Govindan Dayanithi

A rise in the intracellular Ca2+ concentration of isolated rat supraoptic cells in response to oxytocin.

1. Intracellular Ca2+ concentration ([Ca2+]i) was monitored in single cells isolated from adult rat supraoptic (SO) nuclei. The great majority of cells (85%) were neurones and most were immunoreactive to oxytocin or to vasopressin (AVP). 2. The resting [Ca2+]i of the majority (80%) of the neurones remained stable while 20% of the neurones displayed spontaneous [Ca2+]i oscillations which disappeared in low‐Ca2+ (100 nM) EGTA buffer. 3. Addition of 100 nM oxytocin increased the [Ca2+]i in both stable and oscillating cells. Two types of responses were observed: (i) a sustained response with [Ca2+]i being maintained at an elevated level and (ii) a brief response with [Ca2+]i quickly returning to a near‐resting level. Responses were reproducible, dose dependent and blocked with a specific oxytocin antagonist. 4. Removal of extracellular Ca2+ did not block the oxytocin response. In EGTA buffer, application of thapsigargin (200 nM) onto oxytocin‐sensitive cells induced an increase in [Ca2+]i and inhibited the oxytocin response. These effects were not induced by other intracellular Ca2+ mobilizers such as tBuBHQ (see Methods) or caffeine. 5. In conclusion, half of the SO cells respond to oxytocin with a rise in [Ca2+]i. The effect is mediated by oxytocin receptors and results from release of Ca2+ from thapsigargin‐sensitive stores.

Hormone release from isolated nerve endings of the rat neurohypophysis.

1. Isolated neurosecretory nerve endings were prepared from rat neurohypophyses. The amount of vasopressin (AVP) and oxytocin released was measured by radioimmunoassay. 2. The amount of hormone release under resting conditions was not affected by external calcium (Ca2+o). Secretion decreased by ca. 50% when external sodium (Na+o) was replaced by choline or sucrose. 3. Ouabain did not modify the basal AVP release. 4. The Na+ ionophore monensin increased the release of AVP only in the presence of Na+o. This increase was maintained during prolonged exposure to the ionophore and occurred in the presence of Ca2+o only. 5. In the presence of Ca2+o, the amount of evoked hormone release was dependent on the external K+ concentration. Half‐maximal activation was achieved with ca. 40 mM‐K+. The K+‐induced secretion was potentiated in Na+‐free solution. 6. Prolonged 100 mM‐K+‐induced depolarization in the presence of Ca2+o gave rise to a large increase in hormone secretion which decreased with time (t1/2 = 2.5 min). The release could be reactivated after permeabilization of the nerve terminals in the presence of micromolar concentrations of Ca2+. 7. A stepwise paradigm in which Ko+ is incrementally increased to 25, 50, 75 and then 100 mM released more AVP than a prolonged exposure to 100 mM‐K+. 8. Veratridine increased the amount of AVP released. This effect was considerably reduced in the absence of Nao+ and abolished in the presence of D600. 9. The depolarization‐induced AVP release was blocked by different Ca2+‐antagonists. Their effectiveness was nitrendipine = nicardipine greater than Cd2+ greater than Gd3+ greater than Co2+ = Mn2+. 10. The dihydropyridine Bay K 8644 potentiated both the basal and the K+‐evoked AVP release. Its maximal effect was obtained with 25‐50 mM‐Ko+. 11. In conclusion, the isolated neurohypophysial terminals which have both Na+ and Ca2+ channels and release AVP and oxytocin upon depolarization might be an excellent system to study further the mechanisms leading to secretion of neurohormones.

