Whether Ca 2ϩ released from stores within the presynaptic nerve terminals also contributes to the Ca 2ϩ elevation evoked by action potentials was tested in intact bullfrog sympathetic ganglia. Intraterminal Ca 2ϩ transients (⌬[Ca 2ϩ ] i ) were evoked by electrical shocks to the presynaptic nerves at 20 Hz and were monitored by fura-2 fluorimetry. Ca 2ϩ released through intraterminal ryanodine-sensitive channels accounted for 46% of the peak Ca 2ϩ elevation. Moreover, in half of the terminals when intraterminal release was blocked by ryanodine, ⌬[Ca 2ϩ ] i reached a plateau at 200 Ϯ 24 nM. Because 20 Hz is a frequency favorable for the release of a neuropeptide, luteinizing hormone releasing hormone (LHRH) from these presynaptic nerve terminals, and because the threshold level for LHRH release is 186 nM, intraterminal Ca 2ϩ release during nerve firing is likely to play a major role in regulating LHRH release. The intraterminal ryanodine channels were facilitated by caffeine as in other tissue. The releasable ryanodine-sensitive store could elevate the intraterminal [Ca 2ϩ ] by an amount as high as 1.6 M at a rate as fast as 250 nM/sec. The store could be refilled within 100 sec after a maximal discharge of its content by 20 Hz firing. Oscillation of [Ca 2ϩ ] i evoked by 20 Hz nerve firing occurred in normal Ringer solution, in ryanodine, and in caffeine with a periodicity of ϳ10 sec. Besides the facilitatory effects on the ryanodine-sensitive channels, caffeine also had inhibitory effects on ⌬[Ca 2ϩ ] i via its action on a different process.
Effects of different patterns of presynaptic stimulation upon release of leuteinizing hormone releasing hormone (LHRH) were studied by monitoring LHRH-induced slow currents from individual postsynaptic neurons in bullfrog sympathetic ganglia. LHRH-mediated synaptic currents in ganglionic B and C neurons were recorded by a single- electrode voltage-clamp technique. Using continuous stimulation, release increased with frequency between 2 and 20 Hz, then declined. Though bursts of stimuli always evoked more release than continuous stimuli of the same average frequency, they were invariably less effective than continuous stimulation at the intraburstal frequency. This demonstrates that frequency, not bursting structure, governs peptide release. The dependence of release upon stimulus frequency was altered when extracellular Ca2+ concentration was changed, implying that release does not depend intrinsically upon stimulation frequency, but simply on the availability of Ca2+.
The rate and the total amount of Ca2+ elevation in the presynaptic terminals of bullfrog sympathetic ganglia depend on the firing frequency of the terminals. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, was used for testing whether mitochondrial Ca2+ uptake is one of the mechanisms that underlie this frequency dependence. Fura-2 fluorimetry was used for measurement of intraterminal Ca2+. When stimulations of different durations (30 and 1.5 s) and frequencies (4 and 20 Hz) evoked Ca2+ transients with similar peak amplitudes (264 +/- 22 nM vs. 251 +/- 18 nM, means +/- SE), CCCP augmented the responses to the 4-Hz stimulation 8.9 times more strongly than it did the responses to the 20-Hz stimulation (249.7 +/- 81.5% vs. 25.3 +/- 10.2%). When stimulations delivered at the two frequencies had the same durations (1.5, 3, 6, 10, 20, and 30 s), CCCP enlarged the responses to the 4-Hz stimulations up to 4.2 times more than it did the responses to the 20-Hz stimulations. When the same number of stimuli (120) was delivered at the two frequencies, the effects of CCCP on the responses evoked by the 4-Hz train were again 6.8 times stronger than its effects on the responses to the 20-Hz stimulation. Therefore neither the peak amplitudes of the responses nor the durations of the stimulations dictated the extent to which the mitochondria modulated the peak [Ca2+]i. Instead, the extent of the modulation was governed by the frequency of stimulation. Specifically, the less frequent the Ca2+ influx, the stronger the mitochondrial modulation. Also, during nerve firing Ca2+ release from the ryanodine-sensitive store had a higher potential to influence the [Ca2+]i transients than did Ca2+ removal by the mitochondria for the first 6 s of the responses. On cessation of stimulation, CCCP reduced the initial rapid rate of Ca2+ decay. Thus uptake by the mitochondria was an important mechanism for Ca2+ removal after repetitive firing at the presynaptic terminals.
Amphibia, like most vertebrate species, have two forms of GnRH, namely [Arg8]GnRH (mammalian GnRH) and [His5, Trp7, Tyr8]GnRH (chicken GnRH II). The differential distribution of the two peptides in the amphibian brain suggests that they may play different roles. Mammalian GnRH, which is found predominantly in the hypothalamus, is most likely the prime regulator of gonadotropin release, while chicken GnRH II, which occurs predominantly in the midbrain and hindbrain, may play a neuromodulatory role. In amphibian sympathetic ganglia, GnRH has been demonstrated to be a neurotransmitter where its release from the presynaptic nerve terminals reversibly inhibits M current, a time- and voltage-dependent potassium current. The occurrence of GnRH in sympathetic ganglia extracts from two amphibian species was investigated. Chicken GnRH II-like immunoreactivity was detected in extracts of bullfrog (Rana catesbeiana) and platanna (Xenopus laevis) sympathetic ganglia after high performance liquid chromatography. Under the chro-matographic conditions used, a second unknown peptide co-eluted with synthetic mammalian GnRH, but showed no cross-reactivity with specific mammalian GnRH antisera. To test the possibility of the presence of a chicken GnRH II receptor in sympathetic ganglion neurones, competition binding of membranes extracted from the sympathetic ganglia of the two amphibian species was investigated with 125I-labelled GnRH agonists. The binding of 125I-[His5, D-Arg6, Trp7, Tyr8]GnRH (a chicken GnRH II agonist) to membranes from the sympathetic ganglia of both amphibian species was specific and had a higher affinity than chicken GnRH II, mammalian GnRH and a mammalian GnRH agonist [D-Ala6, NMe-Leu7, Pro9-NHEt]GnRH. These findings suggest that endogenous chicken GnRH II may play a role in synaptic transmission in the sympathetic ganglia via a receptor specific for chicken GnRH II.
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