Effects of toxins on Ca2+ currents and peptide release from nerve terminals.
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Alkaloids↗
Biomedical subjects
J R Lemos
Publications and source records attributed to J R Lemos.
Ethanol directly modulates gating of a dihydropyridine-sensitive Ca2+ channel in neurohypophysial terminals.
Ingestion of ethanol results in a decreased level of plasma vasopressin, which appears to be caused by inhibition of arginine vasopressin (AVP) release from the neurohypophysis. Activation of membrane voltage-gated Ca2+ channels plays an important role in triggering this neurohormone release. In this article, single-channel recordings are used to demonstrate that ethanol, at concentrations constituting legal intoxication, inhibits dihydropyridine-sensitive "L-type" Ca2+ channels in isolated nerve terminals of the rat neurohypophysis. Ethanol reduced the channel open probability in a concentration-dependent manner. To allow finer resolution of channel openings and to better characterize the mechanisms of ethanol action, Bay K 8644 was used to prolong the openings of L-type Ca2+ channels. In the presence of this dihydropyridine (DHP), the reduction of the channel open probability by concentrations of ethanol of 25 mM or higher could be determined to be due primarily, although not completely, to a shortening of the open duration of this L-channel. Channel conductance was unaffected by ethanol, even at high concentrations. These results are consistent with previous macroscopic data indicating that calcium channels in these peptidergic terminals are targets for ethanol action, and indicate that ethanol acts directly on the gating characteristics of the L-type channel. Furthermore, examination of open and closed state transitions, as well as Hill plot analysis, suggests that ethanol's effects on gating are consistent with the interaction of a single drug molecule with a single target site, possibly the L-channel itself.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
A novel method for identification of peptide contents from individual neurohypophysial terminals after patch-clamp recordings.
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Animals↗
Single channel recordings of Nt- and L-type Ca2+ currents in rat neurohypophysial terminals.
1. Ca2+ currents through single channels in acutely dissociated nerve terminals from rat neurohypophyses were recorded using cell-attached patch recordings with 110 mM Ba2+ as the charge carrier. 2. One type (Nt, where the t denotes terminal) of single Ca2+ channel current was evoked only by depolarizing steps from holding potentials less negative than -50 mV. Because this channel opened primarily at the beginning of a 180-ms-long voltage pulse, the averaged ensemble current decayed rapidly (approximately 30 ms). Infrequently, the channel opened throughout such a long pulse, resulting in a long-lasting averaged ensemble current. The averaged channel open time constant (tau) was 0.34 ms and the two averaged closed time constants were 1.78 (tau 1) and 86.57 (tau 2) ms. The mean unitary slope conductance for this channel was 11 pS and its threshold for activation was approximately -10 mV. 3. The other type (L) of single Ca2+ channel current could be evoked in isolation by depolarizations from holding potentials more positive than or equal to -50 mV. This channel opened throughout an entire 180-ms-long voltage pulse. The averaged ensemble current, therefore, showed little inactivation. The averaged channel open-time constant was 0.49 ms and the two average closed time constants were 2.02 (tau 1) and 79.91 (tau 2) ms. The mean unitary slope conductance for this channel was 25 pS. 4. Bay K 8644 (5 microM), a dihydropyridine (DHP) Ca2+ channel agonist, increased the open probability of the larger-conductance L-type Ca2+ channel by prolonging the average duration (to 2.79 ms) of channel openings, but did not alter the single channel slope conductance. In contrast, the same concentration of Bay K 8644 did not affect the smaller-conductance Nt-type Ca2+ channel. The DHP Ca2+ channel antagonist nicardipine (5 microM), but not nifedipine (5 microM), reduced the open probability of the large-conductance L-type Ca2+ channel by shortening the duration (to 0.36 ms) of channel openings. 5. The voltage- and time-dependent properties of these two types of single Ca2+ channel currents are in close agreement with those of the two components of macroscopic Ca2+ currents previously reported using the "whole-terminal" recording method. Therefore these two types of single channels appear to underlie the macroscopic current. 6. Our studies suggest that the terminal Nt-type Ca2+ channel differs from the conventional somatic N- and T-type Ca2+ channels in some respects, and that the terminal L-type Ca2+ channel is similar to the conventional somatic L-type Ca2+ channel.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Comparison of effects of tetrandrine on ionic channels of isolated rat neurohypophysial terminals and Y1 mouse adrenocortical tumor cells.