Oxytocin: Crossing the Bridge between Basic Science and Pharmacotherapy

Is oxytocin the hormone of happiness? Probably not. However, this small nine amino acid peptide is involved in a wide variety of physiological and pathological functions such as sexual activity, penile erection, ejaculation, pregnancy, uterus contraction, milk ejection, maternal behavior, osteoporosis, diabetes, cancer, social bonding, and stress, which makes oxytocin and its receptor potential candidates as targets for drug therapy. In this review, we address the issues of drug design and specificity and focus our discussion on recent findings on oxytocin and its heterotrimeric G protein‐coupled receptor OTR. In this regard, we will highlight the following topics: (i) the role of oxytocin in behavior and affectivity, (ii) the relationship between oxytocin and stress with emphasis on the hypothalamo–pituitary–adrenal axis, (iii) the involvement of oxytocin in pain regulation and nociception, (iv) the specific action mechanisms of oxytocin on intracellular Ca2+ in the hypothalamo neurohypophysial system (HNS) cell bodies, (v) newly generated transgenic rats tagged by a visible fluorescent protein to study the physiology of vasopressin and oxytocin, and (vi) the action of the neurohypophysial hormone outside the central nervous system, including the myometrium, heart and peripheral nervous system. As a short nine amino acid peptide, closely related to its partner peptide vasopressin, oxytocin appears to be ideal for the design of agonists and antagonists of its receptor. In addition, not only the hormone itself and its binding to OTR, but also its synthesis, storage and release can be endogenously and exogenously regulated to counteract pathophysiological states. Understanding the fundamental physiopharmacology of the effects of oxytocin is an important and necessary approach for developing a potential pharmacotherapy.

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.

Interaction of SNX482 with Domains III and IV Inhibits Activation Gating of α1E (CaV2.3) Calcium Channels

We have investigated the action of SNX482, a toxin isolated from the venom of the tarantula Hysterocrates gigas, on voltage-dependent calcium channels expressed in tsa-201 cells. Upon application of 200 nM SNX482, R-type alpha(1E) calcium channels underwent rapid and complete inhibition, which was only poorly reversible upon washout. However, upon application of strong membrane depolarizations, rapid and complete recovery from inhibition was obtained. Tail current analysis revealed that SNX482 mediated an approximately 70 mV depolarizing shift in half-activation potential, suggesting that the toxin inhibits alpha(1E) calcium channels by preventing their activation. Experiments involving chimeric channels combining structural features of alpha(1E) and alpha(1C) subunits indicated that the presence of the domain III and IV of alpha(1E) is a prerequisite for a strong gating inhibition. In contrast, L-type alpha(1C) channels underwent incomplete inhibition at saturating concentrations of SNX482 that was paralleled by a small shift in half-activation potential and which could be rapidly reversed, suggesting a less pronounced effect of the toxin on L-type calcium channel gating. We conclude that SNX482 does not exhibit unequivocal specificity for R-type channels, but highly effectively antagonizes their activation.

Vasopressin-induced intracellular Ca2+ increase in isolated rat supraoptic cells.

1. The intracellular Ca2+ concentration ([Ca2+]1) was monitored in single magnocellular neurones freshly isolated from rat supraoptic nucleus. Application of 100 nM vasopressin increased [Ca2+]1. Two types of [Ca2+]1 responses were observed: (i) a transient response, displayed by 86% of the vasopressin‐sensitive neurones, and (ii) a sustained response displayed by 14% of the vasopressin‐sensitive neurones. 2. Among responding neurones, 52% were vasopressin sensitive, 44% were oxytocin sensitive and 4% were sensitive to both peptides. 3. Responses to vasopressin were dose dependent, showed a progressive desensitization after successive applications, were specifically blocked by the V1a vasopressin receptor antagonist, SR 49059, and were unaffected by the oxytocin receptor antagonist, d(CH2)5OVT. 4. Vasopressin responses were completely suppressed by the removal of external Ca2+. 5. The intracellular Ca2+ mobilizers, caffeine and tBuBHQ, did not affect resting or vasopressin‐induced [Ca2+]1 changes. Thapsigargin (200 nM) on its own evoked an increase in [Ca2+]1, and reduced the [Ca2+]1 increase evoked by vasopressin by 52%, suggesting that thapsigargin‐sensitive Ca2+ stores are partially involved in the vasopressin response. 6. Immunocytochemical identification revealed that vasopressin‐responding neurones synthesize vasopressin whereas oxytocin‐responding neurones synthesize oxytocin. 7. In conclusion, vasopressin‐ (partially external Ca2+ dependent) and oxytocin (totally external Ca2+ independent)‐induced [Ca2+]1 changes are mediated by specific receptors. In addition, vasopressin and oxytocin neurones are specifically autoregulated by their own peptides.

