Gang Wang

Role of Q-type Ca2+ Channels in Vasopressin Secretion From Neurohypophysial Terminals of the Rat

1 The nerve endings of rat neurohypophyses were acutely dissociated and a combination of pharmacological, biophysical and biochemical techniques was used to determine which classes of Ca2+ channels on these central nervous system (CNS) terminals contribute functionally to arginine vasopressin (AVP) and oxytocin (OT) secretion. 2 Purified neurohypophysial plasma membranes not only had a single high‐affinity binding site for the N‐channel‐specific ω‐conopeptide MVIIA, but also a distinct high‐affinity site for another ω‐conopeptide (MVIIC), which affects both N‐ and P/Q‐channels. 3 Neurohypophysial terminals exhibited, besides L‐ and N‐type currents, another component of the Ca2+ current that was only blocked by low concentrations of MVIIC or by high concentrations of ω‐AgaIVA, a P/Q‐channel‐selective spider toxin. 4 This Ca2+ current component had pharmacological and biophysical properties similar to those described for the fast‐inactivating form of the P/Q‐channel class, suggesting that in the neurohypophysial terminals this current is mediated by a ‘Q’‐type channel. 5 Pharmacological additivity studies showed that this Q‐component contributed to rises in intraterminal Ca2+ concentration ([Ca2+]i) in only half of the terminals tested. 6 Furthermore, the non‐L‐ and non‐N‐component of Ca2+‐dependent AVP release, but not OT release, was effectively abolished by the same blockers of Q‐type current. 7 Thus Q‐channels are present on a subset of the neurohypophysial terminals where, in combination with N‐ and L‐channels, they control AVP but not OT peptide neurosecretion.

Rat supraoptic magnocellular neurones show distinct large conductance, Ca2+-activated K+ channel subtypes in cell bodies versus nerve endings

1 Large conductance, Ca2+‐activated K+ (BK) channels were identified in freshly dissociated rat supraoptic neurones using patch clamp techniques. 2 The single channel conductance of cell body BK channels, recorded from inside‐out patches in symmetric 145 mM K+, was 246.1 pS, compared with 213 pS in nerve ending BK channels (P < 0.01). 3 At low open probability (Po), the reciprocal of the slope in the ln(NPo)‐voltage relationship (N, number of available channels in the patch) for cell body and nerve ending channels were similar: 11 vs. 14 mVper e‐fold change in NPo, respectively. 4 At 40 mV, the [Ca2+]i producing half‐maximal activation was 273 nM, as opposed to > 1.53 μM for the neurohypophysial channel, indicating the higher Ca2+ sensitivity of the cell body isochannel. 5 Cell body BK channels showed fast kinetics (open time constant, 8.5 ms; fast closed time constant, 1.6 and slow closed time constant, 12.7 ms), identifying them as ‘type I’ isochannels, as opposed to the slow gating (type II) of neurohypophysial BK channels. 6 Cell body BK activity was reduced by 10 nM charybdotoxin (NPo, 37 % of control), or 10 nM iberiotoxin (NPo, 5 % of control), whereas neurohypophysial BK channels are insensitive to charybdotoxin at concentrations as high as 360 nM. 7 Whilst blockade of nerve ending BK channels markedly slowed the repolarization of evoked single spikes, blockade of cell body channels was without effect on repolarization of evoked single spikes. 8 Ethanol reversibly increased neurohypophysial BK channel activity (EC50, 22 mM; maximal effect, 100 mM). In contrast, ethanol (up to 100 mM) failed to increase cell body BK channel activity. 9 In conclusion, we have characterized BK channels in supraoptic neuronal cell bodies, and demonstrated that they display different electrophysiological and pharmacological properties from their counterparts in the nerve endings.

