Immunocytochemical analysis of sex differences in calcitonin gene-related peptide in the rat dorsal root ganglion, with special reference to estrogen and its receptor.

Previous studies have shown that the calcitonin gene-related peptide (CGRP) immunoreactivity in the central nervous system (CNS) of adult rats is sexually dimorphic and regulated by sex steroid. In the present study, we used immunocytochemistry to investigate the sex difference in CGRP-immunoreactive (IR) neurons in rat dorsal root ganglia (DRG). The numbers of CGRP-IR neurons at the cervical, lumbar and sacral levels in the female rats were significantly lower than those of the male rats. We also found that the number of CGRP-IR neurons at the lumbar level was increased in ovariectomized (OVX) rats, but was decreased in estradiol (E2)-treated rats (OVX+E2). A large number of estrogen receptor (ER)-IR neurons at the lumbar level were found in the female rats, and its number was greater than that in the male rats. We also investigated the change in the number of ER-IR neurons of OVX rats after estrogen treatment. The number of ER-IR neurons in the OVX+E2 rats was consistent with that of the intact female rats, but was significantly increased in the OVX rats. As shown by a double-labeling immunocytochemical method, over 80% of the CGRP-IR neurons at the lumbar level showed ER immunoreactivity in the female, OVX and OVX+E2 rats, compared to only about 46% in the male rats. These results indicate that there is a gender difference in CGRP expression in the rat DRG, and that this CGRP expression might be downregulated by estrogen (at least in part) through its receptor.

A novel phasic contraction induced by dithiothreitol in frog skeletal muscle.

1. Dithiothreitol (DTT), at 50-100 mM, induced a phasic reversible contraction of frog skeletal muscle. 2. Exposure of single fibers to nifedipine (20 microM), an L-type Ca2+ antagonist, blocked the twitch and tetanus tensions but never affected the DTT-induced contraction. 3. DTT also produced a phasic contraction in fibers where voltage sensors were inactivated in the presence of high K+ concentration (190 mM). 4. A fiber was mechanically skinned after observation of DTT-induced contraction. The skinned fiber contracted in response to a DTT concentration similar to that required to produce contraction in intact fibers before skinning. 5. In skinned fibers, DTT, at 100 or 200 mM, inhibited the accumulation of Ca2+ by SR, but not Ca2+ ATPase activity. 6. These results suggest that a high concentration of DTT triggers Ca2+ efflux from the SR through action on the Ca2+ release channel and/or closely associated proteins, such as triadin and FK-506 binding protein.

Gold ion inhibits silver ion induced contracture and activates ryanodine receptors in skeletal muscle.

Effects of Au3+ on Ag(+)-induced contractures and Ca2+ release channel activity in the sarcoplasmic reticulum were studied in frog skeletal muscles. Single fibres spontaneously produced phasic and tonic contractures upon addition of 5-20 microM Ag+ or more than 50 microM Au3+. Simultaneous application of 5 microM Ag+ and 20 microM Au3+ inhibited contractures induced by Ag+. Au3+ applied immediately after development of Ag(+)-induced contractures shortened the duration of the phasic contracture and markedly decreased the subsequent tonic contracture. Pretreatment of fibres with Au3+ inhibited the Ag(+)-induced phasic contracture. Ca2+ release channels incorporated into planar lipid bilayers were activated in response to Au3+ at 20 to 200 microM. A close relationship was observed between Ca2+ release channel open probability and amplitude of the Au(3+)-induced tonic contracture. Channel activity was inhibited by 5 microM ruthenium red. We conclude that extracellular Au3+ at low concentrations modifies the interaction of Ag+ with voltage sensors in the transverse tubules to inhibit the Ag(+)-induced contracture and, if it enters the cell, Au3+ may directly activate the sarcoplasmic reticulum Ca2+ release channel to partially contribute to the tonic contracture.

Possible involvement of Ca(2+)-induced Ca2+ release mechanism in Ag(+)-induced contracture in frog skeletal muscle.

