State-dependent nickel block of a high-voltage-activated neuronal calcium channel.

Effect of nickel ions (Ni2+) on noninactivating calcium channels in squid giant fiber lobe (GFL) neurons were investigated with whole cell voltage clamp. Three different effects of Ni2+ were observed to be associated with distinct Ca2+ channel activation states. 1) Nickel ions appear to stabilize closed channel states and, as a result, slow activation kinetics. 2) Nickel ions block open channels with little voltage dependence over a wide range of potentials. 3) Block of open channels by Ni2+ becomes more effective during an extended strong depolarization, and this effect is voltage dependent. Recovery from this additional inhibition occurs at intermediate voltages, consistent with the presence of two distinct types of Ni2+ block that we propose correspond to two previously identified open states of the calcium channel. These results, taken together with earlier evidence of state-dependent block by omega-agatoxin IVA, suggest that Ni2+ generates these unique effects in part by interacting differently with the external surface of the GFL calcium channel complex in ways that depend on channel activation state.

Depolarization-induced slowing of Ca2+ channel deactivation in squid neurons.

Properties of squid giant fiber lobe (GFL) Ca2+ channel deactivation (closing) were studied using whole-cell voltage clamp. Tail currents displayed biexponential decay, and fast and slow components of these tails exhibited similar external Ca(2+)- and voltage-dependence. Both components also shared similar inactivation properties. Increasing duration pulses to strongly depolarizing potentials caused a substantial slowing of the rate of deactivation for the fast component, and also led to an apparent conversion of fast tail currents to slow without an increase in total tail amplitude. A five-state kinetic model that computed the closing of channels differentially populating two open states could simulate the kinetic characteristics of GFL Ca2+ pulse and tail currents over a wide voltage range. The kinetics of the proposed state transition was very similar to the time course of relief of omega-Agatoxin IVA Ca2+ channel block with long pulses. A similar model predicted that the relief of block could occur via faster toxin dissociation from the second open state. Thus, GFL Ca2+ channels possess a unique form of voltage-dependent gating modification, in which maintained prior depolarization leads to a significant delay to channel closure at negative potentials. At the nerve terminal, amplified Ca2+ signals generated by such a mechanism might alter synaptic responses to repetitive stimulation.

All-or-none contraction and sodium channels in a subset of circular muscle fibers of squid mantle.

Motor function in squid (Loligo) mantle reflects the highly coordinated activity of two motor pathways associated with giant and non-giant motor axons that respectively produce all-or-none and graded contractions in mantle muscle. Whereas both types of axons innervate circular mantle muscle fibers, precise nerve-muscle relationships remain unclear. Are squid like most invertebrates, in which single muscle fibers receive dual innervation from giant and non-giant motor axons, or is squid mantle configured more like vertebrates, in which parallel motor axon systems innervate distinct fast and slow muscle fibers? In this report, we describe giant and nongiant motor pathways that appear to control different pools of circular muscle fibers in squid. A subset of circular muscle fibers possesses large Na currents, and these fibers are proposed to employ Na-dependent action potentials to produce fast, all-or-none muscle twitches associated with giant axon stimulation.

Spatial localization of calcium channels in giant fiber lobe neurons of the squid (Loligo opalescens).

Whole-cell voltage clamp was used to investigate the properties and spatial distribution of fast-deactivating (FD) Ca channels in squid giant fiber lobe (GFL) neurons. Squid FD Ca channels are reversibly blocked by the spider toxin omega-Agatoxin IVA with an IC50 of 240-420 nM with no effect on the kinetics of Ca channel gating. Channels with very similar properties are expressed in both somatic and axonal domains of cultured GFL neurons, but FD Ca channel conductance density is higher in axonal bulbs than in cell bodies at all times in culture. Channels presumably synthesized during culture are preferentially expressed in the growing bulbs, but bulbar Ca conductance density remains constant while Na conductance density increases, suggesting that processes determining the densities of Ca and Na channels in this extrasomatic domain are largely independent. These observations suggest that growing axonal bulbs in cultured GFL neurons are not composed entirely of "axonal" membranes because FD Ca channels are absent from the giant axon in situ but, rather, suggest a potential role for FD Ca channels in mediating neurotransmitter release at the motor terminals of the giant axon.