Regulation of activity-dependent dendritic vasopressin release from rat supraoptic neurones

Magnocellular neurones of the hypothalamus release vasopressin and oxytocin from their dendrites and soma. Using a combination of electrophysiology, microdialysis, in vitro explants, and radioimmunoassay we assessed the involvement of intracellular Ca2+ stores in the regulation of dendritic vasopressin release. Thapsigargin and cyclopiazonic acid, which mobilize Ca2+ from intracellular stores of the endoplasmic reticulum, evoked vasopressin release from dendrites and somata of magnocellular neurones in the supraoptic nucleus. Thapsigargin also produced a dramatic potentiation of dendritic vasopressin release evoked by osmotic or high potassium stimulation. This effect is long lasting, time dependent, and specific to thapsigargin as caffeine and ryanodine had no effect. Furthermore, antidromic activation of electrical activity in the cell bodies released vasopressin from dendrites only after thapsigargin pretreatment. Thus, exposure to Ca2+ mobilizers such as thapsigargin or cyclopiazonic acid primes the releasable pool of vasopressin in the dendrites, so that release can subsequently be evoked by electrical and depolarization‐dependent activation. Vasopressin itself is effective in inducing dendritic vasopressin release, but it is ineffective in producing priming.

ATP-evoked increases in [Ca2+]i and peptide release from rat isolated neurohypophysial terminals via a P2X2 purinoceptor.

1 The effect of externally applied ATP on cytosolic free Ca2+ concentration ([Ca2+]i) was tested in single isolated rat neurohypophysial nerve terminals by fura‐2 imaging. The release of vasopressin (AVP) and oxytocin (OT) upon ATP stimulation was also studied from a population of terminals using specific radioimmunoassays. 2 ATP evoked a sustained [Ca2+]i increase, which was dose dependent in the 1‐100 μM range (EC50= 4·8 μM). This effect was observed in only ≈40 % of the terminals. 3 Interestingly, ATP, in the same range (EC50= 8·6 μM), evoked AVP, but no significant OT, release from these terminals. 4 Both the [Ca2+]i increase and AVP release induced by ATP were highly and reversibly inhibited by suramin, suggesting the involvement of a P2 purinergic receptor in the ATP‐induced responses. Pyridoxal‐5‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), another P2 purinergic receptor antagonist, strongly reduced the ATP‐induced [Ca2+]i response. 5 To further characterize the receptor, different agonists were tested, with the following efficacy: ATP = 2‐methylthio‐ATP > ATP‐γ‐S > α,β‐methylene‐ATP > ADP. The compounds adenosine, AMP, β,γ‐methylene‐ATP and UTP were ineffective. 6 The ATP‐dependent [Ca2+]i increase was dependent on extracellular Ca2+ concentration ([Ca2+]o). Fluorescence‐quenching experiments with Mn2+ showed that externally applied ATP triggered a Mn2+ influx. The ATP‐induced [Ca2+]i increase and AVP release were independent of and additive to a K+‐induced response, in addition to being insensitive to Cd2+. The ATP‐induced [Ca2+]i increase was strongly reduced in the presence of Gd3+. These results suggest that the observed [Ca2+]i increases were elicited by Ca2+ entry through a P2X channel receptor rather than via a voltage‐dependent Ca2+ channel. 7 We propose that ATP, co‐released with neuropeptides, could act as a paracrine‐autocrine messenger, stimulating, via Ca2+ entry through a P2X2 receptor, the secretion of AVP, in particular, from neurohypophysial nerve terminals.

