Adolfo E. Cuadra

Modulation/physiology of calcium channel sub-types in neurosecretory terminals.

The hypothalamic-neurohypophysial system (HNS) controls diuresis and parturition through the release of arginine-vasopressin (AVP) and oxytocin (OT). These neuropeptides are chiefly synthesized in hypothalamic magnocellular somata in the supraoptic and paraventricular nuclei and are released into the blood stream from terminals in the neurohypophysis. These HNS neurons develop specific electrical activity (bursts) in response to various physiological stimuli. The release of AVP and OT at the level of neurohypophysis is directly linked not only to their different burst patterns, but is also regulated by the activity of a number of voltage-dependent channels present in the HNS nerve terminals and by feedback modulators. We found that there is a different complement of voltage-gated Ca(2+) channels (VGCC) in the two types of HNS terminals: L, N, and Q in vasopressinergic terminals vs. L, N, and R in oxytocinergic terminals. These channels, however, do not have sufficiently distinct properties to explain the differences in release efficacy of the specific burst patterns. However, feedback by both opioids and ATP specifically modulate different types of VGCC and hence the amount of AVP and/or OT being released. Opioid receptors have been identified in both AVP and OT terminals. In OT terminals, μ-receptor agonists inhibit all VGCC (particularly R-type), whereas, they induce a limited block of L-, and P/Q-type channels, coupled to an unusual potentiation of the N-type Ca(2+) current in the AVP terminals. In contrast, the N-type Ca(2+) current can be inhibited by adenosine via A(1) receptors leading to the decreased release of both AVP and OT. Furthermore, ATP evokes an inactivating Ca(2+)/Na(+)-current in HNS terminals able to potentiate AVP release through the activation of P2X2, P2X3, P2X4 and P2X7 receptors. In OT terminals, however, only the latter receptor type is probably present. We conclude by proposing a model that can explain how purinergic and/or opioid feedback modulation during bursts can mediate differences in the control of neurohypophysial AVP vs. OT release.

P2X purinergic receptor knockout mice reveal endogenous ATP modulation of both vasopressin and oxytocin release from the intact neurohypophysis.

Bursts of action potentials are crucial for neuropeptide release from the hypothalamic neurohypophysial system (HNS). The biophysical properties of the ion channels involved in the release of these neuropeptides, however, cannot explain the efficacy of such bursting patterns on secretion. We have previously shown that ATP, acting via P2X receptors, potentiates only vasopressin (AVP) release from HNS terminals, whereas its metabolite adenosine, via A1 receptors acting on transient Ca2+ currents, inhibits both AVP and oxytocin (OT) secretion. Thus, purinergic feedback‐mechanisms have been proposed to explain bursting efficacy at HNS terminals. Therefore, in the present study, we have used specific P2X receptor knockout (rKO) mice and purportedly selective P2X receptor antagonists to determine the P2X receptor subtype responsible for endogenous ATP induced potentiation of electrically‐stimulated neuropeptide release. Intact neurohypophyses (NH) from wild‐type (WT), P2X3 rKO, P2X2/3 rKO and P2X7 rKO mice were electrically stimulated with four 25‐s bursts (3 V at 39 Hz) separated by 21‐s interburst intervals with or without the P2X2 and P2X3 receptor antagonists, suramin or pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS). These frequencies, number of bursts, and voltages were determined to maximise both AVP and OT release by electrical stimulations. Treatment of WT mouse NH with suramin/PPADS significantly reduced electrically‐stimulated AVP release. A similar inhibition by suramin was observed in electrically‐stimulated NH from P2X3 and P2X7 rKO mice but not P2X2/3 rKO mice, indicating that endogenous ATP facilitation of electrically‐stimulated AVP release is mediated primarily by the activation of the P2X2 receptor. Unexpectedly, electrically‐stimulated OT release from WT, P2X3, P2X2/3 and P2X7 rKO mice was potentiated by suramin, indicating nonpurinergic effects by this ‘selective’ antagonist. Nevertheless, these results show that sufficient endogenous ATP is released by bursts of action potentials to act at P2X2 receptors in a positive‐feedback mechanism to ‘differentially’ modulate neuropeptide release from central nervous system terminals.