UNLABELLED: The effects of tetrandrine on the voltage-gated Ca2+, K+, and Na+ channels of the isolated rat neurohypophysial nerve terminals and Y1 mouse adrenocortical tumor cells were studied using standard and perforated patch-clamp techniques. Among the inward ionic currents, the T-type Ca2+ channel current (I(Ca)) in the Y1 cell line was strongly inhibited (by 52.9%) by a low concentration (13 mumol.L-1) of externally applied tetrandrine. The L-type I(Ca) in the isolated nerve terminals was also strongly inhibited (by 54.2%) by externally applied tetrandrine 33 mumol.L-1, whereas the N-type I(Ca) and Na+ current were not significantly affected by the same dose of the alkaloid. While it had no effect on the fast, transient K+ channels, external application of tetrandrine 1 mumol.L-1 blocked a slowly-gating, maxi Ca(2+)-activated K+ (K+(Ca)) channel in the nerve terminals, decreasing its P(o) by 84.4%. CONCLUSIONS: (1) The order of the sensitivities of ionic channels to tetrandrine is K+(Ca) > T- > L- > N-type Ca2+ > Na+; (2) tetrandrine serves as a specific blocker of the slowly-gating K+(Ca) channel.
Adenoma↗
Tetrandrine blocks a slow, large-conductance, Ca(2+)-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 Ca(2+)-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 microM. IK(Ca) was elicited by depolarizations when approximately 10 microM Ca2+ was present on the cytoplasmic side. Only externally applied tetrandrine, at 1 microM, 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-Ca(2+)-activated K+ channels. Tetrandrine decreased the channel-open probability, within bursts, with an IC50 = 0.21 microM. Kinetic analysis of the channel activity showed that the open-time constant decreased linearly with increasing tetrandrine concentrations (0.01-3 microM), giving an association rate constant of 8.8 x 10(8) M-1 s-1, whereas the arithmetic mean closed time did not change, giving a dissociation rate constant of 136.6 s-1. These results show that tetrandrine is a high-affinity blocker of the type II, maxi-Ca(2+)-activated K+ channel of the rat neurohypophysial terminals.
Alkaloids↗
Possible role during exocytosis of a Ca(2+)-activated channel in neurohypophysial granules.
Ion channels from bovine neurohypophysial granules were incorporated into artificial lipid bilayers. The larger amplitude channel is permeable to cations and exhibits multiple conductances. The channel opens only in the presence of free Ca2+, but is inhibited by relatively high Ca2+ concentrations. Release of vasopressin from permeabilized neurohypophysial terminals also shows a similar biphasic dependence on Ca2+. Release is selectively inhibited by low concentrations of the long-chain alcohol octanol, but not by high concentrations of ethanol, as is the neurosecretory granule Ca(2+)-activated cation channel. Furthermore, Ca(2+)-evoked release and channel activity are both inhibited by the long-chain tetraethylammonium analogs decamethonium and decyl-triethyl ammonium bromide. The close correlation between channel and release properties lead us to conclude that the Ca(2+)-activated channel is involved in peptide secretion.
Alcohols↗
Two types of high-threshold calcium currents inhibited by omega-conotoxin in nerve terminals of rat neurohypophysis.
1. The neurohypophysis comprises the nerve terminals of hypothalamic neurosecretory cells, which contain arginine vasopressin (AVP) and oxytocin. The secretory terminals of rat neurohypophyses were acutely dissociated. The macroscopic calcium currents (ICa) of these isolated peptidergic terminals were studied using 'whole-cell' patch-clamp recording techniques. 2. There are two types ('Nt' (where the subscript 't' denotes terminal) and 'L') of high-threshold voltage-activated ICa in the terminals, which can be distinguished by holding at different potentials i.e. -90 and -50 mV. Replacement of Ca2+ in the bathing solution by Ba2+ increased the amplitude of ICa, primarily due to an increase in the L-type component. Both inward currents were eliminated by adding 50 microM-Cd2+ or when in a Ca(2+)-free bathing solution. 3. omega-Conotoxin GVIA (omega-CgTx) has been widely used as a Ca2+ channel blocker. However, whether this toxin can discriminate between different types of Ca2+ channels is still a subject of controversy. We applied omega-CgTx over a wide range of concentrations (0.01-2 microM) to examine its effects on both Nt- and L-type ICa in these terminals. At a concentration of 30 nM, omega-CgTx selectively reduced, by 48%, the amplitude of Nt-type ICa. In contrast, a higher concentration (300 nM) of omega-CgTx was necessary to inhibit the L-type ICa. 4. omega-CgTx inhibited both Nt- and L-type ICa in a dose-dependent manner, and the half-maximum inhibition (IC50) of the ICa by the toxin was 50 and 513 nM, respectively, which was approximately a tenfold difference. The reduction in both types of currents did not result from any shift in their current-voltage or steady-state inactivation relationships. 5. In contrast, omega-CgTx, at a concentration of 300 nM, had no effect on the tetrodotoxin-sensitive sodium current (INa) of the isolated peptidergic nerve terminals. Furthermore, omega-CgTx did not reduce the long-lasting, non-inactivating ICa in the isolated non-neuronal secretory cells of the pars intermedia (PI) (intermediate lobe of the pituitary). 6. Our studies suggest that omega-CgTx might exert specific blocking effects on both Nt- and L-type Ca2+ channels, but that in the isolated peptidergic nerve terminals, the Nt-type component is more susceptible to this toxin.