Postsomatostatin hypersecretion of growth hormone from perifused rat anterior pituitary cells is dependent on calcium influx.

The role of ionic calcium (Ca2+) in the rebound secretion of growth hormone (GH) following termination of somatostatin (SRIF) administration was investigated in vitro by perifusion of acutely dispersed rat anterior pituitary cells. Treatment with 10 nM SRIF for 40 min significantly reduced the mean GH secretory rate by 3.3 +/- 0.2 ng min-1 representing a 58% decrease from baseline (p < 0.01). Following the withdrawal of SRIF treatment, GH levels surged 3- to 5-fold relative to baseline with the mean secretion rate increasing by 4.5 +/- 0.99 ng min-1 (p < 0.05). GH rebound secretion following SRIF removal from the perifusion medium was completely abolished (p < 0.01) when zero calcium medium (0 Ca2+) or medium containing 2 mM cobalt chloride (Co2+) were administered after SRIF termination. Perifusion with 0 Ca2+ caused the GH release rate to return to above baseline levels. In contrast, Co2+ perifusion caused the GH secretion rate to remain at the level observed during SRIF treatment (-4.52 +/- 0.38 ng min-1 relative to baseline; p < 0.01). Similarly, when cells were exposed to Co2+ alone, a reduction in the rate of GH secretion (-3.96 +/- 0.56 ng min-1; p < 0.01) was evident. After termination of Co2+ treatment, either by itself or following SRIF pretreatment, and upon changing from 0 Ca2+ to normal calcium-containing medium following SRIF pretreatment, a significant overshoot in GH release similar to SRIF withdrawal-induced GH release was observed (p < 0.05 and 0.01, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of pulsatile secretion of thyrotropin and growth hormone in the hypothyroid rat.

To characterize the role of TRH in the generation of TSH pulsatility as well as the effect of hypothyroidism on episodic GH secretion, blood was constantly withdrawn (30-60 microliters/min) from rats treated with 0.02% methimazole in the drinking water for 8-10 days. This treatment significantly reduced circulating levels of both T3 and T4 and elevated plasma TSH; however, since thyroid hormone titers were still detectable (T3, 39.6 +/- 5.3 vs. 89.8 +/- 5.3 ng/dl in euthyroid animals), methimazole-treated rats were referred to as being mildly hypothyroid. TSH was found to be secreted in secretory bursts, consisting of one to several peaks in these rats. Pulsar analysis of TSH secretory profiles revealed a mean pulse frequency of 2.8 pulses/h, a mean pulse amplitude of 10 ng/pulse, and a mean pulse duration of 0.2 h. Euthyroid rats exhibited similar fluctuations of circulating TSH levels; however, due to the variability of the TSH RIA in the range of euthyroid TSH titers, no significant pulsatility was detected by Pulsar. Mean plasma TSH levels in eu- and hypothyroid rats were 2.3 +/- 0.3 and 14.6 +/- 1.8 ng/ml, respectively. To confirm that the TRH antiserum (TRH-AS) used in the present study for passive immunization had sufficient binding capacity to absorb endogenous TRH release, euthyroid rats were pretreated with either normal rabbit serum or TRH-AS, followed by the injection of clonidine (100 micrograms/kg BW, iv). This alpha 2-adrenergic agonist caused a significant (P < 0.01) 12.7-fold rise in plasma TSH levels in normal rabbit serum-treated animals, which was completely abolished by TRH-AS pretreatment, indicating that clonidine stimulates TSH secretion via activation of hypothalamic TRH release. When TRH-AS was slowly infused into hypothyroid rats that were sampled frequently for the detection of TSH pulsatility, it caused a significant (60.3%; P < 0.01) decrease in mean TSH levels, with TSH titers approaching euthyroid concentrations 1 h after the infusion of TRH-AS. The antiserum treatment also caused the disappearance of statistically significant (Pulsar) TSH secretory pulses. Mild hypothyroidism shifted the GH secretory profiles from a low frequency, high amplitude in euthyroid animals to a high frequency, low amplitude pattern in hypothyroid rats. Mean GH levels in hypothyroid rats were 76% lower than those in euthyroid controls. These findings show that TSH is secreted in a pulsatile fashion in the hypothyroid rat and that TRH is predominantly responsible for the generation of TSH pulsatility.(ABSTRACT TRUNCATED AT 400 WORDS)