Tetrandrine: A new ligand to block voltage-dependent Ca2+ and Ca2+-activated K+ channels

Extensive pharmacological investigations on tetrandrine, one of the traditional medicinal alkaloids, are reviewed. Tetrandrine has been used clinically in China for centuries in the treatment of many diseases. A recent series of studies has revealed major mechanisms underlying its multiple pharmacological and therapeutic actions. One of the most interesting discoveries is that tetrandrine is a new kind blocker of the voltage-activated, L-type Ca2+ channel in a variety of excitable cells, such as cardiac, GH3 anterior pituitary and neuroblastoma cells, as well as in rat neurohypophysial nerve terminals. Although tetrandrine does not belong to any of the three classical Ca2+ channel blocker groups, electrophysiological and radioligand binding studies show that tetrandrine is an L-type Ca2+ channel blocker with its binding site located at the benzothiazepine receptor on the alpha 1-subunit of the channel. In addition, tetrandrine is a blocker of the voltage-dependent T-type Ca2+ channel. It is clear that tetrandrines actions in the treatment of cardiovascular diseases, including hypertension and supraventricular arrhythmia, are due primarily to its blocking of voltage-activated L-type and T-type Ca2+ channels. Furthermore, this alkaloid is a potent blocker of the Ca(2+)-activated K+ (K(Ca)) channels of neurohypophysial nerve terminals. The blocking kinetics of tetrandrine on the K(Ca) channel is quite different from that of typical K(Ca) channel blockers such as tetraethylammonium and Ba2+. Although the clinical role of tetrandrine as a blocker of the K(Ca) channels is unclear, it is a promising ligand for the study of K(Ca) channel function.

A novel large-conductance Ca(2+)-activated potassium channel and current in nerve terminals of the rat neurohypophysis.

1. Nerve terminals of the rat posterior pituitary were acutely dissociated and identified using a combination of morphological and immunohistochemical techniques. Terminal membrane currents were studied using the ‘whole‐cell’ patch clamp technique and channels were studied using inside‐out and outside‐out patches. 2. In physiological solutions, but with 7 mM 4‐aminopyridine (4‐AP), depolarizing voltage clamp steps from different holding potentials (‐90 or ‐50 mV) elicited a fast, inward current followed by a slow, sustained, outward current. This outward current did not appear to show any steady‐state inactivation. 3. The threshold for activation of the outward current was ‐30 mV and the current‐voltage relation was ‘bell‐shaped’. The amplitude increased with increasingly depolarized potential steps. The outward current reversal potential was measured using tail current analysis and was consistent with that of a potassium current. 4. The sustained potassium current was determined to be dependent on the concentration of intracellular calcium. Extracellular Cd2+ (80 microM), a calcium channel blocker, also reversibly abolished the outward current. 5. The current was delayed in onset and was sustained over the length of a 150 ms‐duration depolarizing pulse. The outward current reached a peak plateau and then decayed slowly. The decay was fitted by a single exponential with a time constant of 9.0 +/‐ 2.2 s. The decay constants did not show a dependence on voltage but rather on intracellular Ca2+. The time course of recovery from this decay was complex with full recovery taking > 190 s. 6. 4‐AP (7 mM), dendrotoxin (100 nM), apamin (40‐80 nM), and charybdotoxin (10‐100 nM) had no effect on the sustained outward current. In contrast Ba2+ (200 microM) and tetraethylammonium inhibited the current, the latter in a dose‐dependent manner (apparent concentration giving 50% of maximal inhibition (IC50) = 0.51 mM). 7. The neurohypophysial terminal outward current recorded here corresponds most closely to a Ca(2+)‐activated K+ current (IK(Ca)) and not to a delayed rectifier or IA‐like current. It also has properties different from that of the Ca(2+)‐dependent outward current described in the magnocellular neuronal cell bodies of the hypothalamus. 8. A large conductance channel is often observed in isolated rat neurohypophysial nerve terminals. The channel had a unit conductance of 231 pS in symmetrical 150 mM K+.(ABSTRACT TRUNCATED AT 400 WORDS)

Tetrandrine blocks a slow, large-conductance, Ca2+-activated potassium channel besides inhibiting a non-inactivating Ca2+ current in isolated nerve terminals of the rat neurohypophysis