Action Potentials↗
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)
Animals↗
Ethanol reduces vasopressin release by inhibiting calcium currents in nerve terminals.
Ingestion of ethanol (EtOH) is known to result in a reduction of plasma arginine-vasopressin (AVP) levels in mammals. We examined the basis for this effect using a combination of biochemical and electrophysiological techniques. Release of AVP from nerve terminals isolated from the rat neurohypophysis was very sensitive to EtOH, with significant reductions in AVP release evident in 10 mM EtOH. However, EtOH did not affect the release of AVP from terminals which had been permeabilized with digitonin, suggesting that voltage-gated calcium channels might be the target of EtOH's actions. Patch clamping of these terminals indicated that both inactivating and long-lasting calcium currents were reduced in EtOH, but the long-lasting currents were more sensitive (significant reductions in 10 mM EtOH). EtOH-induced decreases in plasma AVP levels can be explained by EtOH's inhibition of calcium currents in the nerve terminals.
Animals↗
Direct identification of individual vasopressin-containing nerve terminals of the rat neurohypophysis after 'whole-cell' patch-clamp recordings.
The membrane currents in rat neurophypophysial nerve terminals, which contain either vasopressin or oxytocin, have been previously recorded using the 'whole-cell' patch-clamp technique. Interpretation of the electrophysiological data would be significantly strengthened by the ability to correlate them with knowledge of the peptide contents of the terminals being studied. Here, a novel method for detection of the peptide hormone, arginine vasopressin, in those individual isolated terminals is described. The unique aspect of this procedure is that the contents of the terminal are aspirated into the recording electrode after 'whole-cell' patch-clamp recording, and then a highly sensitive dot immunobinding assay allows identification of the peptide contents in the terminals.
Animals↗
The action of luteinizing hormone on the testis.
Luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) receptors are coupled to intracellular effector systems, most notably adenylate cyclase, through guanyl nucleotide-binding proteins or G-proteins. The molecular mechanism involved in the dynamic coupling of the LH/hCG receptor however, are not known. It has been postulated that receptor aggregation at the molecular level plays a critical role in this process. There have been attempts to understand the receptor association and dissociation phenomena at the molecular level. One of them involves the participation of the major histocompatibility complex (MHC) class I antigen in the mechanism of receptor activation and/or expression. One molecular basis for these mechanisms consists of a physical interaction between MHC proteins and receptors to form "compound receptors" able to transfer a hormonal signal to the cell. Using a photo-reactive probe we demonstrated that the LH/hCG receptors and the class I antigens are closely associated in the membrane. Thus, it is possible to form covalent complexes of hCG and class I antigens through the binding of the hormone to specific receptors. These findings imply that LH/hCG receptors and the MHC class I antigens may interact at the level of the plasma membrane in the mechanism of LH action. We also performed experiments using a single cell and limiting stimulation to a patch of membrane. The results stimulating the cell in a localized area suggested that even if all components are entirely free to float there is a constraint in the localization of the receptor, G-protein, and/or the effector, supporting the constraint dissociation model. Within a limited area subunits could dissociate, but they would not be free to diffuse throughout the membrane. Moreover the concept of compartmentalization that has been utilized to explain some inconsistencies in second-messenger action now can be proved by experimental design.
Animals↗
Effects of ethanol on calcium channels, potassium channels, and vasopressin release.
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Acetylcholine↗
Possible role for neurosecretory granule channel that resembles gap junctions.
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Animals↗
A fast, transient K+ current in neurohypophysial nerve terminals of the rat.