To determine if an Ag(+)-induced contracture is associated with the Ca(2+)-induced Ca2+ release mechanism in the sarcoplasmic reticulum, effects of Ca(2+)-induced Ca2+ release modulators on the Ag(+)-induced contracture were studied with single fibers of frog toe skeletal muscle. The fiber treated with 1 mM caffeine contracted significantly much more than controls without caffeine at Ag+ concentrations below 1 microM. Procaine shifted the Ag+ concentration-tension curve to the right, dose-dependently. When 10 mM procaine was applied to contracting fibers not treated with caffeine, the duration of 5 microM Ag(+)-induced contracture was shortened with a little decrease in tension amplitude, that was different from the effect of procaine on caffeine contracture. In caffeine solution, 0.5 microM Ag+ caused a long-lasting contracture with sometimes two peaks. 2 mM procaine led to disappearance of such two peaks, resulting in shortening of the contracture. K+ contracture was potentiated by 1 mM caffeine only at lower concentrations of K+, and inhibited by 10 mM procaine. These results suggest that the Ag(+)-induced contracture is composed of two components: Ca(2+)-induced Ca2+ release-dependent and -independent. 5 microM Ag(+)-induced contracture slowly relaxed with a wavy tension pattern to the resting level when 0.05 mM dithiothreitol was applied around peak of the tension. This relaxation was accelerated by procaine application. These findings may be explained by attributing a portion of Ag(+)-induced contracture to the effect of Ca2+ released through the Ca(2+)-induced Ca2+ release mechanism in the sarcoplasmic reticulum.

Involvement of thiols in the induction of inward current induced by silver in frog skeletal muscle membrane.

Exposure of voltage-clamped frog skeletal muscle fibres to silver caused a maintained inward current which could be carried by Ca2+, Mg2+ or Na+. Inorganic Ca2+ channel blockers and dithiothreitol (SH reducing agent) diminished this current, but a Na+ channel blocker did not. Thus, silver activates the Ca2+ channel by acting on SH groups in a Ca2+ channel protein.

Gold ion induces contraction in frog skeletal muscle fibers.

In Ringer solution, gold ions (Au3+) at concentrations more than 50 microM produced a phasic and subsequent tonic contraction spontaneously in single toe muscle fiber of frog. When 1.8 mM Ca2+ in Ringer solution was replaced by 3 mM Mg2+, tonic contraction was no longer provoked in response to Au3+. Only phasic contraction was potentiated by 10 mM perchlorate (an L-type Ca2+ channel activator) irrespective of external Ca2+, and both phasic and tonic contractions were blocked by 10 microM nifedipine (an L-type Ca2+ channel blocker). Upon application of 5 mM dithiothreitol to the contracting fiber, the Au(3+)-induced tension disappeared rapidly. The fiber pretreated with 0.05% H2O2 for 10 min did not respond to Au3+ with visible contraction. Treatment of H2O2-paralyzed fibers with dithiothreitol (to reduce oxidized sulfhydryl groups) fully restored the Au(3+)-induced contraction. These results suggest that the phasic contraction induced by Au3+ probably is mediated through sulfhydryl groups in the L-type Ca2+ channel (dihydropyridine receptor) on the transverse tubular membrane. Sustained contraction was produced by Ca2+ application to Au(3+)-treated fibers in Mg(2+)-Ringer solution, and Au3+ caused membrane depolarization in a dose-dependent manner. These effects of Au3+ may explain tonic contraction development.

Muscle contraction and inward current induced by silver and effect of Ca2+ channel blockers.

Single fibers from toe or anterior tibialis muscle contracted transiently and then tonically in the presence of 1.8 mM Ca2+ on addition of 10 microM Ag+. Exposure of fibers to Cd2+ completely inhibited tonic contraction and modified phasic contraction to some extent. Nifedipine at 10 microM initially potentiated and then completely inhibited twitch tension; subsequently, fibers no longer contracted phasically in response to 20 microM Ag+, whereas slight tonic contraction still occurred. Fibers with membrane potential clamped at -90 mV produced maintained inward current on application of Ag+. Simultaneous administration of 1 mM Cd2+ and 10 microM Ag+ to fibers voltage clamped with the double mannitol gap technique almost completely blocked the inward current. Removal of Cd2+ elicited a rapid and large inward current. Ag(+)-induced inward current was inhibited when 1 mM Cd2+ was applied to fibers during development of the inward current. In fibers paralyzed with 10 microM nifedipine, the inward current induced by 10 microM Ag+ was partially inhibited. These results suggest that phasic contraction induced by Ag+ is controlled by L-type Ca2+ channels (probably voltage sensors) located in the T-tubular membrane, whereas tonic contraction involves Ca2+ channels sensitive and/or insensitive to dihydropyridine in the surface and T-tubular membranes.