Adenosine trisphosphate appears to act via different receptors in terminals versus somata of the hypothalamic neurohypophysial system.

ATP‐induced ionic currents were investigated in isolated terminals and somata of the hypothalamic neurohypophysial system (HNS). Both terminals and somata showed inward rectification of the ATP‐induced currents and reversal near 0 mV. In terminals, ATP dose‐dependently evoked an inactivating, inward current. However, in hypothalamic somata, ATP evoked a very slowly inactivating, inward current with a higher density, and different dose dependence (EC50 of 50 μm in somata versus 9.6 μm in terminals). The ATP‐induced currents, in both the HNS terminals and somata, were highly and reversibly inhibited by suramin, suggesting the involvement of a purinergic receptor (P2XR). However, the suramin inhibition was significantly different in the two HNS compartments (IC50 of 3.6 μm in somata versus 11.6 μm in terminals). Also, both HNS compartments show significantly different responses to the purinergic receptor agonists: ATP‐γ‐S and benzoyl‐benzoyl‐ATP. Finally, there was an initial desensitisation to ATP upon successive stimulations in the terminals, which was not observed in the somata. These differences in EC50, inactivation, desensitisation and agonist sensitivity in terminals versus somata indicate that different P2X receptors mediate the responses in these two compartments of HNS neurones. Previous work has revealed mRNA transcripts for multiple purinergic receptors in micropunches of the hypothalamus. In the HNS terminals, the P2X purinergic receptor types P2X2, 3, 4 and 7 (but not 6) have been shown to exist in AVP terminals. Immonohistochemistry now indicates that P2X4R is only present in AVP terminals and that the P2X7R is found in both AVP and oxytocin terminals and somata. We speculate that these differences in receptor types reflects the specific function of endogenous ATP in the terminals versus somata of these central nervous system neurones.

P2X7 receptors in neurohypophysial terminals: evidence for their role in arginine-vasopressin secretion.

Arginine‐vasopressin (AVP) plays a major role in maintaining cardiovascular function and related pathologies. The mechanism involved in its release into the circulation is complex and highly regulated. Recent work has implicated the purinergic receptor, P2X7R, in a role for catecholamine‐enhanced AVP release in the rat hypothalamic‐neurohypophysial (NH) system. However, the site of P2X7R action in this endocrine system, and whether or not it directly mediates release in secretory neurons have not been determined. We hypothesized that the P2X7R is expressed and mediates AVP release in NH terminals. P2X7R function was first examined by patch‐clamp recordings in isolated NH terminals. Results revealed that subpopulations of isolated terminals displayed either high ATP‐sensitivity or low ATP‐sensitivity, the latter of which was characteristic of the rat P2X7R. Additional recordings showed that terminals showing sensitivity to the P2X7R‐selective agonist, BzATP, were further inhibited by P2X7R selective antagonists, AZ10606120 and brilliant blue‐G. In confocal micrographs from tissue sections and isolated terminals of the NH P2X7R‐immunoreactivity was found to be localized in plasma membranes. Lastly, the role of P2X7R on AVP release was tested. Our results showed that BzATP evoked sustained AVP release in NH terminals, which was inhibited by AZ10606120. Taken together, our data lead us to conclude that the P2X7R is expressed in NH terminals and corroborates its role in AVP secretion. J. Cell. Physiol. 229: 333–342, 2014.