1. Nerve terminals of the rat posterior pituitary were acutely dissociated and identified using a combination of morphological and immunohistochemical techniques. Macroscopic terminal membrane currents and voltages were studied using the whole-cell patch clamp technique. 2. In physiological solutions, depolarizing voltage clamp steps, from a holding potential (-80 mV) similar to the normal terminal resting potential, elicited a fast, inward followed by a fast, transient, outward current. 3. The threshold of activation for the outward current was -60 mV. The outward current quickly reached a peak and then decayed more slowly. The decay was fitted by two exponentials with time constants of 21 +/- 2.9 and 143 +/- 36 ms. These decay constants did not show a dependence on voltage. The time to peak of the outward current decreased and the amplitude increased with increasingly depolarized potential steps. 4. The outward current was blocked by the substitution of K+ with Cs+ and its reversal potential was consistent with a potassium current. 5. The transient outward current showed steady-state inactivation at more depolarized (than -80 mV) holding potentials with 50% inactivation occurring at -47.9 mV. The time course of recovery from inactivation was complex with full recovery taking greater than 16 s. 6. 4-Aminopyridine (4-AP) blocked the transient outward current in a dose-dependent manner (approximately IC50 = 3 mM), while charybdotoxin (4 micrograms/ml) and tetraethylammonium (100 mM) had no effect on the current amplitude. 7. Lowering external [Ca2+] had no effect on the fast, transient outward current nor did the calcium channel blocker Cd2+ (2 mM). 8. The neurohypophysial outward current reported here corresponds most closely to IA, and not to the delayed rectifier or Ca2(+)-activated K+ currents. Neurohypophysial IA, however, appears to be different from the outward currents found in the cell bodies in the hypothalamus which project their axons to the posterior pituitary. 9. Under current clamp, evoked action potential duration increased (122%) upon application of 5 mM-4-AP, indicating that IA is involved in neurohypophysial spike repolarization. 10. The existence of this current could help explain why maximal peptide release only occurs in response to bursts of electrical activity invading the nerve terminals.
4-Aminopyridine↗
Stimulation of an individual cell with peptide hormone in a prescribed region of its plasma membrane results in a compartmentalized cyclic AMP-dependent protein kinase response.
This work describes the stimulation by a peptide hormone of an individual cell in a prescribed region of its plasma membrane. When Leydig cells were stimulated via a section of membrane tightly sealed to an electrode containing LH, a very localized area exhibited the morphological change known as 'rounding up', which is a cyclic AMP-dependent protein kinase-mediated response. This localized stimulation did not produce a wider response through intracellular, intermembranous or extracellular signals. Each individual cell responded to peptide stimulation gradually, with an increase over time and with dose. In contrast, when the stimulation was accomplished using a non-hydrolysable cyclic AMP analogue in the patch electrode, a general response throughout an individual cell was produced. Locally stimulated peptide hormone receptors, adenylate cyclases and cyclic AMP-dependent protein kinases appear to be closely associated so that second messenger production and the effects it mediates are compartmentalized.
Cell Compartmentation↗
Calcium currents and peptide release from neurohypophysial terminals are inhibited by ethanol.
The effects of EtOH on peptide release and on high-threshold, voltage-activated calcium (Ca++) channels were examined in acutely dissociated rat neurohypophysial terminals. These terminals release the peptide hormones, arginine vasopressin (AVP) and oxytocin. Release of AVP from isolated intact neurohypophyses, induced by either electrical stimulation or elevated potassium, was inhibited by clinically relevant concentrations of EtOH. "Whole-cell" patch-clamp recording methods were used to study the effects of EtOH on voltage-activated Ca++ currents (ICa) in the peptidergic nerve terminals. Amplitudes of both fast-inactivating ICa and long-lasting ICa were reduced in EtOH, and the reduction in ICa did not result from a shift in its current-voltage or steady-state inactivation relationships. Only the fast-inactivating component recovered after removal of EtOH. The effects of EtOH on ICa could not be attributed to changes in osmolarity. In contrast to ICa, the fast, transient K+ current was insensitive to EtOH. These results suggest that EtOH-induced reduction of ICa in the peptidergic nerve terminals produces a decrease in AVP release, resulting in lowered plasma AVP levels.
Animals↗
Sodium-activated cation channels in peptidergic nerve terminals.
The patch-clamp technique was utilized to characterize a cation channel in peptidergic nerve terminals isolated from a crustacean neurosecretory system. The cation channel exhibits the unique property of being activated by [Na+]. Distributions of open times demonstrate the presence of two open states with a shift of the distribution from predominantly short open times at [Na+] less than or equal to 10 mM to a predominantly long open state at [Na+] greater than or equal to 40 mM. Desensitization of channel activation occurs on prolonged exposure to [Na+] greater than 40 mM. Open probability increased steeply with [Na+] but was largely independent of membrane potential. Comparison of current-voltage relationships from single dissociated terminals and from those in the intact system show no differences in conductance or selectivity with nearly equal permeability to Na+ and K+, and impermeability to Cs+, divalent cations and anions. Flickering block occurred with [Ca2+]i greater than 1 microM. We propose that Na-activated cation (NAC) channels are activated by Na+ entering during action potentials and provide a sustained depolarizing current that can help sustain repetitive or bursting activity and subsequent facilitation of secretion from these nerve terminals.
Animals↗