The effects of tetrandrine, a bis-benzyl-isoquinoline alkaloid, on voltage-gated Ca2+ currents (ICa) and on Ca2+-activated K+ current (IK(Ca)) and channels in isolated nerve terminals of the rat neurohypophysis were investigated using patch-clamp techniques. The non-inactivating component of ICa was inhibited by external tetrandrine in a voltage- and dose-dependent manner, with an IC50=10.1 μM. IK(Ca) was elicited by depolarizations when approximately 10 μM Ca2+ was present on the cytoplasmic side. Only externally applied tetrandrine, at 1 μM, decreased the amplitude of IK(Ca), whereas the fast inward Na+ current and transient outward K+ current were not affected. Tetrandrine, applied to the extracellular side of outside-out patches excised from the nerve terminals, induced frequent and short closures of single type II, maxi-Ca2+-activated K+channels. Tetrandrine decreased the channel-open probability, within bursts, with an IC50=0.21 μM. Kinetic analysis of the channel activity showed that the open-time constant decreased linearly with increasing tetrandrine concentrations (0.01–3 μM), giving an association rate constant of 8.8×108 M−1s−1, whereas the arithmetic mean closed time did not change, giving a dissociation rate constant of 136.6s−1. These results show that tetrandrine is a high-affinity blocker of the type II, maxi-Ca2+-activated K+ channel of the rat neurohypophysial terminals.

Excitatory versus inhibitory modulation by ATP of neurohypophysial terminal activity in the rat

Much is now known about the electrophysiological properties of the magnocellular neurones of the hypothalamus. Oxytocin neurones are characterized by an intermittent high frequency discharge during suckling that leads to the pulsatile release of oxytocin into the blood and to subsequent milk ejection. Vasopressin neurones are characterized by their asynchronous phasic activity (bursting) during maintained vasopressin release and the subsequent regulation of water balance. In both cases, it is the clustering of spikes, albeit with different time courses for each peptide, that facilitates hormone release. The mechanism underlying this differential facilitation is one of the major unanswered questions in neuroendocrinology. This paper considers recent evidence that indicates that ATP, co‐secreted with vasopressin and oxytocin, may play a key role in the regulation of stimulus—secretion coupling in the neurohypophysis. The activity of the type (II) Ca2+‐activated K+ (Kca) channel found in the nerve terminals was significantly increased in the presence of ATP on the cytoplasmic side of the channel. Extracellular ATP, in contrast, inhibited the type II Kca current in a dose‐dependent manner. Thus, intracellular and extracellular ATP exert opposite effects on the type II Kca channel of neurohypophysial terminals. Furthermore, ATP opens P2±2 channels to increase intracellular [Ca2+] in the nerve terminals and subsequent arginine vasopressin (AVP) release. In contrast, adenosine, acting via A1 receptors, specifically inhibits only the N‐type Ca2+ channel, thus decreasing neuropeptide release. These multiple, conflicting effects of ATP and its metabolite adenosine could explain the patterns of AVP release observed during physiological stimulation in vivo.

Effects of funnel web spider toxin on Ca2+ currents in neurohypophysial terminals.

Funnel web spider toxin (FTX) is reportedly a specific blocker of P-type Ca2+ channels. The effects of FTX on the Ca2+ currents of isolated neurohypophysial nerve terminals of the rat were investigated using the whole-cell patch-clamp technique. Both the transient and long-lasting Ca2+ current components were maximally elicited by depolarization from a holding potential equal to the normal terminal resting potential (-90 mV). Externally applied FTX inhibited the high-voltage-threshold, transient component of the Ca2+ current in a concentration-dependent manner, with a half-maximal inhibition at a dilution of approximately 1:10000. FTX also shifted the peak current of the I-V relationship by +10 mV. The long-lasting Ca2+ current component, which is sensitive to L-type Ca2+ channel blockers, was insensitive to FTX. The transient current, which is sensitive to omega-conotoxin GVIA, was completely blocked by FTX. These results suggest that there could be a novel, inactivating Ca2+ channel in the rat neurohypophysial terminals which is affected by both N-type and P-type Ca2+ channel blockers.