ATP appears to act via different receptors in terminals vs. somata of the Hypothalamic Neurohypophysial System

ATP‐induced ionic currents were investigated in isolated terminals and somata of the hypothalamic neurohypophysial system (HNS). Both terminals and somata showed inward rectification of the ATP‐induced currents and reversal near 0 mV. In terminals, ATP dose‐dependently evoked an inactivating, inward current. However, in hypothalamic somata, ATP evoked a very slowly inactivating, inward current with a higher density, and different dose dependence (EC50 of 50 μm in somata versus 9.6 μm in terminals). The ATP‐induced currents, in both the HNS terminals and somata, were highly and reversibly inhibited by suramin, suggesting the involvement of a purinergic receptor (P2XR). However, the suramin inhibition was significantly different in the two HNS compartments (IC50 of 3.6 μm in somata versus 11.6 μm in terminals). Also, both HNS compartments show significantly different responses to the purinergic receptor agonists: ATP‐γ‐S and benzoyl‐benzoyl‐ATP. Finally, there was an initial desensitisation to ATP upon successive stimulations in the terminals, which was not observed in the somata. These differences in EC50, inactivation, desensitisation and agonist sensitivity in terminals versus somata indicate that different P2X receptors mediate the responses in these two compartments of HNS neurones. Previous work has revealed mRNA transcripts for multiple purinergic receptors in micropunches of the hypothalamus. In the HNS terminals, the P2X purinergic receptor types P2X2, 3, 4 and 7 (but not 6) have been shown to exist in AVP terminals. Immonohistochemistry now indicates that P2X4R is only present in AVP terminals and that the P2X7R is found in both AVP and oxytocin terminals and somata. We speculate that these differences in receptor types reflects the specific function of endogenous ATP in the terminals versus somata of these central nervous system neurones.

P2X Purinergic Receptor Knockout Mice Reveal Endogenous ATP Modulation of Both AVP and OT Release from the Intact Neurohypophysis

Bursts of action potentials are crucial for neuropeptide release from the hypothalamic neurohypophysial system (HNS). The biophysical properties of the ion channels involved in the release of these neuropeptides, however, cannot explain the efficacy of such bursting patterns on secretion. We have previously shown that ATP, acting via P2X receptors, potentiates only vasopressin (AVP) release from HNS terminals, whereas its metabolite adenosine, via A1 receptors acting on transient Ca2+ currents, inhibits both AVP and oxytocin (OT) secretion. Thus, purinergic feedback‐mechanisms have been proposed to explain bursting efficacy at HNS terminals. Therefore, in the present study, we have used specific P2X receptor knockout (rKO) mice and purportedly selective P2X receptor antagonists to determine the P2X receptor subtype responsible for endogenous ATP induced potentiation of electrically‐stimulated neuropeptide release. Intact neurohypophyses (NH) from wild‐type (WT), P2X3 rKO, P2X2/3 rKO and P2X7 rKO mice were electrically stimulated with four 25‐s bursts (3 V at 39 Hz) separated by 21‐s interburst intervals with or without the P2X2 and P2X3 receptor antagonists, suramin or pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS). These frequencies, number of bursts, and voltages were determined to maximise both AVP and OT release by electrical stimulations. Treatment of WT mouse NH with suramin/PPADS significantly reduced electrically‐stimulated AVP release. A similar inhibition by suramin was observed in electrically‐stimulated NH from P2X3 and P2X7 rKO mice but not P2X2/3 rKO mice, indicating that endogenous ATP facilitation of electrically‐stimulated AVP release is mediated primarily by the activation of the P2X2 receptor. Unexpectedly, electrically‐stimulated OT release from WT, P2X3, P2X2/3 and P2X7 rKO mice was potentiated by suramin, indicating nonpurinergic effects by this ‘selective’ antagonist. Nevertheless, these results show that sufficient endogenous ATP is released by bursts of action potentials to act at P2X2 receptors in a positive‐feedback mechanism to ‘differentially’ modulate neuropeptide release from central nervous system terminals.