L‐, N‐ and T‐ but neither P‐ nor Q‐type Ca2+ Channels Control Vasopressin‐Induced Ca2+ Influx in Magnocellular Vasopressin Neurones Isolated from the Rat Supraoptic Nucleus

1 The role of voltage‐dependent Ca2+ channels during vasopressin and oxytocin actions on their respective neurones has been analysed by measuring intracellular Ca2+ concentration ([Ca2+]i) in individual, freshly dissociated magnocellular neurones from rat supraoptic nucleus (SO) using microspectrofluorimetry. 2 Pre‐incubation of vasopressin‐sensitive neurones with Cd2+ (100μM), a non‐discriminatory high‐voltage‐activated Ca2+ channel antagonist, or Ni2+ (50μM), a blocker of T‐type Ca2+ current, reduced [Ca2+]i responses by 77 and 19%, respectively. When Cd2+ was given together with Ni2+, the response was blocked by 92%. Similarly, when Ni2+ was pre‐incubated with Cd2+, the response was blocked by ∼84%. 3 Exposure of vasopressin‐sensitive neurones to a specific Ca2+ channel blocker, nicardipine (L‐type) reduced vasopressin responses by 48% at 1μM and 62% at 5μM. Similarly, ω‐conotoxin GVIA (ω‐CgTX, N‐type; 500 nm) inhibited the response by 46% with a stronger inhibition (75%) at 800 nm. By contrast, neither ω‐agatoxin IVA (ω‐Aga IVA; 300 nm), which blocks both P‐ and Q‐type channels, nor synthetic ω‐conotoxin MVIIC (ω‐MVIIC; 100 or 500 nm), a Q‐type blocker, affected vasopressin‐induced [Ca2+]i responses. These antagonists, given together (nicardipine 5μM +ω‐CgTX 800 nm+ω‐Aga IVA 300 nm), decreased vasopressin‐induced [Ca2+]i responses by 76 %. 4 In vasopressin‐sensitive neurones, the presence of both nicardipine and ω‐CgTX, reduced the K+‐evoked [Ca2+]i increase by 61 %. This blockade was increased by a further 21 % with ω‐Aga IVA, suggesting that N‐, L‐ and P‐type channels contribute to the depolarization‐induced [Ca2+]i rise. In addition, ω‐MVIIC alone reduced the K+‐evoked [Ca2+]i release by 24%. Also the remaining K+ responses were further reduced by 60% when pre‐incubated with L‐ N‐ and P‐type blockers, suggesting the involvement of Q‐type channels. 5 In oxytocin‐sensitive neurones, the peak amplitude of the [Ca2+]i response was not affected by Cd2+ alone, by combined Cd2+ and Ni2+, or by the mixture of nicardipine, ω‐CgTX and ω‐Aga IVA. By contrast, the responses evoked by depolarization with K+ were blocked by Cd2+. Both nicardipine and ω‐CgTX blocked 65% of K+ response and an additional block of ∼18% was obtained with ω‐Aga IVA, suggesting the involvement of L‐, N‐ and P‐type channels. In combination, these antagonists strongly inhibited (∼80% reduction) the K+ responses. Further reduction to 18% was made by the Q‐type blocker ω‐MVIIC. Pre‐incubation with L‐, N‐ and P‐type blockers caused an additional block of 71 %. 6 Some supraoptic neurones (5–10%) responded to both vasopressin and oxytocin, with only the [Ca2+]i responses induced by vasopressin blocked (> 90% inhibition) by the mixture of Ca2+ channel antagonists. 7 In conclusion, both vasopressin and oxytocin magnocellular SO neurones have been shown to express T‐, L‐, N‐, P‐, Q‐ and R‐type Ca2+ channels in their somata. Our results show that the vasopressin‐induced [Ca2+]i increase in vasopressin‐sensitive neurones is mediated by L‐, N‐and T‐type Ca2+ channels and not by P‐ and Q‐type channels; Ca2+ channels are not involved in oxytocin action on oxytocin‐sensitive neurones and L‐, N‐, P‐ and Q‐type channels control the K+‐induced [Ca2+]i increase in SO neurones.

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 EtOHs 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 EtOHs inhibition of calcium currents in the nerve terminals.