Effects of Toxins on Ca2+Currents and Peptide Release from Nerve Terminals

Voltage-activated calcium channels in nerve terminals of the neurohypophysis are instrumental for triggering the exocytotic release of the neuropeptides, oxytocin (OT) and vasopressin (AVP). In depolarization-secretion coupling, Ca2+, acting as a second messenger, flows into the terminals through these channels and consequently evokes release..2 In neurons, the major voltage-gated Ca2+ channels were originally named and distinguished as the T(low-voltage-threshold), N-, and L(high-voltagethreshold) type^.^,^ Both of the high-voltage Ca2+ channel types may be involved in the process of neurotransmitter or neuropeptide A number of pharmacological agents, including toxins, have been used to further

Calcium channels do not play a role in the steroid response to ACTH IN Y1 adrenocortical cells.

Y1 cells derived from mouse tumor zona fasciculata cells (ZF) were used to assess the importance of extracellular (EC) calcium availability via voltage-dependent calcium channels (VDCC) on steroidogenesis. The steroidogenic response to ACTH was investigated in the presence of blockers known to affect both calcium and potassium channels in Y1 cells. Y1 cells respond to either ACTH (100 pM) or cAMP (300 microM) at low EC Ca2+ (1 microM) suggesting that EC Ca2+ is not absolutely necessary for a steroidogenic response. However, increases in Ca2+ from 0.05-2.2 mM induced a small but significant biphasic response, first stimulating then inhibiting steroidogenesis. Nickel and amiloride, blockers of T-type Ca2+ channels in Y1 cells, did not depress ACTH-induced steroidogenesis. The dihydropyridine, nifedipine, which is an L-type channel antagonist did not affect ACTH-induced steroidogenesis while the agonist, Bay K 8644 was consistently inhibitory. Neither pimozide nor omega-conotoxin which suppressed Ca2+ currents in Y1 cells inhibited ACTH-induced steroidogenesis. Depolarization of the membrane which would activate VDCCs was inhibitory rather than stimulatory. The present studies using blockers of both voltage-dependent Ca2+ and K+ channels suggest that EC Ca2+ plays a modulatory role in ACTH-induced steroidogenesis in Y1 cells but the data do not support the concept that activation of voltage dependent calcium channels are an important mechanism for steroidogenesis.

Voltage dependent calcium and potassium currents in Y-1 adrenocortical cells are unresponsive to ACTH

In this report we use both whole cell and perforated patch clamp recording techniques to characterize calcium and potassium channels in Y-1 adrenocortical cells in order to assess their responsiveness to ACTH. Both transient and long-lasting components of an inward calcium current were identified which were similar to T and L-type Ca2+ currents. With Ba2+ as the charge carrier, the transient current activated at voltages more hyperpolarized than -50 mV with V1/2 for activation at -78.1 mV, and for steady state inactivation at -52.3 mV. The L-type current activated at -20 mV, with a V1/2 for activation at -29.9 mV and steady state inactivation at -44.2 mV. Under perforated patch conditions the response was shifted to more depolarized voltages. Both currents were responsive to agents which usually affect T- or L-type Ca2+ currents. The transient current was completely blocked by 50 microM lanthanum or 200 microM nickel and partially blocked by 300 mM amiloride. Cadmium (100 microM) and nifedipine (300 nM) completely blocked the long-lasting current while omega-conotoxin GVIA (1992 nM) inhibited the current by only 20-25%. The agonist, Bay K 8644 was stimulatory at 50 nM. Both transient and sustained outward potassium currents similar to A-type and delayed rectifier currents, respectively, were present. The transient current demonstrated fast activation at voltages more positive than -10 mV, inactivation with continued depolarization and steady state inactivation at V1/2 = -50 mV. The sustained current activated rapidly and had minimal inactivation with continued depolarization. The transient current was blocked by 5 mM 4AP and the sustained by 25 mM TEA. While Y-1 cells contain both calcium and potassium currents similar to those found in other adrenocortical cells, none of the currents were affected by ACTH or AII, secretagogues which stimulate steroidogenesis. These data, combined with the inability of both Ca2+ and K+ channel blockers to alter ACTH-induced steroidogenesis as reported earlier, suggests that neither calcium nor potassium currents are responsive to ACTH in Y-1 cells.