P2X Purinergic Receptor Knockout Mice Reveal Endogenous ATP Modulation of Both Vasopressin and Oxytocin Release from the Intact Neurohypophysis: P2XR KOs reveal ATP modulation of neurohypophysial release

Bursts of action potentials are crucial for neuropeptide release from the hypothalamic neurohypophysial system (HNS). The biophysical properties of the ion channels involved in the release of these neuropeptides, however, cannot explain the efficacy of such bursting patterns on secretion. We have previously shown that ATP, acting via P2X receptors, potentiates only vasopressin (AVP) release from HNS terminals, whereas its metabolite adenosine, via A1 receptors acting on transient Ca2+ currents, inhibits both AVP and oxytocin (OT) secretion. Thus, purinergic feedback‐mechanisms have been proposed to explain bursting efficacy at HNS terminals. Therefore, in the present study, we have used specific P2X receptor knockout (rKO) mice and purportedly selective P2X receptor antagonists to determine the P2X receptor subtype responsible for endogenous ATP induced potentiation of electrically‐stimulated neuropeptide release. Intact neurohypophyses (NH) from wild‐type (WT), P2X3 rKO, P2X2/3 rKO and P2X7 rKO mice were electrically stimulated with four 25‐s bursts (3 V at 39 Hz) separated by 21‐s interburst intervals with or without the P2X2 and P2X3 receptor antagonists, suramin or pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS). These frequencies, number of bursts, and voltages were determined to maximise both AVP and OT release by electrical stimulations. Treatment of WT mouse NH with suramin/PPADS significantly reduced electrically‐stimulated AVP release. A similar inhibition by suramin was observed in electrically‐stimulated NH from P2X3 and P2X7 rKO mice but not P2X2/3 rKO mice, indicating that endogenous ATP facilitation of electrically‐stimulated AVP release is mediated primarily by the activation of the P2X2 receptor. Unexpectedly, electrically‐stimulated OT release from WT, P2X3, P2X2/3 and P2X7 rKO mice was potentiated by suramin, indicating nonpurinergic effects by this ‘selective’ antagonist. Nevertheless, these results show that sufficient endogenous ATP is released by bursts of action potentials to act at P2X2 receptors in a positive‐feedback mechanism to ‘differentially’ modulate neuropeptide release from central nervous system terminals.

Adenosine Trisphosphate Appears to Act via Different Receptors in Terminals Versus Somata of the Hypothalamic Neurohypophysial System: Differential ATP effects on terminals versus somata

ATP‐induced ionic currents were investigated in isolated terminals and somata of the hypothalamic neurohypophysial system (HNS). Both terminals and somata showed inward rectification of the ATP‐induced currents and reversal near 0 mV. In terminals, ATP dose‐dependently evoked an inactivating, inward current. However, in hypothalamic somata, ATP evoked a very slowly inactivating, inward current with a higher density, and different dose dependence (EC50 of 50 μm in somata versus 9.6 μm in terminals). The ATP‐induced currents, in both the HNS terminals and somata, were highly and reversibly inhibited by suramin, suggesting the involvement of a purinergic receptor (P2XR). However, the suramin inhibition was significantly different in the two HNS compartments (IC50 of 3.6 μm in somata versus 11.6 μm in terminals). Also, both HNS compartments show significantly different responses to the purinergic receptor agonists: ATP‐γ‐S and benzoyl‐benzoyl‐ATP. Finally, there was an initial desensitisation to ATP upon successive stimulations in the terminals, which was not observed in the somata. These differences in EC50, inactivation, desensitisation and agonist sensitivity in terminals versus somata indicate that different P2X receptors mediate the responses in these two compartments of HNS neurones. Previous work has revealed mRNA transcripts for multiple purinergic receptors in micropunches of the hypothalamus. In the HNS terminals, the P2X purinergic receptor types P2X2, 3, 4 and 7 (but not 6) have been shown to exist in AVP terminals. Immonohistochemistry now indicates that P2X4R is only present in AVP terminals and that the P2X7R is found in both AVP and oxytocin terminals and somata. We speculate that these differences in receptor types reflects the specific function of endogenous ATP in the terminals versus somata of these central nervous system neurones.