei. Thus radial regrowth seems to differ from de novo hypertrophy in that nuclei are only added during the latter. We speculate that the number of myonuclei might reflect the largest size the muscle fibers have had in its previous history.

Volume-sensitive Cl- current in bovine adrenocortical cells: comparison with the ACTH-induced Cl- current.

In a previous study performed on zona fasciculata (ZF) cells isolated from calf adrenal glands, we identified an ACTH-induced Cl- current involved in cell membrane depolarization. In the present work, we describe a volume-sensitive Cl- current and compare it with the ACTH-activated Cl- current. Experiments were performed using the whole-cell patch-clamp recording method, video microscopy and cortisol-secretion measurements. In current-clamp experiments, hypotonic solutions induced a membrane depolarization to -22 mV. This depolarization, correlated with an increase in the membrane conductance, was sensitive to different Cl- channel inhibitors. In voltage-clamp experiments, hypotonic solution induced a membrane current that slowly decayed and reversed at -21 mV. This ionic current displayed no time dependence and showed a slight outward rectification. It was blocked to variable extent by different conventional Cl- channel inhibitors. Under hypotonic conditions, membrane depolarizations were preceded by an increase in cell volume that was not detected under ACTH stimulation. It was concluded that hypotonic solution induced cell swelling, which activated a Cl- current involved in membrane depolarization. Although cell volume change was not observed in the presence of ACTH, biophysical properties and pharmacological profile of the volume-sensitive Cl- current present obvious similarities with the ACTH-activated Cl- current. As compared to ACTH, hypotonic solutions failed to trigger cortisol production that was weakly stimulated in the presence of high-K+ solution. This shows that in ZF cells, membrane depolarization is not a sufficient condition to fully activate secretory activities.

Two types of pharmacologically distinct Ca(2+) currents with voltage-dependent similarities in zona fasciculata cells isolated from bovine adrenal gland.

Voltage-activated Ca(2+) currents, in zona fasciculata cells isolated from calf adrenal gland, were characterized using perforated patch-clamp recording. In control solution (Ca(2+): 2.5 mm) a transient inward current was followed, in 40% of the cells, by a sustained one. In 20 mm Ba(2+), 61% of the cells displayed an inward current, which consisted of transient and sustained components. The other cells produced either a sustained or a transient inward current. These different patterns were dependent upon time in culture. Current-voltage relationships show that both the transient and sustained components activated, peaked and reversed at similar potentials: -40, 0 and +60 mV, respectively. The two components, fully inactivated at -10 mV, were separated by double-pulse protocols from different holding potentials where the transient component could be inactivated or reactivated. The decaying phase of the sustained component was fitted by a double exponential (time constants: 1.9 and 20 sec at +10 mV); that of the transient component was fitted by a single exponential (time constant: 19 msec at +10 mV). Steady-state activation and inactivation curves of the two components were superimposed. Their half activation and inactivation potentials were similar, about -15 and -34 mV, respectively. The sustained component was larger in Ba(2+) than in Sr(2+) and Ca(2+). Ni(2+) (20 microm) selectively blocked the transient component while Cd(2+) (10 microm) selectively blocked the sustained one. (+/-)Bay K 8644 (0.5 microm) increased the sustained component and nitrendipine (0.5-1 microm) blocked it selectively. The sustained component was inhibited by calciseptine (1 microm). Both components were unaffected by omega-conotoxin GVIA and MVIIC (0.5 microm). These results show that two distinct populations of Ca(2+) channels coexist in this cell type. Although the voltage dependence of their activation and inactivation are comparable, these two components of the inward current are similar to T- and L-type currents described in other cells.

Functional coupling between the cyclic adenosine monophosphate pathway and cholecystokinin secretion in RIN cells.

The cellular events associated with cAMP-dependent cholecystokinin (CCK) release were investigated with an X-ray induced rat pancreatic tumor cell line (RIN 1056 E). Forskolin dose-dependently stimulated the release of CCK. Agents that increase [Ca2+]i (thapsigargin, Bay K 8644, ionomycin) also stimulated the release of CCK. Conversely, absence of extracellular Ca2+ or cell treatment with various calcium channel blockers strongly reduced the forskolin-induced CCK release. Finally, the cAMP-kinase inhibitor H89, the calmodulin antagonist W7 and the Ca/calmodulin-dependent protein-kinase II inhibitor KN62 strongly inhibited the forskolin-evoked CCK secretion. We conclude that the release of CCK via a cAMP-dependent pathway is dependent on the activation of voltage-dependent calcium channels and may implicate protein kinase A, calmodulin and the Ca/calmodulin-dependent protein kinase II.

Role of calcium in the bombesin-induced intestinal CCK release in rats.

In the isolated vascularly perfused rat duodenojejunum, vascular infusion of bombesin (100 nM) provoked an early, transient (6 min) release of CCK (500% of basal), followed by a sustained response (400% of basal). The calcium chelator EGTA (2 mM) reduced the early peak and abolished the second phase of CCK release. A similar variation was evoked by verapamil (10 microM), whereas diltiazem (100 microM), nifedipine (50 microM), and omega-conotoxin (100 nM) had no significant effect. It is concluded that bombesin-induced CCK release from rat intestine is dependent on the availability of extracellular calcium and on the activation of calcium channels sensitive to blockers of the phenylalkylamine family.

Role of cyclic nucleotides and calcium in the nutrient-induced release of cholecystokinin-like immunoreactivity in rats.

1. This study was undertaken with an isolated vascularly perfused rat duodenojejunal preparation to investigate the mechanisms of the release of cholecystokinin measured by immunoassay (cholecystokinin-like immunoreactivity, CCK-LI). 2. Intra-arterial infusion of forskolin (2-20 microM) evoked a prompt and well-sustained secretion of CCK-LI which was increased to a mean value of 600% of basal with the highest dose tested. 3-Isobutyl-1-methylxanthine (IBMX) (10(-6)-10(-4) M) stimulated the secretion of CCK-LI (maximal increase of 400% of basal at 10(-4) M). 3. Intra-arterial infusion of beta-phorbol 12-myristate 13-acetate (5 x 10(-7)-5 x 10(-6) M) and calcium ionophore A23187 (10(-6)-10(-5) M) alone or in combination provoked only a transient increase in the release of CCK-LI. 4. Luminal infusion of a 5% ovalbumin hydrolysate solution produced an immediate release of CCK-LI followed by a well-sustained secretion at 580% of basal. Intra-arterial infusion of IBMX (10(-5) or 10(-4) M) in combination with luminal peptone induced a release of CCK-LI which was equal to the sum of the CCK responses evoked by IBMX and peptone given separately. 5. Intra-arterial infusion of EGTA (2 mM) abolished the forskolin- and peptone-induced CCK secretion while luminal EGTA (2 mM) had no inhibitory effect. Verapamil (5 x 10(-5)-10(-4) M) or nifedipine (10(-5)-5 x 10(-5) M) inhibited the peptone-evoked CCK secretion. A high concentration of trifluoperazine (10(-4) M) strongly reduced the release of CCK-LI induced by intraluminal peptone while 10(-5) M was ineffective. 6. It is concluded that the peptone-induced secretion of CCK-LI involves a cyclic AMP-dependent mechanism and the activation of calcium channels possibly located at the basolateral side of the CCK cell.